British Phycological Journal Life history and morphology of ...

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Life history and morphology of Chondrus nipponicus (Gigartinales, Rhodophyta) from Japan Juliet Brodie

a d

b

, Michael D. Guiry & Michio Masuda

c

a

Smithsonian Marine Station , 5612 Old Dixie Highway, Link Port, Fort Pierce, Florida, 34946, USA b

Department of Botany , University College Galway, The National University of Ireland , Galway, Ireland c

Department of Botany, Faculty of Science , Hokkaido University , Sapporo, 060, Japan d

Bath College of Higher Education , Newton St. Loe, Bath, BA2 9BN, UK Published online: 17 Feb 2007.

To cite this article: Juliet Brodie , Michael D. Guiry & Michio Masuda (1991) Life history and morphology of Chondrus nipponicus (Gigartinales, Rhodophyta) from Japan, British Phycological Journal, 26:1, 33-50, DOI: 10.1080/00071619100650041 To link to this article: http://dx.doi.org/10.1080/00071619100650041

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Br. phycol. J. 26:33-50 1 March 1991

Life

History and Morphology o f Chondrus nipponicus Rhodophyta) from Japan

(Gigartinales,

By JULIET BRODIE*t, MICHAEL D. GUIRY~ and MICHIO MASUDA§


*Smithsonian Marine Station at Link Port, 5612 Old Dixie Highway, Fort Pierce, Florida, 34946, USA ~Department of Botany, University College Galway, The National University of Ireland, Galway, Ireland §Department of Botany, Faculty of Science, Hokkaido University, Sapporo, 060 Japan The vegetative and reproductive morphology of Chondrus nipponicus from Japan is described. This species is dichotomously or irregularly branched with marginal and surface proliferations. Plants are dioecious; spermatangia develop singly or in pairs from outer cortical cells; the carpogonial branches are three-celled and borne on a large supporting cell. An enveloping tissue is absent in the mature cystocarp. Tetrasporangia are transformed from intercalary or terminal branches produced secondarily from medullary cells. In culture, tetraspores from plants collected in Oshoro Bay, Hokkaido, and Iwamuro, Niigita Prefecture, gave rise to dioecious male and female gametophytes, but only under short-day conditions at 15°C. In the presence of male plants, female plants formed carposporophytes that released carpospores, which germinated in turn to form tetrasporangial plants under both long- and short-day conditions at 15°C. The species thus has a Polysiphonia-type life history. Gametangial formation is under short-day photoperiodic control, whereas tetrasporangial production is not, an unusual situation in the algae. In Japan, C. nipponicus is distributed mainly on the Sea of Japan coasts of Honshu and Hokkaido and on the Pacific coasts of northern Honshu and southern Hokkaido. Crossability studies indicate that Japanese populations of this species are entirely sexually compatible. Atlantic C. crispus is, however, not interfertile with Japanese C. nipponicus. Retention of C. nipponicus in the genus Chondrus is recommended for the time being.

Yendo (1911, p. 599) described a red alga from Japan under the Japanese name "Maruba-tsunomata", meaning a Chondrus with round blades. He later published a valid description (Yendo, 1920, p. 4) based on plants from five localities on the coast of the Sea of Japan. Okamura (1936, p. 656), who considered that C. ocellatus Holmes was highly variable, included C. nipponicus as a form of C. ocellatus. C. nipponicus has not been mentioned in the literature since, and a state of confusion surrounds the presence of this species and that of C. crispus Stackhouse in Japan. C. crispus was first reported from Japan in a brief description by Okamura (1902, tPresent address: Bath College of Higher Education, Newton St. Loe, Bath BA2 9BN, UK 0007-1617/91/010033+ 18 $03.00/0

p. 24). Yendo (1911, p. 595) provided a more detailed description, which was followed by another brief mention by Okamura (1916, p. 29). Later, Okamura (1932, p. 85) treated C. crispus from Japan as C. ocellatus f. crispus Okamura, and this opinion was accepted subsequently by Japanese phycologists (see Mikami, 1965, p. 244). Mikami (1965), however, concluded that C. ocellatus f. crispus was identical to C. crispus, and other authors have followed his example (e.g. Tazawa, 1975; van den Hoek, 1982). More recently, L/ining, Guiry & Masuda (1987) investigated the upper temperature tolerance of North Atlantic and North Pacific Chondrus species, and produced some evidence to support the notion that C. nipponicus is not the same © 1991 British PhycologicalSociety


34

J. Brodie, M. D. Guiry and M. Masuda

entity as C. crispus from the Atlantic. They believed that C. crispus was only known with certainty from the North Atlantic, and that reports of the species in Japan were erroneous, as the morphology and reproduction of these plants were different from Atlantic plants. They also commented that male and female isolates of Japanese plants referred to as C. crispus were not reproductively compatible with C. crispus isolates from the Atlantic. An examination of the vegetative and reproductive morphology of field-collected and cultured plants of C. nipponicus has been made, and crossability studies undertaken, in an attempt to define this species more clearly.

