Eur. J. Phycol. (2002), 37: 385–401. # 2002 British Phycological Society 385 DOI: 10.1017\S0967026202003736 Printed in the United Phylogenetic relationships within the (Phaeophyceae): rbcL, RUBISCO spacer and morphology

STEFANO G. A. DRAISMA1, JEANINE L. OLSEN2, WYTZE T. STAM2 AND WILLEM F. PRUD’HOMME VAN REINE1

" Section Phycology, Nationaal Herbarium Nederland, Universiteit Leiden branch, PO Box 9514, 2300 RA Leiden, The Netherlands # Department of Marine Biology, Centre for Ecological and Evolutionary Studies, Biological Centre, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands

(Received 3 July 2001; accepted 10 March 2002)

Phylogenetic relationships within the Sphacelariales sensu stricto were investigated using both a molecular and a morphological approach. Twenty species were included, representing all three families (i.e. Cladostephaceae, and Stypocaulaceae) and six of the eight genera. The outgroup consisted of six species representing the Syringodermatales, , Choristocarpaceae and Onslowiaceae. DNA sequences of partial rbcL [1255–1375 nucleotides (nt)] and the adjacent RUBISCO spacer (40–842 nt) were determined in order to assess the molecular phylogeny. Only the 3h-end of the spacer (112 nt positions) was alignable for the ingroup taxa. Partition homogeneity testing showed that rbcL and RUBISCO spacer sequences could be combined. However, analysis of rbcL alone or in combination with the RUBISCO spacer gave the same results with only slight differences in support (bootstrap, jackknife, decay). Support was low at the base of the ingroup. Four basal clades could be discerned: (1) Stypocaulaceae, (2) Sphacelaria radicans, (3) Sphacelaria caespitula and (4) all other included taxa: (Sphacella subtilissimajthe Sphacelaria subgenus Propagulifera) and (Cladostephus spongiosusjSphacelaria nanajthe Sphacelaria subgenera BattersiajPseudochaetopteris). The independent morphological analysis (using 23 unordered morphological characters) revealed high homoplasy and an almost completely unresolved tree in which only the subgenus Propagulifera was supported. The morphological characters were subsequently mapped onto the rbcL tree in order to identify diagnostic or phylogenetically informative characters. Blackening in response to bleach and the presence of secondary segments were found to be basal synapomorphies for the Sphacelariales; presence of propagules with a lenticular central apical cell defines the Propagulifera; and strict acroblastic branching mode and axillary zoidangia define the Stypocaulaceae. The remaining characters have been gained or lost multiple times. This study highlights the problem of extreme morphological convergence and\or plasticity. Four options for a new circumscription including nomenclatural changes are discussed. It is concluded that none of the options will lead to greater clarity with respect to either identification or classification.

Key words: Choristocarpaceae, Cladostephaceae, morphology, Onslowiaceae, phylogeny, rbcL, RUBISCO spacer, Sphacelariaceae, Sphacelariales, Stypocaulaceae

Introduction diplohaplontic life history and asexual reproduction from distinctively shaped vegetative propagules in The order Sphacelariales (Phaeophyceae) was some genera. Oltmanns (1922) divided the order erected by Migula (1909) and is characterized by into three families based on modes of growth and blackening of the cell walls when treated with distinctive branching patterns (Fig. 1). These were bleaching liquid and growth by conspicuous apical the Cladostephaceae, the Sphacelariaceae and the cells (Prud’homme van Reine, 1993). Transverse Stypocaulaceae. The family Choristocarpaceae divisions of the subapical cells (or primary Kjellman 1891 was also placed in the Sphacelariales segments) produce secondary segments which may by Fritsch (1945) based on growth from prominent undergo longitudinal and secondary transverse apical cells. However, members of the Choristo- divisions. In this manner the distinctive parenchy- carpaceae do not have the characteristic ordinal matous construction is produced (Fig. 1). Other character of transverse division of subapical cells, general features of the order include an isomorphic nor blackening of their cell walls with bleach treatment. Correspondence to: S. G. A. Draisma. Fax: j13 (0)71 5273511. In a recent molecular phylogenetic study of the e-mail: Draisma!NHN.LeidenUniv.NL Phaeophyceae, Draisma et al. (2001) have shown S. G. A. Draisma et al. 386

van Reine was not aware of the description of Sphacelaria kovalamensis V. Krishnamurthy & Baluswami in Krishnamurthy (1992), which had been previously described in Krishnamurthy & Baluswami (1988) without a Latin diagnosis and was therefore initially invalid. Its membership in the Sphacelariales is not clear. The aim of the present paper is to expand the phylogenetic analysis within the Sphacelariales s.s. by including representatives of all families and as many species as possible. We take both a molecular and a morphological approach. First we assess phylogenetic relationships based on sequences of the chloroplast-encoded large subunit of the RUBISCO gene (rbcL) and its adjacent RUBISCO spacer; next we assess phylogenetic relationships based on unordered morphological characters; and finally we compare the two analyses. Our objectives were to determine the best estimate of phylogeny, to establish which morphological features are useful for identification versus those that are useful for phylogenetic reconstruction, and to assess nomen- Fig. 1. Diagrammatic representation of anatomical clatural consequences under different circum- characters used in taxonomic descriptions of the scriptions. Sphacelariales. Materials and methods that the Sphacelariales is paraphyletic. In a re- analysis of the entire Phaeophyceae (including nine Taxon sampling species and genera and all families of the Spha- celariales), using both chloroplast and nuclear Taxa used in the phylogenetic analyses are listed in Table encoded DNA sequences, they were able to identify 2, including taxa with previously published sequences. Table 3 lists additional specimens for which (partial) rbcL three clades: the Sphacelariales s.s. (i.e. including and RUBISCO spacer sequences were obtained that were the families Cladostephaceae, Sphacelariaceae and (nearly) (see Results) identical to sequences of specimens Stypocaulaceae); a clade containing the genera listed in Table 2 and were therefore not included in the Onslowia and Verosphacela; and a clade containing analyses. Choristocarpus by itself at the base of the rooted phaeophycean tree. Therefore, it is clear that the DNA extraction, DNA amplification, cloning and Choristocarpaceae do not belong to the Spha- plasmid isolation, DNA sequencing celariales. Draisma & Prud’homme van Reine All steps from DNA extraction to DNA sequencing are (2001) have separated Onslowia and Verosphacela described in Draisma et al. (2001). Field material was from the Choristocarpaceae and placed them in the used for Sphacelaria plumigera and Sphacelaria mirabilis. newlycreatedfamilyOnslowiaceae.BoththeChoris- DNA from the encrusting species S. mirabilis was tocarpaceae and the Onslowiaceae are considered extracted in only 1 ml CTAB-PVPP extraction buffer, # incertae sedis. because only a tiny piece of crust (p6mm) was available. The Sphacelariales s.l. (Table 1) have been com- The same protocol was followed as described by Draisma prehensively reviewed by Prud’homme van Reine et al. (2001) up to and including the three CIA cleaning steps. From this step onwards, DNA was directly (1993). Newly described species since Prud’homme precipitated. DNA extracts of S. mirabilis, Sphacelaria van Reine’s review (1993) include Sphacelaria nip- caespitula 4, Sphacelaria nana 2 and 3, Halopteris filicina ponica Kitayama (1994), Sphacelaria tsengii 3 and Stypocaulon scoparium 3 were PCR-amplified with Draisma et al. (1998) and Sphacelaria recurva Keum the BirF forward primer (l BLSrbcL1277F in Siemer et et al. (2001), all belonging to the subgenus Pro- al., 1998) (5h-CAG CTA ACC GTG TTG C-3h) instead of pagulifera. Prud’homme van Reine (1993) did not the RbcL68F or RbcL188F forward primers (see include Halopteris congesta (Reinke) Sauvageau, Draisma et al., 2001) which were used for the other taxa. but this species would have been included in the genus Stypocaulon. The type species of the genus Alignment and phylogenetic analysis Phloiocaulon (i.e. Phloiocaulon squamulosum [Suhr] Sequences were aligned manually and edited using Se- Geyler), is now called Phloiocaulon suhrii (J. quence Navigator version 1.0 (PE Applied Biosystems, Agardh) P. Silva (Silva et al., 1996). Prud’homme Foster City, CA, USA). Phylogeny of the Sphacelariales 387

