Eur. J. Phycol. (2004), 39: 83 – 92.
Analysis of rDNA ITS1 indels in Caulerpa taxifolia
(Chlorophyta) supports a derived, incipient species status for the
invasive strain
I S A B E L L E M E U S N I E R 1, 2, M Y R I A M V A L E R O 1, 3, J E A N I N E L . O L S E N 4
AND WYTZE T. STAM4
1
Laboratoire Ge´ne´tique et Evolution des Populations Ve´ge´tales, UPRESA CNRS 8016, Bât. SN2, Universite´ de Lille I, 59655
Villeneuve d’Ascq cedex, France
2
Coastal Oregon Marine Experiment Station, Hatfield Marine Science Center, Oregon State University, 2030 SE Marine Science
Drive, Newport, OR 97365-5229, USA
3
Equipe Evolution et Ge´ne´tique des Populations Marines, UMR UPMC-CNRS 7127, Station Biologique de Roscoff, BP 74, Place
Georges Tessier, 29682 Roscoff cedex, France
4
Department of Marine Biology, Centre for Ecological and Evolutionary Studies, Biological Centre, University of Groningen, PO
Box 14, 9750 AA Haren, The Netherlands
(Received 15 May 2003; accepted 20 October 2003)
We analysed insertion-deletion patterns in 159 published sequences of ITS1 for Caulerpa taxifolia (Vahl) C. Agardh
collected from 55 localities throughout the species’ range. Five indelotypes (I) were identified that represented a sequential
loss of insertions from the ancestral type (I3) to the most derived type (I0). The I3 consists of the complete ITS1 sequence,
which is also characteristic of three outgroup species. In contrast, the I0 has lost three inserts from the complete sequence
and is associated with the invasive forms found in the Mediterranean, California and southeastern Australia. The I2 was
found in samples from the Red Sea and Jakarta, whereas the I1 was associated with samples from Australia and New
Caledonia. When mapped onto location and habitat, these ordered character-states reveal a widespread distribution of I3
and (probably) I2, which are associated with offshore coral reefs and clear oligotrophic waters. The I1 and I0 comprise a
paraphyletic assemblage of the more derived types harbouring two or three deletions and occurring together along mainland
Australian coasts in more turbid environments. The presence of I0, I1a and I1b along the Australian coast indicates that the
ecological transition that gave rise to the coastal ecotype has been present at least since the time of the mutation between I2
and I1. These types of fixed differences confirm that C. taxifolia consists of at least two incipient species—the coastal form
being an offshoot derived from the clear-water ecotypes. The finding of indelotype I1a in an isolate from Sousse (Tunisia)
confirms a second Mediterranean introduction and highlights the urgency for further research in the evolutionary
diversification of one of the most intriguing and troublesome seaweeds.
Key words: Caulerpa taxifolia, invasive species, incipient species, indels, ITS
Introduction
Comparative phylogenetic analyses of the internal
transcribed spacers (ITS) of the nuclear rDNA
cistron have played a crucial role in elucidating the
invasive biology of Caulerpa taxifolia by providing
unequivocal taxonomic identification within the
genus (Olsen et al., 1998); establishing the genetic
uniformity of the invasive aquarium strain (Jousson et al., 1998); and in documenting its spread
within the Mediterranean, to southern California
(Jousson et al., 2000; Kaiser, 2000) and to the
greater Sydney, Australia, area (Schaffelke et al.,
2002).
Correspondence to: W.T. Stam. e-mail: [email protected]
The biogeographic source of the invasive
Mediterranean strain has been narrowed down
to the Moreton Bay area around Brisbane,
Australia (Meusnier et al., 2001, 2002; Famà et
al., 2002), which is also the probable source of
populations found in the greater Sydney area
(Schaffelke et al., 2002). These investigations have
also shown that C. taxifolia is probably a
complex of subspecies with at least two widespread clades. First, the presence of an intron in
the rbcL gene of the chloroplast DNA is
restricted to ‘tropical’ areas (Caribbean, Red
Sea, SE India) (Famà et al., 2002). Absence of
the intron was characteristic of the invasive and
introduced populations found in the Mediterranean and in California, as well as in natural
