New Record of the Distribution of Saccharina Japonica (Laminariales, Phaeophyceae) in Hokkaido, Japan

Research Note Algal Resources (2019) 12：67-72

New record of the distribution of Saccharina japonica (Laminariales, Phaeophyceae) in Hokkaido, Japan

Norishige YOTSUKURA1 *, Takashi MAEDA2 and Tadashi KAWAI3

Abstract : The distribution of a saccharinan kelp that is similar to Saccharina japonica was confirmed in Hidaka region, Hokkaido. As a result of amplified fragment length polymorphism (AFLP) analysis for the study individuals from Utoma in Hidaka region and the four varieties of S. japonica, genetic similarity (S) shows significantly higher val-

ue and genetic differentiation coefficient (GST) shows lower value both between the study individuals and S. japonica var. japonica. The structure analysis based on the limited AFLP data about the study individuals and S. japonica var. japonica showed two clus- tersinallsamples.Theclusterdominantinthestudyindividualswasthesameasthe one dominant in the individuals collected from the inner part of the Uchiura Bay. Accordingly, the study individuals should be treated as S. japonica var. japonica and it is inferred that the individuals were introduced from the vicinity of boundary of Iburi and Oshima regions to Hidaka region.

Keywords : AFLP, distributional area, Hidaka region, Saccharina japonica var. japonica

The coasts of Hokkaido have species diversity with S. japonica var. religiosa (Miyabe) N. of saccharinan kelps that is among the highest Yotsukura, S. Kawashima, T. Kawai, T. Abe & in the world. Along the coastline of Hidaka L. D. Druehl S. japonica var. ochotensis (Miyabe) region on the Pacific, Saccharina angusutata N. Yotsukura, S. Kawashima, T. Kawai, T. Abe (Kjellman) C. E. Lane, C. Mayes, L. D. Druehl & & L. D. Druehl and S. japonica var. diabolica G. W. Saunders forms marine forests, suppor- (Miyabe) N. Yotsukura, S. Kawashima, T. Kawai, ting localmarineecosystemsaswellasserving T. Abe & L. D. Druehl being the local varieties as important fishery resources (Kawashima 2004, (Yotsukura et al. 2008). The varieties in Saccharina Yotsukura et al. 1999). In recent years, in the sea japonica lack stable morphological characteris- west of Cape Erimo in Hidaka region, a con- tics and these significantly vary depending on siderable amount of kelp similar to S. japonica where they grow; therefore, it is difficult to (Areschoug) C. E. Lane, C. Mayes, L. D. Druehl & visually differentiate them from one another in G. W. Saunders but clearly morphologically dif- an objective manner (Sanbonsuga 1978, San- ferent from S. angusutata has been confirmed bonsuga and Torii 1973, 1974, Funano 1991, (Figure 1) and harvested by fishermen. For S. Yotsukura et al. 2008). Yotsukura et al. (2016) japonica, the standard variety (var. japonica) investigated genetic structure of S. japonica var. of the species is known to grow on the coasts japonica and its varieties collected from vari- off Iburi and Oshima regions around Hokkaido, ous locations on the coasts of Hokkaido using

1 Field Science Center for Northern Biosphere, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo, Hokkaido 060-0809, Japan 2 Hakodate Fisheries Research Institute, Hokkaido Research Organization, 20-5 Benten-cho, Hakodate, Hokkaido 040-0051, Japan 3 Central Fisheries Research Institute, Hokkaido Research Organization, 238 Hamanaka-cho, Yoichi, Hokkaido 046-8555, Japan ＊Corresponding author : Tel: +81-11-706-2535, fax: +81-11-706-3450, e-mail: [email protected]

67 Norishige YOTSUKURA, Takashi MAEDA and Tadashi KAWAI

Fig. 1. Saccharinan kelps in Hidaka region; A: Sporophytes resembling that of Saccharina japonica characterized by the linear-lanceolate form, the undulate marginal portion of blade, the broad median fascia, and the developed mucilage canals in both stipe and blade, B: Sporophytes of S. angustata characterized by the linear form, the non- or slightly-undulate marginal portion of blade, the narrow median fascia, and the underdeveloped mucilage canals in both stipe and blade microsatellite marker and amplified fragment length polymorphism (AFLP) analyses. Although the authors used individuals collected from locations off Hidaka region, they did not re- port findings useful for species identification. In the present study, therefore, AFLP analysis was performed on individuals found in the study area, Hidaka region, to identify them and esti- mate their origins. The sporophytes were collected on the coast of Utoma in Samani Town - S. japonica var. japonica and the three varieties religiosa, ochotensis and diabolica collected from various locations of Hokkaido were used for comparison (Figure 2, Table 1). These samples were taken to the laboratory immediately after collection, Fig. 2. Map of Hokkaido showing the collecting sites of samples used in this study (●). I: Hidaka where an immature section of the lamina region, II: Iburi region, III: Oshima region around the meristem was cut using a razor (sample size ca. 15 cm2). The cut section was

