Antithamnion nipponicum (Ceramiaceae, Rhodophyta), incorrectly known as A. pectinatum in western Europe, is a recent introduction along the North Carolina and Pacific coasts of North America

Abstract

Female and male reproductive structures in material from California known as Antithamnion hubbsii are illustrated and described for the first time. This partly prostrate species is characterized vegetatively by pinnae arranged in distichous opposite pairs, bearing adaxial and abaxial pinnules, with the distal-most pinnules restricted to the abaxial side of pinnae. Basal cells of the pinnae produce multicellular rhizoids with digitate holdfasts as well as indeterminate lateral axes, and gland cells originate adaxially alongside the lower pinnule cells. Antithamnion nipponicum has been placed in synonymy with Antithamnion pectinatum, a species described from Auckland I., New Zealand. The latter is here recognized as a separate southern hemisphere species bearing adaxial and abaxial distal-most pinnules and new indeterminate lateral axes in place of pinnae. The correct name for the invasive species known in western Europe as A. pectinatum is A. nipponicum. Our phylogenetic analyses of rbcL sequence data also indicate that Californian A. hubbsii and A. nipponicum are conspecific, but distinct from A. pectinatum and A. aglandum. The distribution of A. nipponicum includes the Pacific coast of California and the Atlantic coast of North Carolina, USA. Its presence is inferred in the Mediterranean Sea. Historical reports suggest that this species was recently introduced from Japan.

Keywords:

• Antithamnion
• Antithamnionella
• Antithamnion hubbsii
• A. nipponicum
• A. pectinatum
• Atlantic Ocean
• Antithamnieae
• California
• Ceramiaceae
• invasive species
• morphology
• North Carolina
• rbcL
• Rhodophyta
• taxonomy

Introduction

The circumscription of the genus Antithamnion Nägeli (1847) in the tribe Antithamnieae Hommersand (1963) has been modified several times. Feldmann-Mazoyer's (1941) broad definition of the genus included Pterothamnion Nägeli, Antithamnionella Lyle and Platythamnion J. Agardh. Wollaston (1968, 1971, 1972) restricted the genus Antithamnion to those species with axes completely lacking rhizoidal cortication, determinate pinnae arranged in opposite pairs, presence of a small quadrate cell at the base of each pinna, presence or absence of gland cells, tetrasporangia cruciately divided, and carpogonial branches borne singly on the basal cells of the pinnae. Maggs & Hommersand (1993) further described in great detail the pre- and post-fertilization reproductive morphology that characterizes the genus and the tribe. The branching pattern of the pinnae and the position of gland cells are useful characters for discriminating between species (e.g., L'Hardy-Halos, 1968; Athanasiadis, 1996).

Antithamnion hubbsii Dawson was described from vegetative material collected in Melpomene Cove, Isla Guadalupe, Baja California, Mexico (Dawson, 1962, p. 16, pl. 5, fig. 2; pl. 6, fig. 3) (holotype: Dawson No. 8302, LAM 500043). A fragment of the holotype and a lateral indeterminate branch (axis) were photographed by Athanasiadis (1996, p. 146, fig. 66A, B). Previously the only reproductive structures recorded were tetrasporangia (Young, 1981), although recently we collected thalli of A. hubbsii from California exhibiting all reproductive stages. Antithamnion hubbsii has been reported from California, USA (Abbott & Hollenberg, 1976), Japan (Itono, 1969), south-east South Africa (Norris, 1987), and New Zealand (Adams, 1994, p. 243), and the taxonomy of this species is still confused (Athanasiadis, 1996). Recent collections of a hitherto unreported Antithamnion species from Atlantic North Carolina, USA, confirmed that, on the basis of thallus morphology and rbcL sequence, the Californian and North Carolinian specimens were conspecific.