MATERIALS AND METHODS Herbarium material examined The following herbarium specimens, deposited in the Herbarium, Department of Botany, Faculty of Science, Hokkaido University, Sapporo, Japan (SAP), were used to describe the gross morphology and the geographical distribution of C. nipponicus. Pacific coast. Iwate Pref. (Prefecture): Otsuchi, 24 July 1979, leg. M. Kurogi et al. 049331; Miyako, 3 August 1951, leg. S. Kawashima, 027077; Fudai, 21 July 1952, leg. S. Kawashima, 027082. Aomori Pref.: Shiriyazaki, 6 August 1956, leg. S. Kawashima, 049773; Shiriya, 26 June 1987, leg. M. Masuda, 053279. Hokkaido: Osatsube, 26 July 1938, leg. Y. Yamada & Y. Nakamura, 023598; Rebunge, 4 July 1985, leg. K. Kogame, 050160; Toyoura, 1 August 1985, leg. K. Kogame, 050158; Usu, 10 September 1969, leg. M. Masuda, 053287, 11 April 1986, leg. K. Kogame, 050159. Tsugaru Straits. Aomori Pref.: Asamushi, 5 August 1959, leg. H. Mikami, 028921, 048992; Wakinosawa, 25 June 1987. leg. M. Masuda, 053271; Sai, 27 June 1987, leg. M. Masuda, 053277. Hokkaido: Hakodate, 19 February 1982, leg. T. Shimizu, 053284, 4 June 1984, leg. M. D. Guiry & M. Masuda, 053283. Seto Inland Sea. Ehime Pref.: Ikata, August 1981, leg. H. Ishibashi, 042101, 048084.

Sea of Japan. Fukuoka Pref.: Wakamatsu, 17 August 1959, leg. S. Segawa, 049555-6; Moji, 6 April 1978, leg. T. Yoshida, 049557. Shimane Pref.: Uppurui, 28 March 1983, leg. Y. lkoma, 014443. Kyoto Pref.: Maizuru, 7 July 1950, leg. I. Umezaki, 049772. Fukui Pref.: Takanosu, 8 August 1942, leg. Y. Nakamura, 023637-40, 023643. Ishikawa Pref.: Shiga, 18 July 1989, leg. M. Masuda, 053272; Nanao, 25 August 1987, leg. S. Arai, 053276. Toyama Pref.: Kokubu, 3 March 1984, leg. Y. Ohno, 053282; Karashima, 4 August 1929, leg. K. Oshima, 049325. Niigata Pref.: Aikawa, 12 June 1987, leg. K. Ikehara, 053266; Senkaku-wan, 26 August 1971, leg. M. Masuda, 053281; Murakami, 9 July 1987, leg. K. Ikehara, 053265. Yamagata Pref.: Atsumi, 2 July 1984, leg. K. Ikehara, 053268-9, 20 July 1989, leg. M. Masuda, 053273; Kamo, July 1931, leg. T. Hirohashi, 012112, 13 July 1984, leg. K. Ikehara, 049699; Yusa, 13 July 1984, leg. K. Ikehara, 049700. Akita Pref.: Konoura, 20 July 1989, leg. M. Masuda, 053270; Kosagawa, July 1934, leg. T. Muraoka, 020021; Funagawa, 10 October 1985, leg. S. Arai, 049694. Aomori Pref.: Fukaura, 10 August 1988, leg. M. Masuda, 053275; Kodomari, 10 December 1980, leg. T. Kudo, 053289; Tappizaki, 11 December 1980, leg. T. Kudo, 053278. Hokkaido: Kojima, 8 September 1946, leg. Y. Hasegawa, 049005, 7 August 1950, leg. Y. Hasegawa, & E. Fukuhara, 049002, 049004; Okushiri, 6 October 1943, leg. Y. Hasegawa, 025266; Esashi, 6 September 1985, leg. M. Masuda, 053264; Tomari, 15 April 1984, leg. K. Kobayashi, 050013, 25 June 1985, leg. K. Kobayashi, 050014; Kamuenai, 1 August 1954, leg. Y. Tsuji, 028482; Yobetsu, 6 June 1984, leg. K. Kobayashi, 050012, 053274; Oshoro, 19 July 1959, leg. H. Mikami, 028925; Asari, 14 August 1959, leg. H. Mikami, 028923, 048990-1; Zenibako, 15 July 1956, leg. H. Mikami, 048993, 25 July 1956, leg. H. Mikami, 049989, July 1956, leg. H. Mikami, 049771; 31 July 1958, 028924, 0489964; Rumoi, 17 February 1982, leg. M., Masuda, 053280.

Specimens used for morphological and life history studies The following specimens of C. nipponicus were used in reproductive and life history studies: Oshoro Bay, Hokkaido, Japan, 5 December 1981, M. Masuda, including the tetrasporophytes from which strains 421 male and female and 422 male and female were cultured. Iwamuro, Niigata Pref., 10 January 1982, K. Kobayashi, identified by M. Masuda, including the tetrasporophyte from which the strain 431 male and female were cultured.

