Taxonomic Studies in the Schizymeniaceae (Nemastomatales, Rhodophyta):Onthe Identity of Schizymenia Sp

Phycologia (2011) Volume 50 (2), 109–121 Published 3 March 2011

Taxonomic studies in the Schizymeniaceae (Nemastomatales, Rhodophyta):onthe identity of Schizymenia sp. in the Azores and the generic placement of Nemastoma confusum

1,3 4 2,5 6,7 2,8 DANIELA GABRIEL *, TOM SCHILS ,MANUELA I. PARENTE ,STEFANO G.A. DRAISMA ,ANA I. NETO 3 AND SUZANNE FREDERICQ 1CIBIO-Açores, 2Departamento de Biologia, Universidade dos Açores, 9501-801 Ponta Delgada, Açores, Portugal 3Department of Biology, University of Louisiana at Lafayette, Lafayette, LA 70504-2451, USA 4Marine Laboratory, University of Guam, Mangilao, GU 96923, USA 5Departamento de Cieˆncias e Engenharia do Ambiente, Instituto do Mar – IMAR, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal 6Nationaal Herbarium Nederland – Universiteit Leiden, 2333 CC Leiden, The Netherlands 7Institute of Ocean & Earth Sciences, University of Malaya, Kuala Lumpur 50603, Malaysia 8CIIMAR (Centro Interdisciplinar de Investigaçaõ Marinha e Ambiental), Universidade do Porto, Rua dos Bragas, 289-4050-123 Porto, Portugal

GABRIEL D., SCHILS T., PARENTE M.I., DRAISMA S.G.A., NETO A.I. AND FREDERICQ S. 2011. Taxonomic studies in the Schizymeniaceae (Nemastomatales, Rhodophyta): on the identity of Schizymenia sp. in the Azores and the generic placement of Nemastoma confusum. Phycologia 50: 109–121. DOI: 10.2216/09-67.1

Comparative rbcL sequence analysis indicates that the species going under the name Schizymenia dubyi in the Azores should be referred to as S. apoda. Sequences of Schizymenia specimens from China and Namibia were also identified as S. apoda, of which the type locality is the Cape Province in South Africa. Schizymenia dubyi, described from Atlantic France, is clearly a distinct species that we here report for Japan and Sicily in the Mediterranean Sea. Both Schizymenia species, along with an unreported species from Japan, are distinct from S. pacifica described from Washington, in the Pacific Coast of North America. Secondary pit connections were observed in gametophytes of S. apoda from the Azores, a previously unknown character for the Nemastomatales. Examination of type material of Nemastoma confusum indicates that this species, currently placed in the Nemastomataceae, should be transferred to the genus Platoma in the Schizymeniaceae. A morphological comparison between Platoma confusum (Kraft & John) comb. nov. with descriptions of P. cyclocolpum and P. chrysymenioides suggests that the three species are closely related.

KEY WORDS: Azores, Nemastoma, Nemastomatales, Platoma, rbcL, Rhodophyta, Schizymenia, Schizymeniaceae, Systematics, Taxonomy