A review of the literature indicated that species of Antithamnion with creeping filaments that grow attached to the substratum by means of digitate multicellular rhizoids, and have distichous pinnae bearing gland cells, encompass several names. Such species have typically been referred to as A. hubbsii in the eastern Pacific (Baja California and Pacific Mexico: Dawson, 1962; California: Abbott & Hollenberg, 1976; Young, 1981) and as Antithamnion nipponicum Yamada et Inagaki in the western Pacific (Japan: Yoshida et al., 1985; Yoshida, 1998; Kamiya & Kawai, 2002; Mine et al., 2003; Korea: Kang, 1966; Lee & West, 1980; Lee et al., 2001). Examining reproductive material of A. nipponicum from the vicinity of Otaru, Japan, Abbott (1999) concluded that certain Antithamnion collections from Hawaii were A. nipponicum. Kim et al. (1996) described a closely related species, Antithamnion aglandum Kim et Lee for specimens from Cheju Island, Korea, which lacked gland cells and produced hair cells in female gametophytes, while Lee et al. (2001) suggested that the species referred to as A. nipponicum by Lee & West (1980) was actually A. aglandum.

Although Itono (1969) referred to the Japanese material he studied as A. hubbsii, he expressed doubt that A. hubbsii and A. nipponicum were distinct species because Dawson's (1962, p. 10) critical character for discriminating between the two species was not clear-cut; i.e., secondary determinate branchlets in A. nipponicum were predominantly pectinate or secund, whereas they were predominantly pinnate or forked in A. hubbsii.

Athanasiadis & Tittley (1994) and Athanasiadis (1996, p. 147) placed A. nipponicum in synonymy (with a question mark) with Antithamnion pectinatum (Montagne) Brauner, a species described from Auckland I., New Zealand. Athanasiadis (1996, p. 146) recognized A. hubbsii as a species distinct from A. pectinatum on the basis of the regular development of new indeterminate lateral axes originating from basal cells of sparsely branched pinnae in the former, while new axes replace a pinna in a pair in the latter. Wollaston & Womersley (1998) dropped the term whorl-branches for the Antithamnieae, using the older term pinna (pinnae) for the opposite determinate laterals and the term pinnule (pinnules) for the branchlets borne on the pinnae; their revised terminology is used here.

Prior to Athanasidis & Tittley's (1994) merger of A. nipponicum into A. pectinatum, an invasive Antithamnion species in the Mediterranean was first recorded as A. nipponicum (Verlaque & Riouall, 1989), and subsequently, the name was changed to A. pectinatum, a taxon that has since been reported to have spread further in western Europe (Curiel et al., 1996, 1998, 2002; Verlaque, 2001; Boudouresque & Verlaque, 2002). Antithamnion pectinatum was recently recorded from the US Atlantic coast off Connecticut (Athanasiadis & Tittley, 1994) and from Australia (Womersley, 1998, p. 106). Adams (1994) did not mention A. pectinatum or Callithamnion pectinatum Montagne, but instead used the name Antithamnion applicitum (Harvey) J. Agardh to refer to all the samples she studied from New Zealand, referring to a smaller plant from the warmer waters of the Kermadec Islands with “gland cells, very conspicuous, sessile on short pinnule” as A. hubbsii. Neither species were critically described and no comparisons are possible based on her descriptions (Hommersand, pers. comm.). Norris's (1987) depiction of a species he referred to as A. hubbsii from Natal, South Africa, includes minute glandless specimens that clearly represent another species, distinct from both A. nipponicum and A. pectinatum.

This paper explores the suggestion that specimens from California and Western Europe known as A. hubbsii or A. pectinatum are really A. nipponicum, and that A. pectinatum from New Zealand is a distinct species. As an invasive species, the distribution of A. nipponicum has extended into North Carolina. We briefly comment on the distribution of A. nipponicum vis-à-vis that of Antithamnionella spirographidis (Schiffer) Wollaston of the tribe Heterothamnieae.

Materials and methods

Molecular study

DNA samples were prepared using the DNeasy Plant Mini Kit (QIAGEN, Valencia, CA, USA) from fresh, field-collected silica-gel dried specimens. The rbcL gene was amplified using the primer combinations: F7-R753 and F645-RrbcSstart as listed in (Lin et al., 2001). Sequencing primers used were: F7, F645, F993, R367, R753, R1150, RrbcS start (Freshwater & Rueness, 1994; Lin et al., 2001; Gavio & Fredericq, 2002). Amplification conditions followed Cho et al. (2003). Sequences were determined for both forward and reverse strands using an ABI Prism 3100 Genetic Analyzer (PE Applied Biosystems, Foster City, CA) with the ABI Prism BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems). Silica gel-dried specimens and extracted DNA samples are deposited at the University of Louisiana at Lafayette, stored at −20°C. Seven taxa representing four species of Antithamnieae, and four taxa representing two species of Heterothamnieae, were sequenced for inclusion in the phylogenetic analysis (Fig. 1); Ceramium californicum J. Agardh of the Ceramieae was used as the outgroup based on phylogenetic hypotheses derived from earlier global analyses of the Ceramiaceae (data not shown). DNA sequences have been deposited in GenBank. GenBank accession numbers, species identification and authors, information concerning origin, date and collectors are listed in Table 1.

