J. Phycol. 34, 682–691 (1998) MOLECULAR EVIDENCE FROM nrDNA ITS SEQUENCES THAT LAMINARIOCOLAX (PHAEOPHYCEAE, ECTOCARPALES SENSU LATO) IS A WORLDWIDE CLADE OF CLOSELY RELATED KELP ENDOPHYTES1 Elke Burkhardt 2 Botanisches Institut, Christian-Albrechts-Universität, 24098 Kiel, and Institut für Meereskunde, Abteilung Meeresbotanik and AG Marine Pathologie, Düsternbrooker Weg 20, 24105 Kiel, Germany and Akira F. Peters 3 Institut für Meereskunde, Abteilung Meeresbotanik and AG Marine Pathologie, Düsternbrooker Weg 20, 24105 Kiel, Germany rine macrophytes. They are unicellular or filamentous and generally are morphologically reduced. Infecting their basiphyte populations in prevalences up to 100%, they live as commensalistic or hemiparasitic symbionts but may become pathogens that cause morphological disturbances, reducing their hosts’ value as food or raw material for extraction of chemical contents (Yoshida and Akiyama 1979, Apt 1988, Correa et al. 1988, 1994, 1997, Peters and Schaffelke 1996, Sánchez et al. 1996, Ellertsdóttir and Peters 1997). Many endophytic red algae are strictly parasitic. Recent studies have demonstrated that these symbionts may be able to transform host cells and that many taxa of adelphoparasites probably evolved from their basiphytes (Goff and Zuccarello 1994, Goff et al. 1996) via sympatric speciation. This paper deals with the systematics and evolution of endophytic Phaeophyceae. Except for the strictly parasitic Herpodiscus durvillaeae (Lindauer) South (South 1974, Peters 1990), all brown endophytes so far described are pigmented. Most species are similar in morphology. They are microscopic and possess a branched, filamentous thallus with diffuse growth, phaeophycean hairs, and plastids with pyrenoids. They are thus included in the ‘‘simple brown algae,’’ that is, the ‘‘Ectocarpales sensu lato’’ (Fritsch 1945, Peters and Burkhardt 1998). Many brown endophytes were originally classified in Ectocarpus Lyngbye and are now commonly accommodated in Streblonema Derbès et Solier in Castagne (Derbès and Solier 1851). Several other genera, however, have also been proposed to classify endophytic taxa. These are Entonema Reinsch (1875), Phycocelis Stroemfelt (Kuckuck 1894), Myrionema Greville (Sauvageau 1898), Pilocladus Kuckuck (1954), Gononema Kuckuck et Skottsberg in Skottsberg (Pedersen 1981), Microspongium Reinke (Pedersen 1984), and Laminarionema Kawai et Tokuyama (Kawai and Tokuyama 1995). A family, Streblonemataceae, for endophytic taxa was mentioned by Kylin (1947, as Streblonemaceae) and formally described by Zinova (1953). ABSTRACT Marine brown algae living as endophytes in macroalgae are morphologically simple and their taxonomy is particularly difficult. A molecular phylogeny for endophytic taxa isolated from kelps and red algae, and for putative epiphytic and free-living relatives, was inferred from partial small subunit and complete internal transcribed spacer nuclear ribosomal DNA sequences. It has revealed the following results. (1) Three species of endophytes isolated from members of the Laminariales are closely related. They form a clade together with the epi-endophytic species Laminariocolax tomentosoides (Farlow) Kylin. Members of the clade possess uniseriate plurilocular sporangia, and they may form erect filaments. Laminariocolax eckloniae sp. nov., occurring in the South African host Ecklonia maxima (Osbeck) Papenfuss, is described. The new combinations, Laminariocolax aecidioides (Rosenvinge) comb. nov. and L. macrocystis (Peters) comb. nov., are proposed for two taxa previously classified in Gononema and Streblonema, respectively. (2) The genus Laminariocolax occurs worldwide in temperate areas, and the phylogeny of the taxa studied is in agreement with biogeographic distribution. (3) Laminariocolax belongs to the Ectocarpales sensu lato. The genus is more closely related to Chordaria than to Dictyosiphon, Ectocarpus, or Scytosiphon. (4) Two brown endophytes (Streblonema spp.), isolated from red algae, are closely related to each other and may form a sister clade to Laminariocolax. Key index words: biogeography; endophyte; internal transcribed spacer; Laminariales; Laminariocolax; nuclear ribosomal DNA; pathogen; small subunit; taxonomy Abbreviations: ITS, internal transcribed spacer; MPT, most parsimonious tree; NJ, neighbor-joining; PCR, polymerase chain reaction; SSU, small subunit Marine endophytic algae have evolved in all major seaweed groups. Their habitat is the interior of ma1 Received 6 May 1997. Accepted 14 April 1998. Present address: Department of Botany, University of Cape Town, Private Bag, Rondebosch, 7701 South Africa. 