ISSN 0967-0262 print/ISSN 1469-4433 online # 2004 British Phycological Society
DOI: 10.1080/09670260310001646531
I. Meusnier et al.
populations ranging from subtropical NE Australia (Queensland) to temperate SE Australia
(New South Wales). Second, a combined nucleoplasmic analysis of nuclear ITS and intron 2 of
the chloroplast 16S rDNA (Meusnier et al., 2002)
revealed two well-defined lineages: the first clade
grouped nontropical invasive and introduced
populations with inshore-mainland populations
from Australia, whereas the second clustered all
‘offshore-island’ tropical populations. Finally,
using ITS sequencing, Schaffelke et al. (2002)
have shown that samples collected from six
different sites on the Great Barrier Reef (tropical
NE Australia) were genetically distinct from
mainland coastal areas and grouped with other
tropical reef populations coming from the Caribbean and Red Sea. They referred to these as
the ‘Reef Clade’, which is equivalent to the
preceding ‘offshore-island’ (Meusnier et al., 2002)
and ‘tropical groups’ (Famà et al., 2002) referred
to above. A second general finding from these
studies is the correlation between the molecular
data and distinct morphologies and habitat
differences. The tropical individuals (regardless
of whether they are inshore-mainland or offshoreisland) are small (5 10 cm tall) and delicate,
whereas the more cold-tolerant individuals (including subtropical and Mediterranean, Australian and invasive populations) are large (25 –
50 cm or more) and robust, with thick stolons,
wide fronds and large pinnules. A third general
finding is that regional Australian populations of
C. taxifolia have been brought into contact with
each other. Contact between different species
(Arnold, 1997) as well as between normally
disjunct populations of a single species (Ellstrand
& Schierenbeck, 2000) can lead to several outcomes—all of which are relevant to the fate of
the species and their potential invasiveness
(Levin, 2000; Kolar & Lodge, 2001; Sakai et
al., 2001; Grosholz, 2002).
In the case of C. taxifolia, Meusnier et al.
(2002) hypothesized that at least two consecutive
founder events have occurred that involved the
invasive strain. First, the Moreton Bay samples
are probably derived from the North Queensland
populations; and second, the invasive Mediterranean strains are, themselves, derived from the
Moreton Bay populations. The success of the
invasive strain of C. taxifolia in the Mediterranean and elsewhere has thus been facilitated by
the initial founding events in combination with
selection for cold-adapted genotypes. We, therefore, hypothesized that the native tropical strain
was the ancestral type that gave rise to the more
cold-tolerant invasive strain. Unfortunately, the
Meusnier et al. (2002) study was unable to
determine which populations belonged to the
84
ancestral type, thus preventing a test of our
hypothesis.
Most genetic variation in ITS is expressed as
single point mutations, which are not phylogenetically informative although they may contribute to
homoplasy and long terminal branches. In contrast, indels (involving two or more nucleotides) are
generally few but often highly phylogenetically
informative. They are also less subject to homoplasy because there is a lower probability of
reversion. Consequently, indels are more discriminatory. Slippage replication is thought to be the
dominant mechanism for the generation of indels
in ITS as compared with transposition, crossingover and gene conversion (Levinson & Gutman,
1987; Li, 1997). In particular, slippage tends to lead
to deletions on one strand and duplications on the
opposite strand, which then become fixed. The
ITS1 of C. taxifolia has three indel regions of
interest.
In the present paper, we analyse the step-wise
loss of the three inserts in ITS1 sequences using
all currently available data. We will show that
ITS indelotypes are strongly correlated with both
habitat and geography, and test our hypothesis
that the loss of genetic variation associated with
invasive types is coupled with incipient speciation.
Materials and methods
DNA sequences
The internal transcribed spacer sequences (ITS1) of the
nuclear rDNA cistron were retrieved from GenBank/
EMBL (sequences deposited by August 2002) and
aligned using BioEdit version 5.0.9 (Hall, 1999).
GenBank accession numbers can be found in Jousson
et al. (1998, 2000), Olsen et al. (1998), Meusnier et al.
(2001, 2002), Famà et al. (2002) and Schaffelke et al.
