Integrative and Comparative Biology, volume 51, number 3, pp. 492–504
doi:10.1093/icb/icr075
SYMPOSIUM
Speciation in Red Algae: Members of the Ceramiales as
Model Organisms
Christine A. Maggs,1,* Hugh L. Fletcher,* David Fewer,*,† Louise Loade,* Frédéric Mineur* and
Mark P. Johnson*,‡
*School of Biological Sciences, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL,
Northern Ireland; †Department of Applied Chemistry and Microbiology, University of Helsinki, Finland; ‡Martin Ryan
Institute, National University of Ireland Galway, Galway, Ireland
From the symposium ‘‘Speciation in Marine Organisms’’ presented at the annual meeting of the Society for Integrative
and Comparative Biology, January 3–7, 2011, at Salt Lake City, Utah.
1
E-mail: [email protected]
Synopsis Red algae (Rhodophyta) are an ancient group with unusual morphological, biochemical, and life-history
features including a complete absence of flagella. Although the red algae present many opportunities for studying speciation, this has rarely been explicitly addressed. Here, we examine an aspect of paternal gene flow by determining
fertilization success of female Neosiphonia harveyi (Ceramiales), which retains a morphological record of all successful and
unsuccessful female gametes. High fertilization rates were observed except when there were no males at all within the
tidepool, or in a submerged marina environment. Small numbers of reproductive males were able to saturate fertilization
rates, suggesting that limited availability of sperm may be less significant in red algae than previously thought. In another
member of the Ceramiales, Antithamnion, relatively large chromosomes permit karyological identification of polyploids.
The Western Pacific species Antithamnion sparsum is closely related to the diploid species Antithamnion defectum, known
only from the Eastern Pacific, and appears to have evolved from it. Molecular evidence suggests that A. sparsum is an
autopolyploid, and that the European species known as Antithamnion densum is divergent from the A. sparsum/defectum
complex.
Introduction
The red algae (Rhodophyta) are related to the green
plants, as members of the Archaeplastida supergroup
(Hampl et al. 2009). They differ from green plants by
a suite of unusual biochemical and ultrastructural
features: stalked phycobilosomes containing the
purple and red accessory pigments phycocyanin
and phycoerythrin are borne on unstacked thylakoid
membranes; and carbohydrates are stored in the cytoplasm in unique floridean starch grains. The most
striking feature of red algae in an evolutionary perspective is the complete absence of flagella, centrioles
or any other 9 þ 2 structures (Pueschel 1990; Ragan
and Gutell 1995). Comparative analyses with sister
groups show that these structures have been lost
during evolution (Morrow 2004).
Red algae were traditionally divided into two
groups based on morphological, anatomical, and
life-history differences: the Bangiophycidae were
defined mainly by the absence of characters present in the Florideophycidae and considered to be
‘‘primitive.’’ The Bangiophycidae is now seen to
be paraphyletic, and the class Bangiophyceae is
well-resolved as sister to the large monophyletic
class Florideophyceae (Verbruggen et al. 2010).
Bangiomorpha fossils from the 1200 Ma Hunting
Formation, which are very similar to members of
the Bangiophyceae, are regarded as the oldest taxonomically resolved eukaryotic fossil and the earliest
fossil evidence for multicellular eukaryotic life
(Butterfield 2000). Bangiomorpha is also the earliest
known example of sexual reproduction in the fossil
record (Butterfield 2000).
The Rhodophyta includes 6000 recognized species in 700 genera (Woelkerling 1990; Guiry and
Guiry 2011) and they exhibit diverse morphologies
Advanced Access publication July 8, 2011
ß The Author 2011. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved.
For permissions please email: [email protected].
Speciation in red algae
ranging from tiny filaments or parasitic pustules to
leafy forms a meter or more in length. The great
majority of these species have been identified and
described using the morphological species concept,
which has dominated algal systematics since its
Linnaean starting point (John and Maggs 1997).
The ‘‘pure’’ biological species concept (BSC), regarding species as ‘‘groups of actually or potentially interbreeding natural populations which are
reproductively isolated from other such groups’’
(Mayr 1963) has not been widely used in seaweeds,
for practical reasons (Wattier and Maggs 2001).