3 Author for reprint requests; e-mail afpeters@ifm.uni-kiel.de. 2 682 MOLECULAR SYSTEMATICS OF KELP ENDOPHYTES 683 FIG. 1. Geographic origin of isolates used. Numbers as in Table 1. 1–4 5 Laminariocolax aecidioides, 5 5 L. eckloniae, 6 5 L. macrocystis, 7–8 5 L. tomentosoides, 9 5 Streblonema radians, 10 5 Streblonema sp., 11 5 Gononema pectinatum. Dangeard (1970) and Pedersen (1984) proposed that the endophytic brown algae are not independent taxa, but rather life-history stages of macroalgae, or asexual species recently evolved from such stages. This hypothesis was based on, and has been supported by, culture experiments that discovered ‘‘streblonematoid’’ microscopic stages or generations in many species of Ectocarpales sensu lato (Pedersen 1984, Peters and Müller 1985, Peters 1987, 1988, 1992). Occasionally, microscopic epiphytic brown algae isolated from the field developed into macrothalli in culture (Loiseaux 1969, 1970). Cultures of endophytes isolated from the field, however, failed to form macrothalli and showed either a direct reproduction of microthalli (Pedersen 1984) or an alternation of two microscopic generations (Peters 1991, Peters and Ellertsdóttir 1996). Due to the simple thalli of endophytic brown algae, the question of whether or not morphological similarities among taxa are the result of parallel evolution or of common descent is difficult to address with classical methods. The present paper describes phylogenetic analyses, based on DNA sequence data, of a group of endophytic brown microalgae that infect members of the Laminariales, and of two isolates from red algal hosts. Sequences analyzed are partial small subunit (SSU) as well as entire internal transcribed spacer (ITS) regions of the nuclear ribosomal cistron. The SSU sequences are conserved among brown algae and are considered to be of limited value for phylogenetic inferences below the ordinal level, or even among closely related orders (Saunders and Kraft 1995, Tan and Druehl 1996). The faster evolving ITS regions, by contrast, have been used to distinguish brown algal taxa within families and genera as well as among geographical subpopulations of species (Saunders and Druehl 1993a, Peters et al. 1997, Stache-Crain et al. 1997). MATERIALS AND METHODS Algae. Representatives of three taxa of brown endophytes growing in members of the Laminariales, two endophytic species from red algal hosts, and two putative relatives, Gononema pectinatum and Laminariocolax tomentosoides (Fig. 1, Table 1), that grow epiendophytically (Skottsberg 1921, Kylin 1937, Russell 1964), were isolated and purified by A.F.P. and cultivated under standard culture conditions (Peters 1991). Replicate isolates were used for L. tomentosoides and L. aecidioides to confirm isolation and identification of these microscopic algae and to examine intraspecific DNA variation. DNA extraction, amplification, and sequencing. Total DNA was extracted and purified from ca. 0.5 g fresh weight cultivated algae following the protocol of Van Oppen et al. (1993). PCR amplification parameters and primers used are reported in Peters and Burkhardt (1998). The SSU, ITS1, 5.8S, and ITS2 were amplified in three overlapping fragments using the primer pairs JO2FTW7CSR, TW5F-5.8S1R, and ITS1F-ITS4R. Direct sequencing of PCR products purified with QIAquick columns (Qiagen) was performed for both DNA strands, with 35S-dATP (Amersham) and the T7 DNA polymerase kit (Pharmacia) using the primers TW1F, ER, TW3F, AFP3R, TW7CSF, AFP2F, JO3R, JO3F, ITS1R, ITS1F, AFP1F, DIC2R, DIC1R, ITS2CR, ITS3F, 5.8S1R, MO11R, MO11F, JO6R, and ITS4R (sequences and positions of primers in Peters and Burkhardt 1998). Sequences were read by eye from X-ray films after exposure for 2 to 28 days. ITS1 and ITS2 were sequenced entirely. For the SSU, only those motifs identified by Tan and Druehl (1996) as the most variable among brown algae were sequenced. They included loop 10, loops E21–1 and E21– 