J. Phycol. 34, 850–856 (1998)
MEDITERRANEAN CAULERPA TAXIFOLIA AND C. MEXICANA (CHLOROPHYTA)
ARE NOT CONSPECIFIC1
Jeanine L. Olsen2
Department of Marine Biology, Centre for Ecological and Evolutionary Studies, University of Groningen, Biological Centre, Postbus 14,
9750 AA Haren, The Netherlands
Myriam Valero, Isabelle Meusnier
Laboratoire de Génétique et Evolution des Populations Végétale, URA-CNRS 1185, Bat. SN2, Université de Lille 1, 59655 Villeneuve
D’Ascq Cedex, France
Stella Boele-Bos and Wytze T. Stam
Department of Marine Biology, Centre for Ecological and Evolutionary Studies, University of Groningen, Biological Centre, Postbus 14,
9750 AA Haren, The Netherlands
folia; introduced species; ITS; Mediterranean invasions;
rDNA
ABSTRACT
In 1984, Caulerpa taxifolia (Vahl) C. Agardh was
reported along the coast of Monaco. Over the past decade
it has spread along 60 km of the Mediterranean coastline
and presently represents a potential risk to biodiversity. Several explanations have been advanced regarding the presence of C. taxifolia in the Mediterranean. One hypothesis
maintains that the alga was introduced accidentally into
the sea at Monaco, where it has been used as a decorative
alga in aquaria. Caulerpa taxifolia has not been reported
in earlier marine floras of the Mediterranean, and its sudden appearance has suggested that it may be a recent introduction. Another hypothesis proposes that C. taxifolia
and Caulerpa mexicana Sonder ex Kützing are morphological variants of one another and hence conspecific
taxa. Caulerpa mexicana has been found in the eastern
Mediterranean since at least 1941. In order to establish
the taxonomic identities of these taxa, individuals from five
populations of C. taxifolia and four populations of C.
mexicana were collected from within and outside of the
Mediterranean. Comparative DNA sequence analysis of the
nuclear ribosomal cistron, including the 39-end of the 18S,
ITS1, 5.8S, and ITS2 regions, show clear phylogenetic separation of the two taxa using parsimony and maximum
likelihood analyses. Separation is maintained whether the
analyses are based on just the more conserved 18S data or
just the fast- evolving spacers. The two species are thus not
conspecific. For specimens of uncertain identity (i.e. taxifolia–mexicana intermediates), a PCR diagnostic amplification can easily be performed because the ITS1 in C.
taxifolia is 36 nucleotides shorter than the ITS1 in C.
mexicana. Whether or not C. taxifolia has been present
for a longer period of time in the marine flora, either as a
cryptic endemic species or as the result of one or more introductions, represents an additional hypothesis that will
require identification of biogeographic populations from
throughout the world, as well as a population-level study
of the Mediterranean region.
Abbreviations: CIA, chloroform-isoamyl alcohol; CTAB, cetyltrimethylammonium bromide; ITS, internal transcribed
spacer; NaAc, sodium acetate; TE, Tris-EDTA buffer
More than 60 species of exotic macrophytic algae
have been documented in the Mediterranean Sea—
more than 50% of them since 1970 (Boudouresque
and Ribera 1995). Since the beginning of the 19th
century, seven species of Caulerpa have been recorded, including C. mexicana Sonder ex Kützing (5 C.
crassifolia (C. Ag.) J. Ag.), C. ollivieri Dostal, C. prolifera (Forsskål) Lamour., C. racemosa (Forsskål) J. Ag.,
C. scalpelliformis (Brown ex Turn.) C. Ag., C. sertularioides (S.G. Gmelin) Howe, and C. taxifolia (Vahl) C.
Agardh (Gallardo et al. 1993). Most of these species,
however, are restricted to the warm waters of the
east and southeastern Mediterranean and are considered to have entered the Mediterranean since the
opening of the Suez Canal in 1869. The only exceptions are C. prolifera, which is found throughout the
Mediterranean and recorded by Lamouroux (1809),
and the 1984 appearance of C. taxifolia off the coast
of Monaco (Meinesz and Hesse 1991). Over the past
decade, C. taxifolia has spread between Toulon,
France, and Genoa, Italy, occurring at 70 sites and
covering nearly 3000 hectares of subtidal area (De
Villèle and Verlaque 1995). About 60 km of coastline are currently affected and there is concern
about potential reduction in biodiversity (Boudouresque et al. 1995, Romero 1997), demise of seagrass
beds (Posidonia oceanica (L.) Delile) (De Villèle and
Verlaque 1995), effects on local fisheries (Francour
et al. 1995), and general negative effects on the
coastal ecosystem (Académie des Sciences–Paris
1997).
