A Systematic Re-Examination of Four Laminaria Species: L. Japonica, L. Religiosa, L. Ochotensis and L. Diabolica

植物研究雑誌 J. Jpn. Bot. 83: 165–176 (2008)

A Systematic Re-Examination of Four Laminaria Species: L. japonica, L. religiosa, L. ochotensis and L. diabolica

Norishige YOTSUKURAa, Shoji KAWASHIMAb,Tadashi KAWAIc,＊, Tsuyoshi ABEd and Louis D. DRUEHLe

aMuroran Marine Station, Field Science Center for Northern Biosphere, Hokkaido University, Muroran, Hokkaido, 051-0003 JAPAN; b , Hakodate, Hokkkaido, 041- JAPAN; c,＊Hokkaido Wakkanai Fisheries Experimental Station, Wakkanai, Hokkaido, 097-0001 JAPAN; E-mail: [email protected] dThe Hokkaido University Museum, Hokkaido University, Sapporo, Hokkaido, 060-0810 JAPAN; eBamfield Marine Science Centre, Bamfield, BC, V0R 1B0, CANADA ＊Corresponding author (Received on September 18, 2007)

The systematics of Laminaria japonica J. E. Areschoug, L. religiosa Miyabe, L. ochotensis Miyabe and L. diabolica Miyabe, which are similar morphologically and restricted to the coastline of northern Japan, was re-examined. From the morphological observations, it was concluded that the discrimination of the four species based on their morphological features was problematic. The results of crossbreeding experiments and molecular phylogenetic analyses suggest that these species are not genetically isolated. Consequently, we concluded that these four species should be revised as four varieties of one species: L. japonica J. E. Areschoug var. japonica, var. religiosa, var. ochotensis and var. diabolica.

Key words: Japanese kelp, Laminaria, Saccharina, systematic re-examination.

About thirty species of Laminaria (includ- delineating features. Okamura (1896), Ueda ing Saccharina) are known worldwide, oc- (1933), and Yabu (1964) have debated the curring in temperate to frigid zones (Druehl validity of these species. 2000). In Japan, 13 species of this genus are In this report, the systematics of these four currently distributed mainly along the coast- species was re-examined based on our results lines of Hokkaido and of Pacific in northern and published findings of morphological ob- Tohoku (Kawashima 1989, Yoshida 1998, servations, crossbreeding experiments and Yoshida et al. 2005). These Japanese molecular phylogenetic analyses. laminarians can be classified roughly into four groups based on morphological charac- Morphological approach teristics and their distribution (Kawashima Laminaria japonica was first described by 2004, Yotsukura et al. 2006). Among the Areschoug (1851). Subsequently, detailed Japanese Laminaria, four species belonging morphological characteristics of sporophyte to the L. japonica group (L. japonica J. E. of this species were investigated and also Areschoug, L. religiosa Miyabe, L. L. religiosa, L. ochotensis and L. diabolica ochotensis Miyabe and L. diabolica Miyabe) were described as new species by Miyabe are morphologically similar and lack striking (1902). The four species of the L. japonica