(2002). The full alignment is available from WTS.
Phylogenetic analyses
Phylogenetic analysis of the partial ITS1 alignment given
in Fig. 1 (upper panel) was performed using maximum
parsimony (MP) in PAUP* 4.0b10 (Swofford, 2002)
under the heuristic search option, 50 random sequence
additions, unweighted and unordered characters, and
TBR branch swapping. Gaps were coded as ‘missing
data’ and the three indels recoded as independent events
in a gap matrix ensuring equal weight. This is preferred
to counting gaps as ‘fifth base’ in which case, long gaps
(as is the case with insert 2) disproportionately affect the
analysis. Bootstrap resampling (1000 times) could only
be performed on a reduced data set in which identical
sequences were only represented once. Caulerpa prolifera
(Forsskål) J.V. Lamouroux, C. mexicana Sonder ex
Kützing and C. racemosa (Forsskål) J. Agardh were used
as outgroup taxa (Fig. 1).
Caulerpa taxifolia indel evolution
5
15
....|....|....|
C. prolifera TTC-AAACTACTACT
outgroups C. mexicana TTC-AAACAAACACT
C. racemosa TTCATAACTACAA-I3
taxausmr
TTCTATATGTGTATA
I3
taxaussr
TTCTATATGTGTATA
I3
taxaushtr
TTCTATATGTGTATA
I3
taxausar
TTCTATATGTGTATA
I3
taxaushc
TTCTATATGTGTATA
I3
taxausmyr
TTCTATATGTGTATA
I3
taxauskr
TTCTATATGTGTATA
I3
taxphi
TTCTATATGTGTATA
I3
taxmart
TTCTATATGTGTATA
I3
taxgua
TTCTATATGTGTATA
I3
taxpuer
TTCTATATGTGTATA
I3
taxjap
TTCTATATGTGTATT
I3
taxegy
TTCTATATGTGTATA
I3
taxtah
TTCTATATGTGTATA
I2
taxegy
TTCTATATGTGTATA
I2
taxdja
TTCTATGTGTGTACA
I1a
taxauskp
TTCTATATGTGTATA
I1a
taxausfi
TTCTATATGTGTATA
I1a
taxausgl
TTCTATATGTGTAYA
I1a
taxausmb
TTCTATATATGTACA
I1a
taxauslhi
TTCTATATGTGTATA
I1a
taxauslm
TTCTATATGTGTATA
I1a
taxauscb
TTCTATATATGTACA
I1a
taxausph
TTCTATATGTGTATA
I1a
taxtun
TTCTATATGTGTATA
I1a
taxncal
TTCTATATGTGTATA
I1a
taxaq
TTCTATATGTGTATA
I1b
taxauscb
TTCTAT--GTGTATA
I0
taxauskp
TTCTAT--GTGTATA
I0
taxausfi
TTCTAT--GTGTATA
I0
taxausmb
TTCTAT--GTGTATA
I0
taxausbr
TTCTAT--GTGTATA
I0
taxauslc
TTCTAT--GTGTATA
I0
taxncal
TTCTAT--GTGTATA
I0
taxmed
TTCTAT--GTGTATA
I0
taxbal
TTCTAT--GTGTATA
I0
taxcro
TTCTAT--GTGTATA
I0
taxcarl
TTCTAT--GTGTATA
I0
taxaq
TTCTAT--GTGTATA
Great Barrier Reef
Townsville
(19°15' S)
Brisbane
(27°33' S)
85
65
75
85
95
105
115
125
|....|....|....|....|....|....|....|....|....|....|....|....|....|.