Chondrus nipponicus from Japan


Morphology Field-collected plants were fixed whole in the field in 10% formalin in sea-water and preserved in 4% formalin in sea-water. Cultured material was fixed in 5-10% formalin in sea-water at room temperature (20°C). After 24 h, the fixative was replaced with 4% formalin in sea-water. Because of the highly mucilaginous nature of cultured material, better fixation was achieved if these plants were cut into pieces 1-5 mm in length prior to being fixed. To improve nuclear staining, fixed plants were bleached by exposing them to light for several days. Sections 15-30 ~tm in thickness were cut using a freezing microtome, or thicker sections were cut by hand. For general morphology, sections were stained with 1% aqueous aniline blue for 10-60 min, mordanted with 10% HC1, and mounted in 80% Karo (commercial corn syrup). Other sections, for nuclear staining, were placed in aceto-iron-haematoxylin-chloral hydrate (Wittman, 1965) for 1-4 h, destained with 45% acetic acid, and mounted in 1:1 Hoyer's mounting medium, according to the procedure of Hommersand & Fredericq (1988). Drawings were made with the aid of a camera lucida attachment. Herbarium specimens have been deposited in the Phycological Herbarium, Department of Botany, University College, Galway, Ireland (GALW), and SAP.

35

were also initiated between C. nipponicus and an isolate of C. crispus from Fife, Scotland.

Spore size Carpospore and tetraspore size were investigated for three tetrasporangial and three cystocarpic plants collected on 2 December 1989 at Oshoro. The diameter of 100 freshly released spores from each plant was measured. To test whether there was a significant difference between the size of tetraspores and carpospores, a comparison of means and standard deviations was performed between the means of the diameters of tetraspores and carpospores.

Spore development Spore development was investigated by inoculating spores onto a series of glass microscope slides in medium-enriched sea-water. After 24 h, and then every alternate day, a slide was fixed in 5% formalin in sea-water.

RESULTS

Life history studies

Typification

Cultures were established from tetraspores released by tetrasporophytes collected in the field. The tetraspores were inoculated onto glass microscope slides using a sterilized Pasteur pipette, and grown in 100% enriched sea-water medium, as compounded by Guiry & Cunningham (1984), in glass culture vessels. The cultures were maintained at 15___1°C, with irradiance levels of 10-25 ~tmol photons m-2s zl, using cool-white fluorescent tubes. Cultivation methods were as previously described (Guiry & Maggs, 1982; Guiry & Cunningham, 1984; Brodie & Guiry, 1988a). Life histories were completed at 15°C, 16:8 h light:dark cycle (long d a y = LD) and 15°C, 8:16 h light:dark cycle (short day = SD).

Yendo's voucher specimens of C. nipponicus were examined on loan from the Herbarium of University Museum, University of T o k y o (TI). They were collected at five localities along the coast of the Sea of J a p a n and each sheet was designated as "specimen original" except (3) and (4): (1) Tsueyama, Tajima (Hyogo Prefecture). August 1895 (Herbarium Imperial Museum, No. 16; Fig. 1); (2) Oshoro, Hokkaido, July 1908 (2 sheets; Figs 2, 3; (3) Iwanai, Hokkaido, undated; (4) Yoichi, Hokkaido, August 1915; (5) Yagishiri Island, Hokkaido, August 1910 (Fig. 4). Specimens (1), (2) and (5) can be treated as syntypes and one of these must be chosen as a lectotype specimen. Two tetrasporangial specimens on sheet 5 are similar in morphology to the habit illustration of this species published by Yendo (1911) and cited in the original description. This sheet is here designated as the lectotype.

Crossability studies Three female plants of each strain were cocultured with all other strains of C. nipponicus at 15°C, SD. All crosses were taken to the point of tetraspore release from tetrasporophytes. Four representative hybrids of these were grown to the next generation of gametophytes until cystocarps were formed and carpospores released. Crosses


36


4 FIGS 1-4. Syntypesand lectotype of Chondrus nipponicus. Fig. 1. Syntype from Tsueyama, Tajima; August 1895. Figs 2, 3. Syntypes from Oshoro, Hokkaido (undated). Fig. 4. Lectotype;tetrasporophytes from Yagishiri Island, Hokkaido; August 1910. Geographical distribution Figure 5 shows the distribution of C. nipponicus in Japan. The species occurs along the coast of the Sea of Japan from Fukuoka Prefecture to the west coast of Hokkaido, which is under the influence of Tsushima Warm Current. It also grows on the coast of Seto Inland Sea (Ehime Prefecture) and along the Pacific coasts of north-eastern (Iwate Prefecture, Aomori Prefecture and southern Hokkaido) Japan, which are influenced by the Tsushima Warm Current and its terminal branch, Tsugaru Warm Current.

Phenology Periodic observations of a population of C. nipponicus were made at Oshoro on the west coast o f Hokkaido during 1977-78,

1984-85 and 1988-89. Plants of this species grow abundantly on rocks near the lowwater mark. Macroscopic young plants appear in October and grow vegetatively during the winter and spring months. Tetrasporangial plants first appear in early June and continue to form tetrasporangial sori until late January. Spermatangial plants become recognizable in late June and persist until December. Cystocarpic plants are evident in mid-July and continue to produce cystocarps until February or sometime in March. Reproductive blades become •eroded after spore or spermatium release and thus become smaller. These reproductive plants also continue to form new erect axes from the basal holdfasts; at any season young uprights less than 3 mm tall are present amongst the large blades. Plants are thus perennial and may become fertile in the next season.