INTRODUCTION (Kylin) Kylin (DeCew et al. 1992) revealed a heteromorphic life history where the tetraspophytic phase was identified as Schizymenia J. Agardh (Schizymeniaceae) has the largest a crustose species originally attributed to the generitype of geographical distribution of all nemastomatalean genera. Haematocelis, H. rubens J. Agardh. The 10 species of Schizymenia that are currently recognised Kylin (1956) circumscribed the genus Schizymenia as are found in temperate, subtropical and tropical waters, follows: ‘with blade-like thalli that bear gland cells in the ranging from the subantarctic islands to Alaska (Guiry & cortex, produce carpospores towards the exterior of the Guiry 2009). The genus Schizymenia is distinguished from thallus, with the base of the carposporophyte lying deep other genera in the Schizymeniaceae by the presence of inside the medulla, and in which nearly all cells of the diagnostic gland cells resembling large, inverted tears, gonimoblasts become carpospores. Additional characters located terminally on cortical filaments (Dixon & Irvine include weakly developed sterile filaments, or lack thereof, 1977). surrounding the carposporophyte, and a weakly defined The type of the genus, Schizymenia dubyi (Chauvin ex but distinct ostiole located above the slightly raised Duby) J. Agardh, was established by Agardh based on carposporangial mass’. The shape and size of the blades, Halymenia dubyi Chauvin ex Duby from Atlantic France, along with vegetative features such as short cortical branch and it is the most studied and best-characterised member of systems with subspherical inner cells, may also serve to distinguish species (Abbott 1967; Womersley 1994). the Schizymeniaceae. The genus also contains the only Schizymenia known tetrasporophyte of Schizymeniaceae found in nature The species of occurring in the Azores were originally reported by Trelease (1897) as S. undulata J. (Masuda & Guiry 1994). Extensive culture studies per- Agardh (Terceira Island) and S. obovata J. Agardh (Corvo formed on S. dubyi (Ardre´ 1977, 1980) and S. pacifica Island), both species currently regarded as synonyms of S. dubyi, described from Atlantic France, and S. apoda (J. * Corresponding author ([email protected]). Agardh) J. Agardh described from Atlantic South Africa, 109 110 Phycologia, Vol. 50 (2), 2011 respectively (Guiry & Guiry 2009). After extensive studies deposited in the Herbarium of the Department of Biology, on the Azorean marine flora, Neto (1994) reported that University of the Azores. Additional samples of Nemasto- only S. dubyi occurred in this archipelago. matales from the Ghent University Herbarium (GENT, The macroalgal communities of the Azores have been Belgium) and the Herbarium of the University of Louisiana studied since the mid-1800s (Seubert 1844), but early at Lafayette (LAF, USA) were used for comparison. publications were restricted to species inventories. Since The observations of N. confusum presented in this work Feldmann (1942), more attention has been given to the are based on five well-preserved slides of the isotype from ecology of Azorean macroalgae, and several papers have Ghana (John no. 7260) used for the original description of been published by Neto and coauthors over the past this species (Kraft & John 1976, p. 332). The material was 15 years (see Tittley et al. 2001, 2009; Tittley & Neto 2005). obtained through the kindness of Dr Gerry Kraft Life history studies and morphological information (Par- (University of Melbourne Herbarium – MELU) as a loan ente et al. 2003a, b; Toste et al. 2003a, b; Gabriel et al. 2009, to Dr Willem Prud’homme van Reine from the National 2010) are scanty. The floristic studies reveal a mixed flora Herbarium of the Netherlands, Leiden (L). with a strong component of cold-water species together A combination of stereo- and compound microscopes with a few tropical and subtropical taxa (Neto 1997). was used to describe morphological and anatomical The recently resurrected order Nemastomatales (Kylin) characters and reproductive structures for the diagnosis. Saunders & Kraft comprises two families, the Nemasto- Species identifications were based on the original descrip- mataceae and Schizymeniaceae (Masuda & Guiry 1995; tions and on a critical analysis of the literature. Represen- Saunders & Kraft 2002; Gavio et al. 2005), both tative collections of microscope slides and photomicro- represented in the marine flora of the Azores. Nemastoma graphs were made. Microscope slides of squash mounts confusum Kraft & John (Fredericq et al. 1992; Neto 1994), were prepared from liquid-preserved material, stained with Itonoa marginifera (J. Agardh) Masuda & Guiry (Larkum 1% aniline blue with HCL acidification, and mounted in 1960; Neto 1994) and Predaea feldmanii Børgesen (Freder- 50% Karo corn syrup–water solution (containing a few icq et al. 1992; Neto 1994; Tittley & Neto 1994) are the drops of 2% phenol). Photomicrographs were taken using three reported species of the Nemastomataceae. Platoma various digital cameras connected to light microscopes. cyclocolpum (Montagne) F. Schmitz (South & Tittley 1986; Measurements of cells and other structures were made Neto 1991, 1994) and S. dubyi (Schmidt 1929; Palminha using a micrometer eye piece (presented in the text as length 1957; South & Tittley 1986; Neto 1994; Tittley & Neto per width). 1994) are the listed species of the Schizymeniaceae. DNA samples were prepared using the DNAeasy Plant The red algal genus Nemastoma J. Agardh (1842) Minikit (Qiagen, Valencia, CA). Silica gel dried specimens currently comprises nine species predominantly inhabiting and extracted DNA samples are deposited at LAF and tropical to subtropical waters worldwide (Huisman 1999; stored at 220uC. Plastid-encoded rbcL was selected to infer Rodriguez-Prieto et al. 2004). Despite its wide distribution a phylogeny of Schizymenia and related nemastomatalean range, representatives of the genus are not abundantly genera. Protocols for DNA extraction, gene amplification present in the respective floras, and most of the species are and sequencing are described in Gavio & Fredericq (2002). known only from their original description (Kraft & John PCR primers (F7-R753, F57-R557, F645-R1150, F993- 1976). The reproductive features of Nemastoma species RrbcSstart) and sequencing primers (F7, F57, F645, F993, other than those described in N. dichotomum J. Agardh R376, R557, R753, R1150, RrbcSstart) are listed in Lin et have been poorly studied and require re-examination al. (2001) and Gavio & Fredericq (2002). (Masuda & Guiry 1994). Nemastoma confusum was A total of 24 rbcL sequences were used in this study, described by Kraft & John (1976) based on gelatinous including newly generated sequences of which the vouchers specimens from Ghana. The specific epithet alludes to are located at LAF, and of sequences downloaded from initial misidentification of the species as Predaea feldmannii GenBank. A data set of available members of Schizymenia when cystocarps could not be observed. was assembled, and two other taxa in the Schizymeniaceae, P. cyclocolpum and Titanophora pikeana (Dickie) Feld- As part of a revision of the Schizymeniaceae, the present mann, were added as the out-group based on a global paper reports on a molecular-based reappraisal of Schizy- phylogenetic analyses of the Nemastomatales (Gavio et al. menia species and provides morphological and ecological 2005). The information about the taxa, collection sites and observations of the Azorean Schizymenia species. We also collectors is listed in Table 1. present reasons for transferring N. confusum to the genus The generated rbcL sequences were compiled, edited and Platoma. aligned using Sequencher software (Gene Codes Corp., Ann Arbor, MI, USA) and exported for phylogenetic analysis in PAUP* v.4.0 beta 10 (Swofford 2003) and MATERIAL AND METHODS MacClade v.4 (Maddison & Maddison 2000). Phylogenetic analyses were conducted with the maximum Algal material used in the present study was collected since parsimony (MP) and maximum likelihood (ML) algorithms 1990 at different sites of the Archipelago of the Azores. as implemented in PAUP and PhyML v2.4.4 (Guindon & Collections were made from April to September, in the Gascuel 2003), respectively, and the Bayesian inference as intertidal. Reference collections were made by storing implemented in MrBayes 3.0 (Hall 2001; Huelsenbeck & samples in a 5% formalin/seawater solution, pressed as Ronquist 2001). Parsimony trees obtained under the Fitch herbarium sheets or dried in silica gel. Collections are criterion of equal weights for all substitutions (Fitch 1971) Table 1. List of species used in the rbcL sequence analysis with collection information and GenBank accession nu mber.