... ATATAA-GCTTT-----------GT-AAAGACGCATATGG--CTAT-----GTAATGTTGATGTTGT
... -TATGG--CTATGACT-GTTGTTGTTAA-GACGCATATGT--CTGT-----GTAATAACAATAGTGA
... -TATGT-----------------------GA-ACATATGT--CTATATGTATTTGTAACAATATTGA
... -TATGTTGCTAT----------TAC-AAAGA-ACATATG--CCTATGTT--GTAATGAATGTGCTGT
... -TATGTTGCTAT----------TAC-AAAGA-ACATATG--CCTATGTT--GTAATGAATGTGCTGT
... -TATGTTGCTAT----------TAC-AAAGA-ACATATG--CCTATGTT--GTAATGAATGTGCTGT
... -TATGTTGCTAT----------TAC-AAAGA-ACATATG--CCTATGTT--GYAATGAATGTGCTGT
... -TATGTTGCTAT----------TAC-AAAGA-ACATATG--CCTATGTT--GTAATGAATGTGCTGT
... -TATGTTGCTAT----------TAC-AAAGA-ACATATG--CCTATGTT--GTAATGAATGTGCTGT
... -TATGTTGCTAT----------TAC-AAAGA-ACATATG--CCTATGTT--GTAATGAATGTGCTGT
... -CATGTTGCTAT----------TAC-AAAGA-ACATATG--CCTATGTT--GTAATGAATGTGCTGT
... -TATGTTGCTAT----------TAC-AAAGA-ACATATG--CCTATGTT--GTAATGACTGTGCTGT
... -TATGTTGCTAT----------TAC-AAAGA-ACATATG--CCTATGTT--GTAATGAATGTGCTGT
... -TATGTTGCTAT----------TAC-AAAGA-ACATATG--CCTATGTT--GTAATGAGTGTGTTGT
... -TATGTTGCTAT----------TGC-AAAGA-ACATATG--CCTATGTT--GTAATGAATGTGCTGT
... -TATGTTGCTAT----------TAC-AAAGA-ACATATG--CCTATGTT--GTAATGAATGTGCTGT
... -TATGTTGCTAT----------TAC-AAAGA-ACATATG--CCTATGTT--GTAATGAATGTGCTGT
... -TATGTT--------------------------------------------GTAATGAATGTGCTGT
... -TATGTT--------------------------------------------GTAATGAATGTGCTGT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGA-TGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTTGCTAT----------TAC-AAAGA-ACATATG--CCTATGTT--GTAATCACTGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GG
... -TATGTT--------------------------------------------GTAAT-AATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
... -TATGTT--------------------------------------------GTAATGAATGT---GT
Hicks Reef
Hastings Reef
Michaelmas Reef
Arlington Reef
Sudbury Reef
Myrmidon Reef
Kelso Reef
Kissing Point
Gladstone
Fraser Island
Moreton Bay
Lord Howe Island
Sydney
(33°54' S)
Careel Bay
Port Hacking
Lake Conjola
Ancestral
type
I3
I2
Offshore coral reefs,
clear water,
small delicate thalli
(native forms)
I1a I1b
I0
Mainland coasts,
turbid water,
large robust thalli
(invasive forms)
Fig. 1. Caulerpa taxifolia. Upper panel: Portion of alignment of ITS1 in which relevant insert sequences (1, 2 and 3) are shown in
red and correspond to positions 7 – 8, 72 – 113 and 127 – 129 in the full ITS1 alignment as determined from prior phylogenetic
analyses. Flanking regions of the alignment are highlighted in grey. The relationships among the five indelotypes (colours) and
sample locations correspond to the symbol colours in the lower panel and to the codes in Table 1. Lower panel: Biogeographic
distribution of ITS1 indelotypes of C. taxifolia.
I. Meusnier et al.
Terminology
We use the term indelotype (I) to refer to the relative
presence and absence of three specific insert-sequences
characteristic of ITS1 in Caulerpa. The designation
‘indelotype’ does not exclude the presence of other point
mutations outside these regions. The distinction we
make is important with respect to intra- and interindividual comparisons, and should be kept in mind
when comparing the present results with those in other
published papers in which all nucleotide polymorphisms
(encountered over the length of the sequence) are
counted as characterizing a new genotype. By way of
illustration: five complete ITS1 sequences might differ by
one or more nucleotides so that the five sequences
correspond to five genotypes. With respect to ITS indels,
however, the five genotypes might all pertain to a single
indelotype.
Results
Published ITS1 sequences from 159 individual C.
taxifolia, collected from 55 localities, were compared. Sequences were easy to align and three
regions in this alignment contain inserts of two, 28
and three bases, respectively (Fig. 1). The sequential loss of these inserts and the corresponding
indelotypes are listed in Table 1, and the geographic distribution of observed indelotypes is
shown in Fig. 1. We note that a MP analysis of
the ITS1 region of all 159 sequences produced
4 12 000 trees with absolutely no resolution in the
consensus tree (not shown).
