Eur. J. Phycol. (2002), 37 : 173–178. # 2002 British Phycological Society
DOI : 10.1017\S0967026202003682 Printed in the United Kingdom
173
Inheritance patterns of ITS1, chloroplasts and mitochondria
in artificial hybrids of the seaweeds Fucus serratus and
F. evanescens (Phaeophyceae)
J . A . C O Y E R1 , A . F . P E T E R S2 , G . H O A R A U1 , W . T . S T A M1 A N D J . L . O L S E N1
" Department of Marine Biology, Centre for Ecological and Evolutionary Studies, University of Groningen, PO Box 14,
9750 AA Haren, The Netherlands
# Marine Oq kologie, Institut fuW r Meereskunde, DuW sternbrooker Weg 20, 24105 Kiel, Germany
(Received 2 October 2001 ; accepted 7 February 2002)
Patterns of nuclear and organelle inheritance among artificial hybrids of the seaweeds Fucus serratus and F. evanescens were
detected using single-strand conformation polymorphism (SSCP). Three alleles were identified in the 231 bp rDNA-ITS1
gene (nuclear) : two in F. serratus and one in F. evanescens. Alleles differed by 1–2 bp and all hybrids possessed one allele
from each parent. Two haplotypes were present in the 288 bp Rubisco spacer (chloroplast), differentiated by a 33 bp indel.
Two haplotypes differing by a single nucleotide were found in a 135 bp region of nad11 gene (mitochondrion). Both
organelles are maternally inherited, as all hybrids contained the haplotypes of the parent contributing the egg. Although
laboratory hybrids among Fucus spp. have been produced previously, this is the first time that both nuclear and
cytoplasmic genetic markers have been used to document inheritance patterns. SSCPs analysed on an automated sequencer
offer a rapid and powerful approach for identifying suspected hybrids from field samples, as well as a screen for
intraspecific and intra-individual variation in DNA regions prior to confirmation of variations by sequencing.
Key words : Fucus serratus, F. evanescens, hybrids, ITS1, nad11, organelle inheritance, Rubisco spacer, SSCP
Introduction
Analysis of the DNA sequence in nuclear and
organellar genes and spacers of seaweeds has been
widely applied at the phylogenetic (Olsen et al.,
1998 ; Draisma et al., 2001) and biogeographical
(Peters et al., 1997 ; Meusnier et al., 2001) scales, as
well as at the population level (Coyer et al., 2001).
Central to their importance as genetic markers,
however, is an understanding of their mode of
transmission from parent to offspring. For example,
transmission of animal mitochondria is through
maternal lines in most species, although several
exceptions exist and, in some species, such as marine
mussels, ‘ paternal leakage ’ is relatively common
(reviewed in Avise, 1994). In plants, mitochondrial
inheritance is usually maternal, whereas chloroplast
inheritance can be maternal (usually), biparental or
paternal (reviewed in Avise, 1994 ; Morgensen,
1996 ; see also Isoda et al., 2000).
Little is known about inheritance patterns of
nuclear and organellar sequences in marine macroalgae. Only one study has documented inheritance
patterns of internal transcribed spacer (ITS) regions
in laboratory crosses using molecular markers
Correspondence to : J. Coyer. e-mail : coyerja!biol.rug.nl
(Liptack & Druehl, 2000). Chloroplast inheritance
has been examined in four species using electron
microscopy (Bouck, 1970 ; Bisalputra et al., 1971 ;
Brawley et al., 1976 a ; Motomura, 1990) and only
one species using genetic markers (Zuccarello et al.,
1999 a). Mitochondria are less commonly used for
phylogeny or population studies in macroalgae and
inheritance patterns have been investigated with
electron microscopy in two species (Brawley et al.,
1976 b ; Motomura, 1990) and with molecular markers in one (Zuccarello et al., 1999 b).