GenBank accession Species Collection identification Collection locality Collection data number Platoma cyclocolpum (Montagne) Schmitz SMG-04-202 Ponta Garc ¸a, Vila Franca, Sa õ D. Gabriel & P. Madeira, 20 Aug. 2004 FJ868809 Miguel, Azores (8-m depth) Schizymenia apoda (J. Agardh) LAF-7-6-93-1-1 Swakopmund, Namibia M.H. Hommersand, 6 Jul. 1993 AY29440 1 J. Agardh Schizymenia apoda (J. Agardh) LAF-6-94-1-1 Taiping Cape, Shandong prov., China M.H. Hommersand, Jun. 1 994 AY294392 J. Agardh Schizymenia apoda (J. Agardh) GRW-04-87 Barro Vermelho, Graciosa, Azores D. Gabriel, 10 Jun. 2004 FJ878 860 J. Agardh Schizymenia apoda (J. Agardh) GRW-04-88 Barro Vermelho, Graciosa, Azores D. Gabriel, 10 Jun. 2004 FJ878 861 J. Agardh Schizymenia apoda (J. Agardh) GRW-06-786 Baı á da Fonte, Graciosa, Azores 9 Jul. 2006 FJ878862 J. Agardh Schizymenia apoda (J. Agardh) SMG-04-164 Praia do Po ´pulo, Saõ Miguel, Azores M.I. Parente, 14 Jun. 2004 FJ878863

J. Agardh Gabriel Schizymenia apoda (J. Agardh) SMG-04-165 Praia do Po ´pulo, Saõ Miguel, Azores M.I. Parente, 14 Jun. 2004 FJ878864 J. Agardh Schizymenia apoda (J. Agardh) SMG-05-145 Sa õ Roque, Saõ Miguel, Azores D. Gabriel, 9 Aug. 2005 FJ878865 J. Agardh al.: et Schizymenia apoda (J. Agardh) SMG-05-226 Mosteiros, Sa õ Miguel, Azores D. Gabriel & M.I. Parente, FJ878866 J. Agardh 07 Sep. 2005 Schizymenia apoda (J. Agardh) SMG-05-259 Sa õ Vicente, Saõ Miguel, Azores D. Gabriel, 21 Sep. 2005 FJ878867 Schizymenia J. Agardh Schizymenia apoda (J. Agardh) SMG-06-84 Sa õ Miguel, Azores D. Gabriel, 2006 FJ878868 J. Agardh Schizymenia apoda (J. Agardh) PG07220 Praia do Po ´pulo, Saõ Miguel, Azores D. Gabriel & M. Parente 2007 GQ495983 J. Agardh Schizymenia dubyi (Chauvin ex Duby) LAF-6-22-93-1-1 Pighet, Brittany, France J. Cabioch, 22 Jun. 1993 AY2943 89 and Azores the in sp. J. Agardh Schizymenia dubyi (Chauvin ex Duby) LAF-7-10-94-1-1 Jodogahama, Miyako, Iwate-ken, M.H. Hommersand & M. Yoshizaki, AY294388 J. Agardh Japan 10 Jul. 1994 Schizymenia dubyi (Chauvin ex Duby) LAF-7-10-94-1-2 Jodogahama, Miyako, Iwate-ken, M.H. Hommersand & M. Yoshizaki, FJ878869 J. Agardh Japan 10 Jul. 1994 Schizymenia dubyi h (Chauvin ex Duby) PG08865 Mondello, Sicily, Italy M. Parente & R. Sousa 2008 GQ495984 J. Agard Schizymenia dubyi (Chauvin ex Duby) PG08887 Mondello, Sicily, Italy M. Parente & R. Sousa 2008 GQ495985 J. Agardh

