AdvBotRes 35 - 2001 Wattier & Maggs - Intraspecific Variation in Seaweeds

Intraspecific Variation in Seaweeds: The Application of New
Tools and Approaches
R I~ M I W A T T I E R * a n d C H R I S T I N E A. M A G G S
School of Biology and Biochemistry, Medical Biology Centre, Queen's

University, Belfast BT9 7BL, Northern Ireland, UK
I. Introduction .................................................................................................... 172

II. Species Concepts and Intraspecific Variation ............................................ 174
III. Life Histories and their Variability .............................................................. 178
IV. Population Genetics ....................................................................................... 181
A. D N A Isolation from Seaweeds: A Technical Nightmare ................... 182
B. Single-locus Nuclear Microsatellites .................................................... 183
C. Multilocus Nuclear Markers (RAPDs, ISSRs, M13-fingerprinting
and AFLPs): Are These the Tools of the Future? .............................. 188
D. Plastid Markers ....................................................................................... 190
E. Breeding Systems .................................................................................... 192
V. Biogeography and Phylogeography .............................................................. 195
A. Physiological Approaches ...................................................................... 195
B. Molecular Approaches: Nuclear Markers ........................................... 196
C. Molecular Approaches: Plastid Markers ............................................. 199
VI. Conclusions and Perspectives ....................................................................... 200
Acknowledgement .......................................................................................... 200
References ....................................................................................................... 201
Seaweeds, marine macroalgae that are individually visible to the naked eye, belong
to three divisions, chlorophytes (green algae), chromophytes (brown algae) and
rhodophytes (red algae), which are broadly convergent in morphology, yet represent a
high proportion of eukaryotic diversity. Despite the commercial value of seaweeds,
biodiversity is still poorly understood, particularly at the infraspecific level. Species-level
*Present address: School of Biological Sciences, University of Wales, Bangor,

Gwynedd LL57 2UW, Wales, UK.
Advances in Botanical Research Vol. 35

incorporating Advances in Plant Pathology Copyright © 2001 Academic Press
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172 c . A . M A G G S and R. W A T T I E R
taxonomy is still centred on the morphological species concept, which is increasing used
in concert with other data, particularly molecular analyses, although phycologists are
still not sufficiently confident in the relatively new fields of molecular taxonomy and
phylogenetic systematics. Seaweeds show an extraordinary diversity of life histories and
offer great potential to theorists interested in ecological and evolutionary processes.
Population genetics has developed slowly in seaweeds compared to that for most other
groups of conspicuous and economically important organisms primarily because of the
problem, now largely overcome by technical developments, of isolating high-quality
DNA from large numbers of individuals. Plastids are believed to be uniparentally
inherited in seaweeds, so their use permits comparison and interpretation with well-
developed mitochondrial markers in animals. The spacer between the genes for the
large and small subunits of rubisco, used as a target for polymerase chain reaction-
single-strand conformational polymorphism (PCR-SSCP), has recently been developed
for four tropical red algal species, allowing the analysis of spatial genetic structure
within populations and at macrogeographic scales. The most widely used marker for
phylogeographic studies, ribosomal DNA internal transcribed spacer sequences, has
provided new interpretations of seaweed biogeography but is unsuitable for population-
level studies. Single-locus microsatellites, often described as the "marker of choice"for
microevolutionary studies, have only recently been developed for one red, one green and
three brown algae. This involved enormous expenditure of time and effort due to very
low genomic frequencies of microsatellites and small proportions of polymorphic loci.
Despite these severe difficulties, microsatellites have already proved to be extremely
useful markers for the seaweed species for which they were available. They enabled
studies of kinship and male gamete dispersal in Gracilaria gracilis, breeding system and
spatial population structure in Cladophoropsis membranacea and Laminaria digitata,
population structure in relation to possible ecotypic differentiation in Fucus serratus,
and genetic neighbourhood size in Ascophyllumnodosum. Thus, despite the increasing
interest in molecular studies during the last decade, assessment of intraspecific genetic
biodiversity of seaweeds is still in its infancy. The most useful markers (microsatellites,
AFLPs, intersimple sequence repeats and SSCPs), of which AFLPs and SSCPs seem to
be the most promising, have been introduced into macroalgal studies only in the last 4
years. Because microsatellites are particularly difficult to develop for seaweeds, one
major challenge of the next 10 years will be to seek alternative highly polymorphic single-
locus/co-dominant nuclear markers as a substitute.
I. INTRODUCTION
The algae are a very diverse, unnatural assemblage of oxygen-producing

photosynthetic organisms. Seven phytogenetic lineages (the chlorophytes,
chromophytes, rhodophytes, dinophytes, euglenophytes, cryptophytes
and glaucophytes) are currently resolved, based principally on analyses of
ultrastructure and 18S ribosomal gene sequence data (Andersen, 1992; John
and Maggs, 1997). Three of these lineages, the chlorophytes (green algae),
chromophytes (brown algae) and rhodophytes (red algae), contain
seaweeds, marine macroalgae that are individually visible to the naked eye.
Seaweeds are typically multicellular, although their highly diverse body
plans include the non-cellular coenocytic construction of some green algae
that consist of interwoven highly multinucleate filaments (van den Hoek et
INTRASPECIFIC VARIATION IN SEAWEEDS 173
al., 1995). Broadly convergent evolution of similar types of thalli is seen in all
three little-related seaweed divisions, the Chlorophyta, Chromophyta (also
known as Heterokontophyta and including the Phaeophyceae) and
Rhodophyta. Their higher classification is in a state of almost constant flux
as new evidence is considered in relation to existing data and previous
classification schemes. While some texts (e.g. Margulis, 1990) place these
lineages into the Protista (or Protoctista), a Kingdom that can no longer be
justified (Corliss, 1994), there are many alternative classification schemes,
some more traditional than others. In the most recent of these (Cavalier-
Smith, 1998), the Chlorophyta and Rhodophyta are attributed to the
kingdom Plantae, in separate subkingdoms (Viridiplantae and Biliphyta)
and the Chromophyta are placed with diverse heterokonts and other related
organisms in the kingdom Chromista. What is clear is that the seaweeds
represent a high proportion of eukaryotic diversity, occupying twigs on
several major branches of the Tree of Life (http://phylogeny.arizona.edu/
tree/phylogeny.html). Phylogenies inferred from nuclear and plastid gene
trees differ greatly because of secondary endosymbioses: gene-cluster
analysis has provided powerful new evidence of the common evolutionary
history of red and brown algal plastids (Stoebe and Kowallik, 1999).
Interest in biodiversity has increased during the last decade (Loreau and
Olivieri, 1999), but seaweeds are among the many groups of organisms that
remain underanalysed. Despite the commercial value of seaweeds as food
and for products such as hydrocolloids, agrochemicals and, increasingly,
paramedical and biomedical supplies (Jensen, 1993; Radner, 1996), their
biodiversity is still poorly known. Species-level studies are in most cases at
the alpha-taxonomy stage, often still in the exploratory phase or the later
systematic phase, in which a wide selection of material of each species is
subject to extensive field and herbarium studies (Stace, 1989). For relatively
few species have we reached the detailed biosystematic phase (Stace, 1989),
but the present review will focus on these species. Clearly, we have not
arrived for any species at the ultimate goal of ideal omega-taxonomy, based
on analysis of all available characters (Turrill, 1935).
The detection and quantification of biological variability at the
infraspecific level is central to investigations of ecologists and evolutionary
biologists in order to elucidate key elements of processes such as breeding
systems, speciation and adaptation. However, morphological variation in
relation to taxonomy remains the best-studied aspect of intraspecific
variation in seaweeds. W. H. Harvey, in his monumental study Phycologia
Britannica (1846-1851), laid the foundation for detailed herbarium-based
studies of variation, which were resumed in the 20th centuries in such algal
floras as Seaweeds of the British Isles (initiated by Dixon and Irvine, 1977) and
a comprehensive seaweed flora of southern Australia (e.g. Womersley,
1998). Intraspecific physiological variation has mostly been covered
under the topic of "ecotypes", with experimental procedures often highly
174 c.A. MAGGSand R. WATTIER
dependent on the development over the last four decades of laboratory

culture techniques for seaweeds. Similarly, studies of intraspecific life-
history variability in seaweeds were stimulated by the feasibility of laboratory
cultures and they have been extended to a wide range of species, although
very few of them have been examined in detail.
Despite the increasing interest in molecular studies during the last decade,
assessment of intraspecific genetic biodiversity of seaweeds is still in its
infancy. Although a large set of DNA genetic markers is now available and
being extensively used in evolutionary biology of animals and land plants, to
answer questions ranging from phylogeography to fine spatial structure or
mating systems, such DNA markers as random amplified polymorphic
DNAs (RAPDs; Patwary et al., 1993), M13-fingerprinting (Coyer et al.,
1994), microsatellites (Wattier et al., 1997), amplified fragment length
polymorphisms (AFLPs; Donaldson et al., 1998, 2000), intersimple sequence
repeats (ISSRs; Vis, 1999) and SSCPs (Zuccarello et al., 1999a) have
been applied to seaweeds only recently. As a result, mechanisms for the
maintenance and distribution of genetic diversity within macroalgal species
(i.e. mating patterns and gene flow) are largely unknown (van Oppen et
al., 1996a).
The present review, rather than attempting to include all literature
relevant to intraspecific variation in seaweeds, focuses on the best-known
species for which diverse types of information, particularly molecular data,
are available. These are primarily species of commercial interest, either
because they are harvested (e.g. some members of the red algal genera
Gracilaria and Porphyra and the carrageenophytes Chondrus and
Mastocarpus, and the brown kelps) or because of their nuisance value, such as
the green algae Ulva and Enteromorpha and the highly invasive Caulerpa
taxifolia. Nevertheless, some of the most complete studies to date concern
two common and widespread genera of tropical mangrove-inhabiting red
algae, Caloglossa and Bostrychia. Our review is organized into different
temporal and spatial scales of variation, and covers the limited information
on processes that may operate over these scales. Acknowledging that
questions outnumber answers, we review current viewpoints and information
on appropriate molecular methodology for intraspecific studies, and suggest
ways forward using markers not yet applied to macroalgae. Central to the
examination of the intraspecific diversity of any group of organisms is to set
out the species concepts employed, which we discuss in the next section.
II. SPECIES CONCEPTS AND INTRASPECIFIC VARIATION
The idea of a species, and how it can be defined, has inspired a voluminous,
and often controversial, literature dating back even to before Darwin's
(1859) Origin of Species. Up to 22 different species concepts can be identified
(Mayden, 1997), and in each revision of an existing concept a new emphasis

or interpretation is proposed (e.g. Mallet, 1995). Every conceivable
approach has been taken, from suggestions that there is only one justifiable
or workable species concept for a particular group of organisms to the view
that a combination of several concepts should be employed (Ridley, 1993).
The search for the ideal species concept has been likened to the quest for the
Holy Grail (Cracraft, 1997)! The paradox at the heart of the problem has
been described lucidly by Hull (1997). While scientists would like their
species concepts to be both generally applicable and theoretically
significant, these goals tend to conflict with each other: "Theoretically
significant species concepts tend not to be very operational. Attempts to
make them more operational result in their being theoretically less
significant". One particularly thorny aspect of the problem of species
concepts is the question of whether or not species are of greater significance
("more real") than other taxa (Claridge et al., 1997) or instead are an
artificial construct (Bachmann, 1998).
For algae, species-level taxonomy is still centred on the morphological
species concept, based explicitly or implicitly on the detection of
morphological discontinuities in sets of field-collected or cultured organisms
(John and Maggs, 1997). Even where other data are considered, they are
always evaluated in the context of morphological characters. For example,
two geographical clades of the ceramialean red alga Phycodrys rubens,
separated by 1.8% divergence of ribosomal internal transcribed spacer
(ITS) sequences (for molecular details see Section IV.D), were identified in
the eastern Atlantic Ocean and Baltic Sea entrance (van Oppen et al.,
1995a,b). Despite allozyme evidence suggesting that the two clades are not
interfertile and may represent sibling species, the absence of any identifying
morphological features prevented the proposal of an additional species
of Phycodrys. Similarly, discontinuities in the variation of rubisco spacer
sequences (described in Section V.C) and intersterility between lineages of
the red alga Caloglossa leprieurii (Kamiya et al., 1998; Zuccarello et al., 2000)
suggested the existence of two sibling species but taxonomic changes were
not proposed because of the lack of discriminating morphological
characters. In this case, the study is ongoing, further sibling species may
be detected, and caution is probably appropriate. In the brown seaweed
Sphaerotrichia divaricata, Peters et al. (1993) found that there was an
"intraspecific" sterility barrier between isolates from Japan and elsewhere,
but the Japanese entity was not recognized at the species level because it
exhibited no consistently different morphological features. In fact, as far as
we can determine, newly discovered sibling species of seaweeds have never
been formally described on the basis of non-morphological characters:
phycologists are exploring the relatively new fields of molecular taxonomy
and phylogenetic systematics, and are still unsure how to treat cryptic species
even when they are supported by breeding data.
On the other hand, when morphological or life-history characters are

discovered to be congruent with even very minor levels of molecular
divergence, species-level separation is sometimes proposed. Examples are
seen in the recognition of new species in the genera Porphyra and Ulva,
based primarily on single substitutions in spacer regions (Brodie et al., 1996;
Coat et al., 1998; Dion et al., 1998). In order to segregate additional species,
it is of paramount importance to determine the suitability of particular
genetic markers as systematic tools at the species level, to check that
marked discontinuities are present in the data set and, conversely, that
intraspecific diversity will have no impact on supraspecific analyses. This
applies equally to DNA markers based on plastid (cp) DNA (Wattier et al.,
2001), ribosomal DNA ITS regions (Pillman et al., 1997; Serrfio et al., 1999)
and isozymes (Sosa and Lindstrom, 1999).
Surprisingly, in view of the centrality of the morphological species concept,
morphological plasticity is rarely evaluated by field experiments such as
transplantation. Despite the great potential of this approach to studies of
systematics and evolution (Mathieson et al., 1981), the topic appears to
be currently unfashionable! A study of two morphotypes of the California
elk kelp Pelagophycus porra provides some insight into one of the possible
reasons for the rarity of transplant studies. Two forms of P.porra have been
recognized, sometimes at the species level (Miller et al., 2000). They differ
in morphology (e.g. size of buoyancy aids) as well as phenologically and
demographically. The morphotypes have some characteristics of ecotypes,
growing in habitats of differing wave exposure, and are genetically distinct
using RAPD markers. Transplant experiments suggested that ecological
responses were genetically based, but were marred by losses of transplanted
algae. Despite the practical difficulties of transplants, Littler and Littler's
(1992) study of populations of all western Atlantic species of Avrainvillea
demonstrated, for example, thatA, levis develops a bulbous holdfast in sand
or soft sediment and a conical holdfast when growing on hard substrata. In
several other species of Avrainvillea, qualitative characters considered of
taxonomic importance (e.g. stipe length) were discovered to be unreliable
since they were influenced by light and depth conditions.
For many groups of organisms, the biological species concept has been the
most influential one, regarding species as "groups of actually or potentially
interbreeding natural populations which are reproductively isolated from
other such groups" (Mayr, 1963). The problem at the heart of this concept is
that it can be applied only to sympatric populations and, because of the
paucity of good species-specific morphological markers in seaweeds, it can
be tested only using macromolecular or other biochemical markers. Thus
there have been very few studies in which the biological species concept
has been strictly tested on seaweeds. In the kelp genus Alaria, restriction
mapping of the rapidly evolving ribosomal intergenic spacer (IGS) region
suggested natural hybridization between A. rnarginata and A. tenuifolia,
which are normally morphologically distinct and occur in slightly different