In marine macroalgae, verification of hybrid
status for individuals with morphologies intermediate between two co-occurring species currently
depends upon crossing experiments, which are
difficult, labour-intensive and often not feasible. In
the Kattegat and Baltic Sea, populations of Fucus
serratus L. and F. evanescens C. Ag. (Phaeophyceae)
have become sympatric since F. evanescens was
introduced to the Oslofjord in the 1890s (Schueller
& Peters, 1994). Putative hybrids of the two species
were observed in Oslofjord in the late 1970s (Lein,
1984 ; Rice & Chapman, 1985) and from 1998 to
2000 wherever the two species co-occurred in the
Kattegat to western Baltic Sea (Coyer and Peters,
unpublished data). Therefore, it was of interest to
J. A. Coyer et al.
develop appropriate genetic markers that could be
used to rapidly confirm the existence of hybrids and
hybridization in field populations. The aims of the
present study were to : (1) verify hybridization in
laboratory crosses of F. serratus and F. evanescens
using genetic markers and single-strand conformational polymorphism (SSCP), and (2) assess the
inheritance patterns of nuclear and organellar markers in Fucus.
Materials and methods
Artificial hybrids were produced in the laboratory using
fertile parental individuals collected from Blushøj (near
Elsega/ rde), Denmark (56m 10h N, 10m 43h E) in April 2000.
Individual receptacles were placed in separate plastic
bags and transported to the laboratory on ice.
The sex of individual Fucus serratus was determined
microscopically. In the dioecious F. serratus, all receptacles of an individual contain either antheridia or
oogonia. Receptacles from mature F. serratus thalli were
stored in plastic bags overnight at 0–4 mC. The addition of
ice-cold sterilized seawater induced conceptacles to release antheridia or oogonia, which were collected and
washed once in sterile seawater before further use.
As conceptacles of F. evanescens contain both antheridia and oogonia (l hermaphroditic), collecting oogonia
after release from conceptacles was inappropriate, because eggs from such preparations already were fertilized
by conspecific sperm. Hence, to prevent selfing in F.
evanescens, oogonia were collected from conceptacles
immediately after sectioning them with a razor blade, and
washed (i3) in sterile seawater before use. Clusters of F.
evanescens antheridia were obtained in the same manner.
Oogonia of F. serratus or F. evanescens isolated as
described above were inoculated with or without antheridia. A reciprocal crossing design was used (Table 1) in
which F. evanescens females were crossed with F. serratus
males and males of the same F. evanescens individuals
were crossed with F. serratus females. A set of crosses
consisted of these two combinations, plus negative
controls consisting of eggs only (no sperm added) and
positive controls consisting of conspecific crosses. Both
negative and positive controls were performed for each of
the species involved.
Crosses were performed in sterile plastic dishes containing 3 ml sterile seawater at 5 mC. We made nine
different sets of crosses by combining nine individuals of
F. evanescens with nine male and nine female individuals
of F. serratus. To allow quantification of fertilization
success for an unpublished study, each cross and control
was replicated four times using 10 oogonia in each
replicate. Replication also served to increase the probability of obtaining isolates not contaminated by small
filamentous algae, because unialgal cultures were required for subsequent raising of thalli to the size necessary
for DNA extraction. After inoculation, dishes were
incubated at 20 µmol m−# s−" white light at 5 mC. Three
days after the start of the experiments, the seawater was
replaced by culture medium (half-strength Provasoli’s
Enriched Seawater ; Provasoli, 1963) containing 6 mg l−"
GeO to prevent growth of diatoms. Replenishment of
#
culture medium occurred at 14 day intervals, but without
GeO .
