Rhodymeniocolax mediterraneus sp. nov. (Rhodymeniales, Rhodophyta), parasitic on Rhodymenia ardissonei from the western Mediterranean Sea

A. Vergés, C. Izquierdo and M. Verlaque. 2005. Rhodymeniocolax mediterraneus sp. nov. (Rhodymeniales, Rhodophyta), parasitic on Rhodymenia ardissonei from the western Mediterranean Sea. Phycologia 44: 510–516. Rhodymeniocolax mediterraneus Vergés, Izquierdo & Verlaque sp. nov., a sublittoral adelphohemiparasite of Rhodymenia ardissonei from the western Mediterranean Sea, is described. The distinctive vegetative and reproductive characteristics of the new species are: plants generally less than 6 mm in height with terete to compressed axes up to 1(−2) mm broad; a cortex composed of one to three layers of cells; spermatangia arranged in sori located on the subapical part of the thallus; cystocarps laterally positioned on branches; and cruciate tetrasporangia borne in sori. A comparison is made with other parasitic genera of Rhodymeniales and other species of Rhodymeniocolax.


INTRODUCTION
As a part of the 'Flora Phycologica Iberica' project, which started with the monographs of Fucales (Gómez Garreta 2001) and Kallymenia J. Agardh (Vergés 2001), the monograph of Rhodymeniales is being carried out in the Iberian Peninsula at present (Izquierdo Ramírez 2003;Sánchez 2003). During the study of the genus Rhodymenia Greville (1830), a new species of parasitic Rhodymeniales was discovered on Rhodymenia ardissonei Feldmann. This species, previously reported from Corsica as Rhodymeniocolax sp. by Verlaque (1987), is described herein as Rhodymeniocolax mediterraneus Vergés, Izquierdo & Verlaque sp. nov.

MATERIAL AND METHODS
This study is based on Mediterranean specimens collected subtidally by scuba diving in Corsica (France) during May 1981 and July 1982, and intertidally in Catalonia (Spain) from September 2002 to May 2004. Specimens were collected from 0 to 7 m depth. Specimens were preserved in buffered 5% formaldehyde-seawater until further treatment. Transverse and longitudinal sections were made with a Criocut-1800 (Reicher-Jung) freezing cryotome, stained in acidified 1% anilineblue distilled water, and mounted permanently in 50% Karo corn syrup. Drawings were made using a Labophot 2 Nikon microscope with a camera lucida and photographs using a Zeiss Axioskop 2 microscope with a digital camera. Voucher specimens and slides were deposited in the Herbarium of the University of Girona, Spain (HGI), in the algae section with reference letters HGI-A, and the Herbarium of Marc Verlaque, Centre d'Océanologie de Marseille, France, with the reference letter: F (wet specimens).  matosam ex cellulis rotundis aut oblongis 20-80 m in diametro compositam; ad hospitem late affixus, filamentis cellularum parvarum ovoidearum medullam hospitis penetrantibus.
Thallus 1-4(-6) mm high, with irregularly divided terete or compressed branches of 1-5 mm long and 0.2-1(-2) mm broad, verrucose when cystocarps present; hemiparasitic on Rhodymenia ardissonei; multiaxial structure, developing a cortex one to three cells thick, and a pseudoparenchymatous medulla composed of round to oblong cells measuring 20-80 m in diameter; attachment to host contiguous and secondary pit-connected.
ISOTYPES: Isotype collection deposited at the University of Girona, four sporophytes (HGI-A 5836b-e), two gametophytes (HGI-A 5836f-g) and eight microscope slides (S-5836b-1-S-5836b-8) realized with the specimen HGI-A 5836b.   HABITAT AND SEASONALITY: Rhodymeniocolax mediterraneus grows exclusively from 0 to 10 m depth on Rhodymenia ardissonei. The host is either directly attached to the rocky substratum or to sponges, as understory of large erect algae or as monospecific stand in rocky holes. The infected specimens of R. ardissonei are intermixed with uninfected ones. The parasite was never found below 10 m depth although R. ardissonei is frequent in the deep sublittoral assemblages. All the studied specimens were fertile. The gametophytes and tetrasporophytes sometimes occurred on the same host.