habitats (Druehl and Saunders, 1992). In addition to morphologically
intermediate forms, thalli identifiable as one species showed an IGS
restriction profile of a different species, but the extreme morphological
plasticity of these kelps precludes certainty concerning hybridization.
A looser application of the biological species concept, which tests the
ability to interbreed when brought into artificial proximity, has been more
widely used in algae, although many groups suffer from being either entirely
asexual or monoecious, which prevents or severely hampers interfertility
studies (Guiry, 1992; John and Maggs, 1997). Among red algae, mating
complexes/sibling species within traditional morphospecies have been
demonstrated in a few species (e.g. Polysiphonia japonica, Kudo and
Masuda, 1986; Mastocarpus stellatus, Guiry, 1992; Caloglossa leprieurii,
Kamiya et al., 1998; Zuccarello et al., 2000). In Chondrus crispus complete
interfertility was found for 23 isolates from both sides of the North Atlantic
Ocean (Guiry, 1992), and a later molecular study of seven samples showed
little phylogeographic structure (Chopin et al., 1996). Hybridization between
different species and genera of kelps has been reported fairly frequently
(Lewis and Neushul, 1995), based both on culture and field studies. For
example, Lewis and Neushul (1995) reported the formation of a fertile
Pelagophycus x Macrocystis hybrid in culture. Both parent species had
haploid chromosome numbers of n = 16, while the putative F1 was
polyploid with 32 chromosomes. Such claims are complicated, however,
by the ability of kelp gametophytes to form parthenogenetic pseudo-
sporophytes (Le Gal et al., 1996; Liptack and Druehl, 2000) and by
the range of unusual chromosome complements exhibited by kelps (Yabu
and Yotsukura, 1995). Interspecific, and indeed interfamilial, hybridization
between the kelp genera Alaria and Lessioniopsis has now been confirmed
using species-specific primers (Liptack and Druehl, 2000).
The phylogenetic species concept (Cracraft, 1983), which defines a species
as a monophyletic group composed of "the smallest diagnosable cluster of
individual organisms within which there is a parental pattern of ancestry and
descent" is potentially of great importance for algae as it can be applied to
both asexual and allopatric forms. It also offers the opportunities to look at
relationships over time by studying different depths in a phylogenetic tree, as
opposed to the biological species concept which can only be used with living
organisms. Nevertheless, it is not clear exactly how the phylogenetic species
concept should be applied because high-resolution molecular markers can
now "diagnose" such small groups of individuals (Claridge et al., 1997);
advocates of this species concept (e.g. Theriot, 1992) tend to be reticent in
giving examples of its usage for macroalgae. Strict use of phylogenetic
systematics requires a new code of phylogenetic nomenclature, in which taxa
are not assigned to ranks (Cantino, 2000). A draft PhyloCode has recently
been published electronically (http://www.ohiou.edu/phylocode).
178 C.A. MAGGSand R. WATTIER
In practice, as noted above, the morphological species concept is still

used as a basis for species-level and intraspecific studies in macroalgae,
while molecular data assist in calibrating or testing the limits of
morphospecies and defining boundaries between species (Manhart and
McCourt, 1992; John and Maggs, 1997). Breeding studies, involving larger
numbers of putatively conspecific isolates, ideally those for which molecular
data are also available (Kamiya et al., 1998; McIvor et al., 2001), can
strengthen the interpretations separately derived from each class of data.
We have centred the examples in the present review on algae to which
more than one species concept can be applied because different classes of
data are available. In other words, in defining the "species" within which
variation is detected, we have attempted to use a suite of species concepts as
advocated by Ridley (1993). We are also much attracted by the idea that at
some point in the future it may be possible to rank taxa on the basis of the
length of time they have been separated from others that share a common
ancestor - in a "standardized temporal scheme of biological classification"
(Avise and Johns, 1999)- so that all species could encompass a roughly
equal amount of genetic diversity. One of the main drives for this proposal
was to enhance comparability between groups, and certainly the seaweed
Divisions show widely disparate levels of intraspecific molecular
divergence. Evidence of this is the utility of ITS sequences at the familial
level in phylogenetic studies of the Phaeophyceae (Serrfio et al., 1999),
whereas it is often impossible to align ITS sequences of related genera in the
Rhodophyta (e.g. Goff et al., 1994; van Oppen et al., 1995a), or even
of different species groups in the green algal genus Cladophora (Bakker
et al., 1992).
III. L I F E H I S T O R I E S A N D T H E I R V A R I A B I L I T Y
Seaweeds show an extraordinary diversity of life histories (Hawkes, 1990)

compared with that of most other groups of organisms (Stearns, 1992).
There is considerable convergence and overlap of life-history types between
green, brown and red algae, each of which exhibits both single-phase and
haplo-diploid life cycles (van den Hoek et al., 1995). Haplo-diploid algae in
all divisions display life histories that are isomorphic, i.e. haploid and diploid
phases are morphologically closely similar if not identical, or heteromorphic,
in which the phases may be so dissimilar that they can be linked only by
culture studies or molecular methods. In heteromorphic life cycles, one
phase, which may be either the gametophyte (unisexual or bisexual) or the
sporophyte, is cryptic (crustose, filamentous or boring) by comparison with
the other. The morphological phase is not necessarily linked to ploidy
(Pedersen, 1981; Tanner, 1981; Maggs, 1988). Life histories of green and
brown algae are often highly plastic (Pedersen, 1981; Tanner, 1981), and
parthenogenetic and sexual reproduction frequently occur in tandem

(Pedersen, 1981; Tanner, 1981). Red algae are notorious for their complex
life histories. Uniquely in the Florideophycidae, the more highly evolved
group, which includes most of the red seaweeds, the immediate product of
fertilization is not the diploid sporophyte, but a hemi-parasitic diploid tissue
surrounded by female nutritive tissue, collectively called the cystocarp. This
stage, which is regarded as a mechanism compensating for the lack of motile
sperm in the red algae (Searles, 1980), releases numerous genetically
identical diploid spores that give rise to sporophytes. Life-history patterns
vary greatly among red algal species (West and Hommersand, 1981). The
algae - in particular the rhodophytes, which show such a range of forms,
life histories and reproductive strategies - offer great potential to theorists
interested in ecological and evolutionary processes (Hawkes, 1990), but as
yet there have been few such investigations.
In addition to the life-history diversity that occurs among seaweeds,
considerable intraspecific diversity has been observed in each division.
Some of the simplest examples are seen in brown algae, such as the switch to
parthenogenesis in the isomorphic Ectocarpus siliculosus, which is triggered
by temperature (M~ller, 1967), and the influence of salinity on life histories
in various heteromorphic species (Pedersen, 1981). Heteromorphic life
histories in red algae are of particular ecological interest. Typically
the gametophytic phase is erect, often blade-like, while the sporophytic
phase is crustose, which has important ecological implications. In both
florideophyte (reviewed by Maggs, 1988) and bangiophyte red algae, a
heteromorphic life history can take place apomictically at a single ploidy
level. For example, in Mediterranean isolates of Porphyra leucosticta,
a heteromorphic pseudo-sexual life history involving gametophytes and
conchocelis filaments takes place at one ploidy level (n = 3), whereas
in other isolates, sexual and single-phase apomictic life histories occur
(Gargiulo et al., 1994).
The best-known example of intraspecific variation in a heteromorphic life
history is that of the common carageenophyte Mastocarpus stellatus, which
was the subject of an extensive series of field and culture studies, principally
by J. A. West and M. D. Guiry (reviewed by Maggs (1988) and Guiry (1992)).
It grows in lower-shore habitats subject to high levels of both mechanical and
biological disturbance, by water movement and grazers, which selectively
damage the erect gametophyte. The divergent morphologies of crusts and
erect axes can be regarded as adaptations to different sources of mortality
(the chief problem for crusts is overgrowth by competing organisms),
or "bet-hedging" (Lubchenco and Cubit, 1980). Under conditions of
unpredictable variability in grazing pressure or severity of mechanical
damage, survival of a species with a heteromorphic life history is predicted to
be highest if there is continuous production of both phases. Two main life-
history patterns occur: (i) a typical sexual heteromorphic life history; and
180 C.A. MAGGS and R. WATTIER
(ii) an apomictic life history in which diploid females are recycled by the
formation of carpospores. These show distinct geographical distributions. In
the northern part of its geographical range, only the apomictic life history
occurs, towards its southern limit only the sexual type is found and both types
grow sympatrically in the middle of the range. When they are sympatric, they
are spatially segregated with apomicts in rock pools and sexual populations
on the lower shore. As the conditions in intertidal pools vary rapidly and
greatly, this situation apparently contrasts with the classical view that
apomicts are associated with predictable environments (Williams, 1975),
while sexual reproduction is more advantageous in unpredictable
conditions. A further degree of complexity exists: diploid carpospores
released by diploid females can give rise to mixed progeny, consisting of
both crusts and gametophytic blades or even sectored individuals with both
morphologies (Maggs, 1988). This ensures that both life-history phases
can develop from spores of the more resistant one, the sporophyte.
Environmental influence on the phase of progeny may allow the the
proportion of erect plants to respond rapidly to environmental conditions,
and the genetic component of the variability allows for gradual temporal
change in relative frequencies.
Some red algae with isomorphic life histories also have apomictic
populations in a particular part of the species' range, often the northern
part (Hawkes, 1990). In the ceramialean species Dasya ocellata, northern
populations (Ireland) are diploid perennial apomicts, whereas southern
populations (Portugal) consist of ephemeral sexually reproducing thalli
(Maggs, 1998). However, in the most detailed study to date of geographical
variation in life histories (West and Zuccarello, 1999), among 176 isolates
of Bostrychia moritziana) obtained throughout the tropical and warm-
temperate Southern Hemisphere, the proportion of sexual isolates varied
greatly without obvious geographic pattern, from nil (New Zealand and
Victoria, Australia) to 99% (e.g. the rest of Australia). Asexual populations
appear to have arisen repeatedly by loss of meiosis in tetrasporangia of
sexual populations. There are sometimes slight morphological differences
between sexual and asexual populations, which can be used to propose
segregate asexual species (e.g. Bostrychia bispora) but these cannot
normally be justified as they are unlikely to be monophyletic entities (West
and Zuccarello, 1999). These differences may also be accompanied by
contrasting physiological performance. Although this possibility has not yet
been explored experimentally, diploid parthenogenetic gametophytes of
the palmarialean crustose alga Rhodophysema elegans grew faster than
normal haploids (Saunders et al., 1989). Minor morphological differences
between haploids and diploids may be typical of red algae, and in Gracilaria
gracilis are thought to be ecologically significant, potentially playing an
important role in the evolution and maintenance of haplo-diploid life cycles
(Hughes and Otto, 1999).
IV. POPULATION GENETICS
In this section, we describe the current state of knowledge of intraspecific

genetic variation at small spatial scales and short temporal scales, i.e.
microgeographic and mesogeographic genetic structure, determined using
various molecular markers. The genome of all photosynthetic seaweeds
can be subdivided into three components: the organellar genomes,
mitochondrial (mtDNA) and chloroplastic (cpDNA), referred to as
haplotypes, and the nuclear genome (nDNA). Both mtDNA and cpDNA
markers were long thought to be monomorphic at the intraspecific level,
but recent studies described below have shown this assumption to be
false, mtDNA and cpDNA are characterized by uniparental, maternal,
inheritance, although experimental evidence is lacking for most species (van
der Meer, 1990; Zuccarello et al., 1999a). Nuclear DNA is characterized by
Mendelian inheritance, with each homologous chromosome segregating
independently at meiosis. Two major classes of marker must be
distinguished: single-locus/co-dominant vs. multilocus/dominant markers.
Single-locus/co-dominant markers target a unique site within the nuclear
genome; alleles on both homologous chromosomes of diploid individuals
are detected at the same time and homozygous (same alleles) and
heterozygous (different alleles) genotypes (co-dominance) can be
distinguished. Allele frequencies are used to estimate spatial genetic
structure and homozygote-heterozygote deficiency-excess can be seen as a
basic measure of the mating system. In contrast, multilocus/dominant
markers detect so many genomic sites at a time that it is almost impossible to
distinguish homozygotes from heterozygotes. Further details may be
obtained from the many published reviews concerning DNA markers (e.g.
Lessa, 1992; Lessa and Applebaum, 1993; Bachmann, 1994; Karp et al., 1996;
Cruzan, 1998; Haig, 1998; Parker et al., 1998; Ouborg et al., 1999).
Unfortunately, although isozymes are co-dominant markers that have
provided a good basic understanding of gene flow in many groups, they
have often given disappointing results in marine macroalgae owing to a
combination of unreliable detection and/or low levels of polymorphism (see
Sosa and Lindstrom, 1999). Fewer than 15 species have been studied during
the last two decades using these techniques (Sosa and Lindstrom, 1999).
More polymorphic markers, at the DNA level, are needed to elucidate
population genetics of seaweeds. Such markers are described in the following
sections and can be (artificially) subdivided into the following four classes:
(i) Sequence data: the sequence of nucleotides of a DNA fragment is
determined by sequencing techniques.
(ii) Restriction fragment length polymorphism (RFLP): presence vs.
absence of recognition sites for endonucleases (as well as insertion/
deletions) results in restriction fragments of different sizes.
(iii) Single-strand conformational polymorphism: it is assumed that point

mutations will confer differences in spatial conformation of single-
stranded DNA leading to differences in migration patterns in
electrophoresis.
(iv) Variable number of tandem repeat (VNTR). Part of the nuclear
genome consists of 2-6 bp (microsatellite) or 10-50 bp (minisatellite)
units organized in tandem arrays of a few to hundreds of copies. The
number of tandem repeats can be variable, generating size differences.
We begin this section by examining the primary reason for the slow
development of population genetics for seaweeds compared with that for
most other groups of conspicuous and economically important organisms,
i.e. the problem of isolating DNA from large numbers of individuals.
A. DNA ISOLATIONFROM SEAWEEDS:A TECHNICALNIGHTMARE
In general, isolation of good-quality DNA (i.e. of high molecular weight,

without inhibitors) has proven difficult for all groups of algae (Olsen, 1990;
Sosa and Oliveira, 1992). For red algae, the agars and carrageenans co-
isolated with DNA result in highly viscous solutions, and potentially inhibit
endonuclease digestion and PCR (Jin et al., 1997). All reported protocols
focus on reducing or eliminating polysaccharides. In the 1980s such
measures included up to three CsC1 gradient ultracentrifugations as
purification steps to remove polysaccharides. These protocols provide high-
quality DNA but are complex, expensive and very time consuming. An
additional problem is the large amount of seaweed material required,
which effectively precludes investigations of individuals. More recently such
protocols have generally been confined to studies of very small numbers of
samples (Patwary et al., 1993, Kitade et al., 1996, Chopin et al., 1996). In fact,
the 1990s were characterized by intensive research activity around
developing an alternative, simpler, DNA isolation protocol that could meet
all constraints regarding quality, quantity and large-scale assays. More than
10 protocols have been published for red algae alone since Sosa and
Oliveira's (1992) review, all derived from protocols developed for land
plants, fungi or bacteria, either applied straightforwardly or simplified and
specifically modified for red seaweeds. Most remain quite time consuming,
especially when multiple organic solvent extractions and DNA alcoholic
precipitations are required. In addition, many protocols are characterized
by inconsistent quality requiring additional treatment (Stean et al., 1991;
Patwary and van der Meer, 1994; Hong et al., 1995).
To be compatible with population assays, a DNA isolation protocol must
include apparently opposed and incompatible features: on one hand, it must
be cost and time effective to permit the processing of large numbers of
samples (often hundreds for population genetic assays) and on the other
hand it must result in high yield of DNA of high molecular weight, with
a low level of co-isolated contaminants. Although some markers, like
microsatellites or RAPDs, can work on minute amounts of evenly degraded
DNA, others, such as Southern-based RFLPs or AFLPs, require up to 1 mg
of high-molecular-weight DNA.
The Chelex resin-based protocol reported by Goff and Moon (1993) fulfils
the first set of requirements, being very simple and quick. A minute amount
(c. 50 mg fresh weight) of material is crushed in extraction buffer containing
5% w/v Chelex. Samples are boiled for 5 rain and centrifuged for 10 min
and the supernatant is the DNA solution used for PCR. This protocol is
characterized by a very high throughput, with 48 samples extracted in less
than 2 h. However, the major concern about Chelex-isolated DNA is that the
DNA solution is a crude extract and DNA endonuclease and exonuclease
activity have been reported for many animal and plant species, with DNA
totally degraded, sometimes in less than 24 h. In addition, this protocol has a
very poor yield (less than 100 ng in total) and, most of the time, does not
produce high-molecular-weight DNA (most DNA is below 1 kb in size).
Those two last features prevent application of markers such as Southern-
based RFLPs or AFLPs. Nevertheless, a slightly modified version of this
protocol was successfully applied to 1200 individuals of Gracilaria gracilis,
for microsatellite analysis (Wattier et al., 1997, 1998; Wattier, 1998; Engel et
al., 1999; Luo et al., 1999). Five-year-old samples were still suitable for PCR
but had been stored at -20°C, thawed on ice and kept on ice each time they
were used. However, Zuccarello and collaborators reported that an excess of
material can lead to inhibitory effects in PCR (Zuccarello et al., 1999a) and
that DNA of some individuals was not suitable for rubisco spacer PCR-SSCP
analysis (Zuccarello et al., 1999c).
An optimized protocol developed for red algae (Wattier et al., 2000)
provides DNA of high molecular weight with no RNA, and is suitable for
endonuclease restriction, M13cs PCR-fingerprinting, and cpDNA PCR-
RFLP. The yield is at least 5 i~g of DNA from 10 mg of dried algal material.
DNA stored at 4°C for up to 18 months showed no sign of degradation. In
addition, the protocol is time effective (48 samples in 5 man-hours work),
cost-effective (c. 0.3 Euro a sample) and is user friendly, not involving
hazardous chemicals. We have found the Qiagen DNeasy Plant Mini Kit
(Qiagen GmbH, Hilden, Germany) to be very effective in obtaining DNA
for sequencing purposes, but unsuitable for large numbers of samples.
B. SINGLE-LOCUS N U C L E A R MICROSATELLITES
Single-locus nuclear microsatellites (commonly known as microsatellites)