Table 1. Overview of the Sphacelariales sensu lato and notes on their distribution (Prud’homme van Reine, 1993; Kitayama, 1994; Draisma et al., 1998; Keum et al., 2001; Draisma & Prud’homme van Reine, 2001). Genera and subgenera labelled with an asterisk have one or more representatives in the present study (see Table 2)

Family genus No. of subgenus Type species species Distribution

Cladostephaceae Cladostephus* C. spongiosus (Hudson) C. Agardh 1817 1 Bipolar in temperate waters Sphacelariaceae Sphacelaria* S. reticulata Lyngbye 1818 42–47 Cosmopolitan Sphacelaria* S. reticulata 4 Temperate to cold N. Atlantic Pseudochaetopteris* S. plumigera Holmes 1883 4 Temperate to cold N. Atlantic and circumpolar (2 sections) Section Pseudochaetopteris: S. plumigera; section Racemosae S. racemosa Greville 1824 Battersia* S. mirabilis (Reinke ex Batters) 1 Temperate N. Atlantic Prud’homme van Reine 1982 Propagulifera* S. cirrosa (Roth) C. Agardh 1824 17–22 Worldwide temperate to tropical. Some (3 sections) Section Propagulifera: S. cirrosa; section species are endemic in small areas. S. cirrosa Furcigerae: S. rigidula Ku$ tzing 1843; and shows a disjunct distribution in temperate section Tribuloides: S. tribuloides N. Atlantic and S. Australia Meneghini 1840 Bracteata S. bracteata (Reinke) Sauvageau 1900 3 S.E. Australia and New Zealand, except S. sympodiocarpa which is endemic in Gulf of Biscay Reinkea S. reinkei Sauvageau 1900 6 Australia and New Zealand. S. bornetii also in S. America Incertae sedis 7 2 freshwater species (China & USA), other species endemics of Kerguelen, New Zealand, Pacific Mexico and Indonesia Sphacella* S. subtilissima Reinke 1890 1 W. Mediterranean, Canary Islands and S. Australia Stypocaulaceae Alethocladus* A. corymbosus (Dickie) Sauvageau 1903 1 Antarctic and sub-Antarctic regions Halopteris* H. filicina (Grateloup) Ku$ tzing 1843 4 S. Australia, New Zealand, and S. America. Exception is H. filicina which occurs in warm temperate N.E. Atlantic, Korea, and Japan Phloiocaulon P. suhrii (J. Agardh) P. Silva 1996 3 S. Australia and S. Africa Ptilopogon P. botryocladus (Hooker and Harvey) 1 S.E. Australia and New Zealand Reinke 1890 Stypocaulon* S. scoparium (Linnaeus) Ku$ tzing 1843 5 3 in S. Hemisphere and 2 in N. Hemisphere temperate waters Choristocarpaceae Choristocarpus* C. tenellus (Ku$ tzing) Zanardini 1 Mediterranean and Canary Islands Discosporangium D. mesarthrocarpum (Meneghini) 1 Warm temperate N. Atlantic and S. Australia Hauck 1885 Onslowiaceae Onslowia* O. endophytica Searles in Searles & 2 Warm temperate Atlantic USA and Bahamas Leister 1980 Verosphacela* V. ebrachia Henry 1987 1 Endemic in Florida

N., S., W. and E. indicate north(ern), south(ern), west(ern) and east(ern) respectively.