Fig. 1. Maximum likelihood tree inferred from 11 rbcL sequences of Antithamnion and Antithamnionella species, and from one specimen of Ceramium californicum used as the outgroup. The Ln likelihood of the tree was −3992.6321. Bootstrap values (>50%) are shown for all ML (top) and MP (bottom) analyses.

Table 1.  Taxon and collection information for specimens used in the morphological study and rbcL analyses

Species	Location	Collection date	Collector	Habit/attachment	GenBank number
Antithamnion aglandum Kim et Lee	Jeoongdori, Wando, Korea	29.Jan.1999	S.M. Boo, T.O. Cho & H.G. Choi	Creeping on rope	AY594700
Antithamnion hanovioides (Sonder) De Toni	Pennington Bay, Kangaroo Island, S. Australia	7.Sept.1995	M. Hommersand		AY591927
Antithamnion ‘hubbsii’ Dawson	Halfmoon Bay, San Mateo Co., California, USA	3.Jun.2003	T.O. Cho & B.Y. Won	Creeping on decaying wood of floating docks	AY591930
Antithamnion nipponicum Yamada et Inagaki	Hachinohe, Aomori, Japan	21.May.1995	M. Kamiya	Culture strain #1078, Kobe University Research Center for inland seas	AY594699
Antithamnion nipponicum	In front of Duke University Marine Laboratory, Pivers I., Beaufort, Carteret Co, North Carolina, USA	29.Oct.2003	T.O. Cho & B.Y. Won	Creeping on Hypnea sp.	AY591928
Antithamnion nipponicum	Jetty, Beaufort, Carteret Co., North Carolina, USA	29.Oct.2003	T.O. Cho	Creeping on Hypnea sp.	AY591929
Antithamnion nipponicum	Torumni, Kobe, Japan	15.Jul.2004	S. Kawaguchi
Antithamnion pectinatum (Montagne) Brauner	Lee Bay, Stewart Island, New Zealand	3.Oct.2004	W.A. Nelson	Creeping on Laurencia thyrsifera	DQ023481
Antithamnion pectinatum	Island Bay, Wellington, North Island, New Zealand	22.Aug.1990	W.A. Nelson	Creeping on Gelidium sp.	-
Antithamnionella spirographidis (Schiffner) Wollaston	Burghsluis, Netherlands	8.Aug.1997	M. Hommersand		AY591925
Antithamnionella spirographidis	Monterey Bay, California	11.Dec.1999	T.O. Cho		AY591923
Antithamnionella ternifolia (Hooker et Harvey) Lyle	Williamston, Australia	28.Mar.2002	J. West		AY591926
Ceramium californicum J. Agardh	Yaquina Bay, Oregon, USA	13.Jul.1998	T.O. Cho & G.-I. Hansen		AY591931

The generated rbcL sequence data were compiled and manually aligned with Sequencher (Gene Codes Corp., Ann Arbor, MI) and MacClade 4.0 (Maddison & Maddison, 2000) and exported for phylogenetic analysis. Phylogenetic analyses were conducted with Maximum Likelihood (ML) and Maximum Parsimony (MP) algorithms as implemented in PAUP * v.4.0 beta 10 (Swofford, 2002). Only the ML tree is shown here (Fig. 1), as the tree topology was similar to the MP tree. For the ML analyses, the aligned sequences were first analyzed with Modeltest (v.3.0, Posada & Crandall, 1998), which compared different models of DNA substitutions in a hierarchical hypothesis-testing framework, to select a base substitution model that best fits our sequence data. The optimal model for the sequence was a GTR (General Time Reversible model, Rodriguez et al., 1990) + G (Gamma distribution). The parameters were as follows: assumed nucleotide frequencies A = 0.3139, C = 0.1569, G = 0.2096, T = 0.3196; substitution rate matrix with A–C substitutions = 1.5763, A–G = 4.0321, A–T = 4.1766, C–G = 0.9943, C–T = 13.4581, G–T = 1.0000; proportion of sites assumed to be invariable = 0; gamma distribution shape parameter = 0.2024. The ML tree was generated by a heuristic search of 1,000 random additions holding 1 tree at each step under the invoked settings for the respective base substitution model. Support for nodes was determined by calculating bootstrapping proportion values (Felsenstein, 1985) using 1,000 bootstrap replicates for the ML analysis. MP trees were inferred from a heuristic search and option support for nodes was determined by calculating bootstrapping proportion values (Felsenstein, 1985) using 1,000 replicates for MP analyses