The species was apparently absent on R. ardissonei collected in autumn (from October to December). A cryptic form (endoparasitic form?) is suspected living during this period.
HABIT: The pink to pinkish-white plants are adelphohemiparasitic (hemiparasitic because pigmented) on R. ardissonei. They are 1-4(-6) mm high and composed of a cluster of irregularly divided terete or compressed branches that are up to 5 mm long and 1(-2) mm broad, arising from a cushion-like base that penetrates deeply into the host thallus (Figs 1-4). Branches become verrucose when cystocarps are present (Figs 2, 3).
VEGETATIVE STRUCTURE: The thallus structure is multiaxial. The cor-tex consists of one to three layers of rounded to ovoid cells. Outer cortical cells are 5-10 m in diameter. The pseudoparenchymatous medulla, up to 1200 m across, consists of nonpigmented, isodiametric to oblong cells, 100 m long and 20-80 m in diameter in the centre of the thallus and diminishing in diameter towards the cortex (Fig. 5). At the host-parasite interface, cells of the parasite creep between the cortical and medullary cells of the host without apparent modification to the host's structure. Cells of both algae are contiguous and secondarily pit-connected (Fig. 6).
REPRODUCTIVE STRUCTURES: The gametophytes are monoecious. Spermatangia are formed in subapical sori on the branches. The outermost cortical cells elongate and form one or two spermatangial mother cells, up to 8 m in diameter, that cut off outwardly one or two spermatangia measuring up to 3 m in diameter. Spermatia are shed through mucilage that becomes very thick during spermatangial development (Fig. 7).
Plants are procarpic with four-celled carpogonial branches, which are slightly bent and positioned on a modified inner cell that acts as a supporting cell. The first three cells of the carpogonial branch are   (Figs 14, 15). Abbreviations: ac, auxiliary cell; amc, auxiliary mother cell; cb, carpogonial branch; cc, cortical cell; cp, carpogonium; fc, fusion cell; g, gonimoblast; nt, nutritive tissue; p, protein body; pgc, primary gonimoblast cell; s, spermatangium; sc, supporting cell; smc, spermatangia mother cell; 1st, first cell of the carpogonial branch; 2nd, second cell of the carpogonial branch; 3rd, third cell of the carpogonial branch.
Figs 16, 17. Tetrasporangial features. Fig. 16. Surface view of a tetrasporangial sorus with mature tetrasporangia (HGI-A 5800). Fig. 17. Transverse section of a tetrasporangial sorus (HGI-A 5834). Scale bars ϭ 20 m (Figs 16, 17). angular in shape. The carpogonium is ovoid or round with a simple trichogyne (Fig. 8). The supporting cell, which is 17-28 m in diameter, is round or occasionally ovoid and bears the two-celled auxiliary branch, which is composed of the auxiliary mother cell and the auxiliary cell. One or two densely staining bodies, probably proteinaceous in nature (Kylin 1930), are always present in the auxiliary cell (Figs 14, 15). The transfer of the nucleus from the zygote to the auxiliary cell has not been observed after presumed fertilization. From the auxiliary cell, a primary gonimoblast cell is formed and acquires a 'wineglass' shape when the gonimoblast is young (Figs 9, 15). Later, the supporting cell, auxiliary mother cell, auxiliary cell and the primary gonimoblast cell form a large and elongated fusion cell (Figs 10, 11). The protein bodies in the auxiliary cell disappear and a deeply stained ring appears at the basal margin of the primary gonimoblast cell from which develop some thick gonimoblast filaments that form gonimolobes (Fig.   11). Most of the cells of the carposporophyte become carposporangia, which measure 4-8(-10) m in diameter when young and 9-12(-18) m when mature. The basal nutritive tissue is formed by ovoid cells (Figs 10, 13). Around the young gonimoblast there are some vegetative cells that become erect filaments, most of which disintegrate when the gonimoblast matures (Figs 10-12). The cystocarps are laterally inserted over the thallus and often clustered. The mature cystocarps, protruding, hemispherical, ostiolate, reach up to 625 m in height (including the pericarp) and 200-675 m in diameter. The pericarp, 80-125 m thick, is formed by up to 13 layers of small round cells arranged in radial rows (Fig. 13).