Phylogenetic analyses were done with PAUP* version saturation at rbcL third codon sites did not negatively 4.0b4a (Swofford, 1998). Maximum parsimony (MP) affect the phylogenetic signal when comparing phaeo- analysis was done as heuristic searches, using the tree phycean orders. Therefore, mutational saturation at third bisection-reconnection (TBR) branch-swapping algor- codon sites is not considered a problem at the lower ithm, with random sequence addition (100 replicates) and taxonomic level considered in this study. treating gaps as missing data. For the maximum like- Decay analysis of MP trees was performed with lihood (ML) analyses the nucleotide substitution model AutoDecay version 4.0 (Erikson, 1998). Bootstrapping was first determined using MODELTEST version 2.1 (Felsenstein, 1985) and parsimony jackknifing (Farris et (Posada & Crandall, 1998). The general-time-reversible al., 1996) were performed in PAUP* using respectively model of DNA substitution (Rodrı!guez et al., 1990) was five and ten random sequence additions and 10000 used for ML, including a gamma distribution (G) and a replicates. Character deletions per jackknife replicate proportion of invariable sites (I). were set at 37%. For ML bootstrapping only 100 Draisma et al. (2001) concluded that mutational replicates were possible. S. G. A. Draisma et al. 388 (1375 nt) AJ287932 (315 nt) (1372 nt) AJ287946 (255 nt) (1255 nt) AJ287933 (135 nt) (1374 nt) AJ287939 (195 nt) (1374 nt)(1371 nt)(1368 nt) AJ287915 (40 nt) (1373 nt) AJ287905 (161 nt) (1255 nt) AJ287906 (174 nt) (1255 nt) AJ287916 (159 nt) AJ287914 (133 nt) AJ287917 (144 nt) L RUBISCO spacer a a a a a a a a a a rbc EMBL accession no. and length identifier, collection year ller, 1991 AJ287869 (1250 nt) AJ287931 (210 nt) \ $ . (1987). et al Collection site Collector ) in Novaczek , Sweden W. F. Prud’homme van Reine, 1967 AJ287881 (1375 nt) AJ287929 (331 nt) $ (2) St Lunaire, Brittany, France W. F. Prud’homme van Reine, 1968 AJ287895 (1233 nt) AJ287936 (211 nt) Bonne Bay, Newfoundland, Canada E. C. Henry, 1987 AJ287897 (1255 nt) AJ289737 (140 nt) Stypocaulon scoparium . (2001). b Setchell & Gardner Oiso, Awajishima, Japan W. F. Prud’homme van Reine, 1993 AJ287893 (1255 nt) AJ287951 (276 nt) et al ex tzing (1) Dock-do Island, South Korea Y.-S. Keum, 1993 AJ287894 (1375 nt) AJ287934 (196 nt) $ Batters) Prud’homme van Reine St Andrews, Scotland S. G. A. Draisma, 1997 AJ287882 (190 nt) AJ287930 (272 nt) tzing Øystese, Norway W. F. Prud’homme van Reine, 1967 AJ287875 (1255 nt) AJ287923 (367 nt) ) $ tzing Porto Santo, Madeira Archipelago W. F. Prud’homme van Reine, 1994 AJ287866 $ ex s.s. Ku tzing) Zanardini La Nave, Ischia, W. Italy E. C. Henry, 1987 AJ287861 $ ex (Dickie) Sauvageau Astrolabe Island, Antarctica R. Moe, 1985 AJ287860 (Hudson) C. Agardh Oosterschelde, Zeeland, The Netherlands W. F. Prud’homme van Reine, 1966 AJ287863 tzing Galway, Ireland W. F. Prud’homme van Reine, 1966 AJ287883 (1366 nt) AJ287941 (252 nt) tzing Zanzibar, Tanzania O. de Clerck, 1997 AJ287851 (Ku (L.) Ku Henry Henderson Point, BC, Canada A. Whittick, 1982 AJ287868 Sauvageau Meneghini Sesoko, Okinawa, Japan W. F. Prud’homme van Reine, 1993 AJ287891 (1375 nt) AJ287947 (257 nt) $ $ Henry Vero Beach, FL, USA E. C. Henry, 1987 AJ287867 HolmesMontagne St Andrews, Scotland Tsuyazaki, Kyushu, Japan S. G. A. Draisma, 1997 S. Kawaguchi, 1989 AJ287878 (1375 nt) AJ287926 (842 nt) AJ287889 (1374 nt) AJ287949 (238 nt) Lyngbye Espegrend, Norway W. F. Prud’homme van Reine, 1967 AJ287870 (1255 nt) AJ287919 (173 nt) Reinke Fuerteventura, Canary Islands D. G. Mu Searles in Searles & Leister Fort Pierce, FL, USA E. C. Henry, 1981 AJ287864 Greville St Andrews, Scotland W. F. Prud’homme van Reine, 1971 AJ287880 (1374 nt) AJ287928 (423 nt) Segawa Ogi, Sado, Japan T. Kitayama, 1990 AJ287890 (1375 nt) AJ287950 (255 nt) (Reinke (Dillwyn) C. Agardh Øystese, Norway W. F. Prud’homme van Reine, 1967 AJ287874 (782 nt) AJ287922 (201 nt) Lyngbye Espegrend, Norway W. F. Prud’homme van Reine, 1967 AJ287879 (1255 nt) AJ287927 (345 nt) Ku Ku (Hudson) J. V. Lamouroux Oosterschelde, Zeeland, The Netherlands O. de Clerck, 1996 AJ287852 (Ruprecht) Okamura Harvey Asko (Roth) C. Agardh Fife Ness, Scotland S. G. A. Draisma, 1997 AJ287865 (Grateloup) Ku Naegeli Species used in the molecular and morphological phylogenetic analyses Previously published in Draisma The same individual as isolate CAN1 (referred to as a b Sphacella subtilissima Sphacelaria plumigera Sphacelaria plumosa Sphacelaria racemosa Sphacelaria arctica Sphacelaria divaricata Sphacelaria yamadae Sphacelaria tribuloides Sphacelaria californica Sphacelaria mirabilis Sphacelaria cirrosa Stypocaulon scoparium Sphacelaria radicans Sphacelaria caespitula Sphacelaria nana Sphacelaria rigidula Alethocladus corymbosus Halopteris filicina Stypocaulon durum cervicornis Onslowia endophytica Syringoderma phinneyi Verosphacela ebrachia Cladostephus spongiosus Outgroup taxa Choristocarpus tenellus Species Ingroup taxa (Sphacelariales Table 2. Phylogeny of the Sphacelariales 389

Table 3. Specimens with sequences that were (nearly) identical to those in Table 2 and were therefore not included in the phylogenetic analyses

EMBL accession no. for

Species Collection site Collector\identifier, collection year rbcL Rubisco spacer

Sphacelaria caespitula 2 St Andrews, Scotland S. G. A. Draisma, 1997 AJ287871 (1374 nt) AJ287919 (173 nt) Sphacelaria caespitula 3 Cherbourg, Normandy, W. F. Prud’homme van Reine, 1968 AJ287872 (1255 nt) AJ287920 (173 nt) France Sphacelaria caespitula 4 Espegrend, Norway W. F. Prud’homme van Reine, 1967 AJ287873 (190 nt) AJ287921 (173 nt) Sphacelaria nana 2 Espegrend, Norway W. F. Prud’homme van Reine, 1967 AJ287876 (190 nt) AJ287924 (371 nt) Sphacelaria nana 3 Tjomme, Norway W. F. Prud’homme van Reine, 1967 AJ287877 (173 nt) AJ287925 (365 nt) Halopteris filicina 3 Roscoff, Brittany, France W. F. Prud’homme van Reine, 1968 AJ287896 (190 nt) AJ287935 (211 nt) Stypocaulon scoparium 2 Canary Islands I. Germann\E. C. Henry, 1987 AJ287898 (1254 nt) AJ287938 (190 nt) Stypocaulon scoparium 3 Rade de Brest, Brittany, W. F. Prud’homme van Reine, 1967 AJ287899 (190 nt) AJ287940 (195 nt) France

Table 4. Morphological characters used in the morphological phylogenetic analysis. Indented characters depend upon character states of the above characters that are outlined to the left