#
174
Table 1. Reciprocal crossings between Fucus serratus (Fs)
and F. evanescens (Fe)
Cross
Rubisco
ITS1
spacer
nad11
(nuclear) (chloroplasts) (mitochondria)
FeL (2081)iFsK (5000)
L19
L20
L21
αiβ
αβ
αβ
αβ
AiB
A
A
A
1i2
1
1
1
FeL (2087)iFsK (5009)
L58
L60
L62
αiβ
αβ
αβ
αβ
AiB
A
A
A
1i2
1
1
1
FeL (2094)iFsK (5016)
L45
L46
L48
αiβ
αβ
αβ
αβ
AiB
A
A
A
1i2
1
1
1
FeL (2095)iFsK (5017)
L35
L36
L37
Fs L (5003)iFeK (2081)
L24
L25
L26
αiβ
αβ
αβ
αβ
γiα
γα
γα
γα
AiB
A
A
A
BiA
B
B
B
1i2
1
1
1
2i1
2
2
2
Fs L (5018)iFeK (2095)
L38
L41
L42
βiα
αβ
αβ
αβ
BiA
B
B
B
2i1
2
2
2
Fs L (5019)iFeK (2094)
L50
L51
L52
βiα
αβ
αβ
αβ
BiA
B
B
B
2i1
2
2
2
Fs L (5023)iFeK (2087)
L63
L64
L65
βiα
αβ
αβ
αβ
BiA
B
B
B
2i1
2
2
2
FeLiFeK (2081C)
FsL (5023C)iFsK (5009C)
α
β
A
B
1
2
Results are shown from four different crossing experiments
involving four parent individuals of F. evanescens and eight of
F. serratus. Three F1 thalli from each cross are presented.
Individuals of F. evanescens served as females (upper half of
table) or males (lower half ) because they are hermaphroditic.
Values in brackets are individual-specific identification codes and
a subscript c indicates positive control crosses for each species.
Alleles and haplotypes derived from portions of ITS1 (nuclear),
Rubisco (chloroplast) and nad11 (mitochondrial) genes as
determined by SSCP. GenBank accession numbers for sequences
and alignments : α (AY044258), β (AY044260), γ (AY044259), A
(AY044263), B (AY044264), 1 (AY044261) and 2 (AY044262).
After 4 weeks, embryos were identified by their typical
club-shape, the presence of an apical tip with hair pit, and
growth of basal rhizoids. Of the nine original sets of
crosses, two were discarded at this stage because embryos
were present in the negative controls of F. evanescens,
which indicated selfing. Only unialgal cultures (nonaerated) were maintained, in which thalli reached 5–
50 mm in length after 7–11 months at 10–15 mC. Offspring
of four sets of crosses were finally harvested for DNA
(using 5–20 mg of fresh tissue) as previously described
(Coyer et al., 2002).
40
2
F. serratus
(Oudot-Le Secq, unpublished.
data)
175
See Table 1 legend for GenBank accession numbers of primers.
135
F : 5h-TTTGGTAGAGGTAGGTAACG (FAM)
R : 5h-TGTAACAGAAGTAATTCCATA (NED)
42
255, 288
2
F. evanescens (AF102939)
F. serratus (AF102943)
F. distichus (AF195515)
F. vesiculosus (AF132474)
3
47
231
F : 5h-TCGACCAAACGTGTCTGTTT (FAM)
R : 5h-ACGCTAGGCTTCCTTCCTTC (NED)
F : 5h-TGAATATACTTCAACAGATACACC (FAM)
R : 5h-TGGTAAAAATGAAAAACATCCTTG (NED)
rDNA-ITS1
(nuclear)
Rubisco spacer with
rbcL and rbcS flanks
(chloroplast)
nad11
(mitochondrion)
Primers (fluorescent label)
Gene
Table 2. Identity and characteristics of primers used for SSCP analysis
Fragment size (bp)
Ta (mC)
No. of alleles\haplotypes
Reference sequences
for primer design
ITS and organelle inheritance in Fucus
Sequence polymorphisms were revealed by singlestrand conformation polymorphism (SSCP ; Orita et al.,
1989 ; Hayashi, 1992). The technique detects sequence
variation by the differential migration of single-stranded
DNA in non-denaturing, high-resolution, polyacrylamide gels. Using SSCP, differences of one or more
nucleotides can be detected in fragments from 300 to
450 bp with nearly 90 % accuracy (Hayashi, 1991 ; Lessa
& Applebaum, 1993). As mutations may affect the
mobility of only one or both of the two DNA strands
(Hoarau et al., 1999 ; Lescasse, 1999), labelling each
strand independently confers additional sensitivity. Independent labelling was achieved by labelling the forward
and reverse primers (Table 2) with a different fluorescent
dye and using an ABI 377 autosequencer (Applied
Biosystems) for detection. SSCP allows a rapid and
inexpensive means to determine sequence differences (as
revealed by conformational differences) for a large
number of samples (Sunnucks et al., 2000).