were first reported in 1989 (Litt and Luty, 1989; Tautz, 1989; Weber and
May, 1989). Microsatellites are regions of the genome characterized by
184 c . A . MAGGS and R. WATTIER
tandem repeats of very short (1-6 bp) DNA core motifs and seem to be
ubiquitous in eukaryotic organisms (Tautz and Renz, 1984; Wang et al.,
1994). Most microsatellites are characterized by a VNTR between copies on
homologous chromosomes, a source of length polymorphism within the
microsatellite sequence. Using specific PCR primers defined in unique and
putatively conserved flanking sequences, it is possible to detect size variants
(alleles) at one site in the genome (locus) that can be scored by appropriate
high-resolution electrophoresis (Litt and Luty, 1989; Tautz, 1989; Weber
and May, 1989).
The most important attribute of microsatellites is that they are single-
locus nuclear markers with co-dominant alleles. Thus, data can be expressed
as single genotypes or allelic frequencies and can benefit from the
framework of theoretical population genetics analysis developed over the
the last 70 years. Co-dominance allows the definition of homozygote and
beterozygote genotypes in diploid individuals, a crucial factor in population
genetics analysis since homozygote-heterozygote deficiency can be seen as a
basic measure of the mating system and spatial genetic structure. Over the
decade since their discovery, microsatellites have been used extensively
for evolutionary studies including those of kinship and breeding systems,
spatial genetic structure and gene flow (for reviews see Bruford and
Wayne, 1993; Queller et al., 1993; Schl6tterer and Pemberton, 1994).
Microsatellites are often described as the "marker of choice" and primers
are now available for a wide range of organisms including almost all phyla of
animals and divisions of land plants. However, the development of new site
(locus)-specific primers requires prior knowledge of flanking sequences;
availability of DNA sequences in databases is still limited for many
organisms and no or virtually no nuclear microsatellites and their flanking
sequences have been found as a by-product of genome characterization.
Therefore, microsatellites and their flanking sequences have to be
"extracted" directly from the genome. Various types of protocols are
available, commonly based on molecular cloning techniques (Rassmann et
al., 1991). The "standard" cloning procedure includes the construction of
small insert-size genomic libraries, library screening with oligonucleotide
microsatellite probes and sequencing of positive clones to allow definition of
site-specific PCR primers. This part of microsatellite marker development is
laborious and is the drawback of such genetic markers (Schl6tterer and
Pemberton, 1994). Although, once developed, microsatellite markers have a
number of appealing features, such as requiring small amounts of DNA and
showing high reproducibility, an increasing list of limitations has become
apparent, including low levels of polymorphism, scoring difficulties for
dinucleotide loci, null alleles and allele size homoplasy.
Microsatellites are thought to be ubiquitous in eukaryotic organisms
(Tautz and Renz, 1984; Wang et al., 1994). However, single-locus
microsatellite markers have only recently been developed for algae (Wattier
et al., 1997), involving enormous expenditure of time and effort by several

European research groups. To our knowledge, primers have been published
for only three species, the European red alga Gracilaria gracilis (Wattier et
al., 1997; Luo et al., 1999), the Atlantic kelp Laminaria digitata (Billot et al.,
1998) and the tropical green seaweed Cladophoropsis membranacea (van der
Strate et al., 2000). Unpublished population genetic studies, based on
microsatellites, were presented at the second European Phycological
Congress (August 1999) for two additional North Atlantic brown seaweeds,
Ascophyllum nodosum (Olsen and Stam, 1999) and Fucus serratus (Coyer et
al., 1999), primers for which should soon be available.
Development of microsatellites proved to be particularly difficult for all
five species, for three main reasons: (i) very low genomic frequency of
microsatellites; (ii) a small proportion of polymorphic loci; and (iii) a high
proportion of loci with relatively low levels of polymorphism. In addition
to these very serious obstacles, the problems previously mentioned, such
as scoring difficulties for dinucleotide loci, null alleles and allele size
homoplasy, have also been encountered with algae. The haplo-diploid life
cycles of algae further complicate the application of nuclear microsatellites
to algae because genotypes cannot be accurately determined unless the
ploidy is known.
The genomic frequency of nuclear microsatellites in seaweeds appears to
be low: out of 22 000 clones screened for an AG polymer in Gracilaria
gracilis, only 35 positive clones were detected (Wattier et al., 1997). Attempts
to develop microsatellites for other red algae, such as the edible seaweed
PaImaria palmata, have proved to be equally frustrating. It is surprising that
similar problems have been encountered in both red and brown seaweeds in
view of their very distant phylogenetic relationship (in respect of their
nuclear genomes), and at present we are unable to offer any possible
explanation for these observations. The rarity of microsatellites in seaweeds
exacerbates scoring difficulties. Because microsatellites are co-dominant
markers, scoring of heterozygotes is of primary importance. However,
almost all dinucleotide loci are characterized by "stuttering" or "ladder"
patterns (Murray et al., 1993). For a given allele, in addition to the PCR
product corresponding to the "true" allele, artefactual products are
amplified, shorter by n-1 to n-3 repeats (n being the number of repeats in
the "true" allele). For a diploid individual, determining heterozygous status
can be difficult for alleles with a small difference of 1-2 repeats because
multiband patterns are partially superposed. Trinucleotide loci are less
prone to this kind of problem, and it scarcely applies to tetranucleotide
loci. It is therefore tempting to favour them against dinucleotides, but as
the genomic frequency of trinucleotide and particularly tetranucleotide
microsatellites is much below that of dinucleotides, they require much more
effort to develop. In view of the scarcity of all microsatellites in seaweeds,
dinucleotide motifs must be exploited despite the difficulties.
For most organisms, microsatellites are regarded as highly variable

markers and more than 10 alleles within a population is a figure commonly
observed. In algae, by contrast, low levels of polymorphism seem to be the
norm. Microsatellite markers developed for G. gracilis exemplify the
situation (Wattier et al., 1997; Luo et al., 1999). Of 20 loci developed,
nine were monomorphic when tested with 20-24 individuals from several
locations on the English Channel and French Atlantic coasts. The 11 loci
revealed to be polymorphic were further tested in samples of 71-78 haploid
and 89-94 diploid individuals from one population. Five loci exhibited only
2-4 alleles. Four loci were slightly more polymorphic with 5-7 alleles.
Similarly, in Cladophoropsis membranacea, of eight (GT) loci developed
(van der Strate et al., 2000), for five loci there were 2-5 alleles and three loci
had 7-11 alleles. The number of alleles observed for Laminaria digitata
(Billot et al., 1998) was also relatively low. It is speculative to extrapolate
these results, based on only three species, to all algae but it seems this is also
the case for Ascophyllum nodosum, for which 15 of the 16 first loci defined
were monomorphic and the only polymorphic locus exhibited only two
alleles. For well-studied organisms, the numbers of motif repeats and levels
of polymorphism have been related to (i) a low probability below a given
threshold and (ii) a positive correlation above this threshold (Weber, 1990).
This relationship, initially established for CA repeats in humans, has been
confirmed for other types of motifs and organisms (Akkaya et al., 1992; Yang
et al., 1994; Kemp et al., 1995, but see Valdes et al., 1993). All loci identified
for G. gracilis were defined with this possible limitation in mind, having at
least 9 and 6 repeats for di- and trinucleotide motifs respectively. In G.
gracilis only one locus (Gv2CT) was truly highly polymorphic, with 14 alleles.
On the other hand, one locus (GvlCT) was extraordinarily potymorphic with
52 detected alleles. Comparative data for other organisms are not readily
available, but such highly polymorpbic loci with more than 40 alleles per
population appear to be rare, although it is not uncommon to observe 10-20
alleles within populations of animals or land plants.
The problem of null alleles is one area of microsatellite research in which
studies on algae have made a contribution to the field in general. For a single
locus, a null allele is an allele that is not amplified/detected. There are
several sources of non-detection described for microsatellites. One source is
associated with mutations in flanking sequences: either point mutations
(Paetkau and Strobeck, 1995; Ishibashi et al., 1996) or deletions (Callen et
al., 1993; Ede and Crawford, 1995) can prevent primers from binding and
therefore stop amplification of the corresponding non-detected ("null")
allele. Null allele frequencies can be as low as 0.05 (Lehman et al., 1996) but
can reach an amazing 0.5 (Pemberton et al., 1995). For organisms with diploid
life cycles, point mutations or deletions present at very high frequencies can
be easily detected as many individuals (homozygous for the null allele) will
not be amplified. As the frequency of null alleles decreases, they become
INTRASPECIFICVARIATIONIN SEAWEEDS 187
harder to detect because null homozygotes become rare. For low-frequency

null alleles, only pedigree analysis can reveal the presence of a null allele.
Working with haplo-diploid organisms, assuming the haploid stage can
be typed, makes the work simpler as non-amplification of null alleles in
haploids can readily be detected. A further source of non-amplification
results from short allele dominance, where, in heterozygotes including a
short and a long allele, only the short allele is detected. Such a systematic
bias was first experimentally analysed with one dinucleotide locus of
Gracilaria gracilis (Wattier et al., 1998). To test this hypothesis, the intensity
of patterns obtained from haploid individuals with different allele sizes was
compared with those of artificial heterozygotes created by mixtures of two
haploid DNA samples. Long alleles were amplified less when they were
in association with a shorter allele. Standard PCR conditions can even
lead to the non-amplification of the longer alleles, creating an artificial
heterozygote deficiency. The true extent of the problem is not really known
and to our knowledge only one other case has been reported. In addition,
GvlCT is a very peculiar locus characterized by a remarkably large range of
allele sizes (>300 bp) within a population, a very unusual feature.
Size homoplasy occurs because alleles are scored as size variants, so it is
difficult to ascertain whether alleles of identical sizes (identical by state)
have the same "genealogy" and are indeed identical by descent or not. Two
factors favour the possibility of high levels of size homoplasy: the mutation
mechanism and a high mutation rate. Mutation processes at microsatellite
loci are still poorly documented. However, intra-allelic DNA replication
slippage is the most favoured mechanism (Schl6tterer and Tautz, 1992).
DNA replication error can produce slightly shorter or longer alleles (one
to a few repeat(s)). The mutated allele can itself mutate again the next
generation and return to its initial size, resulting in allele size homoplasy;
mutation rates at microsatellite loci can be high (e.g. Kwiatrowski et al.,
1992; Crozier et al., 1999). Combining these two parameters, size homoplasy
is highly probable. Size homoplasy induces underestimation of divergence
between individuals and/or populations because alleles that are not
genealogically identical are considered as such. The quantitative impact of
such homoplasy on data analysis remains to be evaluated. Within humans,
almost all studies reveal size homoplasy between populations or even within
populations (e.g. Adams et al., 1993; Grimaldi and Crouau-Roy, 1997). For
other organisms, homoplasy has been detected at the interpopulation level
for many species (e.g. Estoup et al., 1995, Taylor et al., 1999). In algae,
interpopulation size homoplasy has already been observed for one locus
(Gv2CT) in Gracilaria gracilis (Wattier, 1998) and one locus in Laminaria
digitata (Billot et al., 1998).
Microsatellite loci are claimed to have an additional advantage over many
other markers in that primers designed in one species may successfully
amplify loci in other related species (Rico et al., 1996). This ability to use
heterologous primers in several different species could eliminate the need to

develop new sets of primers for each species. The extension is sometimes up
to taxonomic levels as high as between superorders. There are numerous
examples at an intrageneric level in land plants and at both intrafamily and
intrageneric level in animals (see Wattier et al. (1997) for references) that
show conservation for almost all loci studied. Unfortunately, when loci
developed for Gracilaria gracilis were applied to other congeners or to the
gracilarialean genus Gracilariopsis, almost none were conserved (Wattier et
al., 1997). This is not necessarily surprising because there is a wide genetic
divergence between Gracilaria and Gracilariopsis, with ITS sequences being
unalignable between genera (Goff et al., 1994). By contrast, in the kelps
Martfnez et al. (2000) found that microsatellite loci from Laminaria digitata
(Laminariaceae) could be amplified in two Lessonia species (Lessoniaceae).
The Laminariales appear to be a relatively recently evolved group, with an
estimated divergence time for the families of 8.5-34 million years ago
(Druehl and Saunders, 1992). Although some polymorphism was detected in
Lessonia (Martfnez et al., 2000), all six polymorphic loci developed for Fucus
serratus proved to be monomorphic in Fucus evanescens (Coyer et al., 1999).
Despite these severe difficulties for both microsatellite development and
population assays, microsatellites have already proved to be extremely
useful markers for the seaweed species for which they were available. They
enabled studies of kinship and male gamete dispersal in Gracilaria gracilis
(Engel et al., 1999), breeding system and spatial population structure in
Cladophoropsis membranacea (van der Strate et al., 2000) and Laminaria
digitata (Billot et al., 1998), population structure in relation to possible
ecotypic differentiation in Fucus serratus (Coyer et al., 1999), and genetic
neighbourhood size inAscophyllum nodosum (Olsen and Stam, 1999). In all
these cases, no other marker, especially multilocus markers, could have
supported the analyses.
C. MULTILOCUS NUCLEAR MARKERS (RAPDs, ISSRs, M13-FINGERPRINTING

AND AFLPs): ARE THESE THE TOOLS OF THE FUTURE?
Four classes of multilocus DNA markers have been applied to seaweeds:

RAPDs, ISSRs, M13-fingerprinting and AFLPs. Random amplified
polymorphic DNA (Welsh and McClelland, 1990; Williams et al., 1990) and
intersimple sequence repeats (Zietkiewicz et al., 1994), share the same
technical principle: a unique PCR primer (a random decamer for RAPD
and a microsatellite for ISSR) is used to produce a multilocus-dominant
fingerprint pattern. The three major advantages of both techniques are:
(i) no previous knowledge of the genome is required; (ii) the amount of
genomic DNA needed is limited; and (iii) ease of routine assays, consisting
simply of PCR and agarose gel electrophoresis. However, there have been
and still are major concerns about the reproducibility of both RAPDs
(Hadrys et al., 1992) and ISSR techniques (Weising et al., 1995). Another
serious concern is the lack of informativeness of RAPDs at the population
level (Isabel et al., 1999; van Oppen et al., 1996; Pakker et al., 1996; Coyer et
al., 1997; but see Wright et aI., 2000). At the population level, scoring error
remains approximately constant, but the signal decreases due to increasing
numbers of invariant bands (no phylogenetic information) and
autapomorphic bands (i.e. banding patterns unique to a single individual).
Bulking samples of several individuals can reduce these problems to some
extent, and this method provided evidence of high gene flow between some
populations over a geographical scale in the red alga Gelidium sesquipedale
(Alberto et al., 1997). The same limitations were reported for ISSR in its only
published application to algae, a research note for one freshwater red algal
species, Batrachospermum boryanum (Vis, 1999).
M13-fingerprinting is based on a M13 minisatellite core sequence used as
an oligonucleotide to probe a Southern transfer of genomic DNA. Up to
now this technique has been applied only to two kelp species, Macrocystis
pyrifera (Coyer et al., 1994, 1995) and Postelsia paImaeformis (Coyer et al.,
1997). Sample sizes were limited but these studies show the potential of this
technique for the analysis of genetic structure at various spatial scales.
However, this technique is very time consuming and AFLPs, as described
below, are probably more suitable, especially if large sample sizes are
employed.
AFLP technology was introduced in 1995 (Vos et al., 1995). It is based on
the selective PCR amplification of restriction fragments from a total digest of
genomic DNA. Basically, the technique involves three steps: (i) restriction of
the genomic DNA and ligation of oligonucleotide adaptors; (ii) two rounds
of PCR amplification using pre-selective and selective primers amplifying
decreasing subsets of all the fragments in the total digest; and (iii) sequencing
gel-based analysis of the amplified fragments (Vos et al., 1995). The AFLP
technique requires no prior sequence characterization of the target genome.
Amplification is based essentially on the primer matching the adaptor
sequence. Selectivity is based on a few additional random nucleotides on the 3'
end of primers. The presence of these additional nucleotides results in the
amplification of only a subset of the restriction fragments. Many primer
combinations are possible, amplifying different subsets. Stringent PCR
conditions allow high reproducibility and restriction fragment length
polymorphism is observed. At one locus, alleles differ in size in connection
with point mutations at restriction sites and/or insertion/deletion events.
AFLPs allow the amplification and analysis of many loci at a time. AFLP
markers are co-dominant, that is heterozygotes can normally be distinguished
from homozygotes at a given locus. However, because up to 20 loci are
detected at a time, ascribing a band to a specific locus is often not possible and
banding patterns are analysed in terms of presence or absence of bands. The
first application of AFLP to seaweeds tested the technique on six isolates of
190 C . A . MAGGS and R. WATTIER
the red alga Chondrus crispus (Donaldson et al., 1998). A more extensive
study, involving 10 individuals from 10 Canadian populations (Donaldson
et al., 2000), experienced problems with the sensitivity, reproducibility and
efficiency of the technique. There was no phylogenetic resolution, perhaps
due to inadequate DNA quality. Nevertheless, Kusumo and Druehl (2000),
using CsCl-purified DNA of the Pacific kelp Alaria marginata, detected
variability at each spatial scale tested (from patches a few decimetres in
diameter to a group of stands separated by 185 km), which followed an isolation
by distance model. Despite the technical problems, of all four multilocus
markers presented in this subsection AFLPs seem to be the most promising.
D. PLASTID MARKERS
Plastids are believed to be uniparentally inherited in seaweeds (see next

section). Since the chloroplast genome is transmitted uniparentally without
recombination taking place, data from multiple, physically linked loci can be
combined to give highly informative haplotypes, which can be used to trace
genetic lineages. Plastid markers can reconstruct maternal lineages in the
same way as mitochondrial markers are employed for animals. To date, the
great majority of DNA-based population studies in animals have involved
mitochondrial markers, and a large body of theoretical and practical knowledge
has accumulated, providing unique insights into the genetic structure and
history of natural populations (Avise, 2000). Use of plastid markers for algae
thus permits comparison and interpretation using similar methods of analysis.
For higher plants, RFLP analysis of chloroplast DNA is now recognized
as a powerful tool for the study of evolutionary processes, both at the
intraspecific level and for molecular systematics of species and genera
(reviewed by Soltis et al., 1997). Many examples of intraspecific
polymorphism have been discovered and at least 20 studies have employed
cpDNA-RFLP to study microevolutionary processes, investigating the
genetic structure of various angiosperm species and the genetics of hybrid
zones in genera such as Iris (Young, 1996). cpDNA-RFLP analysis has also
been applied to phylogeographic studies in several species. For algae, by
contrast, the great majority of studies have used cpDNA-RFLP strictly as a
molecular taxonomic tool (e.g. Rice and Bird, 1990; Maggs et al., 1992). The
maximum sample size reported is nine (Goff and Coleman, 1988), each of
which represented numerous bulked individuals. The only algal study
of intrapopulational cpDNA-RFLP, in the red alga Ceramium virgatum
(Wattier et al., 2001), examined the entire chloroplast genome using cpDNA
as a probe. Four haplotypes in total and an average of 1.5 haplotypes per
population were detected. This represents a level of intraspecific and
intrapopulational polymorphism comparable with values obtained in land
plants using similar techniques (purified cpDNA, probes covering the
INTRASPECIFIC V A R I A T I O N IN SEAWEEDS 191
Fig. 1. Sections of gels showing SSCP of the rubisco spacer (haplotypes A-D, L)
of various Australian populations (a-c) of the mangrove-inhabiting red alga
Caloglossa leprieurii, showing different mobilities of fragments even when differing
only by single nueleotides (haplotypes A and D). (Reproduced with permission from
Zuccarello et al. (2000).)
majority of the chloroplast genome and using complete cpDNA as a probe),

and suggests considerable potential for population studies. An important
advantage of this technique for field studies is that it detects DNA of each
species or individual in direct proportion to the amount of that DNA
isolated. Thus contaminating epiphytes, which are often congeneric or even
conspecific when fouling algae such as Enteromorpha spp. are targeted
(Blomster et al., 2000), result in only very faint or undetectable bands.
In red and brown algae the genes for the large (rbcL) and small (rbcS)
subunits of ribulose biphosphate carboxylase/oxygenase (rubisco) are
organized as a co-transcribed operon including a short intergenic spacer (the
rubisco spacer). Sequences of the rubisco spacer have been used extensively
for species-level studies (Parsons et al., 1990; Destombe and Douglas, 1991),
but rarely in population studies, except in conjunction with SSCP, as described
below (Zuccarello and West, 1997; Zuccarello et al., 1999a). However, the
sequencing study by Brodie et al. (1996) of several Porphyra populations found
a single nucleotide substitution between monoecious and dioecious Porphyra
thalli, which were consequently identified as separate species.
SSCP, introduced in 1989 (Orita et al., 1989), is defined as a mobility shift of
single-stranded DNA in neutral polyacrylamide gel electrophoresis due to
single base substitutions. The method is based on the property that the
electrophoretic mobility of single-stranded nucleic acids depends not only on
size but also on sequence. Amplified fragments of the same size but different
sequences will have different single-stranded conformations, which confer
different electrophoretic mobilities. The original method was based on Southern
hybridization but PCR methodology, referred as to PCR-SSCP, is nowadays
the only method used. It is increasingly employed by population geneticists to
detect variation between individuals in various type of organisms with haploid
genomes, although the level of polymorphism observed is highly variable.
The rubisco spacer as a target for PCR-SSCP in algae (Fig. 1) has recently
been developed for four tropical red algal species, Caloglossa leprieurii
(Zuccarello et al., 2000) and three species of Bostrychia (Rhodomelaceae)
(Zuccarello et al., 1999a,b,c). Only one example of each band need be
sequenced, which permits much larger population sample sizes than if the
192 c . A . M A G G S and R. W A T T I E R
fragment is sequenced for all individuals. Great potential and some successful
applications have been reported, including the analysis of spatial genetic
structure within populations (Zuccarello et al. 1999a) or at macrogeographic
scale (Zuccarello et al., 1999b,c, 2000). In Caloglossa leprieurii, over 1000
individuals from 16 populations were examined. Geographically separated
populations are genetically isolated but many populations had only one
haplotype, so small-scale genetic structure could not be investigated.
Development of single-locus nuclear microsatellites for macroalgae has
been very laborious, as described above. By contrast, the recent discovery of
polymorphic mononucleotide repeats in chloroplast genomes has provided
new opportunities for the high-resolution analysis of organellar variation in
plants and algae (for reviews, see Powell et al., 1996; Provan et al., 1999a).
These chloroplast microsatellites have been shown to reveal much higher
levels of cytoplasmic diversity than comparable RFLP studies and
consequently they have allowed the analysis of natural populations with a
finer degree of resolution than had previously been possible (Provan et al.,
1998). In addition, species recalcitrant to analysis using traditional markers
have displayed previously undetected levels of intraspecific variation when
analysed using chloroplast microsatellites (Provan et al., 1999b). Chloroplast
microsatellites generally show a higher proportion of polymorphic loci than
do nuclear microsatellites. The high copy-number of the plastid genome can
provide an ideal target for PCR-based analyses in taxa for which nuclear
markers have been difficult to develop, and plastid microsatellites may prove
in future to be as useful for seaweeds as for higher plants.
E. B R E E D I N G SYSTEMS
The breeding system of an organism can be defined broadly as the mode,

pattern and extent to which it interbreeds with other organisms of the same
or of different taxa (Stace, 1989). Some understanding of the breeding
system for any group of organisms is an important foundation for studies of
morphological variation and evolutionary processes including speciation
and hybridization. Until the development of molecular markers, breeding
systems of seaweeds could generally be studied only in laboratory culture
because heritable morphological markers indicating parentage were lacking.
Culture studies by L~vlie and co-workers on Ulva mutabilis (reviewed by
Tanner, 1981), for example, showed that, although this seaweed is isogamic,
gametophytes represented mt+ or rot- mating types that could mate only with
thalli of the opposite mating type. Mating type appeared to be determined by
single alleles that segregated during meiosis. Gametes produced by different
individuals of the same mating type could not form zygotes.
Three biochemical approaches have, however, yielded information
relevant to breeding systems of seaweeds in their natural environment.
Characterization of the pheromones of a wide range of brown algae showed

that some were identical in different species or even genera (e.g. lamoxirene
is formed throughout the Laminariales; Maier, 1995). This is important
information relevant to the interpretation of reports of interspecific and
intergeneric hybridization in kelps (Liptack and Druehl, 2000). Pyrolysis
mass spectrometry is a technique that exposes the unique chemical
fingerprint (i.e. the chemical phenotype) of a sample by cleaving the
molecules of the sample at their weakest points to produce smaller
fragments. These fragments are then separated on the basis of their mass-to-
charge ratio to produce a pyrolysis mass spectrum. Comparisons of such
spectra provided evidence that, as long suspected, some species of Fucus can
hybridize under natural conditions (Hardy et al., 1998). Chemical races in
the red alga Laurencia nipponica produce different halogenated secondary
metabolites (Masuda et al., 1997; Abe et al., 1999), which provide unusual
genetic markers. All races are interfertile in culture, and some of the
metabolites appear to be encoded by genes on different chromosomes, so
that additional metabolites are produced only by heterozygotes (Masuda et
al., 1997). In the field, hybrids (the laurencin-producing race) are not formed
frequently, as judged by their geographical distribution, but have grown at
the same site in Japan for 25 years (Abe et al., 1999). This suggests that
hybrids are self-propagating, and that most of the surviving spores grow
close to their parents.
A range of PCR-based DNA markers has now been applied to some of the
questions relating to breeding systems in a very limited range of seaweeds. In
red algae, because flagellate stages are entirely lacking, the dispersal of male
gametes has been presumed to be limited, and zygote amplification into a
cystocarp is regarded as a compensating mechanism (Searles, 1980). In order
to test these hypotheses concerning sperm limitation in red algae, Engel et al.
(1999) used two polymorphic microsatellite loci to evaluate both female
and male fertilization success in a natural population of Gracilaria gracilis.
Female fertilization success was estimated by cystocarp yield per unit female
thallus (assuming an even distribution of the sessile female gametes) and
evaluated with respect to the availability of male gametes. Male fertilization
success, estimated by the individual contribution of different males to
zygotes, was assessed by paternity analyses on 350 cystocarps produced in
one reproductive season. Cystocarp yield was not sperm-limited, indicating
that for this species, at least, fertilization is efficient, and the poor dispersal
ability of sperm is not an important factor. The large variation in male
fertilization success could not be explained just by the distance travelled
by the male gamete to find a mate, raising the intriguing possibility that
male-male competition and/or female choice may play a role in shaping
population mating patterns. Studies by Kim and others (Kim et al., 1996;
Kim, 1997) in the ceramialean genus Antitharnnion showed that large
numbers of sperm (up to 300) can bind to each trichogyne (sessile female
gamete) and many male nuclei enter the trichogyne, providing great scope
for sperm competition. A common first-order receptor appeared to operate
at the generic level, permitting attachment of sperm to trichogynes of
congeners, while a second-order receptor prevented interspecific nuclear
fusion. Pickett-Heaps and West (1998) observed sperm nuclei jostling
and even overtaking each other as they travelled down the trichogyne of
Bostrychia towards the female nucleus.
In the kelp Laminaria digitata, RAPD markers can provide unambiguous
genotyping within groups of sporophytes (Billot et al., 1999). Controlled
crosses indicated that RAPD markers were transmitted to the progeny in a
Mendelian fashion. This approach was then applied to parentage analysis
within a set of 30 anonymous sporophytes from a random crossing
experiment involving four parent sporophytes. When the parents of 26
offspring were unambiguously identified, it was seen that, at least in culture,
outbreeding is the norm as opposed to self-fertilization of the gametophytes
derived from each sporophyte. Blocks to polyspermy have been studied in
detail in various Fucus species (Brawley and Johnston, 1992).
Plastid markers are normally presumed to indicate maternal lineages, but
in some higher plants rare or frequent paternal plastid leakage via sperm has
been reported in interspecific hybrids (Yang et al., 2000). The inheritance of
plastids has been assessed in two species of the red algal genus Bostrychia, B.
radicans and B. moritziana, using the rubisco spacer as a target for PCR-
SSCP (Zucarrello et al., 1999a). Paternal transmission was not detected
in the numerous crosses, only the maternal haplotype being found in
sporophytes, but the low number of sporophytes per cross (six or less)
precludes any statistically supported conclusion.
Karyology has rarely been applied to studies of breeding systems in algae;
generally, it has been confined to determining chromosome number as
a particular class of subcellular morphological feature. However, the
discovery that one population of Gracilaria gracilis from Cape Gris-Nez
(northern France) has an anomalous chromosome number of n = 16-18
(Godin et al., 1993; Kapraun et al., 1993) instead of the typical number of
n = 24 found in other populations of G. gracilis (Bird and Rice, 1990,
Kapraun et al., 1993) suggests that this population is genetically isolated.
Differing chromosome complements must prevent interbreeding, or at least
the formation of fertile hybrids, unless or until polyploidization occurs. No
evidence of natural polyploids has been found to date, however, in this well-
studied genus, although artificial polyploids were obtained in cultures of
Gracilaria tikvahiae (van der Meer, 1981). Generally, in red algae, polyploidy
seems common only in the Ceramiales (Maggs, 1988). Studies of
chromosomes in seaweeds are hampered by their very small sizes (typically
<2 t~m even in the Ceramiales), which prevent the application of Giemsa
and flurochrome dyes, standard cytological tools in other groups used to
reveal banding patterns and identify individual chromosomes. As far as we
knOW, newer techniques such as in situ hybridization have not been applied
to seaweeds.
V. BIOGEOGRAPHY AND PHYLOGEOGRAPHY
Biogeography is a comparatively well-studied aspect of seaweed biology,