Five indelotypes were identified among eight
theoretical combinations (Fig. 2, lower left panel).
These represent a sequential step-wise loss of
insertions from the ancestral type (I3) to the most
derived type (I0). The observed indelotypes in Fig.
2 are shown in bold. Determination of the
directionality of the change from ancestral to
derived is based on: (1) the fact that I3-containing
individuals are the most basal within the ingroup
(Fig. 2) of C. taxifolia, i.e., ((((I0, I1a)I2a) I3)
outgroup) and ((((I0, I1b)I2b or c) I3) outgroup) as
well as being characteristic of the three outgroup
species (Table 1); and (2) the observation that
losses are generally easier than gains (Li, 1997).
I2 was found in samples from the Red Sea and
Jakarta, whereas I1 was found in samples from
Australia and New Caledonia. I0 is associated with
the invasive form found in the Mediterranean,
California and parts of Australia. When indelotypes were mapped onto location (Fig. 1), it was
found that I3 and (probably) I2 are the most
widespread and are associated with offshore, coral
reefs and clear oligotrophic waters. In contrast, I1
and I0 represent a paraphyletic assemblage of the
more derived types containing two or three
86
deletions, and occur together along the mainland
Australian coast in more turbid environments.
A single indelotype per individual was found in
all cases (Table 1)—with one exception. Schaffelke
et al. (2002) sequenced a number of clones from
three individuals collected in Careel Bay (Australia)
and found intra-individual polymorphisms in ITS1
in three of the individuals (sensu different genotypes as explained in the Materials and methods).
In one individual, however, two variants of
indelotype I1 (i.e. the widespread I1a and a new
variant, which we have designated as I1b) were
detected. This finding does not change the basic
model of successive deletions, but does signal that
inserts have been lost independently at least twice
starting from I3 to arrive at I1a and I1b (Fig. 2).
Indelotype I0 is characteristic of the aquarium
strain, which also dominates the Mediterranean.
However, a specimen collected from Sousse (Tunisia) carries the I1a indelotype. This is a strong
suggestion that a second introduction of C. taxifolia has occurred in the Mediterranean.
Discussion
Plasticity, ecotypes and incipient species
High levels of morphological plasticity in Caulerpa
are responsible for the proliferation of subspecific
varieties and forms in the taxonomic literature
(Prud’homme van Reine et al., 1996). Plasticity is
defined as the property of a given genotype with a
broad reaction-norm that produces different phenotypes in response to distinct environmental
conditions (Pigliucci, 2001). The extent to which
morphological forms and varieties actually reflect
evolutionary lineages, therefore, remains dubious
without genetic data. The addition of molecular
phylogenetic studies in Caulerpa has clarified some
of the morphological plasticity problem by temporarily removing it from consideration. Phylogenetic analyses of ITS sequences have revealed both
monophyletic and paraphyletic groups within the
C. taxifolia complex. One of these groups consists
of distinct cold-adapted, turbid-water thalli of large
size; the other comprises distinct warm-adapted,