The mode of origin of C. taxifolia along the coasts
of France, Monaco, and Italy is disputed (Académie
des Sciences–Paris 1997, Olsen 1997a). A widely expressed hypothesis is that C. taxifolia was released
accidentally from the Monaco Aquarium, where it
Key index words: Caulerpa mexicana; Caulerpa taxi1
2
Received 29 April 1998. Accepted 23 June 1998.
Author for reprint requests; e-mail [email protected].
850
851
MEDITERRANEAN CAULERPA ITS
was used as a decorative alga in fish tanks (Meinesz
and Hesse 1991, Boudouresque et al. 1995). Subsequent spread of the alga has been facilitated by the
its ecophysiological (Delgado et al. 1996) and reproductive competitive superiority, with regional
dispersal enhanced by attachment to anchors on
pleasure yachts and on fisherman’s nets (reviewed
in Meinesz and Boudouresque 1996). These authors
considered other modes of introduction less likely
because C. taxifolia has never been reported as a
fouling component and because the species has never been reported in the areas around Morocco and
Portugal. Much of the available evidence supports
this view, but an alternative hypothesis was proposed
by Chisholm et al. (1995), who maintained that C.
taxifolia was not a recent introduction but has been
present in the eastern Mediterranean at least since
the opening of the Suez Canal. Their argument,
however, rests on the hypothesis that C. taxifolia is
conspecific with the morphologically similar C. mexicana, which is common in the eastern Mediterranean. They based this on the observation that cultivation of C. mexicana in aquaria led to its morphological conversion into plants resembling C. taxifolia,
and they maintained that the western expansion of
C. taxifolia has been facilitated by changes in the
geostropic current of the western Mediterranean
and increased sea surface temperatures.
Practically all taxonomic studies of Caulerpa discuss problems with intermediate morphologies at
the species level, and this has resulted in a proliferation of infraspecific varieties and forms (Taylor
1960, Coppejans and Prud’homme van Reine 1992,
Prud’homme van Reine et al. 1996). Morphological
plasticity (i.e. ecologically induced phenotypes or
ecads) is known to be widespread and has been
demonstrated in both field and laboratory experiments involving light and water motion in C. racemosa (Peterson 1972, Ohba and Enomoto 1987) and
in C. urvilliana Montagne (Jaubert and Meinesz
1981). Although these studies are compelling, induced morphological similarity is not necessarily sufficient for the recognition of one taxon or separate
taxa, especially when few diagnostic characters are
available. Comparative DNA sequencing or other
molecular data are a necessary supplement (Olsen
1997b).
Species delimitations in Caulerpa are based primarily on morphology of the upright blades (5 assimilators) and rhizoids. Similarities and differences
between C. taxifolia and C. mexicana are discussed
and illustrated in Taylor (1960: pl. 12), Littler et al.
(1989: pls. 40b, 42a), and Meinesz and Boudouresque (1996: fig. 3). Additional discussions can be
found in Coppejans and Prud’homme van Reine
(1992) and Meinesz et al. (1994). In brief, C. taxifolia has regularly opposite, pinnate blades in which
the pinnules are usually markedly upcurved, strongly compressed, contracted at the base, tapering toward the tip, and sharply pointed. Rhizoidal fila-
ments are long (1–10 cm) and separated by less than
a centimeter along the stolon. Caulerpa mexicana
blades are pinnately divided with a flat midrib. Pinnules are closely placed, sometimes overlapping, opposite, ascending, flat oval to oblong or somewhat
arcuate, and basally narrowed to acuminate. Rhizoidal filaments are short (,0.5 cm) and spaced further apart. There is also a gradation in morphology
of the blades in some specimens and intermediates
between the ‘‘typical’’ forms. This has been noted
by many phycologists (Coppejans & Prud’homme
van Reine 1992, Meinesz et al. 1994, Chisholm et al.
1995), including ourselves (following examination
of more than 250 specimens from Rijksherbarium,
Leiden, Netherlands), and certainly provides a legitimate basis for questioning taxonomic circumscriptions.