and the state of knowledge pertaining a decade ago was comprehensively
reviewed by Liining (1990). In the present review we focus on studies that
endeavour to unravel patterns of colonization and genetic divergence at a
macrogeographic scale and on a geological time scale. In macroalgae, this
field of study has been dominated by a research group in the Netherlands at
Groningen founded by Professor C. van den Hoek. Their research initially
centred on physiological studies of numerous conspecific and congeneric
cultured isolates of red, green and brown seaweeds, and was innovative
in extending into molecular investigations as soon as techniques became
available.
A. PHYSIOLOGICALAPPROACHES
For a period of over two decades, physiological tolerances of algae have been
tested in an attempt to interpret present-day distributions (data on 60
species were reviewed by Breeman, 1988), and also to deduce biogeographic
histories of algal species from their thermal tolerances (Wiencke et al., 1994;
Bischoff-Bfismann and Wiencke, 1996). Van den Hoek, Breeman and co-
workers have examined the hypothesis that biogeographic boundaries can
be defined by experimentally examining the relationship between the
distributional extremes of a species and the extremes of temperatures
within which the species can survive or complete its life cycle. Two kinds of
boundaries have been defined: growth/reproduction boundaries (where a
species is not exposed every year to a sufficientlyhigh or low temperature for
growth and reproduction in the favourable season), and lethal boundaries
(where a species is exposed once in several years to a lethal temperature).
When ecotypic variation in temperature responses occurs, it relates mostly
to growth and reproduction boundaries, not to lethal boundaries (van den
Hoek et al., 1990).
Antarctic endemics have growth optima of 0-5°C, upper growth
temperatures of 5-10°C and upper survival temperatures of 11-17°C
(Wiencke et al., 1994). This is believed to be due to long periods of adaptation
(> 14 million years (Ma)) by endemics to Antarctic conditions. Wiencke et al.
(1994) postulate that the first adaptation is an increase in growth rate at low
temperatures, the next, which they suggest takes c. 3 Ma, being the loss of
ability to grow at _15-20°C. ITS data (described below) can now be applied to
evaluate such hypotheses erected on the basis of physiological data. As an
196 c . A . MAGGS and R. WATTIER
example, the brown seaweed Desmarestia viridis (synonym D. wilIii) has a

circumpolar northern hemisphere distribution and is also bipolar. Van den
Hock et al. (1990) suggested that, on the basis of a maximum reproductive
temperature for gametophytes from Japan 8°C higher than for North
Atlantic gametophytes, North Atlantic and North Pacific populations have
been genetically isolated for an extended period. Similarly, they suggested
that the crossing of the equator was likely to have been earlier than the
Pleistocene. However, van Oppen et al. (1993) found that sequences of
the 1073 bp ITS1, ITS2 and 5.8S regions were identical for isolates from
Helgoland (German North Sea) and Japan; Southern Hemisphere isolates
differed from them by only one substitution. They therefore proposed that
transequatorial passage has occurred recently, linked perhaps to Pleistocene
equatorial cooling, which was coupled with stronger winds and and more
upwelling, or perhaps may occur regularly in cooler deep water.
B. MOLECULAR APPROACHES: NUCLEAR MARKERS
Early molecular biogeographic studies in algae employed DNA-DNA

hybridization (Stam et al., 1988; Bot et al., 1989a,b). Although this technique
is rarely used today, it has some advantages over newer methods in
that it examines divergence between whole genomes. A small quantity of
single-stranded radioactive DNA (c. 450 bp long), tracer DNA, from one
geographic location of a species, is combined with a much larger amount of
unlabelled DNA, driver DNA, of the same morphological species from
another geographic location, or from a related species, under conditions
promoting reassociation into double-stranded hybrid DNA (heteroduplex).
Heteroduplexes can be expected to be less stable than homoduplexes,
because of higher mismatch between the base pairs, and to dissociate at
lower temperature, with the temperature difference being a measure of
the DNA sequence divergence between the two geographic populations
compared. Estimates of divergence times based on DNA-DNA
hybridization can only be tentative at best for a variety of reasons, partly
because rates can be calibrated only by using groups with a fossil record
(Olsen et al., 1987; Stam et al., 1988; Bot et al., 1989a,b). In the absence of a
fossil record for most seaweeds, vicariant events have to be be used to date
divergence times, e.g. the postulated time of the Miocene closure of the
Tethys Ocean in the Middle East (14-20 Ma ago) for Atlantic and Pacific
populations of the widespread green seaweed Cladophora aIbida (Bot et al.,
1989a,b). This yielded an average rate of sequence change per Ma of
0.14-0.21% which seemed realistic in comparison to estimated rates of
change of 0.13% (in higher primates) to 0.66% (in Drosophila). Analysis of
ITS and 18S sequences (see next paragraph) supported the findings of the
DNA-DNA hybridization study, providing evidence of diversification in this
clade of Cladophora in the mid-Miocene (Bakker et al., 1995).
The great majority of phylogeographic studies in seaweeds have utilized

ITS sequences, sometimes in association with higher resolution markers
such as RAPD (e.g. van Oppen et al., 1995a,b), which have also been
used successfully as markers for geographical studies (e.g. post-glacial
recolonization in the red alga Palmaria mollis; Lindstrom et al., 1997;
relationships between island populations of Digenea simplex; van Oppen et al.,
1996b). Nuclear ribosomal RNA genes are organized in tandemly repeated
units. The copy number of these units varies from a few hundred to thousands
in plants (Rogers and Bendich, 1987). Each repeat unit contains a sequence
coding for mature 18S, 5.8S and 26S rRNA and transcribed spacers (ITS1 and
ITS2), and is separated from the next repeat unit by an intergenic spacer. The
tandemly arrayed rDNA genes and their spacers are widely used in population
biology, molecular ecology and taxonomy (Hoelzel and Dover, 1991; Arise,
1994). Studies over the past 10 years have revealed that patterns of rDNA
variation are affected both by the "external" forces of selection and drift and
by a variety of "internal" homogenizing processes consequential on a variety
of genomic turnover mechanisms, principal among which are unequal cross-
over, gene conversion and slippage. There is increasing awareness that
the internal mutation and turnover forces acting on DNA, in particular on
repetitive DNA, can dramatically affect the long-term evolutionary histories
of such sequences and hence their usefulness in molecular ecology and
molecular taxonomy (Gray and Jeffreys, 1991; Valdes et al., 1993).
The popularity of the ITS region can be attributed to the relatively high
rate of nucleotide substitution in the transcribed spacer permitting the
systematic comparisons of relatively recently diverged taxa. In addition, the
ITS region can be readily PCR-amplified and sequenced with conserved
primers positioned in the cistronic regions. This repeated gene family
undergoes rapid concerted evolution. As a consequence, the ITS region is
generally homogeneous, or nearly so, within a genome, and a single ITS
sequence can characterize an individual. Furthermore, concerted evolution
and sexual recombination tend to promote ITS region uniformity within
interbreeding populations. It should be noted, however, that recent studies
have detected intra-individual ITS variation in two groups of algae (Fucus
species: Serrfio et al., 1999; Caulerpa racemosa and C. taxifolia: Fam/t et al.,
2000; Jousson et al., 2000) so great as to obscure the phylogeographic signal.
This phenomenon is likely to be more common and ITS studies need to be
carefully approached with this in mind.
The most detailed information on intraspecific ITS variation in seaweeds
has been obtained for species in four green algal genera, Cladophora (e.g.
Bakker et al., 1995), Cladophoropsis (e.g. Kooistra et al., 1992), Enteromorpha
(Blomster et al., 1999, 2000; Tan et al., 1999) and Caulerpa (Pillmann et al.,
1997; Olsen et al., 1998; Jousson et al., 1998, 2000; Famg et al., 2000). Very
different biogeographic histories have been recovered among these green
algal genera. Cladophora and Cladophoropsis are relatively ancient, with high
levels of ITS divergence of up to 15% within morphological species. The large

amount of sequence variation resulted in strong phylogeographic signal. Both
old vicariant events and recent dispersal have been detected (Kooistra et al.,
1992; Bakker et al., 1995). In Caulerpa, by contrast, intraspecific variation
provides little phylogeographic resolution. Phylogeographic relationships in
Caulerpa racemosa were obscured by intra-individual ITS variation (Famg
et al., 2000), and in Mediterranean Caulerpa taxifolia populations the ITS
sequences were monomorphic, possibly implying a severe genetic bottleneck
related to its introduction (Jousson et al., 1998, 2000). In Enteromorpha,
virtually no geographical genetic structure was detected in any of the three
species E. compressa, E. muscoides and E. intestinalis, even on inter-oceanic
scale for E. intestinalis (Blomster et al., 1998, 1999, 2000). This correlates with
their ubiquitous presence as fouling organisms and suggests high gene flow, or
possibly, in the case of E. rnuscoides, rapid and transient recolonization
of coastlines. ITS sequence analysis has proven to be most valuable when
relatively deep phylogeographic structure is being sought. More recent events
can better be detected using higher resolution nuclear markers, such as the
intergenic spacer (IGS) regions.
IGS regions between nuclear rRNA genes are more variable in length and
nucleotide sequence than the adjacent genes. This variation, the occurrence
of hundreds of rRNA gene repeats in each haploid genome, and the ease
with which adjacent conserved regions within the genes can be used as
priming sites for PCR amplification have favoured the use of RFLP and
sequence analysis of IGS regions for resolving interspecific and intraspecific
relationships. Intraspecific variation in IGS length has been reported in
many higher plants. IGS regions from a diverse variety of organisms are
known to be more variable in specific regions that contain arrays of short
(typically less than 0.3 kb) tandemly repeated DNA sequences. Some of
these repeats have been implicated in the regulation of transcription of the
adjoining rRNA genes and some as sites of chromosome crossing over. IGS
sequence data have not been employed in phylogeographic studies of algae,
but length variations and RFLP data were informative for 10 widely disjunct
populations of the green seaweed Acrosiphonia arcta (van Oppen et al.,
1994). Multiple isolates obtained from some sites (e.g. eight from Disko
Island, Greenland) were always identical in length and differed by only 0-1
restriction site, whereas between sites the IGS length varied from 3.5 to
4.2 kb and there were several phylogeographically informative restriction
site mutations. All North Atlantic isolates except Disko were identical
in IGS length and closely related, suggesting recent colonization or
recolonization. Rooting of the dataset, based on analyses of ITS alignments
with related species, suggested that A. arcta has a Southern Hemisphere
distribution, the Antarctic Peninsula having been recolonized post-glacially
from South America. The IGS region appeared to be evolving 5-10 times
faster than the ITS.
Choshi
95O
219
Shimoda Oshoro
1177 1024
: 789%
~ ' D 912 617
5~ 564 56~17~
876f'%
v.- ~ 222 954 ~ 872 ~ r%~60
J __ [ I i I l I I I ~#" • 543
HOKKAIDO
-1159
NC, NZ, ~!~ Akkeshi

Montery i~
Europe,
Nova Scotia
Fig. 2. Minimum spanning tree of Polysiphonia harveyi rbcL haplotypes showing

invasive haplotypes as shaded symbols, Japanese haplotypes as solid symbols, and
hypothetical "missing" haplotypes X, Y and Z, with positions of nucleotide changes
between haplotypes. NC, North Carolina, USA; NZ, New Zealand. (Reproduced
with permission from Mclvor et al. (2001).)
C. MOLECULARAPPROACHES:PLASTIDMARKERS
Plastid markers can be very valuable in phylogeographic studies because of

their broad comparability with widely used mitochondrial markers (Avise,
2000). The gene for the large subunit of rubisco, rbcL, is suitable for
phylogeographic studies in green plants only at the tribal and family levels
(Xiang et al., 1998), but in red algae it shows some intrapopulational as well
as extensive interspecific variability (Nam et al., 2000). A phylogeographic
study of the invasive red alga Polysiphonia harveyi using rbcL sequences
employed the minimum spanning tree methodology developed for
mitochondrial markers (Fig. 2) to show that multiple conspecific invasions
have occurred (McIvor et al., 2001). The four most divergent of six rbcL
haplotypes were observed in Japanese samples from Hokkaido and south-
central Honshu, which are linked by hypothetical "missing" haplotypes that
may occur in northern Honshu. These data are consistent with Japan being
the centre of diversity for and origin of P. harveyi. P. harveyi has been
introduced at least twice into the North Atlantic from presumed different
source populations in Japan or nearby. By contrast, Goff et al. (1992) traced
broad patterns of species invasion in the green alga Codium fragile ssp.
tomentosoides using cpDNA RFLPs, but found no intrapopulational or
interpopulation variation in these markers.
200 C. A. MAGGSand R. WATTIER
VI. CONCLUSIONS AND PERSPECTIVES
John van der Meer, in his 1987 review "Using genetic markers in phycological
research', pointed out that:
"In the coming years we will see a dramatic increase in the use of molecular
genetic techniques (markers at the DNA level), and by the time another decade
passes, it will be hard to imagine we ever tried to examine certain biological
problems without them."
This was far-sighted judging from the perspective provided by 14 years (and
another century!). All of the most useful markers (microsatellites: Wattier et
al., 1997), AFLPs (Donaldson et al., 1998, 2000), ISSRs (Vis, 1999) and
SSCPs (Zuccarello et al., 1999a), except for RAPDs (Patwary et al., 1993)
have been introduced into macroalgal studies only in the last 4 years.
Despite this late start, it is clear from the most recent international
phycological meetings (European Phycological Congress at Montecatini,
1999, and the Phycological Society of America at San Diego, 2000) that
phycologists are keen to be part of the molecular ecology "revolution", and
are not at all conservative in their views or approaches.
Because, as we have noted, single-locus/co-dominant nuclear markers
(microsatellites) are the key markers for population genetics but are
particularly difficult to develop for seaweeds, one major challenge of the
next 10 years will be to develop alternative highly polymorphic single-locus/
co-dominant nuclear markers as a substitute for microsatellite loci. Many
other markers have been reported for various groups of organisms. One
class is similar to microsatellites but is based on minisatellites (Deka et
al., 1994). Alternatively, variation of single copy nuclear DNA (scnDNA)
can be detected by Southern hybridization (Quinn and White, 1987), PCR-
RFLP (Karl and Avise, 1993), PCR-SSCP (Bagley et al., 1997),
heteroduplex analysis (Highsmith et al., 1999) and D G G E (Lessa, 1992).
Availability of macroalgal DNA sequences in electronic databases (i.e.
GenBank, EMBL) is still limited and the number of single-copy nuclear
DNA sequences published for algae is small, not to say extremely small, and
developing any of the above markers will probably imply the construction of
specific genomic libraries, projects that are already underway for several
species of macroalgae.
ACKNOWLEDGEMENTS
Part of this study was supported by EC FAIR research contract PL97-3828,