clear-water thalli of small and delicate architecture
(Famà et al., 2002; Meusnier et al., 2002; Schaffelke
et al., 2002). Reanalysis of the whole data set using
the indelotypes from ITS1 (Fig. 2) sharpens this
picture further because the indelotypes reflect fixed
differences among groups of related clones constituting populations that correspond to the ecotypes, i.e. genetically specialized local populations
adapted to specific environmental conditions (Futuyma, 1998). These ecotypes, in turn, represent
incipient species. Finally, the information from the
Inserts 1, 2 and 3 from the alignment in Fig. 1
AC
AC
AC
RT
1
GCTTTGTAAAGACGCATATGGCTA
GCTATGACTGTTGTTGTTAAGACGCATATGTCTGT
GAACATATGTCTATATG
GCTATTVCAAAGAACATATGCCTATGTT
1
Indelotype
Geographic region
GTT
AGT
Caulerpa prolifera
Caulerpa mexicana
ATT
GYT
Caulerpa racemosa
; Caulerpa taxifolia
1
I3
0
1
1
0
1
1
I2
1
1
1
0
0
0
I1a
Widespread ancestral type associated with offshore reefs and clear water; thalli small and
delicate:
Michaelmas Reef (Australia, near Cairns, 1 sequence);
Sudbury Reef (Australia, near Cairns, 1 sequence);
Hastings Reef (Australia, near Cairns, 1 sequence);
Arlington Reef (Australia, near Cairns, 1 sequence);
Hicks Reef (Australia, north of Cairns, 1 sequence);
Myrmidon Reef (GBR, Australia, 1 sequence);
Kelso Reef (GBR, Australia, 3 sequences);
Bolinao and Sorsogon (Philippines, 4 sequences);
Martinique (7 sequences);
Guadeloupe (3 sequences);
Puerto Rico (4 sequences);
Okinawa (Japan, 1 sequence);
Red Sea (Egypt, 11 of 12 sequences);
Tahiti (2 sequences)
not encountered
Red Sea (Egypt, 1 of 12 sequences);
Jakarta (Indonesia, 1 sequence)
not encountered
Kissing Point (Australia, near Townsville, 15 of 21 sequences);
Fraser Island (Australia, near Brisbane, 12 of 13 sequences);
Gladstone Harbor (Australia, north of Brisbane, 1 sequence);
Moreton Bay (Australia, near Brisbane, 1 of 16 sequences);
Lord Howe Island (Australia, south of Brisbane, 1 sequence);
Lake Macquarie (Australia, near Newcastle, 1 sequence);
Careel Bay (Australia, near Sydney, found in 1 clone among 6 that were sequenced from 1
individual, see Schaffelke et al. (2002), AY034869, clone CTCB3-6 in Schaffelke’s
nomenclature
Port Hacking (Australia, south of Sydney, 5 sequences);
Sequence code used in Fig. 1
Caulerpa taxifolia indel evolution
Table 1. Summary of insert-sequence deletions in ITS1 found in Caulerpa taxifolia from different biogeographic localities. Individual GenBank accession numbers can be found in Jousson et al.
(1998, 2000), Olsen et al. (1998), Meusnier et al. (2001, 2002), Famà et al. (2002) and Schaffelke et al. (2002). Notations: 1 = insert sequence present; 0 = insert sequence absent; Indelotypes (I)
correspond to the combinations of inserts present or absent, i.e. I3 has all three inserts (1,1,1); GBR = Great Barrier Reef; () = provides additional information about the location and the
number of individuals (not clones within individuals) examined that contained that ITS1 indelotype. The exception is Careel Bay (italics) in which one individual was found to contain I1a and
I1b. See text
taxausmer
taxaussr
taxaushtr
taxausar
taxaushcr
taxausmyr
taxauskr
taxphil
taxmart
taxgua
taxpuer
taxjap
taxegy
taxtah
texegy
taxdja
taxauskp
taxausfi
taxausgl
taxausmb
taxauslhi
taxauslm
taxauscb
taxausph
87
(continued )