Morphological study

Details of the specimens examined and their collection sites are given in Table 1. Material was preserved in silica gel upon collection in the field and subsequently transferred to 5% formalin/seawater, or immediately submerged in 5% formalin/seawater. Microscopic observations were made from material stained with 1% aqueous aniline blue, but two minute fragments of the holotype of A. pectinatum from Auckland, New Zealand were not stained. Vouchers are deposited in LAF (herbarium abbreviations follow Holmgren et al., 1990). Photomicrographs were taken with a Polaroid DMC Ie digital camera (Polaroid, Inc., Cambridge, MA) attached to an Olympus BX60 (Olympus, Melville, NY). Images were edited and assembled into plates using Photoshop v.5.0 (Adobe Systems Inc., San Jose, CA). A total of 15 individuals of A. nipponicum (including A. hubbsii) were selected for quantitative measurements. Antithamnionella spirographidis was collected from the Pacific and Atlantic.

Results

Molecular analyses

RbcL sequences of A. nipponicum were generated from four samples: one from California (A. ‘hubbsii’), two from North Carolina, and one from Japan. Of the 1,429-bp portion analyzed (99% sequenced), 1,135 bp are constant, 102 are variable, and 192 are phylogenetically informative. The rbcL sequences between the Pacific (California) and Atlantic (North Carolina) vouchers differed at only one of the sequenced 1,460 sites (position 835: a T in Pacific vs G in Atlantic specimens), whereas the sequences between the individuals from California and Japan were identical. Antithamnion nipponicum differed from A. pectinatum by 5.4–5.5% sequence divergence, from A. aglandum by 2.7% sequence divergence, and from A. hanovioides by 5.7–5.8% sequence divergence.

Antithamnion formed a distinct clade separated from Antithamnionella with strong bootstrap support (Fig. 1) in both ML and MP trees. All samples of A. ‘hubbsii’ and A. nipponicum were also placed in a strongly supported (100% for ML, 100% for MP) monophyletic clade and clearly separated from A. pectinatum, A. aglandum and A. hanovioides. The A. spirographidis sequences from the Atlantic (the Netherlands) and Pacific (Oregon and California) oceans were identical, and all sequenced populations fall in a strongly supported (100% for ML and MP) monophyletic clade.

Morphological observations

Vegetative morphology of A. nipponicum:

The thallus is composed of prostrate and erect axes (Fig. 31) with the prostrate axis consisting of axial segments growing parallel to the substratum, as shown in the North Carolina material. In vouchers from other localities, the prostrate axes were missing from the collecting protocol. We were not able to determine whether the prostrate axes are erect axes that have become secondarily attached to the substratum. The erect axis comprises main and indeterminate lateral axes of unlimited growth bearing opposite pinnae of determinate growth, which in turn bear distichous-opposite pinnules (Fig. 2). Indeterminate lateral axes are irregularly produced from the basal cell of pinnae in erect axes (Figs 6, 35).