68 New record of the distribution of Saccharina japonica (Laminariales, Phaeophyceae) in Hokkaido, Japan

Table 1. Collectoin date and sites of samples used in this study

washed with sterile seawater, placed in a zip- facturer's instructions with slight modifications: pered plastic bag with silica gel and stored (1) 7 l of GLASSMILK suspension was added at -80 ℃. For DNA extraction, the 20-30 mg of regardless of the concentration of DNA extracts; tissue was cut into fragments about 0.5 mm2 in (2) samples were incubated for 10 min after size, which were then placed in a 2-ml micro- addition of GLASSMILK; (3) samples were centrifuge tube to be used as a sample. washed three times with NEW Wash; and (4) Extraction of genome DNA was carried out drying times for GLASSMILK were 8 min at according to the method that combined the cetyl room temperature and 3 min at 65 ℃. Detection trimethyl ammonium bromide (CTAB) and so- of AFLP was performed in accordance with the dium dodecyl sulfate (SDS) methods described method of Maeda et al. (2013) and, for selective by Maeda et al. (2013), and DNA was purified PCR, five selective primer pairs shown in using a GENECLEAN II Kit (Bio 101, Inc., La Yotsukura et al. (2016) were used. Sizing and Jolla, CA, USA) in accordance with the manu- typing of each fragment was carried out using

69 Norishige YOTSUKURA, Takashi MAEDA and Tadashi KAWAI

Gene Mapper ver. 4.1 and the band detection istence of two clusters (Figure 3). In the study threshold was set at 20 rfu and the presence or individuals, one cluster was dominant and this absence of detected fragments larger than 60 bp cluster was also dominant in individuals from was compared. Usu, Rebunge, Shizukari, Nakanokawa, Kotani- Among genetic diversity parameters, genetic ishi and Shiogama. similarity (S) was calculated in accordance with Accordingtopreviousstudies,S and GST the method described by Dice (1945) and calcu- values among the individuals of S. japonica var. lation of genetic differentiation coefficient (GST) japonica collected widely from northern Japan and analysis of molecular variance (AMOVA) to were 0.53 ± 0.1 and 0.27 ±0.113, respectively (ref. evaluate genetic differentiation among popula- Yotsukura et al. 2016). Based on the results of tions were carried out using Arlequin 3.11 the present study, it was appropriate to con- (Excoffier et al. 2005). Genetic structure based on sider the study individuals from Utoma as S. alleles was estimated for up to five samples per japonica var. japonica. The STRUCTURE anal- population using STRUCTURE ver. 2.4.3 ysis showed that the cluster dominant in the (Pritchard et al. 2000). For the STRUCTURE study individuals was the same as the one analysis, burn-in was set at 100,000 and Markov dominant in the individuals collected from Usu, Chain Monte Carlo (MCMC) simulation repeats Rebunge, Shizukari, Nakanokawa, Kotaniishi and was set at 1,000,000. An optimum number of Shiogama - between the study individuals and populations (K) was estimated by comparing the individuals from these locations, S value the likelihoods of K and ΔK (Evanno et al. 2005), was 0.65, 0.65, 0.63, 0.59, 0.58 and 0.46, respec- and ΔK was calculated using STRUCTURE tively, and correspondingly GST value was 0.32, HARVESTER web ver. 0.6.93 (Earl and von 0.18, 0.20, 0.30, 0.33 and 0.48. It was therefore Holdt 2012). suggested that the S. japonica var. japonica that The range of obtained S values between the have recently expanded into the water off Utoma study individuals and S. japonica var. Hidaka region derived from individuals that japonica was 0.44-0.65, and between the study grow in the inner parts of the Uchiura Bay, individuals and S. japonica varieties religiosa, such as the coasts of Rebunge and Shizukari. ochotensis and diabolica were 0.37-0.49, 0.39-0.47 Additionally, the AMOVA of GST value for S. and 0.33-0.48, respectively. The range of GST japonica var. japonica showed no significant dif- values between the Utoma individuals and S. ferences only between Usu and Rebunge. japonica var. japonica was 0.18-0.48, and were Distribution of S. japonica in Hokkaido was 0.44-0.50, 0.51-0.59 and 0.48-0.62 between the study first comprehensively investigated by Miyabe individuals and S. japonica varieties religiosa, (1902) and then by Yendo (1911), Hasegawa ochotensis and diabolica, respectively. Also, when (1959), Nabata and Akino (2003a, 2003b), Nabata STRUCTURE analysis was limited to the study and Abe (2003), Nabata and Takiya (2003) and individuals and S. japonica var. japonica, which Kawashima (1989, 2004). Collectively, these studies show high S values and low GST values with the showed that S. japonica var. japonica was dis- study individuals, the results supported the ex- tributed between Muroran City in Iburi region