I. Meusnier et al.
Table 1. (continued )
Inserts 1, 2 and 3 from the alignment in Fig. 1
Indelotype
0
1
0
I1b
0
0
0
0
1
0
I0
Geographic region
Sequence code used in Fig. 1
Sousse (Tunisia, 1 sequence);
Noumea (New Caledonia, 1 of 7 sequences);
Aquarium strains (3 of 11 sequences)
Careel Bay (Australia, near Sydney, 4 clones from 1 individual from Famà et al. (2002) and 5
clones from 1 individuals from Schaffelke et al. (2002))
not encountered
Invasive derived type associated with mainland, inshore coastlines and turbid waters; thalli large
and robust:
Kissing Point (Australia, near Townsville, 6 of 21 sequences);
Fraser Island (Australia, near Brisbane, 1 of 13 sequences);
Moreton Bay (Australia, near Brisbane, 15 of 16 sequences);
Brisbane (Australia, 4 sequences);
Lake Conjola (150 km south of Sydney, 10 sequences);
Noumea (New Caledonia, 6 of 7 sequences);
Mediterranean (Monaco area, 15 sequences);
Balearic Islands (West Mediterranean, 10 sequences);
Stari Grad Bay (Adriatic Sea, Croatia, 2 sequences);
Carlsbad (California, USA, 5 sequences);
Aquarium strains (8 of 11 sequences)
taxtun
taxncal
taxaq
taxauscb
taxauskp
taxausfi
taxausmb
taxausbr
taxauslc
taxncal
taxmed
taxbal
taxcro
taxcarls
taxaq
88
Caulerpa taxifolia indel evolution
89
111
101
100-I1a
000
Fig. 2. Caulerpa taxifolia. Analysis of ITS1 insertion-deletion patterns. Lower left: Deletion pathways for the three ITS1 inserts
shown in Fig. 1. Of the eight possible indel combinations, five indelotypes (bold) were observed (see also Table 1). The pathway
highlighted in grey follows the observed data and analysis shown in the tree. The I1b indelotype most likely came from one of the
alternate pathways. Note that all three pathways are equally parsimonious. A reversion from I0 to I1, especially involving insert
2, is predicted to be less likely. Upper right: 50% majority-rule consensus tree of 120 equally most-parsimonious trees (69 steps,
CI = 0.812, RI = 0.936, RC = 0.759) based on an analysis of the partial ITS1 sequence alignment and isolates shown in Fig. 1.
Gaps were coded as ‘missing data’ and the three indels recoded as single deletion events. Bootstrap values are shown above
branches (bold and circled) and majority rule scores below branches.
I. Meusnier et al.
indelotypes provides resolution that cannot be
achieved by an analysis of ITS sequence divergence
by itself: the indelotype I3 is definitively the
ancestral type, confirming the hypothesis of two
consecutive founder events. Although more sequencing would undoubtedly reveal the unencountered indelotypes listed in Table 1, the basic
conclusion would not change, i.e. the association
of I3 with clear water and the I0 with coastal, more
turbid waters.
When reproductive barriers are weak
Lineages on their way to becoming separate species
undergo a transition from paraphyly to monophyly
(Avise, 2000). The rate at which speciation occurs
depends upon how fast reproductive barriers are
created. This involves interplay of gene flow,
selection, genetic drift and mutation against the
characteristics of the environment. In reef-building
corals, incipient species (or microspecies) may
persist for very long periods of time as a
consequence of repeated isolation and contact
between reefs (Veron, 1995) which is facilitated
by changing current regimes in conjunction with
mass spawning events. Introgressive hybridization
and the long-term maintenance of microspecies
have been documented in the Acropora aspera
complex on the Great Barrier Reef (Van Oppen et
al., 2002) and within the Madracis decactis complex
in the Caribbean (Diekmann et al., 2001). Reproductive barriers have, therefore, been demonstrated
to be weak.
Caulerpalean taxa are not corals, but some
aspects of their life history and ecology are
sufficiently similar to consider a comparison.
First, paraphyly is very common in Caulerpa—
moderately so within the C. taxifolia complex
and rampantly so in C. racemosa (Famà et al.,
2000; Verlaque et al., 2003). This means that
there is incomplete reproductive isolation. Second, mass spawning is also a feature of Caulerpa.
Clifton (1997) followed several species in Panama
and found subtle differences in timing of gamete
release. Though there is no direct evidence at
present, it is conceivable that a disruption of the
timing of gamete release could result in the loss
of an already weak reproductive barrier, which
could periodically promote hybridization. Phycologists have known for nearly a century that
reproductive barriers can be weak in algae—at
the population, species and even generic levels—
based on laboratory crossing studies as well as
field observations (see Lewis, 1996). However,
these studies have been criticized on the grounds
that what an organism can be made to do in the
laboratory, does not necessarily reflect what
happens in nature. Recently, however, Coyer et
90
al. (2002a,b) have conclusively documented interspecific hybridization in the field between two
species of Fucus in the Kattegat Sea off Denmark. Of particular relevance is the fact that one
of the species involved, F. evanescens, is a known,
century-old introduction to the area, which has
now hybridized with F. serratus. Although this
example involves two different species, rather
than two disjunct populations within a single
species, it shows that if indeed reproductive
barriers are weak within and between seaweed
species, then the potential effects of introductions
(of all kinds) may be disproportionately high on
the local algal communities.