The relatively fast-evolving rDNA internal transcribed spacer (ITS1 and ITS2) sequences are useful
for phylogenetic reconstruction at the inter- and intraspecific levels within the green algae (Bakker et
al. 1992, 1995a, b, Kooistra et al. 1992, 1993, Coleman et al. 1994, Olsen et al. 1994, Van Oppen 1995)
and for Caulerpa in particular. Pillmann et al. (1997)
investigated five species of Caulerpa from South Africa and Australia, including nine geographically
separated populations of C. filiformis (Suhr) Hering.
They were able to show that C. filiformis near Sydney
was not introduced from South Africa because the
ITS sequences of South African and Australian C.
filiformis were very different.
In the present study, we test the hypotheses of
separate species or conspecificity between Mediterranean C. taxifolia and C. mexicana. Partial 18S and
complete ITS sequences from four individuals of C.
mexicana and five individuals of C. taxifolia are compared from within and outside of the Mediterranean.
MATERIALS AND METHODS
Plant material. Samples for DNA extraction were collected in
the field and immediately placed in silica gel. A voucher specimen
also was prepared according to standard herbarium procedure
and has been deposited in Rijksherbarium, Leiden, Netherlands.
Table 1 summarizes the collections used in this study.
DNA extraction and purification. The CTAB method was modified
from Doyle and Doyle (1987) as follows: one or two blades of
material dried in silica gel (grinding or crushing unnecessary)
were added directly to 10 mL preheated (608 C) 2% w/v CTAB
buffer (with 20 mL b-mercaptoethanol) and incubated for 30 min
at 608 C. Repeated extractions with CIA (24:1 v/v) were performed until complete removal of the interphase was achieved.
DNA was precipitated with ⅔ volumes cold isopropanol for 1 h
at 48 C, pelleted by centrifugation (30 min, 10,000 3 g, 48 C),
washed with 80% ethanol, air dried, and dissolved in 500 mL 0.13
TE. RNA was removed by incubation (30 min, 378 C) with 2 mL
RNase (Boehringer Mannheim). DNA was precipitated with an
equal volume of isopropanol in the presence of 1/10 volume 4
M NaAc, washed, dried and redissolved in 200 mL 13 TE. DNAs
were checked on a diagnostic agarose gel. Average yield was 1.0
mg.
PCR amplification. The entire ITS region was amplified as a single fragment (ca. 950 bases long) using TW7-Forward at approximately position 1413 in the 18S gene (59-gag-gca-ata-aca-ggt-ctg-
852
JEANINE L. OLSEN ET AL.
TABLE 1. Isolates of Caulerpa taxifolia and C. mexicana used in the present study.
Isolate
Code
Caulerpa mexicana
mexisr
C. mexicana
mexcan
C. mexicana
mexpan
C. mexicana
mexfla
C. taxifolia
taxmon
C. taxifolia
C. taxifolia
taxelb
taxmes
C. taxifolia ‘‘tax-mex’’
morphotypea
C. taxifolia
taxmex
a
taxaus
Locality
Tel shikmona, Haifa,
Israel
Las Canteras Bch, Las
Palmas, Gran Canaria
San Blas Island, Caribbean Panama
Key Largo, Florida
Preservation
method
Collector
Voucher,
Rijksherbarium,
Leiden
EMBL
accession
number
F. Weinberger (July 1996)
silica gel
RH9801799
AJ007815
Y.S.D.M. de Jong & W. F.
Prud’homme van Reine
(Feb. 1996)
W.H.C.F. Kooistra (Aug.
1997)
? via J. R. M. Chisholm to J.
L. Olsen (Feb. 1995)
J. L. Olsen (Feb. 1995)
silica gel
RH9801944
AJ007816
90% EtOH
RH9801821
AJ007817
silica gel
RH9801943
AJ007818
live, silica gel
RH9801945
AJ007812
silica gel
silica gel
RH9801800
RH9801811
AJ007820
AJ007819
silica gel
RH9801941
AJ007821
silica gel
RH9801946
AJ007823
Monaco Aquarium,
Monaco
Marina di Campo, Elba A. F. Peters (May 1996)
Between Ganzirri and
A. F. Peters (Apr. 1996)
Torre Faro, Strait of
Messina, Sicily
Messina, Sicily
G. Giaconne via, Monaco
Aquarium collection to
Townsville, Australia
J. A. H. Benzie via J. R. M.
Chisholm to J. L. Olsen
(Apr. 1995)
Collected as C. taxifolia and later transformed into ‘‘mexicana’’ morphology (see Chisholm et al. 1995).
tga-tgc-39) and ITS4-Reverse at position 52 in the 28S gene (59tcc-tcc- gct-tat-tga-tat-gc-39). Amplification conditions follow Kooistra et al. (1992) but with reaction volumes of 25 mL instead of
100 mL. Fragments were checked on 1.5% agarose gels and
cleaned with the Qiaquick kit (Westburg).