SSCP alleles\haplotypes were detected by polymerase
chain reaction (PCR) amplification of the ITS1 (nuclear),
ribulose-1,5-bisphosphate carboxylase operon (Rubisco ;
chloroplast), and nad11 (NADH dehydrogenase, subunit
11 ; mitochondrion) (Table 2). A portion of the Fucus
nad11 gene was amplified using PCR primers designed
from the nad11 sequence determined for Pylaiella littoralis (L.) Kjellm (Ectocarpales) (Oudot-Le Secq et al.,
2001). Although microsatellite loci (nuclear) have been
developed for F. serratus and F. evanescens (Coyer et al.,
2002), their high levels of polymorphism would require
numerous samples to discern parental\hybrid patterns.
Consequently, the much less polymorphic ITS1 gene was
used as the nuclear marker in the present study so that
parental\hybrid patterns could be detected with fewer
individuals.
PCR reactions (10 µl total volume) contained 1 µl of
the DNA extract (Coyer et al., 2002), 1iTaq polymerase
buffer (Promega), 2 mM MgCl , 0n2 mM of each dNTP,
#
0n1 µM (ITS1, nad11) or 0n4 µM (Rubisco) of each primer
(one of each primer pair 5h-labelled with FAM, the other
with NED ; Applied Biosystems), and 0n25 or 0n50 U Taq
polymerase (Promega). Additionally, bovine serum albumin (BSA ; 0n8 µg µl−") was added to PCR reactions for
Rubisco and nad11.
PCR was performed with a Mastercycler Gradient
(Eppendorf ) thermocycler (94 mC, 3 min ; followed by
94 mC, 40 s ; annealing temperature (Ta), 40 s ; and
72 mC, 40 s for 40 cycles (42 cycles for Rubisco) ; and a
final extension at 72 mC for 10 min). Amplification products were separated in a 0n4iMDE gel (BMA Bioproducts) in 0n6 % TBE at 19 mC and 45 W on an ABI
377 autosequencer. Samples (0n5–1n0 µl) were loaded
with 1n5 µl of loading buffer containing 67 % deionized
formamide, 11 % GeneScan size standard ROX 350
(Applied Biosystems), 11 % Blue dextran EDTA (Applied
Biosystems) and 11 mM NaOH. Alleles or haplotypes were identified with GeneScan software (Applied
Biosystems).
All unique SSCP polymorphisms for each gene were
verified by direct sequencing of the PCR product (both
strands) from three individuals using the dGTP BigDye
Terminator Kit and ABI 377 autosequencer (Applied
Biosystems). As the sequences of the SSCP polymorphism
were identical in each of the three individuals, the SSCP
polymorphisms correspond to alleles (ITS) or haplotypes
(chloroplast, mitochondrion). Only one of the three
J. A. Coyer et al.
identical sequences for each allele\haplotype was placed
into GenBank (Table 1).
Results
Both sets of reciprocal crosses produced hybrid
individuals, and segregation of alleles or haplotypes
for each gene was clear and unambiguous (Fig. 1).
Three alleles were identified for the 231 bp ITS1
region. Fucus serratus displayed alleles β and γ,
whereas F. evanescens possessed only allele α (Table
1). Subsequent sequencing and alignment (retrievable from GenBank accession numbers in Table 1)
of the three alleles revealed a difference between α
and β at position 87 (A\C) and α and γ at position
199 (G\A). All laboratory hybrids possessed two
alleles, one from each parent.