5, and all positions from loop 44 to the end of the SSU. These sequences are located at positions 60–232, 622–793, and 1486 to the end of the SSU, respectively, in the alignment by Tan and Druehl (1996). Sources of published sequences from type genera of the major subgroups currently recognized in Ectocarpales sensu lato, and of outgroup taxa from Laminariales, Fucales, and Xanthophyceae, are provided in Table 2. Data analysis. Homologous sequences were aligned by eye in the data editor of PAUP 3.1.1 (Swofford 1993). The same program was used for parsimony analyses using the branch-andbound search mode, with noninformative or invariant characters being ignored. For faster analyses, we used alignments consisting only of the informative or invariant positions. Such alignments were produced with the computer program MEGA (Kumar et al. 1993). Gaps were treated as missing values. Alignments are available from A.F.P. Four analyses encompassing different sets of taxa and DNA regions were performed (Table 3). For these same data sets, Kimura two-parameter distances between taxa (Kimura 1980) were estimated in MEGA, and phylogenies were subsequently inferred with the neighbor-joining (NJ) method (Saitou and Nei 1987). Distance trees were similar to those inferred by parsimony, and only the latter are presented. Bootstrapping was performed with 1000 resamplings for both parsimony and distance analyses. 684 ELKE BURKHARDT AND AKIRA F. PETERS TABLE 1. Isolates examined in the present study. No. 1 Source Year of isolation Kiel, western Baltic, Germany 1992 Taxon 2 3 Laminariocolax aecidioides (Rosenvinge) Peters (5Gononema aecidioides (Rosenvinge) Pedersen) L. aecidioides L. aecidioidesa 4 5 L. aecidioides Laminariocolax eckloniae Peters 6 Laminariocolax macrocystis (Peters) Peters (5Streblonema macrocystis Peters) Laminariocolax tomentosoides (Far- Helgoland low) Kylin L. tomentosoidesb Kiel Streblonema radians Howec Los Molinos, Valdivia, Chile 1988 10 Streblonema sp. 1993 11 Gononema pectinatum Kuckuck et Fuerte Bulnes, Brunswick PenSkottsberg in Skottsberg insula, Magallanes, Chile 7 8 9 Fredrikshavn, Jutland, Denmark 1993 Helgoland, North Sea, Germany 1993 Helgoland Kommetjie, Cape Peninsula, South Africa Los Molinos, Valdivia, Chile 1995 1993 1994 1994 1988 Kiel 1988 Host EMBL accession Laminaria saccharina (Linnaeus) Lamouroux Laminaria saccharina Laminaria hyperborea (Gunnerus) Foslie Laminaria hyperborea Ecklonia maxima (Osbeck) Papenfuss Macrocystis pyrifera (Linnaeus) C. Agardh Laminaria digitata (Hudson) Lamouroux L. digitata Grateloupia doryphora (Montagne) Howe Polysiphonia elongata (Hudson) Sprengel Dictyosiphon hirsutus (Skottsberg) Pedersend JA2353/4e JA2355/6e JA2357/8e JA2359/60e Z98566e Z99485–7f Z98581/2e Z98579/80e Z99482–4f Z98575/6e Z99479–81f a Only ITS2 was sequenced for this isolate; it was identical to isolate 4. New record for the western Baltic (see Nielsen et al. 1995). c ITS2 of this isolate was not entirely sequenced. d Gononema pectinatum developed in a culture of an isolate of D. hirsutus (Peters 1992). It was not determined whether G. pectinatum was actually growing as epiphyte or endophyte on field material of D. hirsutus. e ITS. f SSU. b Possible saturation in the sequences used was assessed in MEGA by determining the transition/transversion (ts/tv) ratios between taxa. For an alignment, ts/tv was calculated as the arithmetic mean of all pairwise ts/tv ratios between included taxa, and the alignment was considered saturated when ts/tv , 1 (Bakker et al. 1995). Determination of the borders between spacers and gene regions followed Van Oppen et al. (1993). RESULTS The length of the ITS1 was highly variable among the taxa examined and ranged from 206 to 789 bp. Length variation in ITS2 was comparatively less (273–369 bp; Table 4). Replicate isolates from dif- ferent localities sampled for both Laminariocolax tomentosoides and L. aecidioides had nearly identical sequences. Isolates of L. aecidioides from different hosts were also nearly identical in their ITS sequences (Table 5). The DNA sequences of all endophytes isolated from kelp species were easily alignable over the entire length of the spacers. They differed in only a few positions (Table 5). Nevertheless, several gaps, which were larger in ITS1 than in