No. Character Character states

1 Response to bleach 0: no, 1: yes 2 Transverse division of primary segments into secondary segments 0: no, 1: yes 3 Secondary length growth of secondary segments 0: no, 1: yes 4 Secondary transverse cell walls in secondary segments 0: frequent, 1: absent or scarce 5 Longitudinal cell walls 0: no, 1: yes 6 Longitudinal cell walls in transverse section 0: radial pattern, 1: periclinal pattern 7 Growth 0: leptocaulous, 1: auxocaulous 8 Branching mode 0: hypacroblastic, 1: acroblastic, 2: dichoblastic 9 Branching pattern 0: irregular, 1: regular 10 Acroblastic branching 0: acrohomoblastic, 1: acroheteroblastic 11 Hypacroblastic branching 0: hemiblastic, 1: meriblastic 12 Pericysts 0: absent (rare), 1: present 13 Differentiation in medulla and cortex 0: no, 1: yes 14 Holdfast 0: disc, 1: filamentous, rhizoidal 15 Rhizoids in upper part of plant 0: no, 1: divaricate, 2: appressed to the filament 16 Propagules 0: no, 1: with large apical cell, 2: without large or central apical cell, 3: with lenticular central apical cell 17 Propagules with central apical cell 0: with apical hair, 1: with long arms, 2: with short arms\horns 18 Hairs 0: absent, 1: solitary, 2: in bundles 19 Zoidangia 0: sessile or in thallus, 1: on one-celled stalk, 2: on multicellular stalk 20 Stalked zoidangia 0: on unbranched stalks, 1: on branched stalks or in cymose stands 21 Zoidangia in axils 0: no, 1: yes 22 Plurilocular zoidangia 0: with equal-sized loculi, 1: with different sized loculi, 2: presence of oogonia 23 Life cycle 0: isomorphic, 1: heteromorphic

Congruence of rbcL and RUBISCO spacer sequences allowing combination of the data sets in question. was evaluated using the partition homogeneity test Conversely, P values ! 0n05 indicate a significant dif- (Farris et al., 1995; Cunningham, 1997). This approach ference between the data sets and suggest that they should utilizes a resampling method in order to estimate the not be analysed together. Constant characters were degree to which two data sets (or subsets of data sets) are deleted prior to the analysis, a random sequence addition in agreement (i.e. congruent or homogeneous) or dis- (10 replicates) was applied and 10000 replicates were agreement (i.e. incongruent or heterogeneous). The dif- performed. ference between the numbers of steps required by in- Representatives of the Syringodermatales, Dictyotales, dividual and combined analyses is the incongruence Choristocarpaceae and Onslowiaceae were selected as length difference (ILDMF) (Mickevitch & Farris, 1981). outgroup taxa (Table 2). Draisma et al. (2001) have The distribution of the ILD statistic can be estimated by shown that these taxa branch off prior to the Sphacel- permutation. P values " 0n05 indicate congruence, thus ariales s.s. in a rooted phylogeny of the Phaeophyceae. S. G. A. Draisma et al. 390

Table 5. Data matrix for the morphological analysis

Character number

Taxon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Sphacelaria 110 0 100 0 0 – 1 1 0 0 0 0 – 0 1,21 0 0 0 caespitula S. radicans 1 1 0 0 1 0 0 0,1,2 0 1 1 1 0 0 1 0 – 2 0,1,2 1 0 0 0 S. nana 1 1 0 0,1 1 ? 0 0 0 – ? ? 0 0 1 0 – 1 1, 2 1 0 0 0 S. mirabilis 11– – 1–0 0 – – – – – 0 – 0 – – 1,21 – 0 0 S. plumigera 110 0 110 0 1 – 0 0 1 0 2 0 – 1,22 1 0 0 0 S. plumosa 110 0 110 0 1 – 0 0 1 0 2 0 – 1,21,21 0 0 0 S. arctica 110 0 110 0 0,1– 0 0 1 0 2 0 – 1,22 1 0 0 0 S. racemosa 110 0 110 0 0 – 0 0 1 0 1,20 – 1,22 1 0 0 0 S. cirrosa 1 1 0 1 1 0 0 0 0 – 0 0 0 0,1 1,2 3 0,1 1 1,2 0,1 0 1 0 S. rigidula 1 1 0 1 1 0 0 0 0 – 0 0 0 0, 1 1 3 1 1 1,2 0,1 0 0, 1 0 S. divaricata 110 1 100 0 0 – 0 0 0 1 1 3 1 1 1,20 0 1 0 S. yamadae 110 1 100 0 0 – 0 0 0 0,11 3 1 1 2 0,10 1 0 S. tribuloides 110 1 100 0 0 – 0 0 0 1 0 3 2 1 1,20,10 1 0 S. californica 1 1 0 1 1 0 0 0 0,1 – 0 0 0 0,1 0 3 2 1 2 1 0 ? 0 Sphacella 110 – 0–0 0 0 – – – – 1 0 0 – 0 1 – 0 – 0 Cladostephus 1 1 1 0 1 1 1 0,1 0 1 0,1 1 1 0,1 2 0 – 1,2 1, 2 1 0 1 0 Alethocladus 11? 0 110 1 0 0 – 0 1 1 2 0 – 0 1 0 1 ? 0 Halopteris 1 1 1 0 1 1 0 1 1 1 – 0 1 0,1 2 0 – 1,2 1,2 0 1 1 0 Stypocaulon 111 0 110 1 1 1 – 1 1 1 2 0 – 2 2 0 1 2 0 scoparium S. durum 111 0 110 1 0 1 – 1 1 0 2 0 – 2 2 0 1 ? 0 Syringoderma 00– – 0–0 1 0 0 – – – 1 0 0 – 0 0 – 0 0 1 Onslowia 0 0 – – 1 – 0 0 0 – – – – 1 0 2 – 1 0,1,2 0 0 0 0 Verosphacela 00– – 1–0 0 0 – – – – 1 0 2 – 1 0 – 0 0 0 Dictyota cervicornis 00– – 1–1 2 – – – 1 1 1 0 0 – 2 1 0 0 ? 0 D. dichotoma 00– – 1–1 2 – – – 1 1 1 0 0 – 2 1 0 0 2 0 Choristocarpus 00– – 0–0 1 0 – – – – 0 0 1 – 0 0 – 0 0 0

Character numbers refer to the characters listed in Table 4. Genera with only a single representative (Table 2) are referred to by the genus name only. A dash (–) indicates that the character is not relevant to the taxon under consideration. A question mark (?) indicates that the character state is unknown or doubtful. More than one number for a character means that more than one character state has been reported in the species. Sources of character states are Sauvageau (1900–1914), van den Hoek & Flinterman (1968), Searles & Leister (1980), Prud’homme van Reine (1982, 1993; personal observation), Henry & Mu$ ller (1983), Henry (1987a, b), Womersley (1987), Kitayama (1994), Gibson (1994), Keum et al. (1995, 1999), de Clerck (1998), Kawai & Prud’homme van Reine (1998).