Fig. 3. Genetic structure in Saccharina japonica var. japonica including individuals in Utoma estimated by Bayesian clustering analysis based on AFLP analysis. One pillar corresponds to one individual. Two clusters obtained were classified by different two colors

70 New record of the distribution of Saccharina japonica (Laminariales, Phaeophyceae) in Hokkaido, Japan and Fukushima Town or Matsumae Town in from the Ministry of Education, Science, Sports, Oshima region. The present study confirmed its and Culture, Japan. expansion to the coast of Hidaka region. Previ- ous studies reported that S values obtained from References AFLP analysis roughly reflected the geographi- cal relationships of S. japonica var. japonica (Shan AkinoH,KawaiT,YotsukuraN,KonoT. et al. 2011, Yotsukura et al. 2016). However, the Transportation and spatial distribution of western Pacific coastline of Hokkaido between Saccharina japonica var. religiosa zoospores Uchiura Bay and Hidaka region contains a sec- in surface waters off the coast of Tomari, tion of several dozens of kilometers where dis- Hokkaido, Sea of Japan. Fish. Engineer. 2015; tribution of saccharinan kelps is not confirmed 52 :1-9. (in Japanese) (Kawashima 1989) and, given that the estimated Dice LR. Measures of the amount of ecologic diffusion distance of zoospores of S. japonica on association between species. Ecology 1945 ; the coast of Shiribeshi region is between several 26 :297-302. hundred meters and several kilometers (Akino Earl DA, von Holdt BM. STRUCTURE et al. 2015), it is likely that the expansion in HARVESTER: a website and program for distribution was caused by fishing boats or work visualizing STRUCTURE output and im- boats engaged in port construction. The authors plementing the Evanno method. Conserv. confirmed the growth of similar individuals in Genet. Resour. 2012 ; 4 : 359-361. Shinhidaka and Urakawa Towns in Hidaka Evanno G, Regnaut S, Goudet J. Detecting the region and believe that the detailed process of number of clusters of individuals using the the expansion can be clarified by analyzing these software STRUCTURE: a simulation study. samples and thoroughly investigating port con- Mol. Ecol. 2005 ; 14 :2611-2620. struction works that have taken place at these Excoffier L, Laval G, Schneider S. Arlequin locations. (version 3.0): An integrated software pack- The present and previous studies collectively age for population genetics data analysis. showed that currently along the coastline of Evol. Bioinform. Online. 2005 ; 1 :47-50. Hokkaido, S. japonica var. japonica is distribu- Funano T. Morpholoical and ecological com- ted between Samani Town northwest of Cape parisons of Laminaria religiosa, asurvival Erimo and Fukushima Town southwest of the variation of L. japonica and that of L. Oshima Peninsula. The main producing coasts ochotensis from Oshoro, Sutts and Atsuta. are between Muroran City and western part of Suisanzoshoku. 1991 ; 39 :91-95. (in Japanese) Hakodate City. The AFLP analysis showed that Hasegawa Y. On the distribution of the useful individuals growing in the Japan Sea coasts off Laminariaceous plants in Hokkaido. Hoku- Iwanai Town and Kamoenai Village were ge- suishi-Geppo. 1959 ; 16 : 201-206. (in Japanese) netically close to S. japonica var. japonica but Kawashima S. Illustration book of Japanese the microsatellite marker analysis suggested that Laminariales. Kitanihon-kaiyo center, Sappo- they were not genetically isolated individuals ro. 1989. (in Japanese) (Yotsukura et al. 2016). Further research is re- Kawashima S. Konbu. In Ohno M. (ed) Biology quired to identify these individuals. and technology of economic seaweeds. Uchida Rokakuho, Tokyo, 2004 ; 59-85. (in Acknowledgment Japanese) Maeda T, Kawai T, Nakaoka M, Yotsukura N. The authors thank Mr. N. Sugimoto (Samani Effective DNA extraction method for frag- Town)andMr.S.HamanoandMr.H. ment analysis using capillary sequencer of Katsuragawa (Akkeshi Marine Station, Hokkaido the kelp, Saccharina. J. Appl. Phycol. 2013 ; University) for collecting materials. This re- 25 :337-347. search was supported by a research project Miyabe K. Hokkaido Suisan Chosahokoku 3 grant-in-aid for Scientific Research (25304010) Laminariaceae. Hokkaido Shokuminbu. 1902.

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