ITS2, had to be inserted. Alignment of DNA sequences over the entire spacer regions was also possible between these endophytes and Laminariocolax tomentosoides. TABLE 2. Taxa of free-living algae whose sequences were used for comparison with the endophytes. No. 12 13 14 15 16 17 18 a Taxon Chordaria linearis (Hooker et Harvey) Cotton Dictyosiphon foeniculaceus (Hudson) Greville Ectocarpus siliculosus (Dillwyn) Lyngbye Scytosiphon lomentaria (Lyngbye) Link (5S. simplicissimus (Clemente) Cremades nom. rej. prop.) Alaria marginata Postels et Ruprecht Fucus gardneri Silvac Tribonema aequale Pascherc ITS. SSU. c To date, ITS sequences are not available for these taxa. b Source EMBL accession Peters and Burkhardt (1998) Tan and Druehl (1996), StacheCrain et al. (1997) Kawai et al. (1995), Tan and Druehl (1996) Z98577/8a Z99464–6b Z98573/4a Z99461–3b L43062b U38755a D16558a,b L43066b Saunders and Druehl (1992, 1993b) Bhattacharya et al. (1992) Ariztia et al. (1991) X53987b M55286b Peters and Burkhardt (1998) 685 MOLECULAR SYSTEMATICS OF KELP ENDOPHYTES TABLE 3. Tree statistics. Analysis 1: Analysis of all positions for ITS1 and ITS2. Analysis 2: SSU data. Analysis 3: ITS position alignable among all taxa (5‘‘conserved ITS motifs’’). Includes last 33 positions of 5.8S gene. Analysis 4: All positions used in previous two analyses. In analyses 2–4, the endophytes from the Laminariales and red algae were represented by Laminariocolax tomentosoides and Streblonema sp., respectively. Invariant positions were ignored in analysis 1; noninformative positions were ignored in the other analyses. MPT 5 most parsimonious tree, CI 5 consistency index, RC 5 rescaled consistency index. Analysis 1 2 DNA sequences included Taxa included (numbers from Tables 1, 2) Number of taxa Length of alignment Alignable positions Variable positions Informative positions Transition/transversion ratio Number of MPTs Length of MPTs Number of trees at MPT 1 1 Number of trees at MPT 1 2 Number of trees at MPT 1 3 CI RC Tree in figure no. Full ITS 1, 2, 4–8 7 1640 1640 67 35 2.33 2 70 2 2 2 1 1 2 SSU 7, 10–18 10 597 597 168 56 1.98 1 97 10 25 37 0.763 0.587 3 This species and the endophytes isolated from kelps are henceforth referred to as the ‘‘kelp-endophyte clade.’’ More difficult was the alignment of ITS sequences of the aforementioned isolates with those of two brown endophytes from red algal hosts, Streblonema spp., and with Gononema and Chordaria. The ITS sequences of the two endophytes from red algae were nearly identical. The main difference between them was a 29 bp deletion in the ITS1 of Streblonema radians. Sequences of Dictyosiphon, Ectocarpus, Scytosiphon, and Alaria were unalignable relative to all aforementioned taxa and each other over large regions, mainly in the first part of ITS1 and at the end of ITS2. Nevertheless, there were DNA motifs within the ITS regions that were shared by all taxa, including the outgroup Alaria, and that showed little variation among taxa. These regions are henceforth referred to as ‘‘conserved ITS motifs.’’ They made up 293 of 1640 ITS positions and were used in phylogenetic analyses including more than the kelp endophytes. Traces of other putative common motifs were present in other regions of the spacers, but TABLE 4. Length of ITS1 and ITS2 sequences of Laminariocolax spp., Gononema pectinatum, Streblonema radians, and an unidentified endophyte (Streblonema sp.) isolated from Polysiphonia elongata. Taxon Laminariocolax aecidioides (Kiel isolate) L. aecidioides (Helgoland isolate) L. tomentosoides L. eckloniae L. macrocystis Streblonema radians Streblonema sp. Gononema pectinatum a b From Table 1. Not completely sequenced. Length (bp) Isolate no.a ITS1 ITS2 1 4 7 5 6 9 10 11 688 682 767 789 501 206 279 432 277 279 294 273 290 299b 369 295 3 Partial ITS 8, 10–16 8 1640 293 75 26 1.24 2 51 9 26 85 0.706 0.441 4 4 SSU 1 ITS 8, 10–16 8 2237 890 137 46 1.61 3 89 6 18 27 0.685 0.436 5 they were not alignable among all taxa and were not used in our analyses. Relationships among the isolates of the kelp-endophyte clade were assessed using the entire ITS regions. In the resulting tree (Fig. 2, Table 3), the northern hemisphere taxa (isolates 1–4, 7, 8) were separated from the two southern hemisphere taxa (isolates 5, 6). Within the clade