Morphological characters the alignment was unambiguous. The first 120 nt Morphological characters and their character states are were excluded from the present phylogenetic analy- shown in Table 4 and the data matrix in Table 5. ses because these nucleotides were undetermined for Morphological character states were obtained from the 10 taxa (those that were amplified with the literature. MP analysis based on the matrix of Table 5 RbcL188F primer). EMBL accession numbers of was done using PAUP* under the polymorphism option rbcL sequences are listed in Tables 2 and 3. Only and simple taxon addition (reference taxon Choristo- 782 nt of the rbcL sequence were determined for carpus tenellus ). Bootstrap and parsimony jackknife (JK) Sphacelaria radicans and 190 nt for Sphacelaria analyses were performed with the following settings: 10000 replicates, 5 random sequence additions per mirabilis. The final alignment used 1255 nt (1375 replicate, maxtrees l 100, and for JK nine characters (l minus 120). At the nucleotide level 484 of 1255 nt 39%) were deleted in each replicate. were variable and 313 phylogenetically informative Each morphological character was mapped onto the among 26 taxa (S. mirabilis not included). At the rbcL tree using the MacClade version 3.08a computer amino acid level 84 sites were variable and 44 program (Maddison & Maddison, 1992). phylogenetically informative. Seventy-one sites were informative at the first or second codon Results positions and 242 at third codon positions. Within the Sphacelariales s.s. (20 taxa, S. mirabilis not Alignment properties included) there are 331 variable sites of which 187 In the rbcL is 1467 base pairs in length. are informative, 41 at first and second codon The length of the rbcL alignment in the present positions and 146 at third codon positions. study is 1375 nucleotides (nt), excluding the Average base composition of rbcL was: A 0n30; T RbcL68F primer. No gaps were found; therefore, 0n33; C 0n15; G 0n22. The average transition\ Phylogeny of the Sphacelariales 391

Fig. 2. rbcL tree for the Sphacelariales. One of 21 most parsimonious trees (length l 1312 steps; consistency index l 0n50; retention index l 0n53; rescaled consistency index l 0n27). The 50% majority rule consensus tree is identical except where shown with dotted lines. Branch lengths are proportional to the number of nucleotide changes. Bootstrap percentages are given above branches (MP left and ML right); decay index values are given below branches (left) and parsimony jackknife percentages (right); dashes (–) indicate percentages ! 50% or that the internode did not occur in the ML tree. Decay values of 0 indicate where the strict consensus tree collapses. transversion (ti\tv) ratio for the 1255 nt rbcL sequences were alignable for the species of the alignment was 1n19, for the ingroup taxa alone 1n48, Sphacelaria subgenus Propagulifera only (not and for the outgroup taxa alone 0n80. The ti\tv ratio shown). Spacer sequences of other Sphacelariales is expected to decrease with increasing sequence species differed considerably from each other at the distance because transversions erase the record of 5h-end. The 3h-end of the spacer (94–105 nt) is best the more frequent transitions. Values between 1 and conserved and could be aligned for the 21 taxa of 2 are typical and do not indicate saturation. Values the Sphacelariales s.s. resulting in an alignment of ! 1 do indicate some saturation (Holmquist, 1983; 112 nt positions of which 36 were gapped, 78 Bakker et al., 1995). variable and 51 phylogenetically informative. The The RUBISCO spacer sequences varied in length alignment is submitted to EMBL under accession from 40 nt in the outgroup taxon Choristocarpus number ALIGN 000127. RUBISCO spacer se- tenellus to 842 nt in Sphacelaria plumigera. Average quence lengths and EMBL accession numbers are base composition of the spacers was: A 0n50; T 0n33; listed for each specimen in Tables 2 and 3. All four C0n08; G 0n10. The spacer sequences of the Sphacelaria caespitula specimens showed identical outgroup taxa were unalignable with those of the RUBISCO spacer sequences, as well as the two Sphacelariales s.s. Complete RUBISCO spacer French Halopteris filicina specimens (2 and 3). S. G. A. Draisma et al. 392

Fig. 3. Morphological tree for the Sphacelariales s.l. Parsimony analysis based on 23 unordered morphological characters (Tables 4, 5) with the six most basal taxa used as paraphyletic outgroup. Boxes indicate conventionally recognized Sphacelaria subgenera. (A) 50% majority-rule consensus tree of 119400 most parsimonious trees (length l 92; CI l 0n77; RI l 0n81; RC l 0n63) with group frequencies above branches and characters that change unambiguously below branches (character numbering as in Table 4). (B) Fifty per cent bootstrap and parsimony jackknife tree with support values; bootstrap left, jackknife right.

Alignment of the spacers of the French and the Sphacelaria and is sister to the subgenus Propa- Korean (1) H. filicina specimens (not shown) re- gulifera. The Sphacelaria subgenus Propagulifera is quired 12 gaps (7i one, 4i two, and 1i eight monophyletic. The subgenus Pseudochaetopteris is position(s) in length) and showed 33 substitutions. monophyletic here because Sphacelaria mirabilis The RUBISCO spacer of the Sphacelaria nana (subgenus Battersia) was not included in this analy- specimens only differed by indels (of one and of five sis. An analysis with the 190 nt of Sphacelaria nucleotide positions). Spacer sequences of the Sty- mirabilis grouped it with the species of the subgenus pocaulon scoparium specimens differed by one sub- Pseudochaetopteris. The subgenus Sphacelaria is stitution and one indel of six nucleotide positions. paraphyletic. An analysis excluding the nucleotide positions that are undetermined for Sphacelaria radicans gave the same tree topology. Phylogenetic analysis: rbcL and RUBISCO spacer The maximum likelihood (ML) tree (not shown) Maximum parsimony (MP) analysis of rbcLwas differed from the MP tree only in the position of performed for all three codon positions together Sphacelaria caespitula, which formed a single taxon and resulted in 21 most parsimonious trees (MPTs). clade basal to all the Sphacelariales s.s. However, One of these trees is shown in Fig. 2. A 50% this difference did not occur in the MP and ML majority-rule consensus tree of the 21 MPTs has the bootstrap trees. Additional differences between the same topology as the tree shown in Fig. 2, except ML and MP tree were found within the Stypo- that the branch joining Sphacelaria arctica and caulaceae and Propagulifera, but again these dif- Sphacelaria racemosa collapses. The Stypocaul- ferences were not bootstrap-supported. aceae is monophyletic and the monotypic Cladoste- An analysis of the partial RUBISCO spacer phaceae is nested within the Sphacelariaceae. The sequences (including S. mirabilis and using the genus Sphacella is positioned within the genus StypocaulaceaejS. radicans as outgroups) resulted Phylogeny of the Sphacelariales 393

