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.
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