of northern hemisphere taxa, one branch contained the two isolates of L. tomentosoides, and a second branch contained the three isolates of L. aecidioides. The similarity of ITS sequences among the kelp endophytes and L. tomentosoides was high. Pairwise divergences between isolates ranged from 0.000 to 0.052 (Table 5). The last 330 positions of the SSU were identical in L. aecidioides (isolates 2, 3) and L. tomentosoides (isolates 7, 8). We therefore did not sequence the SSU in more isolates of the kelp-endophyte clade and used the SSU sequence of L. tomentosoides as representative in all analyses involving SSU sequences. The two endophytes from red algae had identical sequences over the last 270 positions of the SSU, and the endophyte from Polysiphonia elongata (Streblonema sp.) was taken as representative of this clade in analyses involving SSU sequences. SSU sequences were easily aligned among all taxa. The position of the endophytes within the brown algae was inferred in analyses using SSU sequences, the conserved ITS motifs, or a combination of both. Analyses of the SSU sequences used Tribonema as the outgroup and gave a single most parsimonious tree (Fig. 3, Table 3). The sequences placed all endophytes among the Ectocarpales sensu lato. This clade of ‘‘simple brown algae’’ was strongly supported by bootstrap values and decay indices. Within this clade, Laminariocolax and Streblonema sp. appeared in a well-supported subgroup together with Gononema, Chordaria, and Dictyosiphon (henceforth referred to 686 ELKE BURKHARDT AND AKIRA F. PETERS TABLE 5. Pairwise divergences for ITS1 and ITS2 between isolates of kelp endophytes. Above diagonal: mean divergence (adjusted for missing data); below diagonal: divergences in absolute nucleotide changes. Numbers behind taxa indicate replicate isolates (Table 1). 1 2 3 4 5 6 7 Laminariocolax aecidioides 1 Laminariocolax aecidioides 2 Laminariocolax aecidioides 4 Laminariocolax tomentosoides 8 Laminariocolax tomentosoides 7 Laminariocolax eckloniae Laminariocolax macrocystis 1 2 3 4 5 6 7 — 0 1 19 16 36 30 0.000 — 1 11 10 23 25 0.001 0.001 — 20 17 37 33 0.020 0.017 0.021 — 4 42 39 0.021 0.016 0.022 0.005 — 38 36 0.039 0.036 0.038 0.041 0.045 — 13 0.040 0.041 0.043 0.048 0.052 0.017 — as ‘‘ingroup’’), whereas Ectocarpus and Scytosiphon were more distant, but related to each other. The internal topology of the ingroup, however, was poorly resolved. Within the endophytes, the conserved ITS motifs were nearly identical. This resulted in 56 most parsimonious trees (MPTs) in a parsimony analysis of these DNA stretches when all endophytic taxa were included (Fig. 4B). Extended calculation times precluded bootstrapping and determination of decay indices. To facilitate these calculations, all endophytes except Streblonema sp. and the Kiel isolate of Laminariocolax tomentosoides were removed from the data set. A parsimony analysis of conserved ITS motifs for the remaining eight taxa (Fig. 4A, Table 3) gave two MPTs. The endophytes appeared in the same branch of the Ectocarpales sensu lato as in the SSU analysis. Within the ingroup, Dictyosiphon was most basal, followed by Gononema, Chordaria, Streblonema, and Laminariocolax. The stability of the ingroup topology, however, remained weak, as indicated by low bootstrap values and decay indices. This data set was the only one in which the NJ tree had a different topology. Scytosiphon and Ectocarpus formed a clade that was more distant to the other species of the Ectocarpales sensu lato than Alaria, and Laminariocolax and Streblonema sp. did not form a clade. The ingroup containing the endophytic taxa, however, was strongly supported by a bootstrap value of 97%, and the basal position of Dictyosiphon in the ingroup by 90% (tree not shown). A combined analysis of the ITS and SSU sequences gave three MPTs, which differed only in the po- FIG. 2. Phylogenetic tree (bootstrap 50% majority rule consensus, unrooted) of isolates of kelp endophytes, inferred by parsimony analysis from entire ITS sequences (Analysis 1). Numbers behind taxa indicate replicate isolates (Table 1). Scale 5 10 steps. Numbers above branches are bootstrap values (parsimony/distance); numbers below branches are decay indices. sition of Chordaria and Gononema relative to the clade of Streblonema