Fig. 2–13. Antithamnion nipponicum from Halfmoon Bay, California, USA. Figs 2–9. Vegetative structure. Fig. 2. Upright thallus. Scale bar 0.5 mm. Fig. 3. Apical region showing successive development of opposite pinnae. Scale bar 100 µm. Fig. 4. Pinnae near apex with mostly abaxial pinnules. Scale bar 50 µm. Fig. 5. Pinnae with basal branched pinnules (arrows) in the middle thallus. Scale bar 100 µm. Fig. 6. Indeterminate lateral axis arising from basal cell of pinna. Scale bar 100 µm. Fig. 7. Gland cell (arrow) developing adaxially from lowermost cells of pinnules. Scale bar 50 µm. Fig. 8. Gland cell developing adaxially from upper side of basal cell of pinnule. Scale bar 5 µm. Fig. 9. Rhizoids developing from basal cell of pinna and terminating in multilobed disc. Scale bar 100 µm. Figs 10–13. Tetrasporic structure. Fig. 10. Erect tetrasporangial thallus with tetrasporangia on the main and lateral axes. Scale bar 0.5 mm. Fig. 11. Pinna bearing tetrasporangial initials (arrows). Scale bar 100 µm. Fig. 12. Pinna bearing mature tetrasporangia. Scale bar 100 µm. Fig. 13. Basal-most cells of pinnules bearing tetrasporangia. Scale bar 40 µm. Abbreviations: AC: apical cell; Ax: axial cell; BC: basal cell; GC: gland cell; IB: indeterminate lateral axis; R: rhizoid; T: tetrasporangium; Pl: pinnule; Pn: pinna.

Apical growth proceeds through transverse divisions of terminal cells producing a straight-to-slightly curved apex (Figs 3, 32). Apical cells are 4–5 µm in diameter and 1.1–1.3 times longer than broad, and subterminal axial cells increase in size away from the apex to 270–340 µm in diameter and 3.5–4.2 times longer than broad. Each axial cell produces a pair of lateral initials on either side; a lateral initial is cut off by oblique longitudinal division on one side and then the other, followed by the initial on the opposite side (Fig. 3), resulting in a pair of upwardly curved opposite pinnae (Fig. 32).

Pinnae are 550–600 µm long, composed of 11–13 cylindrical rachis cells on a quadrangular basal cell. Rachis cells are each 50–70 µm in diameter and 3–3.5 times longer than broad, and the filament (pinna) tapers to an acute apical cell. At maturity, the pinnae on the upper part of each axial cell are distichous-opposite at 45° to the main axis (Figs 4, 5, 33). The basal cell of a pinna is small, about 12–13 µm long and 10–11 µm in diameter, typically subspherical or quadrate in shape (Figs 6, 34), and can initiate rhizoids as well as indeterminate lateral axes (Figs 6, 9).

Pinnules can be produced on the adaxial and abaxial sides of each pinna rachis cell up to the first seven cells. The first pinnules are produced abaxially and the adaxial pinnules form later on. Six to seven pinnules are produced on the abaxial side, and 3–5 on the adaxial side (Figs 4, 5). Pinnules are 20–25 µm in diameter, 2–3 times longer than broad, 5–8 cells long on the adaxial side, 7–9 cells long on the abaxial side, tapering to a small mucronate apical cell. Although most pinnules are simple, lowermost pinnules can bear secondary branchlets on their abaxial side (Fig. 5).

Gland cells (Figs 7, 8, 34) are common and cut off adaxially by a slight concavo-convex division from the upper part of the basal cells of pinnules, and are subtended by the suprabasal cell (Figs 7, 8). They are ovate-to-oblong, about 20 µm long and 12 µm in diameter, pale green or yellowish, and reach the upper boundary of the next cell. Gland cells from specimens from North Carolina contained small darkly staining bodies (Fig. 34).

The uniseriate rhizoids originate from rhizoid initials cut off abaxially from the basal cells of pinnae and continue to grow towards the substratum at 45° to the pinna (Fig. 9). Three-to-four-celled rhizoids terminate in an irregular multicellular lobed disc (Fig. 9) that attaches to the epiphyte host or substratum.

Reproductive structures of A. nipponicum:

Tetrasporangia are scattered in the upper and middle thallus region of the tetrasporophyte (Fig. 10). Tetrasporangial initials are produced singly on the adaxial sides of the basal-most pinnule cells and do not develop beyond the third or fourth pinnule cell (Figs 11, 12). Tetrasporangia are cruciately divided, sessile, spherical to ellipsoidal, 60–70 µm long and 45–55 µm in diameter, excluding the sheath (Fig. 13).