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Fig. 1. Morphological variations of holdfast of four extant laminarian species growing in Hokkaido. A‒B. Laminaria japonica.A.One-year-old sporophyte on 30 July 1986 at Yunokawa, Hakodate. B. Two-year-old sporophyte on 6 August 1984 at Yunokawa, Hakodate. C‒D. L. religiosa.C.One-year-old sporophyte on 24 September at Nagahama, Okushiri. D. Two-year-old sporophyte on 6 June 1996 at Genna, Otobe. E‒F. L. ochotensis.E.One-year-old sporophyte on 25 July 1985 at Horomoi, Shari, F: Two-year-old sporophyte on 17 September 1999 at Akaiwa, Rebun. G‒H. L. diabolica.G.Two-year-old sporophyte on 11 August 1963 at Kyouei, Rausu. H. Two-year-old sporophyte on 2 August 1987 at Aikiappu, Akkeshi. Scale bar=5cm. group share the following characteristics: (basilar shape), degree of the curl around the “Thick fibrous haptera, arranged in a colum- margin, width and thickness of the lamina. nar or pseudo-columnar manner, resulting in However, these morphological characteris- a large and strong holdfast. The stipe is thick tics are inconsistent, and the morphological and lamina are either linear-lanceolate or variability within a species can be dramatic bamboo-leaf surfaced. The median fascia is depending on plant age and environmental distinct and occupies 1/3 to 1/2 the width of characteristics. lamina. The mucilage ducts are well devel- Regarding the holdfast of L. japonica, oped in the stipe and lamina (occasionally in L. ochotensis, and L. diabolica, new haptera two layers)” (Miyabe 1902). Yoshida (1998) arise in tiers, each new tier covering the pre- distinguished these species by holdfast shape ceding tier, forming a conical, bell-shaped, (arrangement of haptera), lamina shape or hemispheric shape (Figs. 1B, F, H). The June 2008 Journal of Japanese Botany Vol. 83 No. 3 167 phyte currently Two-year-old sporophyte on 9 . .A Laminaria japonica Two-year-old sporophyte on 3 October 1983 at Chitorai, D. and 20 cm in D. . ‒ , N Two-year-old sporophyte on 15 June 1993 at Kuzure, Rishiri. .K ‒ . .I and E C ‒ L. diabolica N. L. ochotensis ‒ J. ‒ Akkeshi. Scale bar = 10 cm in A , u pp Two-year-old sporophyte on 6 June 1996 at Genna, Otobe. F. Two-year-old sporophyte on 9 December 2002 at Notsuka, . ust 1987 at Aika .E g te on 2 Au L. religiosa y h H. p ‒ oro p ear-old s y July 1986 at Putofurenai, Muroran. B. Two-year-old sporophyte on 25 July 1999 at Osatsube, Hakodate (formerly Minamikayabe). C. Two-year-old sporo on 9 July 1990 atHakodate). Setarai, E Hakodate (formerly Toi). D. One-year-old (left) and two-year-old sporophyte (right) both on 3 July 1986 at Higashitoi, Toi ( Iwanai. G, H. One-year-old sporophyte on 18 June 1991 at Oshoro, Otaru. I J. Two-year-old sporophyte on 18 September 1983 at Futatsuiwa, Abashiri. K Rausu. L. Two-year-old sporophyte on 25 June 1990 at Mizutoriba, Hamanaka. M. One-year-old sporophyte on 24 June 1990 at Aikappu, Akkeshi. N. Two- Fig. 2. Morphological variations of lamina of four extant laminarian species growing in Hokkaido. A 168 植物研究雑誌第83巻第3号平成20年6月

Fig. 3. Morphological variation of ten two-year-old sporophytes in Laminaria ochoitensis from Horomoi, Shari. haptera of L. religiosa, when observed from In general, during the first-year of growth, directly above, give the appearance of the lamina bases exhibit cuneate or acute spokes of a wheel (Fig. 1C). However, many shapes, while in the second-year of growth, of the former species (L. japonica, L. the bases become species specific. However, ochotensis, and L. diabolica) exhibit irregu- even in the older sporophyte, the morpho- lar sequences, like L. religiosa,atstages logical characteristics for each species are before sufficient growth has occurred, par- not consistent (Fig. 2). ticularly in the first-year of growth (Figs. The shapes of the holdfasts and laminae 1A, E, G) and even the latter species are variable and are not reliable to distin- (L. religiosa) exhibits no significant differ- guish the four species. Moreover, the width ence from the other three species in the sec- and thickness of the lamina cannot be used ond-year of growth (Fig. 1D). as an absolute measure for identification pur- Usually, L. religiosa is considered to be an poses. For example, Yoshida (1998) showed annual species. However, it is known that that the lamina width of L. japonica was even within its area of distribution, biennial 20–30 cm, while that of L. ochotensis was sporophytes can be found. It seems this spe- less than 20 cm. Commercially, L. japonica cies has the genetic potential to be a biennial, is distinguished by its economic properties a trait shared by the other species. which reflect regional differences (lamina The outline of the lamina, the shape of the length, width, and length/width ratio). lamina base, and the width of the median However, in every region, there were second fascia, for these four species vary greatly ac- year sporophytes with lamina widths of less cording to the locality, age, and whether the than 20 cm (Sanbonsuga and Torii 1974). On kelp is wild or cultured (Fig. 2). Especially L. ochotensis, similar regional differences in the vicinity of species overlaping, a num- are observed between individuals that grow ber of individuals for both species will show in the Sea of Japan and the Sea of Okhotsk. specific characteristics of the other species Those individuals that grow in the Sea of (Kawashima 1989). Okhotsk tend to show large lamina widths, June 2008 Journal of Japanese Botany Vol. 83 No. 3 169 and individuals with lamina width of more tions and L. japonica and L. diabolica in two than 20 cm are observed frequently (Fig. 3). combinations). All hybrids showed normal