and Laminariocolax (Fig. 5, Table 3). In all trees, the ingroup was strongly supported, but branches within the ingroup remained poorly resolved. The bootstrap 50% majority rule consensus tree (not shown) had the same topology as in the analysis of conserved ITS motifs (Fig. 4A). DISCUSSION The main conclusions from this study on the phylogenetic relationships of endophytic brown algae that occur in species of the Laminariales can be drawn from the ITS sequences. The three species of kelp endophytes studied form a clade of closely related species that includes the epi-endophytic species Laminariocolax tomentosoides. This kelp-endophyte clade has a worldwide distribution in temperate waters. Genetic distances within the clade reflect the species’ biogeographic distribution. The two southern hemisphere taxa form a southern subclade, and the two North Atlantic taxa form a northern subclade (Figs. 1, 2). Sequence comparisons of variable regions of the SSU nrDNA concur with cytological observations that all endophytic brown algae examined belong to the Ectocarpales sensu lato. Within this strongly supported clade (100% bootstrap support and decay index more than 5), the endophytic taxa isolated from red algae and from the order Laminariales are more closely related to Chordaria and Dictyosiphon than to Ectocarpus and Scytosiphon. Resolution within the lineage that includes the endophytes, however, was limited by the small number of informative positions. As the most variable regions of the SSU were sequenced, it is unlikely that data from the entire SSU FIG. 3. Most parsimonious tree inferred from SSU data (Analysis 2). Tribonema was used as an outgroup. Scale 5 10 steps. Numbers above branches are bootstrap values (parsimony/distance; provided if $ 50%), numbers below branches are decay indices. MOLECULAR SYSTEMATICS OF KELP ENDOPHYTES 687 FIG. 5. Results of parsimony analysis of combined SSU and ITS data (Analysis 4). Alaria was used as an outgroup. One of three MPTs. Scale 5 10 steps. Numbers above branches are bootstrap values (parsimony/distance; provided if $ 50%); numbers below branches are decay indices. FIG. 4. Results of parsimony analysis of conserved ITS motifs (Analysis 3). (A) Endophytes from kelps and red algae represented by Laminariocolax tomentosoides and Streblonema sp., respectively. Bootstrap 50% majority rule consensus tree, Alaria was used as an outgroup. Scale 5 10 steps. Numbers above branches are bootstrap values (parsimony/distance; provided if $ 50%); numbers below branches are decay indices. (B) Ingroup in one of 56 MPTs from parsimony analysis including all endophyte taxa. Scale 5 5 steps. Numbers behind taxa indicate replicate isolates (Table 1). Clades of endophytes from kelps (Laminariocolax spp.) and from red algal hosts (Streblonema spp.) in boxes. will provide improved resolution. Analyses of conserved ITS motifs suggest that the kelp endophytes form a clade together with the endophytes from red algae (Streblonema spp.), Dictyosiphon, Chordaria, and Gononema. The hierarchy in this ingroup was rather unstable and was different from that of the SSU tree. Combined analyses of SSU sequences and conserved ITS motifs resulted in a slightly more stable internal topology for the Ectocarpales sensu lato. As in the tree from conserved ITS motifs, the kelp endophytes are more closely related to Chordaria than to Dictyosiphon. The congruence of ITS sequences indicates that the two taxa of endophytes isolated from red algal hosts are closely related. Most of our analyses suggest that these taxa are sister to the kelp endophytes. Sequences from more brown endophytes from red algae and from more putative relatives among microscopic brown algae are required to test the hypotheses that (1) brown algae endophytic in Rhodophyta form a widely distributed clade similar to kelp endophytes, and (2) the common ancestor of all the endophytic taxa studied was already an endophyte. Nomenclature of endophytic taxa has been controversial. Usually, they have been classified in the genus Streblonema Derbès et Solier. Pedersen (1978, 1984) suggested that S. sphaericum Derbès et Solier is a microthallus of Myriotrichia clavaeformis Harvey. He also recognized that Derbès and Solier (1851) did not validly publish the genus. The