in the form of a post-doctoral fellowship for RW. We thank Jim Provan for
helpful discussions.
REFERENCES
Abe, T., Masuda, M., Suzuki, T. and Suzuki, M. (1999). Chemical races in the
red alga Laurencia nipponica (Rhodomelaceae, Ceramiales. Phycological
Research 47, 87-95.
Adams, M., Urquhart, A., Kimpton, C. and Gill, P. (1993). The human DllS554
locus: four distinct families of repeat pattern alleles at one locus. Human
Molecular Genetics 2, 1373-1376.
Akkaya, M. S., Bhagwat, A. A. and Cregan, P. B. (1992). Length polymorphisms of
simple sequence repeat DNA in soybean. Genetics 132, 1131-1139.
Alberto, F. R., Santos, R. and Leitfio, J. M. (1997). DNA extraction and RAPD
markers to assess the genetic similarity among Gelidium sesquipedale
(Rhodophyta) populations. Journal of Phycology 33, 706-710.
Andersen, R. A. (1992). Diversity of eukaryotic algae. Biodiversity and Conservation
1, 267-292.
Avise, J. C. (1994). "Molecular Markers, Natural History and Evolution". Chapman
& Hall, New York.
Avise, J. C. (2000). "Phylogeography: The History anf Formation of Species".
Harvard University Press, Cambridge, MA.
Avise, J. C. and Johns, G. C. (1999). Proposal for a standardized temporal scheme
of biological classification for extant species. Proceedings of the National
Academy of Sciences of the USA 96, 7358-7363.
Bachmann, K. (1994). Molecular markers in plant ecology (Tansley review no. 63).
New Phytologist 126, 403-418.
Bachmann, K. (1998). Species as units of biodiversity: an outdated concept. Theory of
Biosciences 117, 213-230.
Bagley, M. J., Medrano, J. F. and Gall, G. A. E. (1997). Polymorphic molecular
markers from anonymous nuclear DNA for genetic analysis of populations.
Molecular Ecology 6, 309-320.
Bakker, F. T., Olsen, J. L. and Stam, W. T. (1995). Evolution of nuclear rDNA ITS
sequences in the Cladophora albida/setqcea clade (Chlorophyta). Journal of
Molecular Evolution 40, 640-651.
Bakker, F. T., Olsen, J. L., Stare, W. T. and van den Hoek, C. (1992). Nuclear rDNA
internal transcribed spacer regions (ITS1 and ITS2) define discrete
biogeographic groups in Cladophora albida (Chlorophyta). Journal of
Phycology 28, 839-845.
Billot, C., Rousval, S., Estoup, A., Epplen, J. T., Saumitou-Laprade, P., Valero, M.
and Kloareg, B. (1998). Isolation and characterization of microsatellite DNA
in the nuclear genome of the brown alga Laminaria digitata (Phaeophyta).
Billot, C., Boury, S., Benet, H. and Kloareg, B. (1999). Development of RAPD
markers for parentage analysis in Laminaria digitata. Botanica Marina 42,
307-314.
Bird, C. J. and Rice, E. L. (1990). Recent approaches to the taxonomy of the
Gracilariaceae (Gracilariales, Rhodophyta) and the Gracilaria verrucosa
problem. Hydrobiologia 204/205, 111-118.
Bischoff-Basmann, B. and Wiencke, C. (1996). Temperature requirements for
growth and survival of Antarctic Rhodophyta. Journal of Phycology 32,
525-535.
Blomster, J., Maggs, C. A. and Stanhope, M. J. (1998). Molecular and morphological
analysis of Enteromorpha intestinalis and E. compressa (Chlorophyta) in the
British Isles. Journal of Phycology 34, 319-340.
Blomster, J., Maggs, C. A. and Stanhope, M. J. (1999). Extensive intraspecific

morphological variation in Enteromorpha muscoides (Chlorophyta) revealed
by molecular analysis. Journal of Phycology 35, 575-586.
Blomster, J., Hoey, E. M., Maggs, C. A. and Stanhope, M. J. (2000). Species-specific
oligonucleotide probes for macroalgae: molecular discrimination of two
marine fouling species of Enteromorpha (Ulvophyceae). Molecular Ecology 9,
177-186.
Bot, P. V. M., Holton, R. W., Stare, W. T. and van den Hoek, C. (1989a). Molecular
divergence between North Atlantic and Indo-West Pacific Cladophora albida
(Cladophorales: Chlorophyta) isolates as indicated by DNA-D NA hybridization.
Marine Biology 102, 307-313.
Bot, P. V. M., Stam, W. T., Boele-Bos, S. A., van den Hock, C. and van Delden, W.
(1989b). Biogeographic studies in three North Atlantic species of Cladophora
albida (Cladophorales: Chlorophyta) using DNA-DNA hybridization.
Phycologia 28, 159-168.
Brawley, S. H. and Johnston, L. E. (1992). Gametogenesis, gametes and zygotes: an
ecological perspective on sexual reproduction in the algae. British Phycological
Journal 27, 233-252.
Breeman, A. M. (1988). Relative importance of temperature and other factors
in determining geographic boundaries in seaweeds: experimental and
phenological evidence. Helgol~inderMeersuntersuchungen 42, 199-214.
Brodie, J., Hayes, P. K., Barker, G. L. and Irvine, L.M. (1996). Molecular and
morphological characters distinguishing two Porphyra species (Rhodophyta:
Bangiophycidae). European Journal of Phycology 31, 303-308.
Bruford, M. W. and Wayne, R. K. (1993). Microsatellites and their application to
population genetic studies. Current Opinion in Genetics and Development 3,
939-943.
Callen, D. F., Thompson, A. D., Shen, Y., Phillips, H. A., Richards, R. I., Mulley, J.
C. and Sutherland, G. R. (1993). Incidence and origin of "null" alleles in
the (AC)n microsatellite markers. American Journal of Human Genetics 52,
922-927.
Cantino, P. D. (2000). Phylogenetic nomenclature: addressing some concerns. Taxon
49, 85-93.
Cavalier-Smith, T. (1998). A revised six-kingdom system of life. Biological Reviews of
the Cambridge Philosophical Society 73, 203-266.
Chopin, T., Bird, C. J., Murphy, C. A., Osborne, J. A., Patwary, M. U. and Floc'h, J.-
Y. (1996). A molecular investigation of polymorphism in the North Atlantic
red alga Chondrus crispus (Gigartinales). Phycological Research 44, 69-80.
Claridge, M. F., Dawah, H. A. and Wilson, M. R. (1997). Practical approaches to
species concepts for living organisms. In "The Units of Biodiversity: Species in
Practice" (M. F. Claridge, H. A. Dawah and M. R. Wilson, eds), pp. 1-15.
Systematics Association Special Volume, Chapman and Hall, London.
Coat, G., Dion, P., Noailles, M.-C., de Reviers, B., Fontaine, J.-M., Berger-Perrot, Y.
and Loiseaux-de Go,r, S. (1998). Ulva amoricana (Ulvales, Chlorophyta) from
the coasts of Brittany (France). II Nuclear rDNA ITS sequence analysis.
European Journal of Phycology 33, 73-80.
Corliss, J. O. (1994). An interim utilitarian ("user-friendly") hierarchical
classification and characterization of the protists. Acta Protozoologica 33,
1-51.
Coyer, J. A., Robertson, D. L. and Alberte, R. S. (1994). Genetic variability within
a population and between diploid/haploid tissue of Macrocystis pyrifera
(Phaeophyta). Journal of Phycology 30, 545-552.
Coyer, A. C., Robertson, D. L. and Alberte, R. S. (1995). Genetic variability and

parentage in Macrocystis pyrifera (Phaeophyceae) using multi-locus DNA
fingerprinting. Journal of Phycology 31, 819-823.
Coyer, J. A., Olsen, J. L. and Stare, W. T. (1997). Genetic variability and spatial
separation in the sea palm kelp Postelsia palmaeformis (Phaeophyceae) as
assessed with M 13 fingerprints and RAPDs. Journal of Phycology 33, 561-568.
Coyer, J. A., Peters, A. F., Olsen, J. L. and Stare, W. T. (1999). Population structure
of Fucus sp. in the Skagerrak-Kattegat (Baltic Sea) as determined by
microsatellites. In "Second European Phycological Congress (EPC2)", pp. 77.
Montecatini, Italy.
Cracraft, J. (1983). Species concepts and speciation analysis. In "Current
Ornithology", Vol. 1 (R. Johnson, ed.), pp. 159-187. Plenum Press, New York.
Cracraft, J. (1997). Species concepts in systematics and conservation biology-an
ornithological viewpoint. In "The Units of Biodiversity: Species in Practice"
(M. F. Claridge, H. A. Dawah and M. R. Wilson, eds), pp. 325-340.
Systematics Association Special Volume, Chapman and Hall, London.
Crozier, R. H., Kaufmann, B., Carew, M. E. and Crozier, Y. C. (1999). Mutability of
microsatellites developed for the ant Camponotus consobrinus. Molecular
Ecology 8, 271-276.
Cruzan, M. B. (1998). Genetic markers in plant evolutionary ecology. Ecology 79,
400-412.
Darwin, C. (1859). "On the Origin of Species by Means of Natural Selection". John
Murray & Co., London.
Deka, R., DeCroo, S., Jin, L., McGarvey, S. T., Rothhammer, F., Ferell, R. E. and
Chakraborty, R. (1994). Population genetic characteristics of DIS80 locus in
seven human populations. Human Genetics 94, 252-258.
Destombe, C. and Douglas, S. E. (1991). Rubisco spacer sequence divergence in
the rhodophyte alga Gracilaria verrucosa and closely related species. Current
Genetics 19, 395-398.
Dion, P., de Reviers, B. and Coat, G. (1998). Ulva armoricana nov. sp. (Ulvales,
Chlorophyta) from the coasts of Brittany (France). I. Morphological
identification. European Journal of Phycology 33, 81-86.
Dixon, P. S. and Irvine, L. M. (1977). "Seaweeds of the British Isles", Vol. 1.
"Rhodophyta. Nemaliales, Gelidiales". British Museum (Natural History),
London.
Donaldson, S. L., Chopin, T. and Saunders, G. W. (1998). Amplified fragment length
polymorphism (AFLP) as a source of genetic markers for red algae. Journal of
Applied Phycology 10, 365-370.
Donaldson, S. L., Chopin, T. and Saunders, G. W. (2000). An assessment of the
AFLP method for investigating population structure in the red alga Chondrus
crispus. Journal of Applied Phycology 12, 25-35.
Druehl, L. D. and Saunders, G. W. (1992). Molecular explorations in kelp evolution.
In "Progress in Phycological Research", Vol. 8 (F. E. Round and D. J.
Chapman, eds), pp. 47-83. Biopress Ltd, Bristol.
Ede, A. J. and Crawford, A. M. (1995). Mutations in the sequence flanking the
microsatellite at the KAP8 locus prevent the amplification of some alleles.
Animal Genetics 26, 43-44.
Engel, C. R., Wattier, R., Destombe, C. and Valero, M. (1999). Performance of non-
motile male gametes in the sea: analysis of paternity and fertilisation success in
a natural population of a red seaweed, Gracilaria gracilis. Proceedings of the
Royal Society of London, Series B 266, 1879-1886.
Estoup, A., Taillez, C., Solignac, M. and Cornuet, J. M. (1995). Size homoplasy and
mutational processes of interrupted microsatellites in two bee species, Apis
204 c.A. MAGGS and R. WATTIER
mellifera and Bombus terrestris (Apidae). Molecular Biology and Evolution 12,
1074-1084.
Fame, P., Olsen, J. L., Stare, W.T. and Procaccini, G. (2000). High levels of intra-
and inter-individual polymorphism in the rDNA ITS1 of Caulerpa racemosa
(Chlorophyta). European Journal of Phycology 35, 349-356.
Gargiulo, G. M., de Masi, F., Genovese, G. and Tripodi, G. (1994). Karyology
and effects of temperature and photoperiod on the life-cycle of
Porphyra leucosticta Thuret in Le Jolis (Bangiales, Rhodophyta) from the
Mediterranean Sea. Japanese Journal of Phycology 42, 271-278.
Godin, J., Destombe, C. and Maggs, C. A. (1993). Unusual chromosome number of
Gracilaria verrucosa (Gracilariales, Rhodophyta) in the Cape Gris-Nez area,
Northern France. Phycologia 32, 291-294.
Goff, L. J. and Coleman, A. W. (1988). The use of plastid DNA restriction
endonuclease patterns in delineating red algal species and populations.
Journal of Phycology 24, 357-368.
Goff, L. J. and Moon, D. A. (1993). PCR amplification of nuclear and plastid genes
from algal herbarium specimens and algal spores. Journal of Phycology 29,
381-384.
Goff, L. J., Liddle, L., Silva, P. C., Voytek, M. and Coleman, A. W. (1992). Tracing
species invasion in Codium, a siphonous green alga, using molecular tools.
American Journal of Botany 79, 1279-1285.
Goff, L. J., Moon, D. A. and Coleman, A. W. (1994). Molecular delineation of
species and species relationships in the red algal agarophytes Gracilariopsis
and Gracilaria (Gracilariales). Journal of Phycology 30, 521-537.
Gray, I. C. and Jeffreys, A. J. (1991). Evolutionary transience of hypervariable
minisatellites in man and the primates. Proceedings of the Royal Society
London, SeriesB 243, 241-253.
Grimaldi, M. C. and Crouau-Roy, B. (1997). Microsatellite allelic homoplasy due to
variable flanking sequences. Journal of Molecular Evolution 44, 336-340.
Guiry, M. D. (1992). Species concepts in marine red algae. In "Progress in
Phycological Research", Vol. 8 (F. E. Round and D. J. Chapman, eds), pp.
251-278. Biopress Ltd, Bristol.
Hadrys, H., Balick, M. and Sohierwater, B. (i992). Applications of random amplified
polymorphic DNA (RAPD) in molecular ecology. MolecularEcology 1, 55-63.
Haig, S. M. (1998). Molecular contributions to conservation. Ecology 79, 413-425.
Hardy, F. G., Scott, G. W., Sisson, P. R., Lightfoot, N. F. and Mulyadhi (1998).
Pyrolysis Mass Spectrometry as a technique for studying inter- and
intraspecific relationships in the genus Fucus. Journal of the Marine Biological
Association of the UK 78, 35-42.
Harvey, W. H. (1846-1851). "Phycologia Britannica". Reeve & Co., London.
Hawkes, M. W. (1990). Reproductive strategies. In "Biology of the Red Algae" (K.
M. Cole and R. G. Sheath, eds), pp. 455-476. Cambridge University Press,
Cambridge.
Highsmith, W. E., Jin, Q., Nataraj, A. J., O'Connor, J. M., Burland, V. D., Baunosis,
W. R., Curtis, F. P., Kusukawa, N. and Garner, M. (1999). Use of
DNA toolbox for the characterization of mutation scanning methods. I:
Construction of the toolbox and evaluation of heteroduplex analysis.
Electrophoresis 20, 1186-1194.
Hoelzel, A. R. and Dover, G. A. (1991). Genetic differentiation between sympatric
killer whale populations. Heredity 66, 191-195.
Hong, Y.-K., Kim, H. G., Polne-Fuller, M. and Gibor, A. (1995). DNA extraction
conditions from Porphyraperforata using LiC1.Journal of Applied Phycology 7,
101-107.
Hong, Y.-K., Sohn, C. H., Lee, K. W. and Kim, H. G. (1997). Nucleic acid extraction
from seaweed tissues for polymerase chain reaction. Journal of Marine
BiotechnoIogy 5, 95-99.
Hughes, J. S. and Otto, S. P. (1999). Ecology and evolution of biphasic life cycles.
American Naturalist 154, 306-320.
Hull, D. L. (1997). The ideal species concept - and why we can't get it. In "The Units
of Biodiversity: Species in Practice" (M. F. Claridge, H. A. Dawah and M. R.
Wilson, eds), pp. 357-380. Systematics Association Special Volume, Chapman
and Hall, London.
Isabel, N., Beaulieu, J., Thieriault, P. and Bousquet, J. (1999). Direct evidence for
biased gene diversity estimates from dominant random amplified polymorphic
DNA (RAPD) fingerprints. Molecular Ecology 8, 47%483.
Ishibashi, Y., Saitoh, T., Abe, S. and Yoshida, M. C. (1996). Null microsatellite allele
due to nucleotide sequence variation in the grey-sided vole Clethronomys
rufocanus. Molecular Ecology 5, 589-590.
Jensen, A. (1993). Present and future needs for algae and algal products.
Hydrobiologia 261, 15-23.
Jin, H.-J., Kim, J.-H., Sohn, C. H., deWreede, R. E., Choi, T.-J., Towers, G. H. N.,
Hudson, J. B. and Hong, Y. K. (1997). Inhibition of Taq DNA polymerase
by seaweed extracts from British Columbia, Canada and Korea. Journal of
Applied Phycology 9, 383-388.
John, D. M. and Maggs, C. A. (1997). Species problems in eukaryotic algae: a
modern perspective. In "The Units of Biodiversity: Species in Practice" (M. F.
Claridge, H. A. Dawah and M. R. Wilson, eds), pp. 83-107. Systematics
Association Special Volume, Chapman and Hall, London.
Kamiya, M., West J. A., King, R. J., Zuccarello, G. C., Tanaka, J. and Hara, Y.
(1998). Evolutionary divergence in the red algae Caloglossa leprieurii and C.
apomeiotica. Journal of Phycology 34, 361-370.
Kapraun, D. F., Dutcher, J. A. and Freshwater, D. W. (1993). Quantification
and characterization of nuclear genomes in commercial red seaweeds-
Gracilariales and Gelidiales. Hydrobiologia 261, 679-688.
Karl, S. A. and Avise, J. C. (1993). PCR-based assays of Mendelian polymorphisms
from anonymous single-copy nuclear DNA: techniques and applications for
population gentics. Molecular Biology and Evolution 10, 342-361.
Karp, A., Seberg, O. and Bujatti, M. (1996). Molecular techniques in the assessment
of botanical diversity. Annals of Botany 78, 143-149.
Kemp, S. J., Hishida, O., Wambugu, J., Rink, A., Longeri, M. L., Ma, R. Z., Da, Y.,
Lewin, H. A., Barendse, W. and Teale, A. J. (1995). A panel of polymorphic
bovine, ovine and caprine microsatellite markers.Animal Genetics 26, 299-306.
Kim, G. H. (1997). Gamete recognition and signal transduction during fertilization
in red alga. Algae 12, 263-268.
Kim, G. H., Lee, I. K. and Fritz, L, (1996). Cell-cell recognition during fertilization
in a red alga, Antithamnion sparsum (Ceramiaceae, Rhodophyta). Plant Cell
Physiology 37, 621-628.
Kitade, Y., Yamazaki, S. and Saga, N. (1996). A method for extraction of high
molecular weight DNA from the macroalga Porphyra yezoensis. Journal of
Kooistra, W. H. C. F., Stare, W. T., Olsen, J. L. and van den Hock, C. (1992).
Biogeography of the green alga Cladophoropsis membranacea (Chlorophyta)
based on nuclear rDNA ITS sequences. Journal of Phycology 28, 660-668.
Kudo, T. and Masuda, M. (1986). A taxonomic study of Polysiphonia japonica
Harvey and P. akkeshiensis Segi (Rhodophyta). Japanese Journal of Phycology
34, 293-310.
Kusumo, H. T. and Druehl, L. D. (2000). Variability over space and time in the
genetic structure of the winged kelp Alaria marginata. Marine Biology 136,
397-407.
Kwiatrowski, D. J., Henske, E. P., Weimer, K., Ozelius, L., Gusella, J. F. and Haines,
J. (1992). Construction of a GT polymorphism map of human 9q. Genomics
12, 229-240.
Le Gal, Y., Asensi, A., Marie, D. and Kloareg, B. (1996). Parthenogenesis and
apospory in the Laminariales: a flow cytometry analysis. European Journal of
Phycology 31, 369-380
Lehman, T., Hawley, W. A., Kamau, L., Fontenille, D. and Simard, F. H. (1996).
Genetic differentiation of Anopheles gambiae populations from East and West
Africa: comparison of microsatellite and allozyme loci. Heredity 77, 192-208.
Lessa, E. P. (1992). Rapid surveying of DNA sequence variation in natural
populations. MolecularBiology and Evolution 9, 323-330.
Lessa, E. P. and Applebaum, G. (1993). Screening techniques for detecting allelic
variation in DNA sequence. MolecularEcology 2, 119-129.
Lewis, R. J. and Neushul, M. (1995). Intergeneric hybridization among five genera of
the family Lessoniaceae (Phaeophyceae) and evidence for polyploidy in a
fertile Pelagophycus x Macrocystis hybrid. Journal of Phycology 31,1012-1017.
Lindstrom, S. C., Olsen, J. L. and Stare, W. T. (1997). Postglacial recolonization and
the biogeography of Palmaria mollis (Rhodophyta) along the Northeast
Pacific coast. Canadian Journal of Botany 75, 1887-1896.
Liptack, M. K. and Druehl, L. D. (2000). Molecular evidence for an interfamilial
laminarialean cross. European Journal of Phycology 35, 135-142.
Litt, M. and Luty, J. A. (1989). A hypervariable microsatellite revealed by in vitro
amplification of a dinucleotide repeat within the cardiac muscle actin gene.
American Journal of Human Genetics 44, 397-401.
Littler, D. S. and Littler, M. M. (1992). Systematics of Avrainvillea (Bryopsidales,
Chlorophyta) in the tropical western Atlantic. Phycologia 31, 375-418.
Loreau, M. and Olivieri, I. (1999). Diversitas: an international programme of
biodiversity science. Trends in Ecology and Evolution 14, 2-3.
Lubchenco, J. and Cubit, J. (1980). Heteromorphic life histories of certain marine
algae as adaptations to variations in herbivory. Ecology 61, 676-687.
Ltining, K. (1990). "Seaweeds: their Environment, Biogeography and Eco-
physiology". Wiley-Interscience, New York.
Luo, H., Morchen, M., Engel, C. R., Destombe, C., Epplen, J. T., Epplen, C.,
Saumitou-Laprade, P. and Valero, M. (1999). Characterization of micro-
satellite markers in the red alga Gracilariagracilis.MolecularEcology 8, 700-702.
Maggs, C. A. (1988). Intraspecific life history variability in the Florideophycidae
(Rhodophyta). Botanica Marina 31, 465-490.
Maggs, C. A. (1998). Life history variation in Dasya ocellata (Dasyaceae,
Rhodophyta). Phycologia 37, 100-105.
Maggs, C. A., Douglas, S. E., Fenety, J. and Bird, C. J. (1992). A molecular
and morphological analysis of the Gymnogongrus devoniensis (Rhodophyta)
complex in the North Atlantic. Journal of Phycology, 28, 214-232.
Maier, I. (1995). Brown algal pheromones. In "Progress in Phycological Research",
Vol. 11 (F. E. Round and D. J. Chapman, eds), pp. 51-102. Biopress Ltd,
Bristol.
Mallet, J. (1995). A species definition for the modern synthesis. Trends in Ecology
and Evolution 10, 294-299.
Manhart, J. R. and McCourt, R. M. (1992). Molecular data and species concepts in
the algae. Journal of PhycoIogy 28, 730-737.
Margulis, L. (1990). "Handbook of Protoctista". Jones and Bartlett, Boston, MA.