The decussately or cruciately divided tetrasporangia, 16-33 ϫ 8-18 m, are basally pit-connected to the inner cortical cells and located in extensive sori near the apices of the thallus. The two to three outer cortical cells of tetrasporangial sori are elongated (Figs 16, 17).

DISCUSSION
The four adelphoparasitic (sensu Feldmann & Feldmann 1958) genera presently included in the Rhodymeniales, Faucheocolax Setchell (1923), Rhodymeniocolax Setchell (1923), Gloiocolax Sparling (1957) and Champiocolax Bula-Meyer (1985), are easily distinguishable by their host specificity and anatomical and reproductive features (Table 1). Champiocolax differs from the other genera by having a hollow thallus with monostromatic diaphragms, four-celled carpogonial branches, a tela arachnoidea (persistent distinctive network of cobweblike filaments surrounding mature carposporophytes, Saunders et al. 1999) and tetrahedral tetrasporangia (Bula-Meyer 1985;Womersley 1996). Faucheocolax and Gloiocolax are characterised by anticlinal cortical filaments, three-celled carpogonial branches, the presence of a sterile branch on auxiliary mother cells and a tela arachnoidea (Sparling 1957;Hawkes & Scagel 1986). According to Sparling (1957, p. 345), the arrangement of the carpogonial branches in the parasitic genera of Rhodymeniales is not very valuable as a distinguishing feature, although in Gloiocolax the carpogonial branch is straight and in Faucheocolax is slightly bent. Lastly, Rhodymeniocolax is well characterised by being parasitic on Rhodymenia and in having a pseudoparenchymatous cortex (at least in the young part), four-celled carpogonial branches, no sterile branch on the auxiliary mother cells, disintegrating erect filaments between the gonimoblast and the pericarp and cruciate tetrasporangia.
Two species are currently assigned to the genus Rhodymeniocolax, R. botryoides Setchell (1923, as R. botryoidea), the The habit and anatomical and reproductive structures are very similar for all the species of Rhodymeniocolax; however, they grow on specific host(s) and there are some qualitative and quantitative differences ( Table 2). The thallus is tuberculate with short and thick axes, the cystocarps are terminal and tetrasporangia are scattered over most of the thallus in R. botryoides. In R. austrinus anticlinal cortical filaments are found in old cortices, and tetrasporangia occur in extensive nemathecia. Although poorly known, the unnamed species from the Atlantic differs from the Mediterranean Rhodymeniocolax in having smaller dimensions (length and diameter of axes, medullary cells), a more extended cortex (up to five cortical layers) and cystocarps that are subterminal to terminal. Lastly, R. mediterraneus differs from all the other species in the specific host (Rhodymenia ardissonei), in habit (simple or branched thalli up to 6 mm height, with terete to compressed branches up to 2.0 mm broad), and in having (1) a pseudoparenchymatous cortex, one to three cell layers thick; (2) cystocarps clustered and laterally inserted on branches; and (3) tetrasporangia in subapical sori.
Recent meticulous investigation showed that tetrasporangia are of intercalary origin in most of species of Rhodymeniaceae and reports of terminal tetrasporangia appear to be confined to particular species of the genus Rhodymenia but are seldom if ever substantiated by accurate development studies (cf. the review in Saunders et al. 1999). In Rhodymeniocolax mediterraneus, the material studied did not allow to solve the origin of tetrasporangia and only basal pit-connection of mature tetrasporangia to inner cortical cells was observed.
The genus Rhodymeniocolax is closely related to the anatomy and reproductive structures of its hosts, but differs in having: (1) a probably hemiparasitic way of life; (2) a highly reduced stature; (3) three layers of cortical cells; and (4) a medulla composed of more than two layers of ovoid to elongated cells that are not as large as their host's. There are limited data on the cystocarps of Rhodymenia ardissonei (Codomier et al. 1988) and the reproductive male and female structures are unknown, so comparisons cannot be made with those of Rhodymeniocolax mediterraneus (Table 3).
In conclusion, we propose that these criteria are sufficient to recognize R. mediterraneus as a new species. Saunders et al. (1999, p. 35) emended the definition of the four families of the order Rhodymeniales based on a molecular-systematic investigation. The new species described in this paper fulfils almost all the characters of the emended family of the Rhodymeniaceae.