Spermatangia develop on spermatangial heads of male gametophytes (Fig. 14). Most spermatangial heads replace pinnules in the upper thallus (Figs 15, 16), while some occur on special short branches on the adaxial side of the pinnules in the lower thallus (Fig. 19). The spermatangial head cells cut off spermatangial parent cells in small clusters from their upper end (Figs 17, 20). Each spermatangial parent cell bears one to two spermatangia (Figs 18, 21). Spermatangia are colourless and elliptical-to-spherical, about 3 µm long and 2.5 µm in diameter.

Fig. 14–21. Male structures of Antithamnion nipponicum from Halfmoon Bay, California, USA. Fig. 14. Male thallus. Scale bar 0.5 mm. Fig. 15. Apical region of male thallus. Scale bar 200 µm. Fig. 16. Spermatangial heads replacing pinnules. Scale bar 200 µm. Fig. 17. Spermatangial parent cells (arrow) borne on pinnule-like spermatangial head. Scale bar 10 µm. Fig. 18. Mature spermatangia borne on pinnule-like spermatangial head. Scale bar 10 µm. Fig. 19. Spermatangial heads (arrow) produced on special branches on adaxial side of pinnule. Scale bar 50 µm. Fig. 20. Spermatangial parent cells (arrow) produced on special branches on adaxial side of pinnule. Scale bar 10 µm. Fig. 21. Spermatangia (arrowhead) borne on spermatangial parent cells (arrow) on special branches on adaxial side of pinnule. Scale bar 10 µm. Abbreviations as in Figs 2–13.

In female thalli (Fig. 22), young pre- and post-fertilization stages are found in the upper parts of the thallus (Fig. 23). Procarps are formed singly or in pairs on every segment of the main and indeterminate axes (Fig. 24). The basal cell of a pinna becomes the supporting cell of the carpogonial branch (Fig. 25). After presumed fertilization, the supporting cell enlarges and cuts off an auxiliary cell (Fig. 26). The carpogonium produces a connecting process that fuses to the auxiliary cell (Fig. 26). The auxiliary cell divides into a foot cell and a gonimoblast initial (Fig. 27). The first gonimolobe is cut off terminally from the gonimoblast initial (Fig. 28), followed by the production of one or two additional gonimolobe initials laterally (Fig. 29). The supporting cell, foot cell and axial cell fuse into a fusion cell product (Fig. 29). Mature cystocarps are spherical, about 210–280 µm long and 190–230 µm in diameter (Fig. 30). Morphological features of A. nipponicum from Japan, shown in Figs 36–41, conform to the material from California and North Carolina.

Fig. 22–30. Female structures of Antithamnion nipponicum from Halfmoon Bay, California, USA. Fig. 22. Female thallus. Scale bar 250 µm. Fig. 23. Apical region with procarp (arrow). Scale bar 4.5 µm. Fig. 24. Upper part with procarps (arrows) borne on basal cells of pinna. Scale bar 50 µm. Fig. 25. Procarp composed of carpogonial branch and supporting cell. Fig. 26. Post-fertilization stage showing auxiliary cell borne on supporting cell, and connecting process (arrow) emanating from carpogonium and linking to auxiliary cell. Fig. 27. Formation of gonimoblast initial and foot cell from auxiliary cell. Fig. 28. Formation of first gonimolobes from gonimoblast initial. Scale bars 5 µm. Fig. 29. Formation of fusion cell from foot cell and supporting cell. Scale bar 40 µm. Fig. 30. Mature, naked cystocarp with two gonimobles. Scale bar 100 µm. Abbreviations: Au: auxiliary cell; CB1–3: sequence formation of carpogonial branch cells; Cp: carpogonium; Cy: cystocarp; Ft: foot cell; Fu: fusion cell; G: gonimoblast; Gi: gonimoblast initial; Gl: first gonimolobes; Su: supporting cell.

Fig. 31–35. Antithamnion nipponicum from Beaufort, North Carolina, USA. Fig. 31. Thallus growing on Hypnea sp. Scale bar 500 µm. Fig. 32. Apical region. Scale bar 50 µm. Fig. 33. Pinnae in middle part of thallus. Scale bar 50 µm. Fig. 34. Gland cells. Scale bar 40 µm. Fig. 35. Basal part showing indeterminate lateral axis and rhizoids produced from basal cells of pinnae. Scale bar 50 µm. Abbreviations as in Figs 2–13.