Morphological characteristics used to dis- growth (Yabu 1964) and F2 sporophytes tinguish taxa have been reported to change formed by the self-propagation of F1 sporo- geographically in succession (Ueda 1933) phytes reciprocally of crossed L. japonica, L. and vary markedly depending on growing religiosa, L. ochotensis and L. diabolica environments (Hasegawa 1967, Sanbonsuga occurred in high frequencies (Funano 1978, 1978, Sanbonsuga and Torii 1973, 1974, 1980). Notoya and Kirihara 1989, Funano 1991, The four species are more or less distrib- Nabata et al. 1993). Therefore, it is thus uted continuously around Hokkaido: often difficult to classify these laminarian Laminaria religiosa along the western algae based on lamina using classification shores, L. ochotensis on northern shores, L. system by Miyabe (1902). Similarly, dis- diabolica on eastern shores, and L. japonica crimination of the four species based on their on southeast shores from the Tsugaru Straits morphological characteristics can also be to the Muroran region (Hasegawa 1959, problematic. Kawashima 1989) (Fig. 4). An integrated morphological study of 13 The female gametophytes of the four spe- Japanese Laminaria species, including the cies share the same sexual pheromones: four discussed here, revealed considerable lamoxirene (Müller et al. 1985) and there are overlap of the 10 morphological parameters no marked differences in the period of measured (Druehl et al. 1988). By applying sporogenesis and the time of appearance of multivariate analyses, they resolved five the new generation. It is therefore difficult to morphological groups. Lamina width and assume that there is sufficient reproductive stipe length most strongly discriminated isolation to warrant the differentiation of the these groups. One group contained collec- four species. tions of L. religiosa, L. diabolica and L. japonica and another group contained collec- Molecular phylogenetic approach tions of L. ochotensis and L. diabolica. With In recent years, molecular phylogenetic the exceptions of L. longipedalis Okamura analyses have been conducted on laminarian and L. yendoana Miyabe their analyses could algae to clarify the nucleotide sequence of not resolve individual Japanese species. several genes. For Laminaria in Japan, the nucleotide sequence of ribosomal DNA in- Hybridization approach ternal transcribed spacer regions 1 and 2 Reciprocally crossed L. japonica, L. (ITS-1 and ITS-2), which are encoded in the religiosa, L. ochotensis and L. diabolica nucleus, and the intergenetic region between developed normal sporophytes in all combi- RuBisCo L and RuBisCo S (RuBisCo nations. Furthermore when the hybridized spacer) encoded in the chloroplasts, have sporophytes were tank-cultured in deep- been determined (Yotsukura et al. 1999, seawater at Iwanai, Hokkaido, which is Yotsukura 2005). They found no nucleotide different from the seawater of the four sequence differences among L. japonica, species’ natural habitat, the growth and mor- L. religiosa, L. ochotensis and L. diabolica. phology of all the plants were the same until When the nucleotide sequences of the ITS sporogenesis (Table 1). and RuBisCo spacer of individuals collected Hybridization experiments on the four widely from the habitat of the four species species have been conducted (L. japonica, L. were investigated, the nucleotide arrange- religiosa and L. ochotensis in six combina- ments agreed completely between all 170 植物研究雑誌第83巻第3号平成20年6月 4.5 0.0 0.0 5.2 4.1 0.0 0.0 5.0 5.3 0.0 4.5 4.5 0.0 3.8 5.3 5.5 r 80 50 50 80 60 50 50 60 70 50 70 60 50 60 60 60 72.0 50.0 50.0 73.3 51.7 50.0 50.0 53.6 64.3 50.0 62.0 52.0 50.0 51.4 55.0 55.0 70 50 50 70 40 50 50 50 60 50 60 50 50 50 50 50 Angle of lamina base 3.9 1.1 1.5 0.5 1.2 0.8 0.9 1.5 1.9 0.5 3.0 1.3 1.3 1.2 1.0 5.0 4.0 7.0 5.0 6.0 3.7 5.5 8.0 6.0 3.5 5.5 6.5 6.0 5.0 15.0 10.0 15.0 8.5 2.2 4.6 4.7 4.7 2.8 4.1 4.2 3.9 2.7 6.9 3.4 4.3 4.3 3.8 5.0 MF/LM Thickness ratio of 5.7 1.1 3.0 4.0 3.3 1.6 3.0 2.7 2.0 2.1 3.5 2.0 2.5 3.0 2.6 2.0 1.0 1.0 1.1 1.1 1.0 1.1 1.2 1.1 1.2 1.0 1.1 1.3 1.0 1.1 1.0 1.0 1.4 1.4 1.1 1.7 1.7 1.6 1.4 1.7 1.6 1.6 1.3 1.5 1.6 1.3 1.6 1.7 1.2 1.2 1.1 1.3 1.4 1.3 1.3 1.3 1.3 1.3 1.2 1.4 1.2 1.2 1.2 1.3 LA/LM Length ratio of 0.1 0.1 0.0 0.3 0.3 0.2 0.1 0.2 0.2 0.2 0.1 0.1 0.2 0.1 0.2 0.3 0.8 0.7 1.0 1.3 1.0 0.7 0.3 0.4 0.7 0.6 0.1 0.4 3.3 0.4 0.4 0.9 4.7 5.3 5.0 4.5 4.8 4.0 3.3 3.7 4.6 5.0 1.5 3.3 5.2 3.4 4.3 12.8 3.3 4.6 4.0 2.5 3.7 3.3 3.0 3.2 3.1 4.2 1.4 2.9 4.9 4.6 3.0 3.2 LA/MF Width ratio of 2.8 3.4 2.8 0.8 2.3 2.6 2.6 2.8 2.7 3.7 1.3 2.2 2.5 4.2 2.6 2.1 0.2 2.3 4.0 3.7 1.6 2.6 2.3 2.4 2.0 1.1 1.4 0.7 4.6 1.1 2.0 4.9 6.3 7.5 8.9 7.8 9.1 7.8 6.6 10.1 18.0 18.0 10.2 12.3 13.6 21.1 11.0 16.3 LA 5.9 6.3 5.3 6.5 5.8 6.3 6.1 7.2 7.0 4.9 7.6 12.3 12.3 12.4 11.8 11.7 5.7 4.2 7.8 9.2 2.8 4.6 3.6 4.1 3.1 6.2 6.0 4.6 4.0 5.0 2.6 Length/Width ratio of 10.0 Min Mean Max SD Min Mean Max SD Min Mean Max SD Min Mean Max SD Min Mean Max SD L. japonica. L. religiosa. L. ochotensis. L. diabolica. L. japonica. L. religiosa. L. ochotensis. L. diabolica. L. japonica. L. religiosa. L. ochotensis. L. diabolica. L. japonica. L. religiosa. L. ochotensis. L. diabolica. Female Male Table 1. Measurement of several morphological characters of hybridized sporophytes among four extant laminarian species cultured in deep-seawate L. japonica × L. religiosa × L. ochotensis × L. diabolica × Each character (Angle: °)LA: was lamina; measured MF: as median described fascia; in LM: Kawamata lateral (2001). margin of lamina; Mean: value of mean; SD: standard deviation; Min: value of minimum; Max: value of maximum. June 2008 Journal of Japanese Botany Vol. 83 No. 3 171