37

Straits


Sea of Japan

Pacific Ocean

Seto Inland Sea

200 km

FIG. 5. Geographical distribution of Chondrus nipponicus.

Gross morphology

Vegetative morphology

C. nipponicus shows considerable variation in gross morphology (Figs 6-12). The erect axes are dark red-brown, up to 120 mm tall and 2-20 mm broad, and grow in tufts o f about 20-50 thalli attached to the substratum by a discoid holdfast, approximately 5-10 mm in diameter. The thallus expands from a compressed stipe into a flattened blade which frequently curls. Branching of the main axis is dichotomous or subdichotomous, with opposite or irregularly arranged marginal and surface proliferations, especially on older parts of the thallus. The apices of the branches are rounded.

The thallus consists of a cortex of round or oval cells, an inner cortex of stellate cells, and a filamentous medulla (Fig. 19). Cells of the cortex are uninucleate, and radiate in chains that arise in pairs from small stellate cells at the outer edge of the inner cortex. Each cortical chain is usually composed of five-seven radially elongated cells, which decrease in size from the inner to the outer cortex, are 3-7-5 ~tm long and 1-1.5 ~tm broad. Cortical files may branch dichotomously at every second or third cell. The inner cortex is composed of three-five layers of multinucleate, stellate cells. Filamentous

38



7

.t f,

9

]u

11 ~

1

FIGS6-[2. Morphological variation in Chondrus nippoaicus. Oshoro, Hokkaido; 22 September 1984. Fig. 7. Female; Oshoro; 30 August 1984. Fig. 8. Tetrasporophytes; Oshoro; 6 ]November 1984. Fig. 9. Yebetsu, Shakotan, Hokkaido; 6 June 1984. Fig. 10. Nanao, ]shikawa Prefecture; 25 August 1987. Fig. 11. Females; Oshoro; 26 November 1984. Fig. 12. Oshoro; 21 July 1977.

cells of the medulla (Figs 19, 20) are generally multinucleate, and are 35-100 ~tm long and 2.5-12 txm wide in longitudinal section (LS). Each cell or filament is linked to several other cells in more than one plane to produce the filamentous network of the medulla. A medullary cell protrudes laterally and the terminal portion cuts off an apical cell (Fig. 23) that either links directly or forms a short filament before connecting with a neighbouring medullary cell (Fig. 24).

Gametophyte reproduction

Gametophytes are dioecious (Figs 13, 14). In culture, male plants (Fig. 13) can be distinguished by a white or brownish discolouration at the apices of branches, which represents superficial spermatangial sori. Spermatangia are 2.5-6 Ixm long and 2-2-5 ~m wide, and arise singly or in pairs from the outermost cells of the cortex (Fig. 25). Each spermatangium releases a



39

FIGS 13-18. Gross morphology of parent and cultured plants of Chondrus nipponicus. Fig. 13, Male plant; cultured. Fig. 14. Cystocarpic female plant; cultured. Fig. 15. Tetrasporophyte of C. nipponicus from Oshoro Bay; collected by M. Masuda on 5 December 1981; parent of strain 421. Fig. 16. Mature tetrasporophytes; cultured. Fig. 17. Developing tetrasporophytes in 15°C SD 14 weeks after inoculation of carposporesl Fig. 18. Developing tetrasporophytes at 15°C SD 39 weeks after inoculation ofcarpospores, c, cystocarp; d, disc; p, proliferation; ss, spermatangial sorus; ts, tetrasporoangial sorus; u, upright.

spherical or ovoid, translucent, uninucleate spermatium through the thick, mucilaginous cuticle. Spermatia are 2-5-7.5 ~tm in diameter when freshly released. Carpogonial branches (Figs 26, 27, 31-33) are frequently more numerous on one side of the thallus. A carpogonial branch is threecelled, borne on a large, multinucleate supporting cell that originates in the inner cortex, and is attached by primary and secondary pit connections to surrounding

vegetative cells. Neither the supporting cell nor the carpogonial branch bear sterile side branches. Prominent pit connections link the supporting cell and the first and second cells of the carpogonial branch (Figs 32, 34). The carpogonium is connected laterally to the second cell of the carpogonial branch and lies in close proximity to the supporting cell. It is uninucleate and bears an elongated, often slightly swollen trichogyne (Figs 27, 34). The tip of the trichogyne protrudes



40

FIGS 19-24. Vegetative morphology (LS) of Chondrus nipponicus from Japan and C. crispus from Black Head, County Clare, Ireland, 30 April 1987. Fig. 19. Thallus of C. nipponicus showing cortex, inner cortex and medulla. Fig. 20. Filamentous medulla of C. nipponicus. Fig. 21. Thallus of C. crispus showing cortex, inner cortex and medulla. Fig. 22. Filamentous medulla of C. crispus. Fig. 23. Early stage in filament formation in medulla of C. nipponicus. Fig. 24. Late stage in filament formation in medulla of C. nipponicus, nf, new filament.

beyond the surface of the thallus or is flush with it.