Cloning. PCR products were excised from a 1.5% agarose gel
(C. mexicana) or ligated directly (C. taxifolia–taxmes) into pGEMT and transformed in JM 109 competent cells (both from Promega) using standard blue-white screening according to the manufacturer’s directions. Insert sizes were checked by performing
PCR with either the amplification primers or with the universal
forward and reverse primers. Plasmid isolation was done using
the Flexiprep Kit (Pharmacia).
Sequencing. PCR products were directly sequenced in C. taxifolia
using the amplification primers and one internal primer (C1F
annealing to position 1624 in the 18S 59-gta-cac acc gcc cgt cgc
tcc-39). The PCR products from C. mexicana (and C. taxifolia–taxmes) were first cloned and then sequenced with the universal
forward and reverse sequencing primers as well as the three primers mentioned above. Sequencing was performed on an ABI-310
GeneAnalyzer using the Dye-Terminator cycle sequencing kit
(Perkin Elmer). For direct sequencing, 40 ng of DNA fragments
were used. For cloned fragments, 200 ng of double-stranded plasmid were used.
Data management and alignment. Sequences were managed using Navigator and Factura Software (ABI–Perkin Elmer).
Alignments were performed using Gelassemble in the UWGCG
software package v7.3 (Genetics Computer Group 1991). Properties of the alignment such as GC content and transition:
transversion ratio were computed using MEGA v1.01 (Kumar
et al. 1993).
Phylogenetic analysis. Parsimony analyses were done with PAUP
v3.1.1 (Swofford 1993) using the Branch and Bound search option. Consensus trees were calculated under both strict and majority rule. Bootstrap resampling (Felsenstein 1985) was performed for 1000 replicates under the heuristic search option, random sequence addition. Analyses were done on various portions
of the data set (18S alone, ITS1 alone, ITS2 alone, all data) as
well as within and between clades (Table 2). Maximum likelihood
(ML) was run using DNAML in PHYLIP v3.5c (Felsenstein 1989),
in which analyses were run with ts:tv set at the highest and lowest
computed values from our data set; that is, 1.28 and 0.691, respectively. Empirical base frequencies were used and the global
rearrangement option was applied.
RESULTS AND DISCUSSION
Alignment. Sequences from both species of Caulerpa were easily alignable with many anchoring spots,
despite considerable variation in length (Fig. 1) and
many variable positions (Table 2). Comparative base
composition among the nine sequences was uniform with a mean GC of 47.6 6 0.3% (SD, n 5 9).
This is important because large differences in base
composition among homologous sequences can significantly affect branching order as a result of random associations of nucleotides (Saccone et al.
1993). Transition:transversion ratios were calculated
among all pairwise comparisons to assess the possible role of saturation (Larson 1991). Values of ,1.0
are indicative of saturation. Within the C. mexicana
clade ts:tv averaged 1.08 (n 5 4; range 0.714–1.8)
and within the C. taxifolia clade 1.28 (n 5 5; range
1.0–1.6), indicating no saturation. In contrast, the
ts:tv between the two clades averaged 0.69 (range
0.64–0.74), indicative of some saturation and an old
divergence between the lineages; that is, an underestimation of the divergence. Secondary structure is
evident in the variable domain (V-9) of the 18S gene
but cannot be recovered due to the unsequenced region in the 59 direction. Because the possibility exists
that compensatory base changes need to be accounted for within the 29 informative positions found, we
conducted our analyses with and without the 18S region included. This did not affect the outcome.
Sequence divergence between the two species is
strong with 13% of the positions variable and 12%
informative (Table 2). ITS1 consists of 131–135 nucleotides in C. mexicana but of 99 nucleotides in C.
taxifolia. These are short compared with most ITS1
regions so far reported in green algae (including
853
MEDITERRANEAN CAULERPA ITS
TABLE 2. Data properties and phylogenetic analysis. (A) The alignment. (B) Comparative summary of parsimony analyses under different partitions of
the data. (C) Alternative branching topologies shown in the distribution of most parsimonious trees (MPTs).