Fig. 4. Distribution of four extant laminarian species in this study. samples for each DNA region (Table 2). detected between individuals of L. japonica, Comparison of the ITS and RuBisCo spacers L. religiosa, L. ochotensis and L. diabolica is considered useful for identifying genetic in the ITS and RuBisCo spacer (Yotsukura variation among various species or genus of et al. 1999, Yotsukura 2005), it is presumed brown algae (Stache-Crain et al. 1997, Yoon that the genetic differences are not suffi- et al. 2001). Among laminarialean genera ciently large to justify more than one species. such as Alaria, Halosiphon, Macrocystis, Genetic variation of Japanese laminarian Phyllariopsis, Saccorhiza, the nucleotide algae was investigated by comparing the nu- sequence of ITS and/or RuBisCo spacer has cleotide sequence of 5S rDNA spacer region been used to detect regional variations (Yotsukura 2005, Yotsukura et al. 2006), among the same species (Kraan and Guiry which has a faster rate of nucleotide substitu- 2000, Coyer et al. 2001, Kraan et al. 2001, tion than ITS in land plants (Soltis and Soltis Sasaki et al. 2001). Laminaria are thought to 1999). They reported that L. japonica, have evolved relatively recently (Stam et al. L. religiosa, L. ochotensis and L. diabolica 1988) with divergences observed in the nu- grouped together irrespective of species as- cleotide sequence of the ITS and/or RuBisCo signment or location and that the genetic dis- spacer within species belonging to such tance among these four species was less than various laminarialean taxonomic groups between them and other laminarian species. (Rousseau et al. 2001). Consequently, it is Some individuals could not be sequenced thought that the levels of intraspecies varia- directly and often exhibited two-pattern se- tion seen within Laminaria are relatively quences after cloning (Fig. 5). It is presumed high. However, because no mutations were that these are thalli that had arisen from natu- 172 植物研究雑誌第83巻第3号平成20年6月