Schizymenia pacifica (Kylin) Kylin LAF-7-26-95-1-1 Kanaha Bay, W. Juan Island, WA, M.J. Wynne, 26 Jul. 1995 AY294393 confusum ‘Nemastoma’ USA Schizymenia pacifica (Kylin) Kylin LAF-4-15-94-1-1 Vancouver, British Columbia, S.C. Lindstrom, 15 Apr. 1994 AY294394 Canada (drift) Schizymenia pacifica (Kylin) Kylin LAF-7-24-98-1-1 (TC101) North Boardman St. Park, OR, USA T. O. Cho & G.I. Gayle, 24 Jul. 1998 FJ878870 Schizymenia pacifica (Kylin) Kylin LAF-7-24-98-1-2 (TC130) North Boardman St. Park, OR, USA T. O. Cho & G.I. Gayle, 24 Jul. 1998 FJ878871 Schizymenia sp. cf. novae-zelandiae LAF-9-5-93-1-1 Tokawa, Choshi, Chiba Pref., Japan S. Fredericq & M. Yoshi zaki, 2 Sep. AY294391 J. Agardh 1993 Titanophora pikeana (Dickie) LAF-2-11-01-1-1 Deep Sponge Reef, Sodwana Bay, S. Fredericq & O. DeClerck, 11 Feb. AY294364 J. Feldmann KwaZulu-Natal, South Africa 2001 (27-m depth) 111 112 Phycologia, Vol. 50 (2), 2011 were inferred from a heuristic search, excluding uninfor- that extend into medullary rhizoidal filaments (Figs 6, 7). mative characters consisting of 1000 random sequence Occasionally, inner cortical cells are linked by secondary pit additions holding 10 trees at each step, MULPARS and connections (Fig. 8, arrow). The medulla is composed of tree-bisection-reconnection (TBR) algorithms with the primary filiform filaments as well as secondarily formed MULTREES (saving multiple trees) and STEEPEST rhizoidal filaments that are laxly to densely interwoven with DESCENT option. Support for nodes in the MP and ML one another and that may undergo localised swellings analyses were assessed by calculating bootstrap proportion (Fig. 9). values (Felsenstein 1985) based on 1000 and 100 resam- plings, respectively, retaining groups with a frequency PRE- AND EARLY POSTFERTILISATION STRUCTURES: Thalli greater than 50%, MULPARS and TBR algorithms. are monoecious. Carpogonial branches (Figs 10, 11) develop outwards from an inner cortical cell bearing The optimal models of sequence evolution to fit the data alignment estimated by hierarchical likelihood ratio tests subdichotomies of cortical filaments. The generative performed by Modeltest v.3.6 (Posada & Crandall 1998) cortical cell becomes the supporting cell of a three-celled carpogonial branch (Fig. 10). With maturation, a straight was the GTR + I + G (general time reversible model with a proportion of invariable sites and gamma distribution split trichogyne develops from the carpogonium growing to- into four categories) for the data set. The Akaike wards the thallus surface (Fig. 11). Simultaneously, the information criterion parameters applied in the likelihood cortical cells linked to the supporting cell expand in size and analysis were as follows: assumed nucleotide frequencies become dark staining subsidiary auxiliary cells (a.k.a. A 5 0.3045; C 5 0.1632; G 5 0.2103; T 5 0.3220; nutritive auxiliary cells; Fig. 11). substitution rate matrix A–C substitutions 5 0.0, A–G 5 POSTFERTILISATION STRUCTURES: The formation of con- 6.0541, A–T 5 2.3094, C–G 5 0.8317, C–T5 17.2925, G–T necting filaments was not observed. The generative auxiliary 5 1.0; proportion of sites assumed to be invariable 5 0.485; cell cuts off same-sized gonimoblast cells that are surround- and rates for variable sites assumed to follow a gamma ed by a loose network of vegetative filaments linked to the distribution with shape parameter 5 0.625. auxiliary cell that form an involucrum-like structure around For the Bayesian analysis, four chains of the Markov the carposporophyte (Fig. 12, arrows). A surface ostiole chain Monte Carlo were run, sampling one tree every 100 develops at the distal end of the gonimoblast (Fig. 13). generations for 2,000,000 generations starting with a random tree for the rbcL data sets, with the following MOLECULAR EVIDENCE: All Schizymenia vouchers from the parameters applied: nst 5 6 and Rates 5 invgamma. The Azores belong to a single species, nested in a clade that is first 20,000 generations were discarded as the ‘burn-in’ distinct from, but sister to, S. dubyi from Atlantic France period to reach equilibrium. A 50% consensus tree and Japan (Fig. 14). The Azorean samples are conspecific (majority rule as implemented by PAUP) was computed with Schizymenia apoda from Namibia and China. An from the trees saved after the burn-in point. Reliability of unreported Schizymenia species from Japan is sister to S. the Bayesian consensus tree is given by the frequency at dubyi and S. apoda. A fourth species is S. pacifica (Kylin) which each node appears among all saved trees after the Kylin occurring along the North American West Coast burn-in generation. This frequency corresponds to the (Washington and Oregon, USA, and Canada). The posterior probability of the clades (Hall 2001). uncorrected pairwise difference between S. dubyi and S. apoda is 0.015, between S. dubyi and S. sp. is 0.034, between S. apoda and S. sp. is 0.033, between S. pacifica and S. dubyi is 0.050, between S. pacifica and S. apoda is 0.046 and RESULTS between S. pacifica and S. sp. 0.048.