A. Variation
Length of alignment
Invariable positions
Variable positions
Informative positions
Gaps (.2 nts)
B. Number of MPTs
All taxa (n 5 9)
C. mexicana only (n 5 4)
C. taxifolia only (n 5 5)
All sequences
(bp)
Partial 18S only
(bp)
ITS1 only
(bp)
5.8S only
(bp)
ITS2 only
(bp)
1034
894
140
126 (12%)
10
455
420
35
29 (6%)
3
135
84
51
49 (36%)
2
144
144
0
0
0
300
246
54
48 (16%)
5
18
1, ((mex, can)(pan, fla))
Same as Fig. 2b
6, ((polytomy)aus)
Same as Fig. 2b
1
not done
4
not done
—
—
18
not done
not done
not done
—
not done
always
always
—
always
C. Subset groupings
((tax clade)(mex clade))
alwaysa
C. mexicana clade only within the 18 MPTs
(pan, fla)
18/18 (100%)a
(isr, can)
6/18 (33%)
((pan, fla)(isr, can))
6/18 (33%)
(((pan, fla) isr)can)
6/18 (33%)
(((pan, fla)can)isr)
6/18 (33%)
C. taxifolia clade only within the 18 MPTs
((mextax, elb, mon, mes)aus)
18/18 (100%)
a
Significant at P , 0.01 in maximum likelihood analysis.
ITS1 in Caulerpa filiformis, estimated at 200 nucleotides, Pillmann et al. [1997]), although we have
found an ITS1 in Acetabularia (Dasycladales) of
about 50 nucleotides (our unpubl. data). Further
investigation is required to establish whether short
ITS1 regions are characteristic of siphonous lineages
more generally. ITS2 is longer and more uniform,
being 287–289 nucleotides long in C. mexicana and
290 nucleotides long in C. taxifolia. Pillmann et al.
(1997) found a range in ITS2 lengths among five
species of Caulerpa from 222–311 nucleotides. There
is no variation in the length of the 5.8S gene, although it appears to be about 5–10 nucleotides
shorter than commonly reported.
Within each species, sequence variation is low
as assessed across a broad geographic range. Intraand interindividual variation within a single population were not tested. Amplification of ITS1 and
ITS2 in C. taxifolia produced single, well-defined
bands on gels that presented no problems for direct sequencing. One individual (taxmes) was
cloned for comparison with results from direct sequencing. Cloned material was identical with directly sequenced material. Amplification of ITS in
C. mexicana produced a faint secondary shadow
band and direct sequencing produced poor quality sequences. For all isolates, the principal amplification products were excised from an agarose gel
and cloned. One clone from each individual was
sequenced on both strands. Although microheterogeneity (i.e. intraindividuality) of ITS almost
certainly exists, the high similarity of the C. mexicana sequences from diverse biogeographic loca-
tions suggests that variation is low. For purposes
of this study, however, even modest microheterogeneity would not affect our basic conclusions regarding separate species.
Phylogenetic analysis. Phylogenetic relationships
were inferred from parsimony and maximum likelihood of the full data set (Fig. 2). Maximum likelihood analysis yielded the same topology
(-ln[likelihood] 5 2223.772), in which the position
of the two clades, the position of taxaus (within the
C. taxifolia clade), and the relationship between
mexfla and mexpan (within the C. mexicana clade)
were significantly positive at P , 0.01. Further subanalyses using parsimony of various subsets of the
alignment and taxa are summarized in Table 2. Regardless of the partitions of the data, phylogenetic
signal remained strong. The two species were clearly
separated in all trees with 100% bootstrap values.
Resolution within clades varies, although it tends to
be low. Rearrangements account for the 18 most
parsimonious trees (MPTs), alternative versions of
which are summarized in Table 2. The fact that
more point mutations are seen among individuals
of C. mexicana than among individuals of C. taxifolia
could be related to the broader geographic sampling in C. mexicana.
Whereas the present study shows that C. taxifolia
and C. mexicana are not ecological variants of a single,
phenotypically plastic species, the data offer no clues
about the origins of C. taxifolia in the Mediterranean.
Although the aquarium escape hypothesis continues
to be favored by many scientists, other explanations
cannot yet be ruled out. The possibility that C. taxi-
854
JEANINE L. OLSEN ET AL.