Martinez, E. A., Cardenas, L., Figueroa, C., Vidal, R. and Billot, C. (2000). Inter-
family use of microsatellite markers in Laminariales: the experience between
Laminaria digitata and Lessonia spp. Journal of Phycology 28(Suppl), 46.
Masuda, M., Abe, T., Sato, S., Suzuki, T. and Suzuki, M. (1997). Diversity
of halogenated secondary metabolites in the red ala Laurencia nipponica
(Rhodomelaceae, Ceramiales). Journal of Phycology 33, 196-208.
Mathieson, A. C., Norton, T. A. and Neushal, M. (1981). The taxonomic implications
of genetic and environmentally induced variations in seaweed morphology.
Botanical Review 47, 313-347.
Mayden, R. L. (1997). A hierarchy of species concepts: the denouement in the saga
of the species problem. In "The Units of Biodiversity: Species in Practice" (M.
F. Claridge, H. A. Dawah and M. R. Wilson, eds), pp. 381-424. Systematics
Association Special Volume, Chapman and Hall, London.
Mayr, E. (1963). "Animal Species and Evolution". Harvard University Press,
Cambridge, MA.
McIvor, L., Maggs, C. A., Provan, J. and Stanhope, M.J. (2001). rbcL sequences
reveal multiple cryptic introductions of the Japanese red alga Polysiphonia
harveyi. Molecular Ecology 10, 911-919.
Miller, K. A., Olsen, J. L. and Stam, W. T. (2000). Genetic divergence correlates
with morphological and ecological subdivision in the deep-water elk kelp,
Pelagophycusporra (Phaeophyceae). Journal of Phycology 36, 862-870.
Mfiller, D. G. (1967). Generationswechsel, Kernphasenwechsel und Sexualitfit der
Braunalge Ectocarpus siliculosus im Kulturversuch. Planta 75, 39-54.
Murray, V., Monchawin, C. and England, P. R. (1993). The determination of the
sequences present in the shadow bands of a dinucleotide repeat PCR. Nucleic
Acids Research 21, 2395-2398.
Nam, K. W., Maggs, C. A., McIvor, L. and Stanhope, M. J. (2000). Taxonomy and
phylogeny of Osmundea (Rhodomelaceae, Rhodophyta) in Atlantic Europe.
Olsen, J. L. (1990). Nucleic acids in algal systematics. Journal of Phycology 26,
209-214.
Olseu, J. L. and Stare, W. T. (1999). Genetic neighbourhood size in Ascophyllum
nodosum (Fucales). In "Second European Phycological Congress (EPC2)", p.
76. Montecatini, Italy.
Olsen, J. L., Stare, W.T. , Bot, P. V. M. and van den Hoek, C. (1987). Single
copy DNA-DNA hybridization studies in Pacific and Caribbean isolates
of Dictyosphaeria cavernosa (Chlorophyta) indicate a long divergence.
Helgoliinder Meeresuntersuchungen 4, 377-383.
Olsen, J. L., Valero, M., Meusnier, I., Boele-Bos, S. and Stare, W.T. (1998).
Mediterranean CauIerpa taxifolia and C. mexicana (Chlorophyta) are not
conspecific. Journal of Phycology 34, 850-856.
Orita, M., Suzuki, Y., Seriya, T. and Hayashi, K. (1989). Rapid and sensitive
detection of point mutations and DNA polymorphisms using the polymerase
chain-reaction. Genomics 5, 874-879.
Ouborg, N. J., Piquot, Y. and van Groenendael, J. M. (1999). Population genetics,
molecular markers and the study of dispersal in plants. Journal of Ecology 87,
551-568.
Paetkau, D. and Strobeck, C. (1995). The molecular basis and evolutionary history of
microsatellite null alleles in bears. Molecular Ecology 4, 519-520.
Pakker, H., Breeman, A. M., Prud'homme van Reine, W. F., van Oppen, M. J. H.
and van den Hoek, C. (1996). Temperature responses of tropical to warm-
temperate Atlantic seaweeds. I. Absence of ecotypic differentiation in amphi-
Atlantic tropical-Canary Islands species. European Journal of Phycology 31,

123-132.
Parker, P. G., Snow, A. A., Schug, M. D., Booton, G. C. and Fuerst, P. A. (1998).
What molecules can tell us about populations: choosing and using a molecular
marker. Ecology 79, 361-382.
Parsons, T., Maggs, C. A. and Douglas, S. E. (1990). Plastid DNA restriction analysis
links the heteromorphic phases of an apomictic red algal life history. Journal of
PhycoIogy 26, 495-500.
Patwary, M. U. and van der Meer, J. P. (1994). Application of RAPD markers in
an examination of heterosis in Gelidium vagum (Rhodophyta). Journal of
Patwary, M. U., MacKay, R. M. and van der Meer, J. P. (1993). Revealing genetic
markers in Gelidium vagum (Rhodophyta) through the random amplified
polymorphic DNA (RAPD) technique.Journal of Phycology 29, 216-222.
Pedersen, P. M. (1981). Phaeophyta: life histories. In "The Biology of Seaweeds" (C.
S. Lobban and M. J. Wynne, eds), pp. 52-85. University of California Press,
Berkeley.
Pemberton, J. M., Slate, J., Bancroft, D. R. and Barrett, J. A. (1995). Nonamplifying
alleles at microsatellite loci: a caution for parentage and population studies.
Peters, A. F., Kawai, H. and Novaczek, I. (1993). Intraspecific sterility barrier
confirms that introduction of Sphaerotrichia divaricata (Phaeophyceae,
Chordariales) into the Mediterranean was from Japan. Hydrobiologia 261,
31-36.
Pickett-Heaps, J. and West, J. A. (1998). Time-lapse video microscopy of sexual
plasmogamy in the red alga Bostrychia. European Journal of Phycology 33,
43-56.
Pillmann, A., Woolcott, G. W., Olsen, J. L., Stare, W. T. and King, R. J. (1997). Inter-
and intraspecific genetic variation in Caulerpa (Chlorophyta) based on nuclear
rDNA ITS sequences. European Journal of Phycology 32, 379-386.
Powell, W., Machray, G. C. and Provan, J. (1996). Polymorphism revealed by simple
sequence repeats. Trends in Plant Science 1, 215-222.
Provan, J., Soranzo, N., Wilson, N. J., McNicol, J. W., Forrest, G. I., Cottrell, J. and
Powell, W. (1998). Gene pool variation in Caledonian and European Scots
pine (Pinus sylvestris L.) revealed by chloroplast simple sequence repeats.
Proceedings of the Royal Society of London Series B 265, 1697-1705.
Provan, J., Soranzo, N., Wilson, N. J., McNicol, J. W., Morgante, M. and Powell, W.
(1999a).The use of uniparentally inherited simple sequence repeat markers in
plant population studies and systematics. In "Molecular Systematics and Plant
Evolution" (P. M. Hollingsworth, R. M. Bateman and R. J. Gornall, eds), pp.
3-550. Taylor and Francis, London.
Provan, J., Powell, W., Dewar, H., Bryan, G., Machray, G. C. and Waugh, R. (1999b).
An extreme cytoplasmic bottleneck in the modern European cultivated potato
(Solanum tuberosum) is not reflected in decreased levels of nuclear diversity
Proceedings of the Royal Society of London, Series B 266, 633-639.
Queller, D. C., Strassmann, J. E. and Hughes, C. R. (1993). Microsatellites and
kinship. Trends in Ecology and Evolution 8, 285-288.
Quinn, T. W. and White, B. N. (1987). Identification of restriction fragment length
polymorphism in genomic DNA of the lesser snow goose (Anser caerulescens).
Molecular Biology and Evolution 4, 126-143.
Radner, R. J. (1996). Algal diversity and commercial algal products. BioScience 46,
263-270.
Rassmann, K., Schl6tterer, C. and Tautz, D. (1991). Isolation of simple-sequence

loci for use in polymerase chain reaction-based DNA fingerprinting.
Electrophoresis 12, 113-118.
Rice, E. L. and Bird, C. (1990). Relationships among geographically distant
populations of Gracilaria verrucosa (Gracilariales, Rhodophyta) and related
species. Phycologia 29, 501-510.
Rico, C., Rico, I. and Hewitt, G. (1996). 470 million years of conservation of
microsatellite loci among fish species. Proceedings of the Royal Society of
London, Series B 263, 549-557.
Ridley, M. (1993). "Evolution". Blackwell, Oxford.
Rogers, S. O. and Bendich, A. J. (1987). Ribosomal-RNA genes in plants - variability
in copy number and in the intergenic spacer. Plant Molecular Biology 9, 509-520.
Saunders, G. W., Maggs, C. A. and McLachlan, J. L. (1989). Life history variation in
Rhodophyserna elegans (Rhodophyta) from the North Atlantic and crustose
Rhodophysema species from the North Pacific. Canadian Journal of Botany 67,
285%2872.
Schl6tterer, C. and Pemberton, J. (1994). The use of microsatellites for genetic
analysis of natural populations. In "Molecular Ecology and Evolution:
Approaches and Applications" (B. Schierwater, B. Streit, G. P. Wagner and R.
DeSalle, eds), pp. 203-214, Birkhfiuser Verlag, Basel.
Schlotterer, C. and Tautz, D. (1992). Slippage synthesis of simple sequence DNA.
Nucleic Acids Research 20, 211-215.
Searles, R. B. (1980). The strategy of the red algal life history. American Naturalist
115, 113-120.
Serrfio, E. A., Alice, L. A. and Brawley, S. H. (1999). Evolution of the Fucaceae
(Phaeophyceae) inferred from nrDNA-ITS. Journal of Phycology 35, 382-394.
Soltis, D. E., Gitzendanner, M. A., Strenge, D. D. and Soltis, P. S. (1997).
Chloroplast DNA intraspecific phylogeography of plants from the Pacific
Northwest of North America. Intraspecific chloroplast DNA variation:
systematic and phylogenetic implications. Plant Systernatics and Evolution 206,
353-373.
Sosa, P. A. and Oliveira, M. C. (1992). DNA extraction from macroalgae. Applied
Phycology Forum 9, 7-9.
Sosa, P. A. and Lindstrom, S. C. (1999). Isozymes in macroalgae (seaweeds): genetic
differentiation, genetic variability and applications in systematics. European
Stace, C. A. (1989). "Plant Taxonomy and Biosystematics". 2nd edn. Edward Arnold,
London.
Stam, W. T., Bot, P. V. M., Boele-Bos, S. A., van Rooij, J. M. and van den Hoek, C.
(1988). Single-copy DNA-DNA hybridizations among five species of
Laminaria (Phaeophyceae): phylogenetic and biogeographic implications.
Helgolander Meeresuntersuchungen 42, 251-267.
Steane, D. A., McClure, A., Clarke, A. E. and Kraft, G. T. (1991). Amplification of
the polymorphic 5.8S rRNA gene from selected Australian gigartinalean
species (Rhodophyta) by polymerase chain reaction. Journal of Phycology 27,
758-762.
Stearns, S. C. (1992). "The Evolution of Life Histories". Oxford University Press,
Oxford.
Stoebe, B. and Kowallik, K. V. (1999). Gene-cluster analysis in chloroplast
genomics. Trends in Genetics 15, 344-347.
Tan, I. H., Blomster, J., Hansen, G., Leskinen, E., Maggs, C. A., Mann, D. G.,
Sluiman, H. J. and Stanhope, M. J. (1999). Molecular phylogenetic evidence
for a reversible morphogenetic switch controlling the gross morphology of two