Fig. 4. Morphological trees for the Sphacelariales s.s. Parsimony analysis based on 23 unordered morphological characters (Tables 4, 5) with the four basal taxa used as monophyletic outgroup. Boxes indicate conventionally recognised Sphacelaria subgenera. (A) Fifty per cent majority-rule consensus tree of 19493 most parsimonious trees (length l 73; CI l 0n85; RI l 0n84; RC l 0n71) with group frequencies above branches and characters that change unambiguously below branches (character numbering as in Table 4). (B) Fifty per cent bootstrap and parsimony jackknife tree with support values; bootstrap left, jackknife right.

in 834 MPTs (173 steps, CI l 0n69, RI l 0n71, RC slightly from the tree in Fig. 2 with respect to the l 0n50) (trees not shown). The 50% consensus tree position of Cladostephus spongiosus. In the clade was unresolved except for the Propagulifera clade, with S. nana and the subgenus Pseudochaetopteris, which maintained 100% bootstrap support. The Cladostephus spongiosus became the sister taxon to short length of the alignment provided very little the subgenus Pseudochaetopteris species, but this signal. position is not well supported. The affiliation of The partition homogeneity test (PHT) comparing Sphacella with the subgenus Propagulifera as shown rbcL and RUBISCO spacer (excluding the outgroup in Fig. 2 was confirmed by high support values on taxa and Sphacelaria mirabilis) yielded a P value of the clade leading to these taxa (bootstrap l 93%, 0n196, indicating that the two subsets are not in jackknife l 99%, decay value l 8 steps). Also the conflict and can be combined. support for the clade leading to (Sphacella, Pro- MP analysis of rbcL and RUBISCO spacer pagulifera)j(Pseudochaetopteris, S. nana, Clado- together including the outgroup taxa (having 112 stephus) receives significantly more support (re- uncertain nucleotides in the RUBISCO spacer part spectively 86%, 93% and 4 steps). A similar of the alignment) resulted in 1 MPT (1497 steps, CI analysis with the reduced taxon set (i.e. without the l 0n52, RI l 0n55, RC l 0n29) (tree not shown) original outgroup) with only the rbcL resulted in 9 with the same topology as the tree in Fig. 2. MPTs (679 steps, CI l 0n61, RI l 0n64, RC l 0n39) Bootstrap support values were not higher. When (trees not shown) of which the consensus tree had the analysis was repeated, excluding the original the same topology as in the combined analysis, outgroup and substituting the Stypocaulaceae and except that now Cladostephus stayed with S. Sphacelaria radicans as new outgroups, three MPTs nana. Again here very good support values for (859 steps, CI l 0n62, RI l 0n64, RC l 0n40) were the (Sphacella)j(Propagulifera) and the (Spha- recovered (trees not shown). These trees differed cella, Propagulifera)j(Pseudochaetopteris, S. nana, S. G. A. Draisma et al. 394

Cladostephus) clades were found. Apparently, steps were recovered. The 50% majority-rule con- adding the RUBISCO spacer to the data set did not sensus tree (Fig. 4A) and the bootstrap and par- significantly influence the outcome of the phylo- simony JK tree (Fig. 4B) reveal an unsupported tree genetic analyses. except for the Propagulifera. Ten character state changes (involving eight characters: 5, 7, 10, 12, 14, 15, 18, 22) could be traced unambiguously on the consensus tree in Fig. 4A. However, this was not the Phylogenetic analysis: morphology same subset of characters that changed unam- MP analysis of the ingroup and outgroup taxa using biguously within the ingroup in Fig. 3A (characters: 23 morphological characters (Tables 4, 5) resulted 7, 9, 11, 12, 15, 18, 21, 22). in 119400 MPTs of 92 steps. The 50% majority-rule To further identify and analyse homoplasy, each consensus tree is shown in Fig. 3A and the bootstrap character was mapped onto the topology of the and parsimony jackknife (JK) tree in Fig. 3B. rbcL-based tree (Fig. 5). The tracing displays the Nineteen character state changes (involving 13 ancestral character states that imply the smallest characters) could be traced unambiguously on the number of character state changes. Characters 1, 2 tree in Fig. 3A of which 11 (involving 9 characters) and 23 are constant within the ingroup (synapo- were within the ingroup. When the outgroup was morphic for the order Sphacelariales s.s.) and thus excluded from the MP analysis 19 493 MPTs of 73 not phylogenetically informative. The characters 5,

1. Bleaching response 3. Secondary length growth 2. Secondary segments no no yes yes

4. Secondary transverse 5. Longitudinal walls cell walls frequent no absent or scarce yes polymorphic equivocal equivocal

6. Transverse section 7. Growth radial pattern leptocaulous periclinal pattern auxocaulous Phylogeny of the Sphacelariales 395

8. Branching mode 9. Branching pattern hypacroblastic irregular acroblastic regular dichoblastic polymorphic polymorphic equivocal equivocal

10. Acroblastic branching acrohomoblastic 11. Hypacroblastic branching acroheteroblastic hemiblastic equivocal meriblastic polymorphic

12. Pericysts 13. Differentiation in medulla and cortex absent (rare) present no equivocal yes equivocal

14. Holdfast 15. Rhizoids in upper part of plant disc no filamentous, rhizoidal divaricate polymorphic appressed to the filament equivocal polymorphic equivocal

Fig. 5. (A)–(C) Character mapping on the rbcL-based tree. Each of the 23 morphological characters from Tables 4 and 5 was mapped onto the rbcL tree from Fig. 2. Halopteris filicina 1 and 2 are presented as a single taxon. Each branch is shaded (or patterned) to indicate what states are most parsimoniously placed at the node terminating the branch. Equivocal means that more than one option is equally parsimonious. The boxes just under each terminal taxon name show the state(s) observed in that taxon. If a character is absent in a taxon (indicated with ‘–’ in Table 5) no box is shown and if the state is unknown (‘ ?’ in Table 5) a question mark is shown. S. G. A. Draisma et al. 396

17. Propagules with central 16. Propagules apical cell no with apical hair with large apical cell with long arms without large or central apical cell with short arms (horns) with lenticular central apical cell polymorphic equivocal

18. Hairs 19. Zoidangia absent sessile or in thallus solitary on one-celled stalk in bundles on multicellular stalk polymorphic polymorphic equivocal

20. Stalked zoidangia 21. Zoidangia in axils on unbranched stalks no on branched stalks or yes in cymose stands polymorphic equivocal

22. Plurilocular zoidangia 23. Life cycle with equal sized loculi isomorphic with different sized loculi heteromorphic presence of oogonia polymorphic equivocal