MEDITERRANEAN CAULERPA ITS
folia has been a long-term cryptic resident that was
missed until populations began to expand (for unknown reasons) remains possible. One case in point
is the recent expansion of C. racemosa along the coasts
of Egypt and one year later to southern Italy (Panayotideis and Montesanto 1994). Caulerpa racemosa
has been known from the eastern Mediterranean as
a Lessepsian immigrant, however, so it is not considered to be a recent introduction in the same fashion
postulated for C. taxifolia. A more complex hypothesis involves the possibility of multiple, relatively recent, introductions from aquarium hobbyists. In this
case, multiple original sources could be involved and
could have come from anywhere in the world. At
present there is no support for this hypothesis.
Identification of biogeographic populations of
species using ITS sequences is of practical interest
because it will permit tracking and monitoring of
endemic and nonendemic populations. The Australian sample of C. taxifolia was consistently separated
from the Mediterranean samples, and it is likely that
other global-scale, biogeographic populations can
be identified by their ITS signatures. Within the
Mediterranean samples of C. taxifolia (n 5 4), no
point mutations were found. Although expansion of
the sampling to include individuals from the Balearic Islands and the Adriatic coast is of interest, it is
unlikely that very much intraspecific variation will
be found within ITS sequences unless these (or other populations) truly represent introductions from
non-Mediterranean sources. In that case, ITS may
still prove useful as a tracking marker. Instead, it is
more likely that investigation of genetic differentiation in C. taxifolia, within the regional scale of the
Mediterranean, can be better addressed with a population-level marker. Benzie et al. (1997) used allozyme variation to investigate populational differences
within and between seven Caulerpa species from the
Great Barrier Reef, Australia, including C. taxifolia.
The seven populations of C. taxifolia (corresponding
to isolated patch reefs) were distinguishable and provided evidence for both sexual and asexual reproduction. Allozyme variation among Mediterranean
populations of C. taxifolia and C. racemosa populations
are currently under investigation.
In conclusion, unequivocal species identification
of C. taxifolia and C. mexicana populations in the
Mediterranean is now possible. Where taxonomic
doubts exist (i.e. ‘‘taxmex’’ morphotypes), the differences in length between ITS1 in C. taxifolia (99
bases) and ITS1 in C. mexicana (131–135 bases) can
be determined easily by a simple PCR diagnostic amplification. Finally, the changing geostrophic current and increased sea surface temperatures noted
by Chisholm et al. (1995), as well as the ecology and
phenology of C. taxifolia noted by Boudouresque et
al. (1995), are all likely to further effect the spread
855
FIG. 2. Unweighted parsimony analysis of full data set. (A) One
of the 18 MPTs using the Branch and Bound search option, in
which branch lengths are proportional to the number of changes
(L 5 159, CI 5 0.969, RC 5 0.955). Numbers along branches
correspond to base changes. Trees are unrooted. Bootstrap values
(1000 replicates) are circled. The mexpan– mexfla clade grouped
together 57% of the time and with mexcan only 19% of the time.
Mexpan and mexcan grouped 5% of the time. (B) Strict consensus tree of 18 MPTs. Maximum likelihood topology is the same
(see Results for details).
of C. taxifolia and probably C. racemosa. The fact that
the ranges of Caulerpa species has been (and continues to be) so carefully monitored by Mediterraneanrim workers makes it a model case study. Coupling
these data with other information on the biology
and population genetics of Caulerpa will undoubtedly lead to a better understanding of the dynamics
of marine seaweed invasions and their management.
Note added in proof: Comparison of our partial 18S
data with a complete 18S sequence from Caulerpa
prolifera (courtesy F. W. Zechman, California State
University, Fresno, California) suggests that nucleotides between positions 50 and 150 (Fig. 1) are an
intron. This does not affect our results but may provide additional characters for phylogenetic investigations within the genus.
We thank Florian Weinberger, Yde de Jong, Willem Prud’homme
van Reine, Wiebe Kooistra, Akira Peters, Marco Vicinanza (Messina), and Giulia Ceccherelli (Elba) for assistance with collections.
We also thank John Chisholm for his generous cooperation in
providing additional material of C. taxifolia and the ‘‘taxmex’’
morphotype for inclusion in this study. This research was sup-
←
FIG. 1. Alignment of the 39 end of the 18S gene, ITS1, 5.8S, ITS2 in Caulerpa mexicana and C. taxifolia. Dots, gaps; 1, nucleotides that
are variable within a species; background shading shows those areas that are different between the two species.
856
JEANINE L. OLSEN ET AL.
ported by grants from the French Ministry of the Environment
(195-935-598-000-19 to M.V. and J.L.O.) and the Dutch National
Science Foundation, NWO-GOA Frans-Nederlands Samenwerking (F198-03 to J.L.O. and I.M.).
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