Two haplotypes were identified for the Rubisco
spacer (chloroplast). Fucus serratus displayed haplotype A (255 bp) and F. evanescens haplotype B
(288 bp) (Table 1). Subsequent sequencing of the
176
two fragments revealed a 33 bp deletion (positions
73–106). The hybrids possessed only one haplotype,
which matched the female parent in all cases.
Two haplotypes also were identified for a 135 bp
region of nad11 (mitochondrion) (Table 1). Sequencing of three individuals with haplotype 1 (F.
evanescens) and three individuals with haplotype 2
(F. serratus) revealed a single nucleotide difference
at position 85 (T\C) in the alignment. Once again,
the hybrids contained only one haplotype, which
always corresponded to the female parent.
It is highly unlikely that the single-base difference
observed in the mitochondrial haplotypes was due
to errors in PCR amplification. First, each haplotype was sequenced from three separate individuals
and, as the three sequences were identical, either
there were no PCR errors or errors occurred at the
same base in each of three separate reactions (highly
unlikely). Secondly, only two SSCP patterns (which
are dependent upon sequence) were present among
all 24 F1 thalli. If PCR errors were apparent in our
SSCP analysis, it is improbable that such errors
would occur at the same base in each of the 24
separate reactions.
Discussion
Fig. 1. Hybrid inheritance patterns based on singlestrand conformation polymorphism (SSCP). Each strand
was labelled independently with FAM (grey peaks) or
NED (black peaks) and visualized on an ABI 377
autosequencer. Patterns presented are representative
of all samples. (A) ITS1 (nuclear) ; (B) Rubisco
(chloroplast) ; (C ) nad11 (mitochondrion). Abbreviations :
Fs, Fucus serratus ; Fe, F. evanescens ; numbers refer
to sample codes.
Identification of interspecific hybrids and subsequent study of hybrid zones are of great importance
in understanding speciation and the effects of species
introductions on extant communities ; nevertheless,
the degree and extent of hybridization in marine
macroalgae are essentially unknown. As a prelude
to such studies, we have documented patterns of
gene inheritance in laboratory hybrids of the seaweeds Fucus serratus and F. evanescens, using
crossing experiments and three genetic markers.
The ITS1 region in F. serratus and F. evanescens is
biparentally inherited, as expected from a nuclear
gene. All laboratory hybrids possessed one allele
from each parent. The point mutations in the F.
serratus ITS1 sequences, which formed the basis for
the SSCP polymorphism, were consistent in all of
our F. serratusiF. evanescens crosses, strongly
suggesting that the ITS1 polymorphism is stable
and will be useful in identifying hybrids from the
field. Microsatellite loci (nuclear) should prove
useful for reliable distinction of second-generation
hybrids (F2), backcrosses to either of the parental
taxa, and later-generation hybrids (Allendorf et al.,
2001).
It must also be recognized that the ITS1 is part of
a multi-copy, multi-gene family in which internal
homogeneity of the ITS repeat units is dependent on
concerted evolution (Hillis & Davis, 1988 ; Williams
et al., 1988). Intraspecific and intra-individual polymorphism of ITS is well documented in marine
ITS and organelle inheritance in Fucus
seaweeds (Pillmann et al., 1997 ; Serra4 o et al., 1999 ;
Fama' et al., 2000 ; Coyer et al., 2001), presumably
related to incomplete homogenization under concerted evolution (Dover, 1982) as a result of recent
speciation, hybridization, asexual reproduction,
polyploidy or multi-chromosomal locations. In F.