7 and 10 are not informative within the ingroup subclades. In general, however, all the remaining either (autapomorphic in one species). As can be characters show homoplasy, i.e. character states seen in Fig. 5, a few characters are useful within that have been gained or lost repeatedly. Phylogeny of the Sphacelariales 397

rbcL phylogeny Discussion The rbcL tree in Fig. 2 represents the best estimate RUBISCO spacer of the Sphacelariales s.s. currently available and will serve as the basis in the later discussions. An analysis RUBISCO spacer sequences are, by themselves, not that included both rbcLjpartial RUBISCO spacer informative enough for a phylogenetic analysis of sequences resulted in the same topology. The the Sphacelariales s.s. This is mainly related to bootstrap and parsimony jackknife support per- extreme length variation which affected the align- centages signal that the resolution in the tree is not ment and the subsequent number of usable as good as it appears. This is particularly true in the positions. Lengths ranged from 135 to 842 nt basal portion of the ingroup. We will return to these (Table 2). The general cause of length variation issues later. in spacer sequences is related to duplications, indels or slipped-strand mispairing, which have Morphological phylogeny little effect on these presumptively non-functional regions. In the present case, we were not able to Analysis of the morphological data yielded much detect major duplications, even in the very long less resolution than the molecular data. The only spacers. ingroup clade with moderate bootstrap support was The RUBISCO spacer length of Choristocarpus the Sphacelaria subgenus Propagulifera (Fig. 3B). tenellus (40 nt) is comparable to that of non- The analysis without outgroup (Fig. 4B) of the phaeophycean algae studied to date. morphological data additionally showed weak sup- The RUBISCO spacer is 54 nt long in Heterosigma port for the Stypocaulaceae. The difficulty in es- akashiwo (Hada) Hada ex Sournia (as H. carterae) tablishing monophyletic relationships was in- (Raphidophyceae) (Chesnick et al., 1996), 38–43 nt fluenced by three factors. First, there were relatively in five species of Bacillariophyceae (Daugbjerg & few unambiguous characters and character states, Guillou, 2001), 38–40 nt in two species (three which led to many MPTs and low bootstrap strains) of Bolidomonas () (Daug- support. Of 23 characters, more than 50% showed bjerg & Guillou, 2001) and 38–43 nt in two species homoplasy, i.e. gains and losses of character states (14 and three strains) of (Bailey two or more times. Second, defining taxa by the & Andersen, 1999). This observation is in ac- absence of characters (e.g. Sphacella) and\or auta- cordance with the basal position of C. tenellus in pomorphies (e.g. Cladostephus) are weak criteria. the rbcL-based Phaeophyceae tree presented by Whether or not either of these genera should Draisma et al. (2001). In other brown algae the continue to be recognized is questionable. Third, RUBISCO spacer length ranges from 138–204 nt in natural phenotypic plasticity and\or polymorphism the s.l. (Stache-Crain et al., 1997; in characters among different individuals of a Siemer et al., 1998; Kogame & Masuda, 2001; species reduces utility (e.g. occurrence of more than Peters & Ramı!rez, 2001), 122–200 nt in the one branching mode, solitary vs bundled hairs, (Phillips, 1998; Lee et al., 1999), 144–231 nt in the rhizoidal vs discoid holdfast). Polymorphism oc- (Sasaki et al., 2001; Draisma, un- curred in 11 of the 23 characters. It must be published), 319–438 nt in the concluded, therefore, that high levels of morpho- (Peters & Ramı!rez, 2001), 190–234 nt in the Spor- logical convergence combined with few syn- ochnales (Sasaki et al., 2001), 220–225 nt in the apomorphies greatly restrict the usefulness of mor- (Sasaki et al., 2001), 160 nt in phological characters for phylogenetic classi- Asteronema rhodochortonoides (Boergesen) Mu$ ller fication. Nevertheless, it remains a fact that mor- et Parodi (incertae sedis) (Peters & Ramı!rez, 2001), phology is the basis for identification. 265–289 nt in the advanced Laminariales (i.e. To further explore the reliability of traditional Alariaceae, Laminariaceae and Lessoniaceae) characters, we mapped each morphological charac- (Yoon & Boo, 1999; Kraan & Guiry, 2000; Kraan ter onto the molecular tree (Fig. 5). This was justified et al., 2001; Kawai et al., 2001; Draisma, un- on the grounds that the molecular tree is the best published), and in the other Laminariales estimate of the phylogeny currently available and 148–150 nt, 192 nt, 131 nt, 177–180 nt and 202 nt in on the expectation that at least a few morphological the Chordaceae, the Pseudochordaceae, the Halosi- characters do carry a phylogenetic signal. Bleaching phonaceae, the and the Akkesiphy- response and formation of secondary segments caceae respectively (Yoon & Boo, 1999; Kawai & (characters 1 and 2) are good synapomorphies Sasaki, 2000; Kawai et al., 2001; Sasaki et al., defining the Sphacelariales s.s. Presence of pro- 2001). We have no explanation for these extreme pagules with a lenticular, central apical cell (charac- length differences. Members of the Sphacelariales ter 16) defines the subgenus Propagulifera. Strict s.s. are definitely on the high end with averages acroblastic branching of the main axes (character 8) above 250 nt. and axillary zoidangia (character 21) define the S. G. A. Draisma et al. 398