Schizymenia ‘dubyi’ (Chauvin ex Duby) J. Agardh (1851, Nemastoma confusum Kraft & John (1976, p. 332) from p. 171) from the Azores Ghana (based on isotype J-7260)

HABIT AND VEGETATIVE STRUCTURE: Plants foliose, liver- VEGETATIVE STRUCTURE: The thallus consists of narrow red to brownish-red in colour, simple or broadly lanceolate medullary filaments that are sparsely branched and loosely with irregular lobes and proliferations from the margins, aggregated, sometimes comprising X- and Y-shaped cells, sometimes undulate, soft fleshy, and slippery (Figs 1–4), up and pseudodichotomously branched cortical filaments, with to 20 cm tall. The occasionally perforated blades are cells decreasing in size and becoming more darkly stained attached with a short stipe to the substratum by a small with aniline blue closer to the surface (Fig. 15). Occasion- discoid holdfast, from which other blades can emerge, ally, adventitious rhizoidal filaments are formed in the inner usually growing within dense assemblages of geniculate cortex, growing inward where they intertwine with the Corallinaceae (Fig. 4). Cystocarps on the blade surface are medullary filaments. Gland cells are subspherical and easily distinguished by visible ostioles. intercalary on cortical filaments (Fig. 15), occurring singly The cortex is composed of anticlinal fascicles of at different densities throughout the blade. dichotomously branched moniliform filaments (Figs 5–8, 10), with occasionally darkly staining elongated gland cells PRE- AND EARLY POSTFERTILISATION STRUCTURES: Mature (Fig. 6, arrow) reaching the thallus surface. Innermost three-celled carpogonial branches develop at the distal end cortical cells are spherical, surface cells are more elongated of an inner cortical cell (supporting cell), in an intercalary (Figs 5–8). Inner cortical cells laterally produce primordia position at the dichotomy of a cortical fascicle (Figs 16, 17). Gabriel et al.: Schizymenia sp. in the Azores and ‘Nemastoma’ confusum 113

Figs 1–7. Habit of Schizymenia apoda from the Azores. Fig. 1. Cystocarpic specimen from Saõ Miguel, SMG-01-30; bar 5 1 cm. Fig. 2. Cystocarpic specimen from Saõ Miguel, SMG-04-170; bar 5 1 cm. Fig. 3. Cystocarpic specimen from Graciosa, GRW-04-87; bar 5 1 cm. Fig. 4. Cystocarpic specimen from Saõ Miguel growing on Corallina elongata. Fig. 5. Vegetative morphology showing regularly branched cortical fascicles subtending a medullary filament; bar 5 100 mm. GRW-04-88. Fig. 6. Cortical branches with elongated gland cell (arrow) and filiform medullary filaments; bar 5 40 mm. SMG-06-84. Fig. 7. Primary medullary (m) and secondary rhizoidal filament (r) connected to base of cortical fascicle; bar 5 40 mm. SMG-06-84. 114 Phycologia, Vol. 50 (2), 2011