Instead, a looser application of the BSC, which
tests the ability to interbreed when brought into artificial proximity, has been more frequently applied,
although many groups are either entirely asexual or
monoecious and self-fertile (Guiry 1992; John and
Maggs 1997). Over the past decade, molecular systematics of seaweeds has come to the fore, and a
rapidly increasing number of cryptic or semi-cryptic
species are being identified by DNA barcoding and
other initiatives in biodiversity (Saunders and Le Gall
2010). The fundamental basis of all such endeavors is
the attempt to identify species as separately evolving
lineages of metapopulations (de Queiroz 2007;
Reeves and Richards 2011). When multiple
species-criteria are fulfilled the group is more likely
to be a distinct lineage (de Queiroz 2007).
Clearly, a large number of recognizable species of
red algae have evolved and occur today. In many
groups, well-resolved and taxon-replete phylogenies
include all known species or at least all those within
a given geographical area (e.g., Verbruggen et al.
2007). However, there have been very few studies
explicitly addressing the mechanisms of speciation
that have led to red algal biodiversity by identifying
and measuring reproductive isolation and looking
for causes of prezygotic and post-zygotic
isolation. A notable exception is the demonstration
of allopolyploidy in natural populations following
hybridization between the closely related nori crop
species Porphyra yezoensis and P. tenera (Niwa and
Sakamoto 2010; J. Brodie, personal communication).
In flowering plants, prezygotic barriers that
impede mating or fertilization typically contribute
more to total reproductive isolation than do postzygotic barriers (Reiseberg and Willis 2007).
Reproductive isolation, often involving numerous
barriers, facilitates the accumulation of genetic differences among groups of populations and permits
divergence. Gene flow can be inhibited or completely
stopped by pre-pollination barriers such as mechanical changes to flower structure or color mutations.
These can probably arise rapidly and limit the ability
493
of pollinating insects to transfer pollen from one
species to another (Reiseberg and Willis 2007).
Specialized insect pollinators provide enormous
scope for co-evolution and speciation, and play an
important role in maintaining terrestrial ecosystems
(Ollerton et al. 2011).
In contrast to flowering plants, red algae have no
known mechanisms ensuring that conspecific gametes are brought together or that nonconspecific
gametes are kept separate. The life history of red
algae involves spermcast mating in which male gametes are released and fertilize female gametes retained
by maternal individuals (Serrão and Havenhand
2009). There is no equivalent of pollinators or specialized morphological devices that concentrate gametes of a particular species. Whereas the eggs of many
brown algae produce pheromones that attract motile
sperm, red algal sperm are nonmotile and, not unexpectedly, there are no known pheromones.
Synchronized mass spawning regulated by tidal or
lunar cycles, as seen in fucoid brown algae and
some green seaweeds (Serrão and Havenhand
2009), is likewise unknown in red algae.
Red algal sperm (‘‘spermatia’’) are small, 2–5 mm
in diameter, formed in male sex organs at the end of
a compact system of branched filaments (Dixon
1977). The spermatangia consist largely of massive
mucilage-containing vesicles derived from endoplasmic reticulum and dictyosomes. In vascular plants,
sperm cells’ organization, shape, size, and plasticity
are crucial to the processes associated with fertilization (Lopez-Smith and Renzaglia 2008). Spermatia in
red algae also show phylogenetically significant features despite their lack of flagella and associated
structures (McIvor et al. 2002). When red algal spermatia are released into the surrounding seawater by
discharge of spermatial vesicles, the vesicles’ contents
can form diverse mucilaginous appendages on the
male gametes (Magruder 1984; Broadwater et al.
1991). These vary in shape and size among taxa
and are assumed to affect the hydrodynamic properties of the gametes because movement of released
spermatia depends entirely on water currents. In
view of the unique absence of flagella in this
group, spermatial appendages could be of great functional significance in the fertilization of red algae.
The female red algal sex organ is the carpogonial
branch, the terminal cell of which is the gamete
(‘‘carpogonium’’). The carpogonium consists of an
elongated cell with an enlarged base containing the
nucleus and a hair-like extension called the trichogyne, the receptive surface of the female gamete,
which protrudes beyond the surface of the seaweed
(Dixon 1977). When spermatia adhere to the
494
trichogyne the injected male nucleus can travel down
the trichogyne and internally fertilize a carpogonial
nucleus. Although red algal trichogynes have no mechanical means that prevent access of nonconspecific
spermatia, and sexual encounters apparently rely on
chance, there is evidence for prezygotic isolating
mechanisms in some red algae (in the Ceramiales).