Carposporophytedevelopment After presumed fertilization, the trichogyne contracts, and the carpogonium fuses directly with the supporting cell, which enlarges (Figs 28, 29, 34, 35), and functions as the auxiliary cell. The first two cells of the carpogonial branch are still clearly visible in

early post-fertilization stages (Fig. 29). Sometimes the auxiliary cell may be surrounded by a layer of modified vegetative cells that are linked by pit connections to it (Figs 29, 30). Intercalary divisions of female vegetative cells are occasionally found in the vicinity of the young auxiliary cell. Several gonimoblast initials are cut off inwardly from the auxiliary cell (Figs 30, 36, 37), and these produce gonimoblast filaments that grow into the medulla. Swollen, uninucleate



41

FlGS25-30. Reproductive morphology (TS) of Chondrus nipponicus. Fig. 25. Spermatangial sorus. Fig. 26. Carpogonial branch borne on supporting cell. Fig. 27. Carpogonial branch with carpogonium apparently about to fuse with auxiliarycell. Fig. 28. Fusion ofcarpogonium with auxiliarycell. Fig. 29. Early post-fertilization;auxiliary cells with surrounding femalevegetativecells. Fig. 30. Early post-fertilization;gonimoblast initials and surrounding female vegetative cells, ac, auxiliary cell; cg, carpogonium; gi, gonimoblast initials; r, remnants of carpogonial branch; s, spermatium; sc, supporting cell; sp, spermatangium; sv, surrounding vegetativecell; tr, trichogyne.

gonimoblast cells become linked by secondary pit connections to multinucleate vegetative medullary cells, while differentiating into carposporangia (Fig. 39). Carposporangia are transformed from gonimoblast, and when mature, are round or ovoid, 17.5-30 ~tm long and 15-25 Ixm wide in cultured plants. Gonimoblast tissue extends nearly to the margin of the thallus, without the formation o f a special sterile envelope (Fig. 38). Cystocarps may reach 2-5 mm in diameter at maturity. They are formed near the apices o f branches and tend to bulge more towards one surface of the thallus. Spores are released by a degeneration of the cortical region (Fig. 40), and an organized ostiole is not formed. Gonimo-

blasts continue to branch radially forming new carposporangia at the same time that mature carpospores are being released (Fig. 40). A wound reaction in the form of an inward proliferation of cortical cells appears to take place in the region of spore release (Fig. 40).

Tetrasporophyte reproduction Immature tetrasporophytes are morphologically similar to young gametophytes. At maturity (Figs 15, 16), plants bear tetrasporangia in distinct sori, visible as red, round or oval patches on the lower part of the main axis or on marginal proliferations. Tetrasporangia are transformed from

42

J. Brodie, M. D. Guiry and M. Masuda tr

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.

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57 FIGS 31-37. Reproductive morphology (TS) of Chondrus nipponicus. Figs 31-33. Carpogonial branches and supporting cells. Fig. 34. Carpogonium apparently about to fuse with auxiliary cell. Fig. 35. Fusion of carpogonium with auxiliary cell. Fig. 36. Early post-fertilization auxiliary cell producing gonimoblast initials. Fig. 37. Early postfertilization carposporophyte, ac, auxiliary cell; c, cortical cell; cbl, carpogonial branch 1; cb2, carpogonial branch 2; cg, carpogonium; f, fusion; gi, gonimoblast initial; n, nucleus; pc, pit connection; sc, supporting cell; sv, surrounding vegetative cell; tr, trichogyne; tw, thallus wall; vc, vegetative cell.

uninucleate cells and terminal or intercalary chains of uninucleate cells that are produced secondarily from medullary cells (Figs 41, 43). A tetrasporangial initial enlarges and undergoes transverse division (Fig. 44) followed by longitudinal division resulting in four cruciately arranged spores, by which time pit connections linking tetrasporangia have broken down. A mature tetrasporangial sorus extends across the width of the medulla (Fig. 42). Fully cleaved, mature tetrasporangia are oval, and range in size from 30 to 46 lam long and 25 to 32 lam wide

in cultured plants. In field material they are larger, being 50-65 lam long and 37-52 lam wide. Tetraspores are released in a similar manner to carpospores by breakdown of the cortex. Each sorus initially occupies a distinct region of the medulla; if more than one sorus develops in the same area, then the thallus may become almost completely filled with tetrasporangia. New tetrasporangia develop in a sorus at the same time that others mature and are released, and soft appear to be productive for a long time. The same type of wound response seen in the



43

FIGS 38-42. Reproductive morphology (TS) of Chondrus nipponicus. Fig. 38. Mature carposporophyte with surrounding band of vegetative tissue absent. Fig. 39. Developing carposporangia linked with vegetative cells. Fig. 40. Old carposporophyte, showing area of carpospore release, and reduced numbers of carposporangia in central region. Fig. 41. Early stage in the development of tetrasporangia in medulla of tetrasporangial sorus. Fig. 42. Mature tetrasporangial sorus, dc, developing carposporangium; gf, gonimoblast filament; mt, mature tetrasporangial sorus; rl, area of release; ti, tetrasporangial initial; vc, vegetative cell.

cystocarps occurs in the cortex o f tetrasporophytes wherever tetraspores have been released. Life history All strains o f C. nipponicus exhibit a strict Polysiphonia-type life history in culture. Carpospores inoculated onto glass microscope slides at 15°C L D germinated to f o r m

circular discs. A primary upright, which appeared initially as a dark mass o f cells, developed from the centre o f each disc. Secondary uprights formed f r o m the basal disc, and these uprights increased in n u m b e r as the discs expanded in diameter. Basal discs eventually coalesced on the slide and the rate o f growth o f upright tended to be slower when c o m p a r e d with individuals removed from discs and g r o w n separately.