Table 2. GenBank accession number of the sequence of ribosomal DNA internal transcribed spacer regions 1 and 2 (ITS-1 and ITS-2) and the intergenetic region between RuBisCo L and RuBisCo S (RuBisCo spacer) for four extant laminarian species from northern Japan

Sample ID ITS-1･ITS-2･RuBisCo spacer Species GenBank Accession No. of 5SrDNA spacer in Yotsukura GenBank Accession Number et al. (2006) (Collection date, Collection site)

Laminaria AB255823 (29. i. 2000, Charatsunai, Muroran) AB428798・AB428799・AB428800, japonica AB255814 (15. x. 2000, Kakkumi, Minamikayabe) AB428801・AB428802・AB428803, AB255827 (3. iv. 2000, Kobui, Esan) AB428804・AB428805・AB428806, AB255815 (29. ii. 2000, Furukawa-cho, Hakodate) AB428807・AB428808・AB428809, AB255816 (6. iii. 2000, Shiogama, Fukushima) AB428810・AB428811・AB428812, AB255817 (25. vi. 2002, Ooma, Aomori pref.) AB428813・AB428814・AB428815, AB255825 (26. vi. 2002, Sai, Aomori pref.) AB428816・AB428817・AB428818, AB255818 (8. v. 2002, Odanosawa, Aomori pref.) AB428819・AB428820・AB428821, AB255819 (10. x. 2002, Ishimochi, Aomori pref.) AB428822・AB428823・AB428824, AB255821 (20. iv. 2002, Minmaya, Aomori pref.) AB428825・AB428826・AB428827, AB255820 (21. v. 2001, Omoe. Iwate pref.) AB428828・AB428829・AB428830