Stypocaulaceae. The remaining characters are either a possible Tethyan and north Pacific origin, re- autapomorphies or have been gained or lost mul- spectively. Moreover, conspecificity of the north- tiple times. west Atlantic Stypocaulon sp. and the Pacific S. There is no good basis for the recognition of durum seems apparent because (1) Kawai and Sphacella. It is nested within Sphacelaria. Apart Prud’homme van Reine (1998) determined that the from character 5 there are no other characters that temperature tolerance range of Pacific S. durum is contradict the grouping of Sphacella with Spha- similar to that of the northwest Atlantic Stypocaulon celaria. Character 5 is autapomorphous in sp. and (2) Prud’homme van Reine (unpublished) Sphacella. Most characters that occur in Sphacelaria observed discoid holdfasts (characteristic of S. but are missing in Sphacella (viz. characters 4, 6, 11, durum) on all northwest Atlantic (Canadian) her- 12 and 13) are due to the absence of longitudinal cell barium specimens of Stypocaulon. The observed walls (character 5) in Sphacella. Cladostephus is also difference between the rbcL sequence of the nested within Sphacelaria. Unlike Sphacella, how- Canadian S. durum and the European Stypocaulon ever, it has well-defined morphological features that isolates (Tables 2 and 3, Fig. 2) is in favour of the separate it from Sphacelaria. These specifically hypothesized conspecificity, but the rbcL sequence include the co-occurrence of different branching of a Pacific S. durum isolate is required for con- modes and the arrangement in whorls of deter- firmation. minant laterals. The traditional recognized sub- genus Sphacelaria (which includes the type) is also Halopteris vs. Stypocaulon. The genera Halopteris paraphyletic. Considering S. nana as part of Clado- and Stypocaulon were first described by Ku$ tzing stephus or considering S. radicans as belonging to (1843), but Sauvageau (1903) merged the species of the Stypocaulaceae are untenable on morphological both genera in the single genus Halopteris. grounds but not on molecular grounds. Prud’homme van Reine (1991, 1993) proposed the reinstatement of the genus Stypocaulon, mainly based on the absence (Halopteris) or presence The Stypocaulaceae (Stypocaulon) of oogonia. The rbcL data do not Pacific and Atlantic Halopteris. Halopteris filicina is lead us out of this dilemma. The mostly poorly the single species in the present study for which supported topology within the Stypocaulaceae isolates from different oceanic basins have been makes it impossible to assess the validity of these used. Although the Pacific and the Atlantic two genera. This also applies to the Antarctic genus specimens form a monophyletic clade (Fig. 2), they Alethocladus. could represent two separate cryptic species, be- cause their genetic distance for the rbcL is higher Options for a new circumscription: lumping or than in 22 other pair-wise species comparisons. It is, splitting? however, precarious to base the distribution of two haplotypes solely on one or two isolates per region This part of the Discussion is based on the rbcL (Kooistra et al., 1992; van Oppen et al., 1995). phylogeny in Fig. 2 and redrawn in Fig. 6. Whether or not the two haplotypes of H. filicina Option 1. A radical option would be to transfer all indeed are indistinguishable on the basis of their taxa to the genus Sphacelaria. However, under the morphology needs further investigation. Mathias International Code of Botanical Nomenclature (1935) and Ernst-Schwarzenbach (1957) reported (Greuter, 2000), the name Cladostephus Agardh that gametophytes of Mediterranean H. filicina 1817 would take nomenclatural priority over plants were more slender than sporophytes, whereas Sphacelaria H. C. Lyngbye 1818. Since Sphacelaria Keum et al. (1995) observed that the gametophytes is the best-known name, a proposal for conservation and sporophytes were almost identical in Korean could be submitted. Option 1 would maintain the field and culture specimens. Keum et al. (1995) also greatest stability with respect to nomenclatural reported the occurrence of propagule-like structures changes and preserves monophyly. From the mor- in Korean plants, resembling those of Choristo- phological perspective, however, the lumping of all carpus tenellus. the genera would take some adjustment. Option 2. A second solution would be to recognize Pacific and Atlantic Stypocaulon. Novaczek et al. two groups (Fig. 6), the Sphacelariaceae with one (1989) found that the temperature ranges for the genus Sphacelaria and the Stypocaulaceae with one northern and southern distribution limits of S. genus Halopteris. The main difficulty here is the scoparium were lower in the west Atlantic than in paraphyletic position of S. radicans. There is also the east Atlantic. On the basis of geographic another potential problem. Sphacelaria radicans is distributions and differences in temperature tol- the putative sister-species to S. reticulata Lyngbye erance, they suggested that eastern and western in Hornemann 1818, the type species of Sphacelaria Atlantic populations are two different entities, with (Prud’homme van Reine, 1982). Live material of S. Phylogeny of the Sphacelariales 399

Fig. 6. Four options for a revised classification of the Sphacelariales s.s. based on the topology of the rbcL 50% majority- rule consensus tree. The thickness of horizontal branches indicates the level of bootstrap support. The dashed boxes indicate the traditional classification in Sphacelaria subgenera. The scissors indicate where the generic limits are drawn for each option. See text for a more extensive explanation. reticulata is unlikely to be forthcoming as the last Option 4. This solution is the most divisive. As reported finding occurred near Hofmansgave (Fyn, shown in Fig. 6, the Cladostephus clade in option 3 Denmark) in 1867, and it may be extinct would be further subdivided into three genera. The (Prud’homme van Reine, 1982). Silva (1980) pro- name Chaetopteris F. T. Ku$ tzing 1843 would take posed conserving S. caespitula Lyngbye 1819 as the priority for the four species of the subgenus Pseudo- type species. That proposal was formally rejected in chaetopteris (and S. mirabilis) and a new genus 1986 on the grounds of the priority of S. reticulata. would have to be proposed for S. nana. The genera In a proposal to choose another type species for of the Stypocaulaceae would not be changed. Sphacelaria it could be stated that the combination S. reticulata does not have to be considered as a Conclusion nomen specifico-genericorum (a species description that is at the same time also a genus description), Ideally a classification should reflect shared evol- because it was not the intention of Lyngbye himself. utionary history of the group and simultaneously For the reasons outlined by Silva (1980), S. provide a basis for clear taxonomic identification. caespitula would be the best choice for a new type. It This combination is not always achievable within a is also the most basal species of Sphacelaria. single classification. To continue to recognize Option 3. In this solution Sphacella would con- Cladostephus and Sphacella will result in irrecon- tinue to be recognized, the subgenus Propagulifera cilable paraphyly within Sphacelaria. Circum- would be upgraded to the genus level, the next clade scriptional options 1, 2, 3 and 4 solve this problem (Fig. 6) would become Cladostephus with concomi- by only recognizing monophyletic groups. The tant new combinations and only S. caespitula would selection of any option is largely a matter of be recognized as the true Sphacelaria (including preference rather than of necessity. Therefore, we issues around the type of the genus Sphacelaria as do not propose the adoption of any formal change discussed under option 2). Sphacelaria radicans in nomenclature at this time. would have to become a new genus and the From an evolutionary standpoint, the phylo- remaining clade would be merged under Halopteris. genetic relationships within the Sphacelariales s.s. S. G. A. Draisma et al. 400 reveal a complex paraphyletic assemblage based on References rbcL and RUBISCO spacer sequences, and an A, C.A. (1817). Synopsis Algarum Scandinaviae, pp. xxv. almost completely unresolved tree based on mor- B, J.C. & A, R.A. (1999). Analysis of clonal cultures phology. Highly convergent morphology and often of the brown tide algae Aureococcus and Aureoumbra (Pelago- weak diagnostic characters are a common problem phyceae) using 18S rRNA, rbcL, and RUBISCO spacer in almost all algal groups. For this reason, com- sequences. J. Phycol., 35: 570–574. B, F.T., O, J.L. & S, W.T. (1995). Global phylo- parative molecular data are indispensable. At the geography in the cosmopolitan species Cladophora vagabunda outset of the study, rDNA ITS sequences were (Chlorophyta) based on nuclear rDNA ITS sequences. Eur. J. planned to be used in addition to rbcL and Phycol., 30: 197–208. RUBISCO spacer sequences. 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