44



mf

t

44

j

~~

cl~

FIGS. 43-44. Tetrasporangial development (TS) in Chondrus nipponicus. Fig. 43. Early tetrasporangial development showing terminal and intercalary tetrasporangial initials. Fig. 44. Transverse division of tetrasporangial initials in sorus of tetrasporophyte, c, cortical cell; dr, developing tetrasporangium; i, inner cortical cell; mf, medullary filament; ti, tetrasporangial initial.


Chondrus nipponicus from Japan Secondary uprights continued to form from the discs, with the youngest usually at the expanding margins of the disc. At first, uprights were narrow (0-5 mm), unbranched and blade-like. Later they became broader ( > 1 mm) and paler at the tips and eventually began to branch dichotomously and develop marginal proliferations. Tetrasporangial sori became evident as dark red spots typically located a b o u t half-way up the thallus, or on marginal proliferations. Tetraspores were released 1-2 weeks later. The development of carpospores at 15°C SD was similar to that at LD, but took place over a longer period. Discoid bases developed followed by the formation of primary uprights, one in the centre o f each disc, and later by secondary uprights. As in plants grown from carpospores in LD, secondary uprights appeared along the margin of the expanding disc (Figs 17, 18). The discs usually increased in diameter for a few weeks, followed by a period of very little expansion. Fifty uprights were recorded over a 6-month period on one disc that attained a diameter of 6 mm. Basal discs eventually coalesced on the slides. Uprights began to branch dichotomously at their tips and

45

developed marginal proliferations, followed by formation of tetrasporangial sori. Release of tetraspores took approximately 5 months longer than in tetrasporophytes grown under L D conditions. Tetraspores grown at 15°C L D developed in the same manner as carpospores. Three weeks after inoculation, spores had germinated into circular discs. Primary uprights were visible as dark cell masses in the centres of discs. The uprights grew into blade-like plants and began to branch at the tips and form marginal proliferations from the margins of the thallus. Plants continued to branch becoming bushy in appearance, and were inclined to be brittle. Reproductive structures did not develop at this temperature or daylength over an 8-year period. Germination of tetraspores inoculated at 15°C SD followed much the same pattern as carpospores at L D and SD, and tetraspores at LD. Two weeks after inoculation, the basal discs were circular, and primary uprights were just showing at the centre of each disc. Secondary uprights formed later from the margins of the expanding disc. Uprights became broader at the tips as they increased in length. Marginal proliferations

TABLE I. Summary of the sequence of the life history of Chondrus nipponicus at 15°C. Spm = formation of spermatia; cyst = formation of cystocarps; LD = 16 : 8 h; SD = 8 : 16 h Development from Carpospores

Tetraspores

Summary of events

LD

SD

LD

SD

Primary upright formation (weeks)

2-3

2-3

3-6

1-4

Disc diameter when primary uprights formed (l~m) Presence of hairs on discs (weeks)

100-360

560-360

360-500

86-400

First appearance of secondary uprights (weeks) Disc diameter when secondary uprights formed (mm) First appearance of proliferations (weeks) First appearance of dichotomous branching (weeks) First signs of reproductive structures (weeks) Release of spores (weeks)

8

Length of frond when first reproductive (mm)

- -

- -

- -

3

6-11

--

9

I-1"5

0"5-1"5

--

2

12-16

25-28

19

22

12-20

28-37

19

22

--

36-43

--

21-28

37-45

--

spm34 cyst 34 36

15-18

16-19

--

20

46


TABLE II. Results o f crosses set u p b e t w e e n strains o f Chondrus nipponicus f r o m J a p a n a n d o n e strain o f C. crispus f r o m the Atlantic. + ° = cross a n d offspring released tetraspores; + * = cross a n d offspring released tetraspores a n d these g r e w into v i a b l e g a m e t o p h y t e s ; - = did n o t cross; N D = n o d a t a Females

C. nipponicus

C. crispus

Strain

421

422

431

191

C. nipponicus

421 422 431

+* + o +o

+* + ° +o

+* + ° +,

-ND _

C. crispus

191

--

--

ND


Males

and the beginnings of dichotomous branching at the apices of fronds then followed. Spermatangial sori and cystocarps formed on separate plants followed by release of carpospores. Life history events are summarized in Table I. Crossing studies The results of crossing studies are shown in Table II. All strains of C. nipponicus were compatible. Where tested, carpospores developed into tetrasporophytes that released viable tetraspores. Where tetraspores were grown, these developed into male or female plants; carposporophytes formed on the females, and released carpospores. C. nipponicus did not cross with the isolate of C. crispus from Scotland (Table II). Variation in spore size The diameters of freshly released carpospores ranged between 20 and 30 ktm, with a mean of 25"26 and standard deviation of 1.69. The diameters for freshly released tetraspores were between 19 and 25 Ixm, with a mean of 21.24 and a standard deviation of 1-42. Despite the overlapping ranges, the carpospore diameters were significantly larger than tetraspore diameters. (Comparison of means test, d -- 31.41; P < 0-001.) Spore development Development of carpospores and tetraspores (Figs 45-52) was essentially the same.