situation was remedied by Pringsheim (1863), who described it with S. volubile as the type species. The host of S. volubile, however, is not a kelp but Mesogloia vermiculata (Smith) S. F. Gray, which is a member of the Ectocarpales sensu lato. The multiseriate plurilocular sporangia of S. volubile (Pringsheim 1863) are significantly different from the uniseriate sporangia of the kelp endophytes (Rosenvinge 1893, Russell 1964, Peters 1991). Two genera proposed for kelp endophytes, especially for the comparatively common Laminariocolax aecidioides, were Phycocelis (Kuckuck 1894) and Myrionema (Sauvageau 1898). The type species of these genera (Phycocelis foecunda (5Hecatonema foecundum (Strömfelt) Loiseaux), Strömfelt 1888; Myrionema strangulans, Greville 1827) are epiphytes but have morphological and reproductive characters in common with the endophytes studied. Nevertheless, we consider the habitat difference, which led to the evolution of a specialized mechanism to penetrate the host surface during spore germination (Heesch 1996), sufficient to justify a separate genus for the kelp endophytes. Another genus proposed by Kylin (1947) for Laminariocolax aecidioides was Entonema Reinsch (1875), but Entonema spp. were described by Reinsch from red and green algal hosts. Also, the plurilocular sporangia of the type species, E. penetrans, are multiseriate or uniseriate according to Reinsch’s original drawings. Taken together, this makes it impossible to use Entonema for the classification of the kelp endophytes. A further genus, Gononema Kuckuck et Skottsberg in Skottsberg (1921), type species Gononema pectinatum, was suggested by Pedersen (1981) as a likely place for Laminariocolax aecidioides and a similar endophyte occurring in Alaria esculenta (Linnaeus) Greville. In Pedersen’s cultures, these two endophytes produced phaeophycean hairs and erect branched filaments that resembled erect filaments of Gononema pectinatum. However, similar erect filaments are formed by Laminariocolax tomentosoides. Pedersen considered the lack of phaeophycean hairs in L. tomentosoides an important distinctive character between Laminariocolax and Gononema. Our molecular data 688 ELKE BURKHARDT AND AKIRA F. PETERS FIGS. 6–9. Herbarium and culture material of Laminariocolax spp. and their kelp hosts. FIG. 6. Herbarium specimen of Ecklonia maxima from South Africa with dark spots that contain Laminariocolax eckloniae. Material in the center was excised to isolate the endophyte. FIG. 7. Laminariocolax eckloniae, culture material. P 5 plurilocular sporangium. FIG. 8. Herbarium specimen of Laminaria hyperborea from Helgoland with dark spots that contain Laminariocolax aecidioides. FIG. 9. Laminariocolax aecidioides from Helgoland, culture material, with unilocular (U) and empty plurilocular (P) sporangia on the same thallus. show that this is not the case and that G. pectinatum is as distant from the kelp endophytes as Chordaria linearis. Inclusion of the kelp endophytes in Gononema is therefore not justified. Because it is possible that endophytes evolved from small epiphytic algae, such as Myrionema, Myriactis, Microspongium, or Ulonema, it is desirable to include such microscopic brown epiphytes in future molecular studies. We propose to classify the kelp endophytes studied here in Laminariocolax Kylin (1947) rather than in the problematical genus Streblonema or any other genus mentioned above. Laminariocolax predates another taxon proposed for endophytes, Pilocladus Kuckuck (1954). Morphological similarities between the kelp endophytes studied and the type species Laminariocolax tomentosoides include sporangia that are strictly uniseriate and the capacity to form erect branched filaments (Pedersen 1981, Peters 1991). Within Laminariocolax, phaeophycean hairs are either present (L. aecidioides, L. macrocystis) or absent (L. tomentosoides, L. eckloniae). Apparently, this character is not phylogenetically informative within the kelp-endophyte clade. Our findings support Stache-Crain et al. (1997), who showed that the state of this character changes between the closely related genera Ectocarpus and Kuckuckia. MOLECULAR SYSTEMATICS OF KELP ENDOPHYTES DESCRIPTION Laminariocolax eckloniae Peters sp. nov. Thallus microscopicus, filamentosus, ramosus. Pila et sporangia