Fig. 36–41. Antithamnion nipponicum. Figs 36–38. Field material collected from Kobe, Japan. Fig. 36. Apical region showing successive development of opposite pinnae. Fig. 37. Indeterminate lateral axis arising from basal cell of pinna. Fig. 38. Male thallus. Scale bars 100 µm. Figs 39–41. Female culture strain (strain #1078 in Kobe University Research Center, Japan). Fig. 39. Pinnae. Scale bar 50 µm. Fig. 40. Indeterminate lateral axis produced from basal cell of pinna. Scale bar 40 µm. Fig. 41. Upper part of thallus with procarp born on basal cell (bc). Scale bar 50 µm. Abbreviations as in Figs 2–13.

Fig. 42–47. Antithamnion pectinatum and A. aglandum. Figs 42–44. Fragment of holotype of A. pectinatum from New Zealand. Fig. 42. Vegetative thallus. Scale bar 100 µm. Fig. 43. Middle part of thallus showing indeterminate lateral axis replacing a paired pinna. Scale bar 100 µm. Fig 44. Pinnae showing gland cell (arrow) touching three cells. Scale bar 50 µm. Figs 45–47. A. aglandum from Korea. Fig. 45. Vegetative thallus. Scale bar 300 µm. Fig. 46. Middle part of thallus showing indeterminate lateral axis (arrow) and rhizoid (arrowhead) produced from basal cell of pinnae. Scale bar 50 µm. Fig. 47. Pinnae. Scale bar 100 µm. Abbreviations as in Figs 2–13.

Vegetative morphology of A. pectinatum and A. aglandum:

In type material of A. pectinatum (Figs 42–44) indeterminate lateral axes appear to replace pinnae (Fig. 43) and gland cells are located on determinate 3-celled branchlets (Fig. 44). Material of A. aglandum (Fig. 45) lacks secondary branchlets (Figs 46, 47) and both indeterminate lateral axes and rhizoids are produced from basal cells of pinnae (Fig. 46).

Discussion

Our phylogenetic analyses indicate that the Californian material known as A. ‘hubbsii’ is conspecific with A. nipponicum, but distinct from A. pectinatum and A. aglandum. The specimens from California and North Carolina correspond to the original description and figures of the type of A. hubbsii (Dawson, 1962: 16, pl. 5, fig 2) and to its interpretation in subsequent work (Athanasiadis, 1996: 146, fig. 66), including habit, branching pattern, and the development of new indeterminate lateral axes. In particular, distal pinnules only occur on the abaxial side of pinnae, indeterminate lateral axes are produced from basal cells of the pinnae, and gland cells occur on lower cells of normal pinnules. Rhizoids and determinate axes developing from basal pinna cells have also been illustrated in A. nipponicum from Japan (as A. hubbsii, Itono, 1969) and western Europe (Curiel et al., 1996, fig. 4). These specimens of A. hubbsii also conform to authentic A. nipponicum obtained from culture strains and field collected material from Japan. All reproductive structures conform to those of other published Antithamnion species (Wollaston, 1971; Norris, 1987; Lee & West, 1980; Womersley, 1998).

Although Athanasiadis (1996, p. 147) placed A. nipponicum in synonymy with A. pectinatum, two conspicuous features separate the two: gland cells occur on normal pinnules in A. nipponicum, but only on special determinate 3-celled pinnules in A. pectinatum. Although gland cell production is thought to be environmentally induced in some species of the Antithamnion group (Hansen & Scagel, 1981), the size of the pinnules bearing gland cells has been considered an important character for the circumscription of other species (e.g., Antihamnion diminuatum Wollaston, Antihamnion eliseae Norris) (Wollaston, 1968; Wollaston, 1972; Norris, 1987). Also, although Norris's (1987) record from Natal includes minute glandless specimens of A. nipponicum, gland cells are observed in the pinnules of all our material. Furthermore, indeterminate lateral axes occur on the basal cells of pinnae in A. nipponicum (including A. ‘hubbsii’ ) whereas they are located directly on the axial cells in A. pectinatum (Athanasiadis, 1996, p. 146). In other Antithamnion species, such as Antithamnion verticale (Harvey) J. Agardh, Antithamnion hanovioides (Sonder) De Toni and Antithamnion cruciatum (C. Agardh) Nägeli (Womersley, 1998, p. 105, 110, 117), indeterminate lateral axes are typically produced from the basal cells of the pinnae, as in A. nipponicum. The presence of indeterminate lateral axes replacing pinnae in A. pectinatum may therefore be a diagnostic character of this species. Although Curiel et al. (1996) and Womersley (1998) reported A. pectinatum from Italy and Australia, respectively, as having an unlimited number of lateral axes produced from the basal pinna cells, our observation of the type material shows that indeterminate lateral axes are formed directly on the axial cells. After observing the holotype, Athanasiadis (1996, p. 146) also separated A. nipponicum (as A. ‘hubbsii’ ) from A. pectinatum based on: (i) the regular development of new indeterminate lateral axes from the basal cells of pinnae, and (ii) sparsely ramified pinnae.