L. religiosa AB255804 (19. v. 2000, Genna, Otobe) AB428831・AB428832・AB428833, AB255798 (4. vii. 2000, Isoya, Suttsu) AB428834・AB428835・AB428836, AB255799 (16. xii. 2002, Tomori-gyokou, Tomari) AB428837・AB428838・AB428839, AB255802 (9. xii. 2002, Notsuka, Iwanai) AB428840・AB428841・AB428842, AB255800 (14. ii. 2000, Osyoro, Otaru) AB428843・AB428844・AB428845, AB255809 (18. i. 2000, Minedomari, Atsuta) AB428846・AB428847・AB428848, AB255801 (21. v. 2001, Omoe, Iwate pref.) AB428849・AB428850・AB428851

L. ochotensis AB255790 (8. ii. 2000, Tomamae-gyokou, Tomamae) AB428852・AB428853・AB428854, AB255793 (26. i. 2002, Hourai, Wakkanai) AB428855・AB428856・AB428857, AB255791 (19. iv. 2002, Hakunai, Soya) AB428858・AB428859・AB428860, AB255792 (19. iv. 2002, Boushiiwa, Abashiri) AB428861・AB428862・AB428863

L. diabolica AB255783 (17. xi. 1998, Hunami-cho, Rausu) AB428864・AB428865・AB428866, AB255785 (25. v. 2001, Heinai-cho, Nemuro) AB428867・AB428868・AB428869, AB255784 (27. ii. 2002, Aikappu, Akkeshi) AB428870・AB428871・AB428872 ral hybridizations between individuals with different individuals. Various sequences ana- different DNA sequences. With the aim of lyzed in the L. japonica group appear to sup- verifying this presumption, hybridizations port the notion that active genetic exchanges were conducted using gametophytes with take place in this group. different 5S rDNA spacer sequences. The resulting sporophytes contained both Conclusion sequence pattens. Furthermore, the gameto- Though most morphological characteris- phytes derived from zoospores released from tics of L. japonica, L. religiosa, L. ochotensis these sporophytes showed the same se- and L. diabolica are unstable, several are quences as the original gametophytes (Fig. shared by these species: Mucilage canals 5). This suggests strongly that the individu- present in both stipe and lamina; haptere als with two 5S rDNA spacer sequence pat- mainly in vertical rows (verticillate while the terns are sporophytes that have arisen from frond is young); stipe: short (3–12 cm); natural hybridizations between genetically lamina entire; median fascia thick and broad June 2008 Journal of Japanese Botany Vol. 83 No. 3 173

Fig. 5. DNA banding pattern of polymerase chain reaction amplifying 5S rDNA spacer with primer pairs 5S-F1 and 5S-R1 in Laminaria species (cf. Yotsukura et al. 2006). Lane 1. The sporophyte originated from a female gametophyte (nucleotide length of 5S rDNA spacer: 232bp) and a male gametophyte (232bp) of L. religiosa from Notsuka, Iwanai (lr-n). Lane 2. The sporophyte originated from a female gametophyte of lr-n and a male gametophyte (247bp) of L. japonica from Charatsunai, Muroran (lj-c). Lane 3. The sporophyte originated from a female gametophyte of lj-c (247bp) and a male gametophyte of lr-n. Lanes 4‒13. Subcloning of 5S rDNA spacer in Lane 2. Lane 14. Stable 100bp DNA ladder. Lane 15. The female gametophyte originated from a zoo- spore isolated from the sporophyte treated in Lane 2. Lane 16. The male gametophyte originated from a zoo- spore isolated from the sporophyte in Lane 2. Lane 17. Stable 1kbp DNA ladder. Arrows indicate bands originated from lj-c and arrowheads indicate bands originated from lr-n.