unilocularia absunt. Cum sporangiis plurilocularibus uniseriatis, filamenta et sporangia 6–8 mm crassa. Endophyticus in sporophyto Eckloniae maximae, in lamina hospitis maculas obscuras formans. Locus typicus: Kommetjie, Cape Peninsula, South Africa, 348089 Lat. Merid., 188199 Long. Orient. Holotypus: AFP SA 93–21 in BOL 5 Figure 6. Thallus microscopic, filamentous, branched, devoid of phaeophycean hairs and unilocular sporangia. Plurilocular sporangia uniseriate, filaments and sporangia 6–8 mm in diameter. Endophytic in the sporophyte of Ecklonia maxima, forming dark spots in the host’s blade. Type locality: Kommetjie, Cape Peninsula, South Africa, 348089 S, 188199 E. Holotype: AFP SA 93–21 in BOL 5 Figure 6. The type strain (Fig. 7) was isolated from the interior of a dark spot on a drift sporophyte of Ecklonia maxima. The strain is maintained in culture by A.F.P. Data on natural frequency, distribution, and host range are lacking. The life history of the isolate studied is direct. Laminariocolax aecidioides (Rosenvinge) Peters comb. nov. Basionym: Ectocarpus aecidioides Rosenvinge 1893: 894. The identity of the endophyte associated with dark spots in European Laminaria hyperborea has been unknown (Lein et al. 1991, Ellertsdóttir and Peters 1997). ITS sequences of our isolates from characteristically spotted hosts (Fig. 8) showed that the endophyte is L. aecidioides. In unialgal culture, it had branched filamentous thalli and produced plurilocular and unilocular sporangia (Fig. 9). Laminariocolax macrocystis (Peters) Peters comb. nov. Basionym: Streblonema macrocystis Peters 1991:366. In addition to the three endophyte species from kelps studied by us, several taxa of brown endophytes with uniseriate sporangia have been described from the north Atlantic and Pacific oceans (Saunders 1901, Setchell and Gardner 1922, Pedersen 1981). Future studies may show whether they also belong to the kelp-endophyte clade. A different sporangial morphology occurs in the kelp endophyte Laminarionema elsbetiae Kawai et Tokuyama (Kawai and Tokuyama 1995, Peters and Ellertsdóttir 1996). DNA sequence analyses have shown that this species is only distantly related to Laminariocolax (Peters and Burkhardt 1998). Nomenclatural decisions on the brown endophytes isolated from Rhodophyta are postponed until more taxa have been studied. Also, because of our limited knowledge of the closest relative of Laminariocolax, we refrain from placing the genus into an existing family (Myrionema- 689 taceae, Streblonemataceae) or proposing a new family. Experimental data are lacking on the degree of host specificity of the kelp endophytes studied here. Laminariocolax aecidioides has been reported from different hosts (Laminaria saccharina, Rosenvinge 1893, Peters and Schaffelke 1996; Laminaria hyperborea, Lein et al. 1991, Ellertsdóttir and Peters 1997; Hedophyllum sessile (Areschoug) Setchell, Setchell and Gardner 1922; Undaria sp., Yoshida and Akiyama 1979, Veiga et al. 1997). It is interesting that we did not detect L. aecidioides in Laminaria saccharina at Helgoland, whereas it was frequent in L. hyperborea (Ellertsdóttir and Peters 1997, as Streblonema sp.). Infection studies may show whether each host species harbors a distinctive variety of L. aecidioides. So far, the phylogenetic relationships among the kelps that serve as hosts are unknown, but the close relatedness among the endophytes favors the idea of recent radiation of endophytes onto different hosts over coevolution of hosts and endophytes. We thank Paul C. Silva for nomenclatural information on Streblonemataceae and Poul M. Pedersen for an invitation to the Research Station at Fredrikshavn, Denmark. We also thank Jeanine L. Olsen and Wytze T. Stam for hospitality during work in their laboratory, where some of the sequences were generated. Thanks are further due to Johannes Imhoff, Department of Marine Microbiology at the Institut für Meereskunde, Kiel, Germany, for facilitating sequencing equipment; to Lynda Goff, Gary Saunders, and John Bolton for very helpful suggestions to improve the manuscript; and to Torsten Reincke for drawing Figure 1. Financial support by Deutsche Forschungsgemeinschaft (Pe 346/5) is gratefully acknowledged. Apt, K. E. 1988. 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