vesiculosus, for example, the 1–2 % difference observed in ITS1 and ITS2 sequences within a single
individual was attributed to frequent hybridization
and\or a rate of radiation exceeding the homogenization rate by concerted evolution (Serra4 o et al.,
1999). Our finding of two ITS1 alleles among only
eight individuals of F. serratus illustrates that polymorphism can occur among individuals separated
by a few metres, a degree of intraspecific ITS
polymorphism also found in the kelp, Macrocystis
pyrifera (Coyer et al., 2001). Although we found
no evidence for intra-individual polymorphism in
Fucus (i.e. multiple peaks in SSCP analysis), such
a result cannot be eliminated, especially if sampling
intensity were increased and\or naturally occurring
F. serratusiF. evanescens hybrids were reproductive and backcrosses were prevalent.
Chloroplasts and mitochondria are maternally
inherited in F. serratus and F. evanescens. In all
cases (Table 1), the organellar haplotype found in
the hybrid was identical to that present in the eggcontributing parent. With respect to chloroplasts,
our SSCP-based results obtained from severalmonth old sporophytes differed from previous
electron-microscopy-based studies. Both Bouck
(1970) and Brawley et al. (1976 a) observed sperm
chloroplasts in embryos of F. vesiculosus, which
suggested paternal inheritance. The discrepancy
most likely results from comparing different developmental stages in which older individuals have
lost the traces of paternal chloroplasts. In the kelps,
electron microscopy suggested paternal and maternal inheritance of chloroplasts in Laminaria
ephemera and L. saccharina (Bisalputra et al., 1971),
but essentially maternal inheritance in L. angustata
(Motomura, 1990). Again, the equivocal nature of
the electron microscopy data may be a result of
using different developmental stages for analysis. In
the only other study that has investigated chloroplast inheritance in marine macroalgae, Zuccarello
et al. (1999 a) used SSCP with short fragments of the
Rubisco spacer on mature individuals of the red
alga Bostrychia (Ceramiales) and demonstrated
maternal inheritance of chloroplasts.
Our results support earlier studies investigating
mitochondrial inheritance in three species of marine
macroalgae. Degenerated sperm mitochondria were
observed in 16 h embryos of F. vesiculosus (Brawley
et al., 1976 b) and in pre-division zygotes of the kelp
Laminaria angustata (Motomura, 1990) ; both results were interpreted as evidence for maternal
inheritance. Maternal inheritance of mitochondria
177
was also demonstrated in the red alga Bostrychia
moritziana using sequence variability in the noncoding spacer between the cytochrome oxidase
subunits 2 and 3 genes (Zuccarello et al., 1999 b).
The universality of maternal inheritance of
organelles in marine macroalgae remains to be
determined. Given the phyletic diversity of macroalgae, however, it is probable that both maternal
and paternal inheritance patterns will be found.
SSCPs analysed on an automated sequencer offer
a rapid and powerful approach for surveying
inheritance patterns and polymorphisms in a large
number of samples. For example, selection of
samples for subsequent sequencing can be made
from preliminary SSCP analysis, thereby identifying
polymorphisms that would be missed by direct
sequencing unless expensive and time-consuming
cloning techniques were employed. SSCP analysis
has also verified that our method of producing
artificial hybrids is reliable even in hermaphroditic
taxa such as F. evanescens. Notably, it was not
necessary to immobilize F. evanescens sperm by the
addition of reagents, such as potassium salts,
sodium hypochlorite or methanol (Fritsch, 1945),
which might have affected viability of the eggs as
well. Finally, SSCP can identify field samples
putatively identified as F. evanescensiF. serratus
hybrids, provided that examination of an adequate
number of parental samples reveals species-specificity of the markers and no intraspecific heterogeneity. Subsequent tests of hybrid fitness and
viability in relation to hybrid zones and dispersal
will provide new insights into community structure
and the evolution of seaweeds.
Acknowledgements
We thank M.-P. Oudot-Le Secq for providing
unpublished sequences of mitochondria from F.
serratus and R. Lescasse for technical assistance in
using autosequencers for SSCP analysis. The research was supported by the BIOBASE Project
funded under EU MAST III, Control Number
PL97-1267.
References
A, F.W., L, R.F., S, P. & W, J.K.