common genera of green seaweeds, Ulva and Enteromorpha. Molecular Biology
and Evolution 16, 1011-1018.
Tanner, C. E. (1981). Chlorophyta: life histories. In "The Biology of Seaweeds" (C.
S. Lobban and M. J. Wynne, eds), pp. 86-132. University of California Press,
Berkeley.
Tautz, D. (1989). Hypervariability of simple sequences as a general source for
polymorphic DNA markers. Nucleic Acids Research 17, 6463-6471.
Tautz, D. and Renz, M. (1984). Simple sequences are ubiquitous repetitive
components of eukaryotic genomes. Nucleic Acids Research 12, 4127-4138.
Taylor, J. S., Sanny, J. S. P. and Breden, F. (1999). Microsatellite allele size
homoplasy in the Guppy (Poecilia reticulata). Journal of Molecular Evolution
48, 245-247.
Theriot, E. (1992). Phylogenetic systematics, the theory and practice of taxonomy. In
"Modern Approaches to the Analysis of Algal Morphology and Systematics"
(D. M. John and D. Garbary, eds), pp. 180-207. Progress in Phycological
Research, Vol. 8. Biopress, Bristol.
Turrill, W. B. (1935). The investigation of plant species. Proceedings of the Linnean
Society of London 147, 104-105.
Valdes, A. M., Slatkin, M. and Freimer, N. B. (1993). Allele frequencies at
microsatellite loci: the stepwise mutation model revisited. Genetics 133,
737-749.
van den Hoek, C., Brecman, A. M. and Stam, W. T. (1990). The geographic
distribution of seaweed species in relation to temperature: present and past. In
"Expected Effects of Climatic Change on Marine Ecosystems" (J. J. Brukema
et al., eds), pp. 55-67. Kluwer Academic Publishers, The Netherlands.
van den Hoek, C., Mann, D. G. and Jahns, H. M. (1995). "Algae. An Introduction to
Phycology". Cambridge University Press, Cambridge.
van der Meet, J. P. (1981). Genetics of Gracilaria tikvahiae (Rhodophyceae).
VII. Further observations on mitotic recombination and the construction of
polyploids. Canadian Journal of Botany 59, 787-792.
van der Meer, J. P. (1987). Using genetic markers in phycological research.
Hydrobiologia 151/152, 49-56.
van der Meet, J. P. (1990). Genetics. In "Biology of the Red Algae" (K. M. Cole and
R. G. Sheath, eds.), pp. 103-121. Cambridge University Press, Cambridge
van der Strate, H. J., Olsen, J. L., van de Zande, L., Edwards K. J. and Stare, W. T.
(2000). Isolation and characterization of microsatellite loci in the benthic
seaweed, Cladophoropsis membranacea (Cladophorales, Chlorophyta).
van Oppen, M. J. H, Olsen, J. L., Stam, W. T., van den Hoek, C. and Wiencke, C.
(1993). Arctic-Antarctic disjunctions in the benthic seaweeds Acrosiphonia
arcta (Chlorophyta) and Desmarestia viridis/willii are of recent origin. Marine
Biology 115, 381-386.
van Oppen, M. J. H., Diekmann, O. E., Olsen, J. L. and Stam, W. T. (1994). Tracking
dispersal routes: phylogeography of the Arctic-Antarctic disjunct seaweed
Acrosiphonia arcta (Chlorophyta). Journal of Phycology 30, 67-80.
van Oppen, M. J. H., Olsen, J. L. and Stam, W. T. (1995). Genetic variation within
and among North Atlantic and Baltic populations of the benthic alga
Phycodrys rubens (Rhodophyta). European Journal of Phycology 30, 251-260.
van Oppen, M. J. H, Olsen, J. L. and Stam, W. T. (1995b). Multiple trans-Arctic
passages in the red alga Phycodrys rubens: evidence from nuclear rDNA ITS
sequences. Marine Biology 123, 179-188.
van Oppen, M. J. H., Klerk, H., Olsen, J. L. and Stam, W. T. (1996a). Hidden
diversity in the marine algae: some examples of genetic variation below
the species level. Journal of the Marine Biological Association of the United
Kingdom 76, 239-242.
van Oppen, M. J. H., Klerk, H., de Graaf, M., Stare, W. T. and Olsen, J. L. (1996b).
Assessing the limits of random amplified polymorphic DNAs (RAPDs) in
seaweed biogeography. Journal of Phycology 32, 433-444.
Vis, M. L. (1999). Intersimple sequence repeats (ISSR) molecular markers to
distinguish gametophytes ofBatrachospermum boryanum (Batrachospermales,
Rhodophyta). Phycologia 38, 70-73.
Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Fritjers,
A., Pot, J., Peleman, J., Kuiper, M. and Zabeau, M. (1995). AFLP: a new
concept for DNA fingerprinting. Nucleic Acids Research 23, 4407-4414.
Wang, Z., Weber, J. L., Zhong, G., and Tanksley, S. D. (1994). Survey of plant short
tandem DNA repeats. TheoreticalApplied Genetics 88, 1-6.
Wattier, R. (1998). Microsatellites, development and application to the population
genetics of the red seaweed Gracilaria gracilis. Ph.D. thesis, 285pp. Paris 6
University
Wattier, R., Dallas, J. F., Destombe, C., Saumitou-Laprade, P. and Valero, M.
(1997). Single locus microsatellites in Gracilariales (Rhodophyta): high level
of genetic variability within Gracilaria gracilis and conservation in related
species. Journal of Phycology 33, 868-880.
Wattier, R., Engel, C. R., Saumotou-Laprade, P. and Valero, M. (1998). Short
allele dominance as a source of heterozygote deficiency at microsatellite loci:
experimental evidence at the dinucleotide locus GvlCT in Gracilaria gracilis
(Rhodophyta). Molecular Ecology 7, 1569-1573.
Wattier, R., Prodohl, P. A. and Maggs, C. A. (2000). DNA extaction protocol for red
seaweeds. Plant Molecular Biology Reporter 18, 275-281.
Wattier, R.A., Davidson, A.L., Ward, B.A. and Maggs, C.A. (2001). cpDNA-RFLP
in Ceramium (Rhodophyta): intraspecific polymorphism and species-level
phylogeny. American Journal of Botany (in press).
Weber, J. L. (1990). Informativeness of human (dC-dA)n. (dG-dT) n polymorphisms.
Genomics 7, 524-530.
Weber, J. L. and May, P. E. (1989). Abundant class of human DNA polymorphisms
which can be typed using the polymerase chain reaction. American Journal of
Human Genetics 44, 388-396.
Weising, K., Atkinson, R. G. and Gardner, R. C. (1995). Genomic fingerprinting
by microsatellite-primed PCR: A critical evaluation. PCR Methods and
Applications 4, 249-255.
Welsh, J. and McClelland, M. (1990). Fingerprinting genomes using PCR with
arbitrary primers. Nucleic Acids Research 18, 7213-7218.
West, J. A. and Hommersand, M. H. (1981). Rhodophyta: life histories. In "The
Biology of Seaweeds" (C. S. Lobban and M. J. Wynne, eds), pp. 133-193.
University of California Press, Berkeley.
West, J. A. and Zuccarello, G. C. (1999). Biogeography of sexual and asexual
populations in Bostrychia moritziana (Rhodomelaceae, Rhodophyta).
Phycological Research 47, 115-123.
Wiencke, C., Bartsch, I., Biscoff, B., Peters, A. F. and Breeman, A. M. (1994).
Temperature requirements and biogeography of Antarctic, Arctic and
Amphiequatorial seaweeds. Botanica Marina 37, 247-259.
Williams, G. C. (1975). "Sex and Evolution". Princeton University Press, Princeton,
NJ.
Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A. and Tingey, S. V.

(1990). DNA polymorphisms amplified by arbitrary primers are useful as
genetic markers. Nucleic Acids Research 18, 6531-6535.
Womersley, H. B. S. (1998). "The Marine Benthic Flora of Southern Australia".
Government Printing Division, Adelaide.
Wright, J. T., Zuccarello, G. C. and Steinberg, P. D. (2000). Genetic structure of the
subtidal red alga Delisea pulchra. Marine Biology 136, 439-448.
Xiang, Q. Y., Soltis, D. E. and Soltis, P. S. (1998). The eastern Asian and eastern
and western North American floristic disjunction: congruent phylogenetic
patterns in seven diverse genera. Molecular Phylogenetic and Evolution 10,
178-190.
Yabu, H. and Yotsukura, N. (1995). Notes to nuclear division in the gametophytes
and young sporophytes of Laminaria religiosa Miyabe. Japanese Journal of
Phycology (SorCti) 43, 221-224 [in Japanese, English abstract].
Yang, G. P., Saghai-Maroof, M. A., Xu, C. G., Zhang, Q. and Biyashev, R. M. (t994).
Comparative analysis of microsatellite DNA polymorphism in landraces and
culfivars of rice. Molecular and General Genetics 245, 187-194.
Yang, T. W., Yang, Y. A. and Xiong, Z.G. (2000). Paternal inheritance of
chloroplast DNA in interspecific hybrids in the genus Larrea
(Zygophyllaceae). American Journal of Botany 87, 1452-1458.
Young, N. D. (1996). Concordance and discordance: A tale of two hybrid zones in
the pacific Coast irises (Iridaceae).American Journal of Botany 83, 1623-1629.
Zietkiewicz, E., Rafalski, A. and Labuda, D. (1994). Genome fingerprinting by
simple sequence repeat (SSR)-anchored polymerase chain-reaction
amplification. Genomics 20, 176-183.
Zuccarello, G. C. and West, J. A. (1997). Hybridization studies in Bostrychia:
Correlation of crossing data and plastid DNA sequence within B. radicans and
B. moritziana (Rhodomelaceae, Rhodophyta). Phycologia 36, 293-304.
Zuccarello, G. C., West, J. A., Kamiya, M. and King, R. J. (1999a). A rapid method to
score plastid haplotypes in the red seaweeds and its use in determining
parental inheritance of plastids in the red alga Bostrychia (Ceramiales).
Hydrobiologia 401, 207-214.
Zuccarello, G. C., West, J. A. and King, R. J. (1999b). Evolutionary divergence in the
Bostrychia moritziana/B, radicans complex (Rhodomelaceae, Rhodophyta):
molecular and hybridization data. Phycologia 38, 234-244.
Zuccarello, G. C., West, J. A., Karsten, U. and King, R. J. (1999c). Molecular
relationships within Bostrychia tenuissima (Rhodomelaceae, Rhodophyta).
Phycological Research 47, 81-85.
Zuccarello, G. C., Barlett, J. and Yeates, P. H. (2000). Differentiation of Caloglossa
leprieurii (Rhodophyta) populations in northern and eastern Australia using
plastid haplotypes. European Journal of Phycology 35, 357-364.

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Intraspecific Variation in Seaweeds: The Application of New

Tools and Approaches

School of Biology and Biochemistry, Medical Biology Centre, Queen's

I. Introduction .................................................................................................... 172

*Present address: School of Biological Sciences, University of Wales, Bangor,

Advances in Botanical Research Vol. 35

The algae are a very diverse, unnatural assemblage of oxygen-producing

dependent on the development over the last four decades of laboratory

II. SPECIES CONCEPTS AND INTRASPECIFIC VARIATION

(Mayden, 1997), and in each revision of an existing concept a new emphasis

On the other hand, when morphological or life-history characters are

which are normally morphologically distinct and occur in slightly different

In practice, as noted above, the morphological species concept is still

Seaweeds show an extraordinary diversity of life histories (Hawkes, 1990)

parthenogenetic and sexual reproduction frequently occur in tandem

IV. POPULATION GENETICS

In this section, we describe the current state of knowledge of intraspecific

(iii) Single-strand conformational polymorphism: it is assumed that point

A. DNA ISOLATIONFROM SEAWEEDS:A TECHNICALNIGHTMARE

In general, isolation of good-quality DNA (i.e. of high molecular weight,

Single-locus nuclear microsatellites (commonly known as microsatellites)

et al., 1997), involving enormous expenditure of time and effort by several

For most organisms, microsatellites are regarded as highly variable

harder to detect because null homozygotes become rare. For low-frequency

heterologous primers in several different species could eliminate the need to

C. MULTILOCUS NUCLEAR MARKERS (RAPDs, ISSRs, M13-FINGERPRINTING

Four classes of multilocus DNA markers have been applied to seaweeds:

Plastids are believed to be uniparentally inherited in seaweeds (see next

majority of the chloroplast genome and using complete cpDNA as a probe),

The breeding system of an organism can be defined broadly as the mode,

Characterization of the pheromones of a wide range of brown algae showed

V. BIOGEOGRAPHY AND PHYLOGEOGRAPHY

Biogeography is a comparatively well-studied aspect of seaweed biology,

example, the brown seaweed Desmarestia viridis (synonym D. wilIii) has a

B. MOLECULAR APPROACHES: NUCLEAR MARKERS

Early molecular biogeographic studies in algae employed DNA-DNA

The great majority of phylogeographic studies in seaweeds have utilized

levels of ITS divergence of up to 15% within morphological species. The large

NC, NZ, ~!~ Akkeshi

Fig. 2. Minimum spanning tree of Polysiphonia harveyi rbcL haplotypes showing

Plastid markers can be very valuable in phylogeographic studies because of

VI. CONCLUSIONS AND PERSPECTIVES

Part of this study was supported by EC FAIR research contract PL97-3828,

Blomster, J., Maggs, C. A. and Stanhope, M. J. (1999). Extensive intraspecific

Coyer, A. C., Robertson, D. L. and Alberte, R. S. (1995). Genetic variability and

Margulis, L. (1990). "Handbook of Protoctista". Jones and Bartlett, Boston, MA.

Atlantic tropical-Canary Islands species. European Journal of Phycology 31,

Rassmann, K., Schl6tterer, C. and Tautz, D. (1991). Isolation of simple-sequence

for a reversible morphogenetic switch controlling the gross morphology of two

Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A. and Tingey, S. V.

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AdvBotRes 35 - 2001 Wattier & Maggs - Intraspecific Variation in Seaweeds

AdvBotRes 35 - 2001 Wattier & Maggs - Intraspecific Variation in Seaweeds