In Aglaothamnion large numbers of spermatia can be
bound, saturating at 200–300 per trichogyne (Kim
et al. 1996). Progress in this exciting and challenging
research led by Gwang Hoon Kim (Kongju National
University, Korea) has mostly been reported orally
and in a series of abstracts. ‘‘Rhodobindin’’
gamete-recognition proteins in Aglaothamnion (Kim
and Jo 2005) are involved in binding spermatia to
the trichogyne. The bound spermatia then complete
nuclear division so that the spermatium can contain
more than one male nucleus. Many spermatial nuclei
can enter the trichogyne, providing for the possibility
of competition among sperm. A common first-order
receptor appears to operate at the generic level, permitting attachment of spermatia to trichogynes of
congeners, while a second-order receptor prevents
interspecific nuclear fusion (Kim et al. 1996; Ryu
et al. 2003). Mutations in the genes coding for this
receptor could facilitate rapid speciation, as seen in
various plants and invertebrates (other papers, this
volume). Sperm competition has been demonstrated
in another member of the Ceramiales, Bostrychia.
Multiple sperm nuclei jostle and even overtake each
other as they travel down the trichogyne towards the
female nucleus (Pickett-Heaps et al. 2001).
The Rhodophyta have complex haplo-diploid life
histories. Uniquely in the Florideophyceae, the group
that includes the Ceramiales and most of the other
red seaweeds, the immediate product of fertilization
is not the diploid sporophyte, but a hemi-parasitic
diploid tissue (‘‘carposporophyte’’) usually surrounded by female nutritive tissue, collectively called the
cystocarp. In the majority of lineages an additional
zygote-amplification stage results in thousands of
spores from a single fertilization (Fierst et al. 2005).
This stage, which is usually regarded as a mechanism
compensating for the lack of motile sperm in the red
algae (Searles 1980), releases numerous genetically
identical diploid carpspores that give rise to free-living
diploid tetrasporophytes. Tetrasporangia undergo
meiosis, releasing four haploid spores that give rise
to male and female gametophytes.
In this article, we report on our studies of red
algal speciation, using members of the Ceramiales
as model species. The Ceramiales is the largest red
algal order, which contains approximately half of
the genera and one-third of the species in the
C. A. Maggs et al.
Rhodophyta (Bold and Wynne 1985). Within the
Ceramiales, the Ceramiaceae is one of the most
basal lineages and the Rhodomelaceae is derived
(Verbruggen et al. 2010). The Ceramiaceae is an
ideal group for studies of speciation, having a relatively well-resolved phylogeny, highly speciose
genera, and small thalli (petri-dish-sized) with
rapid completion of the life history, thereby allowing
multiple generations to be studied in culture. They
are also the only red algae known to have
gamete-recognition proteins (Kim and Jo 2005).
Members of the Rhodomelaceae exhibit a valuable
feature for studying breeding systems: the female
gametes are borne in macroscopic organs (procarps)
that either remain on the thallus as a record of
unfertilized gametes or develop into large
post-fertilization structures (cystocarps).
Two aspects of red algal speciation are addressed
here. (1) We examine spermatial distribution and
sperm limitation in natural populations of
Neosiphonia harveyi (Rhodomelaceae), by comparing
fertilization success of female thalli in tidepools with
and without males, and in a continually submerged
habitat. (2) A reproductively isolated autopolyploid
species of Antithamnion (Ceramiaceae), A. sparsum,
is shown to have arisen from a lineage within a diploid species, A. defectum; the European species
A. densum is not closely related.
Materials and methods
Success of fertilization in Neosiphonia harveyi
Field collections
Neosiphonia harveyi was collected from nine tidepools at Crawfordsburn Country Park shore, Co.
Down, Northern Ireland, in November 2006, and
from seven pools at Doaghbeg, Fanad, Donegal,
Ireland in December 2006 (Fig. 1; Table 1). All
thalli were removed from each pool. Each pool was
closely examined to ensure every N. harveyi plant
within the pool was removed, which was possible
because most of the N. harveyi were epiphytic on
larger species, particularly Codium fragile subsp.
fragile, and the rock surface itself was covered with
light pink coralline algae, providing a good color
contrast with N. harveyi (Fig. 1). At these sites, N.
harveyi is confined to pools and does not occur on
the open rock. The approximate surface area (m2) of
the tidepools was recorded and the approximate
volume was calculated using a mean pool depth for
each site. Algae from each pool were placed in seawater in separate labeled containers. Neosiphonia harveyi was also collected from Sandy Point Marina,
495
Speciation in red algae
Fig. 1 Shore in Ireland (Donegal) with tidepools (A and B) where fertilization success was evaluated in N. harveyi (C). Fertilized female
organs (cystocarps, D) and unfertilized procarps (E).