Figs 8–13. Schizymenia apoda from the Azores. Fig. 8. Secondary pit connection (arrow) linking inner cortical cell with cell in neighboring cell file; bar 5 25 mm. GRW-04.88. Fig. 9. Filiform rhizoidal filaments and medullary cells connected by secondary pit connections (arrow); bar 5 100 mm. GRW-04.88. Fig. 10. Immature carpogonial branch (icb) borne on supporting cell (arrow) cut off laterally from inner cortical cell; bar 5 25 mm. GRW- 04.88. Fig. 11. Squashed three-celled carpogonial branch consisting of rounded basal cell, flat hypogenous cell and carpogonium with trichogyne (t). Note that neighboring cortical cells have expanded and started to stain darkly, which marks their transformation into subsidiary auxiliary cells (sac); bar 5 25 mm. GRW-04.88. Fig. 12. Young gonimoblasts (g) borne on auxiliary cell (aux), and incoming connecting filament (cf). Note that the basal cells of cortical filaments surrounding the gonimoblasts have elongated (arrows), forming a ‘rudimentary’ pericarp; bar 5 25 mm. GRW-04.88. Fig. 13. Surface ostiole of the carposporophyte showing carposporangia; bar 5 100 mm. SMG-04-164. Gabriel et al.: Schizymenia sp. in the Azores and ‘Nemastoma’ confusum 115

Fig. 14. One of 276 equally most parsimonious trees based on rbcL sequences showing the position of species of Schizymenia in the Schizymeniaceae, using Platoma cyclocolpum and Titanophora pikeana as the out-group. Branch lengths are proportional to sequence change. The three tiers of numbers at a node, namely represent maximum parsimony and maximum likelihood bootstrap values and Bayesian posterior probabilities, respectively (top to bottom).

The carpogonial branches consist of an oval to rectangular not observed. Connecting filaments traverse the thallus, basal cell, an oval hypogynous cell and a distal conical and each fuses with a generative auxiliary cell located in an carpogonium with a straight trichogyne (Fig. 16). Immedi- intercalary position, similar to the position of the support- ately before putative fertilisation, the two distal cells ing cell bearing a carpogonial branch, on another cortical connected to the supporting cell and their subtending fascicle (Fig. 19). cortical fascicles become darkly staining (Fig. 17). These cells become swollen, produce bulges on the cell’s sides and LATE POSTFERTILISATION STRUCTURES: Upon diploidisa- become irregular in shape (Fig. 18) and act as subsidiary tion, the auxiliary cell enlarges, and a gonimoblast initial is auxiliary cells upon their fusion with the fertilised cut off distally, in the same manner as a carpogonial branch carpogonium (stage not seen). The connecting filament initial (Fig. 20). The gonimoblast initial continues to divide initials originating from the subsidiary auxiliary cells were forming a single gonimolobe (Figs 21, 23). All gonimoblast 116 Phycologia, Vol. 50 (2), 2011

Figs 15–19. Platoma confusum from Ghana. Fig. 15. Pseudodichotomously branched cortical filaments, intercalary gland cell, X-shaped medullary cell and thin medullary filaments; bar 5 40 mm. J-7260. Fig. 16. Three-celled carpogonial branch borne on inner cortical supporting cell (su), consisting of rectangular basal cell (bs), oval hypogynous cell (hc) and conical carpogonium (c); bar 5 40 mm. J-7260. Fig. 17. Three-celled carpogonial branch with swollen subsidiary auxiliary cells (arrows); bar 5 40 mm. J-7260. Fig. 18. Subsidiary auxiliary cell producing a bulge (arrow); bar 5 40 mm. J-7260. Fig. 19. Cortical fascicles bearing an intercalary pyriform generative auxiliary cell; bar 5 40 mm. J-7260. cells transform sequentially into carposporangia, with the newly record S. apoda for the region, and it is likely that more immature ones observed at the base of the gonimo- many or all of the Chinese S. dubyi records are in fact S. lobe. Concomitantly, vegetative cells adjacent to the apoda. Thus, an in-depth morphological and molecular auxiliary cell elongate forming a primordial involucrum study to differentiate the worldwide species of Schizymenia surrounding the carposporophyte (Figs 22, 23). is called for. Only then will the biogeographic patterns among the Azorean, Namibian and Chinese populations of S. apoda and those of S. dubyi from Atlantic France, the DISCUSSION Mediterranean Sea and Japan be understood. Although Ardre´ (1980) considered gland cells to be As inferred from the rbcL sequence data analysis, the generally present in Australian specimens of S. dubyi,itis Azorean vouchers of Schizymenia are conspecific with S. likely that gland cells are not consistently present in apoda from Namibia, a species described from Table Bay, Australia, as is the case for European specimens of the Cape Province, along the west coast of South Africa, and same species (Womersley 1994). As reported in the present are different from S. dubyi, described from Brittany, study, young gland cells are initially subspherical and Atlantic France. Consequently, S. apoda can be added as elongate when maturing to become ovoid and finally a second Schizymenia species to the algal flora of the North longitudinally elongate when reaching the surface. Atlantic. Published records of the species under the name S. DeCew et al.’s (1992) suggestion that North American S. dubyi from Iceland (Caram & Jo´nsson 1972), Ireland pacifica is probably not distinct from S. dubyi has proved to (Cotton 1912; Newton 1931; Guiry 1978; Morton 1994), be incorrect (Womersley 1994; Fredericq et al. 1996). The Great Britain (Newton 1931; Dixon & Irvine 1977; Hardy sequences of a second Schizymenia species from Japan & Guiry 2003), the north coast of Atlantic Spain (Veiga et (Fig. 14) fits the morphological and anatomical description al. 1998) and Portugal (Ardre´ 1970) need to be critically re- of Schizymenia novae-zelandiae J. Agardh 1878 (p. 677) examined to ascertain their correct identity. Schizymenia from New Zealand as was reported and illustrated in dubyi has also been reported from China, but our results Adams (1994). However, as we have not examined type Gabriel et al.: Schizymenia sp. in the Azores and ‘Nemastoma’ confusum 117