(1/3–1/2 as wide as the total breadth of the Yabu (1964), these four species should be lamina); and bullations present only on grouped as a single biological species: young laminae. Laminaria japonica. While Okamura (1896) These morphological characteristics are divided the four species into two varieties, common to the four species. Above all, the later Yabu (1964) divided them into four. bullations that occur on the leaf surfaces are However, the proposal did not cite a seen only during the first year of sporophyte basionym and thus did not follow the growth. This formation of bullations allows International Code of Botanical Nomen- clear distinction of L. angustata Kjellman clature. group species, which never form bullations, Morphological and genetic differences do from L. cichirioides Miyabe group species, not support the retention of the four species. which always form bullations. The four Laminaria species studied gene- According to the biological species con- rally grow in different areas and each has cept, a species can be defined as “a group of subtle, differentiating morphological charac- actually or potentially interbreeding natural teristics on the morphology of the lamina populations which are reproductively iso- base and the lamina margin (Kawashima lated from other such groups” (Mayr 2000). 2004). Based on the aforementioned previous find- Recently, phylogenetic relationships ings, it appears that there is still evidence of among laminarialean algae were examined in genetic exchange among the four Laminaria detail (Lane et al. 2006). This study broke and that genetic divergence is limited. the genus Laminaria into two clades: Furthermore, the constraint of reproductive Laminaria and Saccharina. Yotsukura isolation does not appear to apply to the spe- (2005), employing molecular phylogenetic cies concerned. Consequently, as has been analyses of Japanese laminarians, distin- proposed previously by Okamura (1896) and guished two lineages (non-digitate type and 174 植物研究雑誌第83巻第3号平成20年6月 digitate type). The non-digitate forms, in- Sist. Rast. [Bot. Inst. Akad. Nauk SSSR] 9: cluding the four species we are reporting on, 51 (1972). belong to the Saccharina clade. Saccharina diabolica (Miyabe) C. E. We propose to treat L. japonica, L. Lane, C. Mayes, L. D. Druehl, & G. W. religiosa, L. ochotensis and L. diabolica as Saunders 2006 in J. Phycol. 42: 509 (2006). follows. The authors thank Mr. T. Muto and Mr. H. Saccharina japonica (J. E. Areschoug) Akino (Hokkaido Nuclear Energy Environ- C. E. Lane, C. Mayes, L. D. Druehl & G. W. mental Research Center), Mr. S. Sakamoto Saunders in J. Phycol. 42: 509 (2006). and Mr. M. Nakaya (Iwanai Local Industry Laminaria japonica J. E. Areschoug, Support Center) and Mr. S. Tomiyasu Phyc. Cat.: 29 (1851). 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四ツ倉典滋a, 川嶋昭二b,川井唯史c, 阿部剛史d, L. D. ドゥリュールe ：日本産コンブ属植物 4 種 (マコンブ, ホソメコンブ, リシリコンブ, オニ遺伝子交流が起こっていると推察される. 従って, コンブ) の分類学的再検討これまで独立種として扱われてきたこれら 4 種は北日本沿岸に分布の中心を持ち, 形態的類似性マコンブ (Laminaria japonica J. E. Areschoug)との高いコンブ属植物 4 種 (マコンブ, ホソメコンして統一し, そのなかに分布域の異なる 4 変種ブ, リシリコンブ, オニコンブ) の分類学的再検 (var. japonica, var. religiosa, var. ochotensis, var. 討を行なった. 4 種はこれまで形態的特徴によっ diabolica) を設けるのが適当と考えられる. て分けられてきたが, 形態形質の多くは不安定で (a北海道大学北方生物圏フィールド科学センター, あり, それぞれを形態的に識別することは難しい. b函館市 , これらコンブ属 4 種はそれぞれ “局所的に” かつ c北海道立稚内水産試験場, “他の何れかの種の分布域と接して” 分布してお d北海道大学総合博物館, り, 交雑実験および分子系統解析の結果から現在 eカナダ・バムフィールド海洋科学センター)