+ *

The spore (Fig. 45) divided in two (Fig. 46), and each cell divided again (Fig. 47). As the number of cells increased (Figs 48, 49), one or two new cells were cut off in a horizontal plane from the central cells. This pattern was repeated by the new cells and a disc was formed (Figs 50, 51). As the disc expanded horizontally, cells in the centre cut off cells in a vertical plane (Fig. 52) to form a primary erect axis. Unicellular hyaline hairs with apical cytoplasm were frequently observed on developing discs. DISCUSSION

C. crispus sensu Mikami (1965), Tazawa (1975) and other Japanese authors is not the same species as C. crispus Stackhouse from the North Atlantic, and should be known as C. nipponicus Yendo. This is confirmed by the differences in morphology between the two species (Table III), as well as the results of crossability studies. In some respects, the two species do resemble one another, but in three main morphological areas, viz gross morphology, internal vegetative anatomy and post-fertilization events in the developing carposporophyte, the distinction between them is clear. In terms of gross morphology, C. nipponicus was characterized by Yendo (1920) as a plant having simple blades with many marginal proliferations. Our observations show that C. nipponicus is not always regularly dichotomously branched, but frequently has a more or less irregular branching pattern. In culture, male plants occasionally have only



45

46

47

48

49

50

51

47

52

50 ~m

I

I

FIGS 45-52. Carpospore development in Chondrus nipponicus. Figs 45-49. One day after inoculation. Fig. 50. Two days after inoculation. Fig. 51. Four days after inoculation. Fig. 52. Six days after inoculation.

one or two branches. Marginal proliferations almost always develop in male, female and tetrasporangial plants in culture especially on the older parts of the thallus. C. crispus, on the other hand, is repeatedly dichoto-

mous, often fan-shaped, and only occasionally proliferous from the margins (Dixon & Irvine, 1977), and growth and dichotomous branching at each apex take place in a very regular manner (Taylor & Chen, 1973).

TABLEIII. Comparison of some morphological features in Chondrus nipponicus from Japan and C. crispus from the Atlantic Species Morphological feature Thallus branching Marginal proliferations Diameter of medullary filaments (~tm) Enveloping tissue in mature carposporophyte Intercalary cell division in vicinity of developing gonimoblast Tetrasporangia: length (~tm) width (~tm)

C. nipponicus

C. crispus

Dichotomous/ subdichotomous Abundant

Dichotomous

2.5-12 Absent

Very occasional 20-25(40) Absent

Present

Absent(?)

50-65 37-52

17-40 11-30


48


Isolates of C. crispus in culture rarely form marginal proliferations and then only from the base of the plants (Guiry, unpubl, data). The medullary filaments of C. nipponicus are generally shorter, narrower and less densely packed than in Atlantic plants of C. crispus (Table III; Figs 19-22). Kim (1976), who studied plants of C. crispus from the Atlantic, listed as a distinguishing feature of this species that the medullary cells are shorter and thicker than those in most members of the Gigartinaceae. Mikami (1965) gave dimensions of the inner diameter of medullary filaments in his material from Japan, which agree tolerably well with our observations. Prior to fertilization, the structure of the procarp of C. nipponicus resembles that of C. crispus and other members of the Gigartinaceae. However, post-fertilization events appear to differ in the two species. In C. nipponicus a layer of cells is sometimes formed around the fertilized procarp. This phenomenon is probably associated with a need to provide nutrition for the developing gonimoblast (see Hommersand & Fredericq, 1990, for an account of carposporophyte nutrition). Such an arrangement of pericarpial cells does not appear to occur in Atlantic plants of C. cripsus. Further investigations, however, are required to clarify the nature and development of the layer of cells in C. nipponicus. C. nipponicus and C. crispus both lack an involucre in the mature cystocarp. The presence or absence of an involucre (also known as an "enveloping tissue", "pericarp", "pericarpial filaments", "pericarpium proprium", "Faserh~lle", and "special medullary filaments") was recognized from an early stage (e.g.J. Agardh, 1851, 1876) as an important generic feature of the Gigartinaceae. J. Agardh (1851) and subsequent authors (e.g. Setchell & Gardner, 1937; Kylin, 1956; Mikami, 1965; Kim, 1976) used this feature to distinguish Chondrus from the other genera of the Gigartinaceae. Hommersand & Fredericq (1990) in a study of the pericarpial filaments of Gigartina teedii (Roth) Lamouroux have concluded that the involucre has a nutritive role in that species.