Figs 20–23. Platoma confusum from Ghana. Fig. 20. Auxiliary cell borne intercalary in inner cortex bearing a gonimoblast initial; bar 5 40 mm. J-7260. Fig. 21. Young gonimoblast develops distally from the opposite side of the incoming connecting filament (cf); bar 5 4.5 mm. J-7260. Fig. 22. Young gonimolobe on an inflated auxiliary cell (ac); bar 5 100 mm. J-7260. Fig. 23. Close-up of Figure 22 showing a connecting filament (cf) and elongated cortical cells surrounding the gonimolobe (arrows); bar 5 40 mm. J-7260. material of S. novae-zelandiae, the Japanese specimen is The presence of nutritive cell clusters on cells adjacent to here referred to as Schizymenia sp. Figure 24 presents an the auxiliary cell, a diagnostic character of the genus updated overview of Schizymenia distributions in the world. Predaea, separates Predaea huismanii from N. confusum In detailed studies of S. dubyi, Ardre´ (1977, 1980) (Sanso´n et al. 1991). Although N. confusum shares features discovered that the species is actually heteromorphic in with both Nemastoma and Platoma, the species was placed which the life cycle comprises an erect gametophyte in the former genus mainly on the basis of the presence of alternating with a crustose tetrasporophyte that bears gland cells (Itono & Tsuda 1980). zonate tetrasporangia and resembles Haematocelis rubens Kajimura (1997) suggested that N. confusum was J. Agardh (Hansen 1989). Until the present study, probably a member of Platoma lacking a stipe and noticed secondary pit connections have been reported only from its resemblance to some specimens of Platoma izunosimense the crustose ‘Haematocelis’ tetrasporophyte in Nemasto- Segawa. Masuda & Guiry (1994) noted that N. confusum matales; it is herein newly reported that they occasionally was closely related to P. cyclocolpum based on the following occur in the gametophyte of S. apoda, linking cells of morphological similarities: supporting cells and auxiliary adjacent inner cortical branches and between medullary X- cells originate from the basal cells of cortical fascicles, the cells. gonimoblast develops directly from the auxiliary cell and In the course of a revision of the Nemastomatales, we gland cells are formed in an intercalary position on cortical examined the vegetative and reproductive morphology of fascicles. the red alga N. confusum, a species whose taxonomic status Based on the illustrations and original description of N. should be reinvestigated (e.g. Masuda & Guiry 1994; Gavio confusum, Gavio et al. (2005) also concluded that the et al. 2005). When this species was erected, all nemastoma- species should be transferred to a related genus once the talean taxa were placed in the Nemastomataceae; the mode of connecting filament initiation has been clarified. morphologically similar genera Nemastoma, Platoma and Gavio et al. (2005) contended that most species placed in Predaea were separated based on a combination of features. Nemastoma may in fact be species of Predaea because the 118 Phycologia, Vol. 50 (2), 2011

Fig. 24. Global distribution patterns of Schizymenia species.