The molecular and morphological evidence strongly suggest that A. hubbsii from California is conspecific with A. nipponicum and distinct from A. pectinatum and A. aglandum.

Taxonomic conclusion

Antithamnion nipponicum Yamada & Inagaki, 1935, p. 38, figs 1–3; type locality: Natudomari-zaki, Mutu Bay, Osyoro, Siribesi Prov., Japan; type not designated.

SYNONYM: Antithamnion pectinatum sensu Curiel et al. (1996).

PYNONYM SYNONYM: Antithamnion hubbsii Dawson, 1962, p. 16, pl. 5, fig. 2; pl., fig. 3; type locality: Melpomene Cove, Isla Guadelupe, Baja California, Mexico.

HOLOTYPE: Dawson. no. 8302, LAM 5000043, reproduced in Athanasiadis, 1996, fig. 66.

Antithamnion nipponicum has been reported from Saghalien to Korea (Tokida, 1954; Kang, 1966) and Japan (Itono, 1969) (western Pacific), and California (Abbott & Hollenberg, 1976) (eastern Pacific), but also from South Africa (Norris, 1987). This paper shows that its distribution extends to North Carolina, Atlantic, USA. Verlaque & Riouall (1989) reported A. nipponicum as an invasive species in the Mediterranean. After Athanasiadis (1996) maintained A. hubbsii as a distinct species but placed A. nipponicum into synonymy with A. pectinatum, all reports of an invasive Antithamnion in western Europe were given as A. pectinatum, especially in the lagoon of Venice (e.g., Curiel et al., 1996, 1998, 2002). Since the mid 1980s (Athanasiadis & Tittley, 1994), A. pectinatum has also been recorded from the western Atlantic (Connecticut, by R. Wilce), about the same time that invasive Codium Stackhouse was reported in North Carolina (USA) by Searles et al. (1984). We agree with Verlaque and Riouall (1989) that the Mediterranean species should be designated as A. nipponicum, as for the Californian, North Carolinian, and western European taxon. We suggest that A. nipponicum became established in central California, the Mediterranean and Adriatic Seas, and the Atlantic Ocean via recent introduction from Japan. The introductions may correlate with the introduction of Codium fragile subs. tomentosoides (van Goor) P.C. Silva along the Mid-Atlantic USA coast (Chapman, 1998), and of Undaria Suringar in western Europe (Curiel et al., 1998). We need to make a molecular comparison of type locality material of A. hubbsii before subsuming this species within A. nipponicum. Antithamnion pectinatum is a related but distinct southern hemisphere species from New Zealand.

Another Ceramiaceae species that has been widely reported from the Pacific and Atlantic Oceans, and the western Mediterranean is A. spirographidis (e.g., Feldmann-Mazoyer, 1941; Wollaston, 1968; Lindstrom & Gabrielson, 1989; Athanasiadis, 1996). Our phylogenetic analyses indicate that the sequences of all the Pacific and Atlantic material are identical. It is possible that a single species is distributed throughout these oceans.

Acknowledgements

This study was supported by NSF PEET grant DEB-0328491. We gratefully acknowledge M.H. Hommersand, who kindly provided specimens used in this study, including two small type fragments of Callithamnion pectinatum from Herb. Montagne by F. Ardré. We thank W.A. Nelson for A. pectinatum from New Zealand, M. Kamiya and S. Kawaguchi for culture and field-collected material of A. nipponicum from Japan, and M.H. Hommersand, P. Gabrielson, I.A. Abbott and J.N. Norris for valuable comments.