(2001). The problems with hybrids : setting conservation guidelines. Trends Ecol. Evol., 16 : 613–622.
A, J.C. (1994). Molecular Markers : Natural History, and
Evolution. Chapman and Hall, New York.
B, T., S, C.M. & M, J.W. (1971). In situ
observations of the fine structure of Laminaria gametophytes and
embryos in culture. I. Methods and the ultrastructure of the
zygote. J. Microsc., 10 : 83–98.
J. A. Coyer et al.
B, G.B. (1970). The development and postfertilization fate of
the eyespot and the apparent photoreceptor in Fucus sperm. Ann.
N.Y. Acad. Sci., 175 : 673–685.
B, S.H., W, R. & Q, R.S. (1976a). Finestructural studies of the gametes and embryo of Fucus vesiculosus
L. (Phaeophyta). II. The cytoplasm of the egg and young zygote.
J. Cell. Sci., 20 : 255–271.
B, S.H., W, R. & Q, R.S. (1976b). Finestructural studies of the gametes and embryo of Fucus vesiculosus
L. (Phaeophyta). I. Fertilization and pronuclear fusion. J. Cell.
Sci., 20 : 233–254.
C, J.A., S, G.J. & A, R.A. (2001). Evolution of
Macrocystis spp. (Phaeophyceae) as determined by ITS1 and
ITS2 sequences. J. Phycol., 37 : 574–585.
C, J.A., V, J.H., S, W.T. & O, J.L. (2002).
Characterization of microsatellite loci in the marine rockweeds,
Fucus serratus and F. evanescens (Heterokontophyta ; Fucaceae).
Mol. Ecol. Notes, 2 : 35–37.
D, G.A. (1982). Molecular drive : a cohesive mode of species
evolution. Nature, 299 : 111–117.
D, S.G.A., P’  R, W.F., S, W.T. &
O, J.L. (2001). A reassessment of phylogenetic relationships
within the Phaeophyceae based on RUBISCO large subunit and
ribosomal DNA sequences. J. Phycol., 37 : 586–603.
F' , P., O, J.L., S, W.T. & P, G. (2000). High
levels of intra- and inter-individual polymorphism in the rDNA
ITS1 of Caulerpa racemosa (Chlorophyta). Eur. J. Phycol., 35 :
349–356.
F, F.E. (1945). The Structure and Reproduction of the Algae :
II. Cambridge University Press, London.
H, K. (1991). PCR-SSCP : a simple and sensitive method for
detection of mutations in the genomic DNA. PCR Method.
Applic., 1 : 38.
H, K. (1992). PCR-SSCP : rapid and easy detection of DNA
sequence changes. Hum. Cell., 5 : 180–184.
H, D.M. & D, S.K. (1988). Ribosomal DNA : intraspecific
polymorphism, concerted evolution, and phylogeny reconstruction. Syst. Zool., 37 : 63–66.
H, G., B, P., B, F. & G, R. (1999).
Ge! ne! tique des populations de Beryx splendens de la zone
e! conomique de la Nouvelle-Cale! donie : distribution des haplotypes du ge' ne du cytochrome b de l’ADN mitochondrial et
analyse phyloge! ne! tique de leurs se! quences. Doc. Sci. Techn. IRD
Noumea Se! r. II, 1 : 1–39.
I, K., S, S., W, S. & K, K. (2000).
Molecular evidence of natural hybridization between Abies
veitchii and A. homolepis ( Pinaceae) revealed by chloroplast,
mitochondrial and nuclear DNA markers. Mol. Ecol., 9 : 1965–
1974.
L, T.E. (1984). Hybrider mellom sagtang (Fucus serratus L.) og
gjelvtang (Fucus distichus subsp. edentatus ( Pyl.) Powell) i indre
Oslofjord. Blyttia., 42 : 71–77.
L, R. (1999). Recherche de variants ge! ne! tiques dans le ge' ne
KCNQ1 codant un canal potassique, dans le cadre d’une e! tude
e! pide! miologique : mise au point de la SSCP fluorescente sur
se! quenceur. DEA Thesis, Universite! Pierre et Marie Curie, Paris.