original drawings by Berthold (1884) of the type N. cyclocolpum and P. chrysymenioides Gavio, Hickerson & dichotomum from the Bay of Naples depict auxiliary cells Fredericq (Gabriel et al. 2010). Platoma cyclocolpum has a encompassing one or two swollen cells toward the ends of great morphological plasticity covering apparently distinct filaments. Such rhizoidal auxiliary filaments were the basis morphotypes even when environmental conditions did not for the recognition of the monotypic Adelophycus (as vary between specimens (Huisman 1999). Phylogenetic Adelophyton) Kraft, a genus that thus may have to be studies showed that specimens going under the name P. merged with Nemastoma (Gavio et al. 2005). cyclocolpum worldwide are often misidentified members of Nemastoma confusum had been considered a ‘male’ plant P. chrysymenioides (Gabriel et al. 2010). The distinction of P. feldmannii described by Børgesen from Saint Helena between P. cyclocolpum, P. heteromorphum, P. izunosimense (Kraft & John 1976) or perhaps a species of Predaea lacking and P. chrysymenioides lies in the behaviour of the fertilised auxiliary nutritive cells (Gavio et al. 2005). Upon examina- carpogonium before fusion with the subsidiary auxiliary tion of type material of N. confusum, we conclude that this cells and the origin of the connecting filament (Schils & species belongs to the Schizymeniaceae instead of the Coppejans 2002; Gavio et al. 2005; Gabriel et al. 2010). Nemastomataceae based on the early prefertilisation events Since we were unable to observe this ephemeral stage in the in which the subsidiary auxiliary cells inflate, become darkly slides we had access to, we suggest the new combination staining, produce bulges and become irregular in outline, the Platoma confusum, transferring the species to the genus diagnostic feature of this family (Gavio et al. 2005). Platoma while keeping the specific epithet. Among the Schizymeniaceae (i.e. Platoma, Schizymenia, Titanophora and Wetherbeella), N. confusum is readily Taxonomic conclusions distinguished from species of Titanophora by a lack of Schizymenia apoda (J. Agardh) J. Agardh 1851, p. 175 calcified thallus incrustation and from Schizymenia by a lack of a compact cortex bearing elongate gland cells Figs 2–9 terminally on cortical fascicles. The genus Wetherbeella was TYPE LOCALITY: Table Bay, Cape Province, South Africa. erected on the basis of DNA sequence analysis to accommodate two ‘Platomas’ from Australia that lacked BASIONYM: Platymenia apoda J. Agardh 1848, p. 47. gland cells and formed a sister lineage to the other three SYNONYMS: Platymenia undulata var. obovata J. Agardh genera in the family (Saunders & Kraft 2002). Since we did 1848, p. 47 (fide Silva 1980; Silva et al. 1996). not have access to suitable material of N. confusum for Schizymenia obovata J. Agardh 1851, p. 175 (fide Silva molecular phylogenetic studies, we were not able to 1980, Silva et al. 1996). compare it with Wetherbeella using rbcL sequences. A comparison between N. confusum and descriptions of DISTRIBUTION: South Africa, Tristan da Cunha (Stegenga Platoma suggests that this species is closely related to P. et al. 1997), Namibia (Fredericq et al. 1996), (as S. dubyi): Gabriel et al.: Schizymenia sp. in the Azores and ‘Nemastoma’ confusum 119

The Azores (Neto 1994; Tittley & Neto 1994), Madeira AGARDH J.G. 1848. Om de Kapska artena af sla¨gtet Iridaea. (Levring 1974). O¨ fversigt af Kongelige Vetenskaps-Akademiens Fo¨rhandlingar, Stockholm 5: 46–49. Schizymenia dubyi (Chauvin ex Duby) J. Agardh 1851, p. 169 AGARDH J.G. 1851. Species genera et ordines algarum, seu descriptiones succinctae specierum, generum et ordinum, quibus algarum regnum constituitur. Volumen secundum: algas flor- TYPE LOCALITY: Cherbourg, Manche, Atlantic France. ideas complectens. Part 1. Lundae [Lund]. XII + 351 pp. BASIONYM: Halymenia dubyi Chauvin ex Duby 1830, AGARDH J.G. 1878. De algis Novae Zelandiae marinis Acta ˚ p. 944. Universitatis Lundensis. Lunds Universitets Arsskrift. Afdelningen fo¨r Mathematik och Naturvetenskap 14: 1–32. ARDRE´ F. 1970. Contribution a` l’e´tude des algues marines du SYNONYMS: Haematocelis rubens J. Agardh 1851, pp. 496– Portugal. I. La flore. Portugalia Acta Biologica Series B 10: 497 (fide Ardre´ 1980). 137–555. 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Açores) for the travel grants M3.2.1/I/133/2007 and M3.2.1/ FITCH W.M. 1971. Toward defining the course of evolution: I/062/2008. The National Science Foundation grants DEB- minimal change for a specific tree topology. Systematic Zoology 0315995, DEB-0328491, DEB 0743024, DEB-0919508 and 20: 406–416. DEB-0937978 are thanked for financial support. We also FREDERICQ S., SERRA˜ O E. & NORRIS J.N. 1992. New records of red algae from the Azores. Arquipe´lago 10A: 1–4. would like to thank Joaõ Brum, Nuno A´ lvaro, Patrıćia FREDERICQ S., HOMMERSAND M.H. & FRESHWATER D.W. 1996. Madeira and Sandra Monteiro for their help in collecting The molecular systematics of some agar-and carrageenan- Azorean specimens and Ian Tittley (Herbarium NHM, containing marine red algae based on rbcL sequence analysis. London), Willem Prud’homme van Reine (NHN, Leiden), Hydrobiologia 326/327: 125–135. 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