Mikami (1965, p. 248, fig. 35C) reported, and we observed, that intercalary cell divisions to produce short, square cells occurred in the medulla of C. nipponicus (as C. crispus). Kim (1976, p. 33), commenting on Mikami's observations, states "Their occurrence in my material [of C. crispus] was extremely rare, and if it [sic] does occur consistently, it seems to be a poorly developed enveloping tissue". Very similar intercalary divisions of medullary cells in the area of gonimoblast formation were also reported by Mikami (1965) in three Gigartina species from Japan (viz G. pacifica fig. 18A, B; G. ochotensis fig. 19B; G. mamillosa fig. 21A) that are now referred to as the genus Mastocarpus (see Guiry et aL, 1984). The occurrence of these cells and the absence of an involucre in the mature cystocarp of species of Chondrus (Gigartinaceae) and Mastocarpus (Petrocelidaceae) suggest a relationship that merits further study at the familial level. "Special absorbent filaments", a term introduced by Mikami (1965, p. 208) to describe long filaments that originated from the gonimoblast and penetrated the special medullary filaments, are absent in C. nipponicus. It is likely that these filaments are involved in the nutrition of the gonimoblast in the same way as the involucral filaments. The whole area of carposporophyte nutrition in the Gigartinaceae, Petrocelidaceae and Phyllophoraceae is clearly of fundamental importance for an understanding of the phylogeny of these closely related procarpial families of the Gigartinales, but in the present confused situation no conclusions are possible as to the familial or generic significance of these features. For the time being, therefore, C. nipponicus is retained in Chondrus and the genus Chondrus is retained. It is noteworthy that carposporangia are frequently seen attached by pit connections to swollen multinucleate female vegetative cells in the developing carposporophyte of C. nipponicus. Kim (1976), on observing the same phenomenon in C. crispus from the Atlantic, questioned whether carposporan-



49

gial mother cells are always derived from photoperiodic response was obtained over a gonimoblast filaments. He suggested that the 10-year period (Guiry, unpubl, data). The swollen female vegetative cells around the occurrence of an SD response in the formagonimoblast filaments transform to produce tion of the gametangia without a similar the carposporangial mother cells. Other response in the initiation of tetrasporangia is workers (Schmitz, 1883; Darbishire, 1902; rare among the algae. To date, it has Kylin, 1923; Rosenvinge, 1931; Marshall, generally been observed that only spore Newton & Orr, 1949; Mikami, 1965) found production on sporophytes is under photothat gonimoblast filaments of C. crispus periodic control (Dring, 1984), or that both produced carposporangial mother cells. gametangia and sporangial production is Although we are not in a position to agree or under daylength and temperature control disagree with Kim (1976) without further (Dring, 1984; Brodie & Guiry, 1988b). investigation, it seems probable that the Further evidence that C. nipponicus from vegetative cells supply nourishment to the Japan and C. crispus from the Atlantic are developing gonimoblast when it is no longer two distinct species is provided by the results possible for the auxiliary cells to supply all of crossability studies. Although strains of their needs, as suggested by Mikami (1965). C. nipponicus were entirely compatible with The development of spermatangia is as each other, C. nipponicus and C. crispus described by Mikami (1965) and Tazawa strains did not produce carposporophytes (1975) in male plants of C. nipponicus (as when crossed. C. crispus) from Japan, and is similar to that Carpospores were significantly larger found in C. crispus from the Atlantic (e.g. overall than tetraspores and had a greater Darbishire, 1902; Prince & Kingsbury, size range. This is, to a certain extent, con1973). sistent with their putative differences in Tetrasporangial development in ploidy level. C. nipponicus takes place by the transformaThe results we have presented show that tion of chains of cells that are formed secon- specimens previously referred to as darily from medullary cells, as in C. crispus C. crispus from Japan represent a separate (Kylin, 1923; Rosenvinge, 1931); however, species, C. nipponicus. The generic placement mature tetrasporangia in C. nipponicus are of this entity, however, requires further longer and wider than those reported for study. C. crispus (Table III) from the British Isles (Dixon & Irvine, 1977). ACKNOWLEDGEMENTS C. nipponicus has a Polysiphonia-type life We are grateful to Dr H. Ohba, University of history, which follows much the same Tokyo, for the loan of Yendo's specimens and Dr sequence of events as C. crispus ( C h e n & H. Mikami, Professor Emeritus of Sapporo McLachlan, 1972). It is likely that there is an University for his helpful discussions, and for SD photoperiodic response in the formation providing herbarium specimens. We also thank Dr E.M. Cunningham, Dr S. Fredericq, of carpogonial branches and carposporophytes in C. nippon&us, which were absent in Professor M.H. Hommersand, Professor T. Yoshida and Dr W. B. Jaeckle. LD conditions at the same terhperature. Tetrasporogenesis occurred in both LD and SD, although development was more rapid REFERENCES and reproductive maturity was reached AGARDH,J. G. (1851). Speciesgeneraet ordinesalgarum. earlier in tetrasporophytes in LD, probably a Vol. 2(1): Species genera et ordinesfloridearum. C. W. K. Gleerup, Lund. reflection of the higher photon exposures in J. G. 0876). Speciesgeneraet ordinesalgarum. LD conditions. Thirty-two isolates of AGARDH, Vol. 3(1): Epicrisissystematisfloridearum. T. O. C. crispus from the North Atlantic (Nova Weigel, Leipzig. Scotia, Iceland, Norway, the British Isles, BRODIE,J. & GUIRV,M. D. (1988a). Life history and reproduction of Botryocladia ardreana sp. nov. Germany, France and Spain) all reproduced (Rhodophyta, Rhodymeniales)from Portugal. under LD conditions and no evidence of a Phycologia, 27: 109-130.


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