Returning to the case in hand, the detection of
two indelotypes in a single individual of C. taxifolia
from Careel Bay (Australia) can be interpreted in
two ways. First, the presence of two distinct ITS
types could signal intraspecific hybridization
among formerly distant populations, which has
been shown in many organisms including plants
(e.g. Sang et al., 1995; Campbell et al., 1997;
Quijada et al., 1997), dipterid flies (Tang et al.,
1996), corals (Van Oppen et al., 2002) and algae
(Coyer et al., 2002a,b). Such contacts provide a
unique opportunity to explore adaptation and
hybridization from the perspective of invasive
biology as well as restoration ecology because the
spatial isolation element is instantly removed when
humans move individuals around (Ellstrand et al.,
1999; Ellstrand & Schierenbeck, 2000; Hufford &
Mazer 2003). Second, the presence of the two ITS
types could be due to recombination events. This
result is consistent with our previous studies
(Meusnier et al., 2002) indicating that sexual
recombination does occur as a stochastic event in
C. taxifolia. Clearly, there is a need to investigate
the role of sexual reproduction in Caulerpa species
as well as the potential for mixing among distant
populations.
When is an introduction an introduction?
When C. taxifolia suddenly appeared in Southern
California (Kaiser, 2000), local management
officials were alarmed because Caulerpa was not
part of the regional marine flora; there was no
doubt about its being an introduction. In
contrast, researchers in Florida have had difficulty in mustering a watch for the invasive strain
of C. taxifolia because many species of Caulerpa,
including C. taxifolia, are part of the native
marine flora. The latter situation has been
mirrored in Australia (Schaffelke et al., 2002)
where a human-mediated range expansion involving the Moreton Bay strain has extended the
range of the alga ca. 1000 km south to the
greater Sydney area.
Caulerpa taxifolia indel evolution
Human-mediated range expansions along long
coastlines already harbouring the species may be
just as serious as those that have crossed entire
ocean basins; we simply do not know. It is,
therefore, essential that monitoring programs
characterize the local genotypes so that intrusion by distant (and even not so distant)
neighbours can be recognized. This has now
paid off in the Mediterranean, where up until
now only the I0 type has been found. We have
identified an I1a, in a specimen from Sousse
(Tunisia), which is also characteristic of Australia and the Moreton Bay region (Fig. 1).
This finding demonstrates that a second introduction has occurred in the Mediterranean and
again highlights the fact that C. taxifolia (as
well as other species of Caulerpa) continues to
flourish within the aquarium trade (Jousson et
al., 1998; Frisch & Murray, 2002).
Our study shows that a simple combination of
primers and a measurement of the amplicon length
can give a rapid classification of indelotypes (within
one working day from start to finish). This
diagnostic test will provide a good basis for
determining whether the original invasive strain is
involved or if new variants have been found, in all
new collections of C. taxifolia.
In conclusion, the invasive strains of C. taxifolia are characterized by successive deletions in
the ITS1 and their more derived status as
incipient species. In contrast, native strains display higher genetic variation, have fewer deletions
in the ITS1 and show evidence for sexual
reproduction and recombination. Up until now,
research in C. taxifolia has been conducted at the
global biogeographic scale. In the future, research
needs to be focused on the landscape scale of
population structure where we may begin to
identify cryptic boundaries as well as areas of
secondary contact among previously isolated
populations. Such an approach will further
enhance our understanding of human-mediated
introductions as well as the natural, underlying
microevolutionary processes that drive adaptation
and speciation.
Acknowledgements
We thank François Bonhomme, Jim Coyer and
Galice Hoarau for their comments on earlier
versions of this manuscript. We also would like to
acknowledge the expert review by Prof. Dr.
Wolfgang Grosz, and we are very sorry to hear of
his untimely death. This research was supported by
the French Ministry of the Environment (195-935598-000-19 to M. Valero and J.L. Olsen) and by a
PhD fellowship to I. Meusnier from the French
Ministry of National Education.
91
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