L, E.P. & A, G. (1993). Screening techniques for
detecting allelic variation in DNA sequences. Mol. Ecol., 2 :
119–129.
178
L, M.K. & D, L.D. (2000). Molecular evidence for an
interfamilial laminarialean cross. Eur. J. Phycol., 35 : 135–143.
M, I., O, J.L., S, W.T., D, C. & V, M.
(2001). Phylogenetic analyses of Caulerpa taxifolia (Chlorophyta)
and of its associated bacterial microflora provide clues to the
origin of the Mediterranean introduction. Mol. Ecol., 10 : 931–
946.
M, H.L. (1996). The hows and whys of cytoplasmic
inheritance in seed plants. Am. J. Bot., 83 : 383–404.
M, T. (1990). Ultrastructure of fertilization in Laminaria
angustata ( Phaeophyta, Laminariales) with emphasis on the
behavior of centrioles, mitochondria and chloroplasts of the
sperm. J. Phycol., 26 : 80–89.
O, J.L., V, M.J., M, I., B-B, S.A. & S,
W.T. (1998). Mediterranean Caulerpa taxifolia and C. mexicana
(Chlorophyta) are not conspecific. J. Phycol., 34 : 850–856.
O, M., I, H., K, H., H, K. & S,
T. (1989). Detection of polymorphisms of human DNA by gel
electrophoresis as single-strand conformation polymorphisms.
Proc. Natl. Acad. Sci. U.S.A., 86 : 2766–2770.
O-L S, M.-P., F, J.-M., R, S., K,
B. & L- G, S. (2001). The complete sequence of a
brown algal mitochondrial genome, the Ectocarpale Pylaiella
littoralis (L.) Kjellm. J. Mol. Evol., 53 : 80–88.
P, A.F., O, M.J.H.,  W, C., S, W.T. &
O, J.L. (1997). Phylogeny and historical ecology of the
Desmarestiaceae ( Phaeophyceae) support a Southern Hemisphere origin. J. Phycol., 33 : 294–309.
P, A., W, G.W., O, J.L., S, W.T. & K,
R.J. (1997). Inter- and intraspecific genetic variation in Caulerpa
(Chlorophyta) based on nuclear rDNA ITS sequences. Eur. J.
Phycol., 32 : 379–386.
P, L. (1963). Growing marine seaweeds. Proc. Int. Seaweed
Symp., 4 : 9–17.
R, E.L. & C, A.R.O. (1985). A numerical taxonomic
study of Fucus distichus L. emend. Powell ( Phaeophyta). J. Mar.
Biol. Assoc. U.K., 65 : 433–459.
S, G.H. & P, A.F. (1994). Arrival of Fucus evanescens
(Phaeophyceae) in Kiel Bight (Western Baltic). Bot. Mar., 37 :
471–477.
S4 , E.A., A, L.A. & B, S.H. (1999). Evolution of
the Fucaceae ( Phaeophyceae) inferred from nrDNA-ITS. J.
Phycol., 35 : 382–394.
S, P., W, A.C.C., B, L.B., Z, K.,
F, J. & T, A.C. (2000). SSCP is not so difficult : the
application and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology. Mol. Ecol.,
9 : 1699–1710.
W, S.C., DB, R.W. & F, J.L. (1988). A commentary
on the use of ribosomal DNA in systematic studies. Syst. Zool.,
37 : 60–62.
Z, G.C., W, J.A., K, M. & K, R.J. (1999a).
A rapid method to score plastid haplotypes in red seaweeds and
its use in determining parental inheritance of plastids in the red
alga Bostrychia (Ceramiales). Hydrobiologia, 401 : 207–214..
Z, G.C., B, G., W, J.A. & K, R.J. (1999b).
A mitochondrial marker for red algal intraspecific relationships.
Mol. Ecol., 8 : 1443–1447.