Table 1 Neosiphonia harveyi in tide pools at Crawfordsburn, Co. Down, Ireland (C) and Fanad, Co. Donegal, Ireland (D), indicating pool
areas, numbers, and biomass of N. harveyi per pool, and fertilization success of N. harveyi females
Tide
pool
Area
(m2)
Approx.
volume
(m3)
Male biomass
per m2 (g)
Female
biomass
per m2 (g)
Male: female
biomass ratio
Male: female
numbers ratio
No. of
females
in pool
Total number
of cystocarps
in pool
Fertilization
success of females
in pool (%)
C1
6
1.2
0.0000
0.0367
0.0000
0:2
2
204
35
C2
10
2.0
0.0750
0.0770
0.9740
1:4
4
10,190
79
C3
0.9
0.2
0.1111
0.7144
0.1556
2:7
7
780
90
C4
1
0.2
0.0500
0.5420
0.0925
1:4
4
814
79
C5
3
0.6
0.3196
0.1300
2.4585
3:2
2
555
85
C6
0.5
0.1
0.0800
1.0200
0.0784
1:2
2
1057
90
C7
2.0
0.0075
0.0230
0.3261
1:3
3
353
76
D1
10
1.5
0.5
0.0000
0.2027
0.0000
0:1
1
230
5
D2
0.5
0.2
0.4620
0.4620
1.0000
3:2
4
143
80
D3
3
1.0
0.0253
0.0223
1.1345
5:2
2
310
76
D4
2.25
0.7
0.1244
0.0809
1.5377
1:4
4
261
74
D5
1.5
0.5
0.0000
0.2587
0.000
0:1
1
92
7
Marina
–
–
–
–
–
–
–
–
5–37 mean: 14.0
Note. For a yacht marina in southern England, only fertilization success of N. harveyi females is provided.
Hayling Island, Hampshire, in February 2007. It grew
in the marina as an epiphyte on Sargassum muticum
in open conditions on the sides of the pontoons.
This marina has no retaining walls and at high tide
is completely open to the sea, where tidal currents
are strong (42 knots).
496
C. A. Maggs et al.
Collection of data
All N. harveyi individuals from each pool were sorted
into male gametophytes, female gametophytes, tetrasporophytes, or non-reproductive thalli, which were
counted and weighed to determine the biomass (g)
of males, females and tetrasporophytes per m2 of
rock pool. Samples from tidepools lacking females
were discarded. The samples from Sandy Point
Marina were also sorted into males, females, and
tetrasporophytes. Females from both sites were processed as follows. Female plants were weighed and
larger thalli were broken into pieces. The pieces of
larger females were spread evenly over a 1-cm2 grid.
Depending on the overall size of the female, one
piece was removed from either every third square
or every other square so that one-third to one-half
of the thallus was examined. These pieces were
placed on a slide and examined under low magnification with a compound microscope (Leitz Dialux).
The numbers of procarps and cystocarps on each
piece (or entire small individual) were recorded.
Growth is apical, and procarps are formed in series
along the axes, the youngest being closest to the apex
(Fig. 1). Any procarps older than developing cystocarps on the same axis have failed to be fertilized.
Percentage fertilization success for each female gametophyte was determined as
Percentage of fertilization success ¼
cystocarps=ðcystocarps þ procarpsÞ 100:
ð1Þ
To estimate the numbers of carpospores formed in
each tide pool, the minimum number of carpospores
per cystocarp was determined by squashing ten
cystocarps of various sizes under cover slips to
allow mature carpospores to escape and be counted.
Multiplying this mean by the number of cystocarps
in the tidepool gave a minimum spore production
per pool.
Data analysis
A single-factor ANOVA test (in Microsoft Excel
2003) established whether fertilization success of females from tidepools differed from those collected
from the marina. Spearman’s rank correlation (in
MINITAB 14) was used to investigate the correlation
between biomass of males per m2 of tide pool and
fertilization success of the females in that pool. The
relationship was analyzed with a non-linear regression in Sigmaplot. Spearman’s rank correlation was
also used to investigate the relationship between
male:female ratio and fertilization success.
Polyploidy in the Antithamnion densum/sparsum
complex
Collection of samples, documentation, and culture
methods
Samples were collected and isolated into culture or
obtained as cultured isolates from the UTEX culture
collection or from other sources as shown in Table 2.
Samples were transported live back to the laboratory
in sterilized seawater, and cleaned and sorted carefully under a dissecting microscope. Cultures were
isolated from vegetative tips placed in sterile seawater, and grown when unialgal in half-strength modified von Stosch medium (Guiry and Cunningham
1984) at 15 or 208C, in a regime of 16:8 h light:dark,
at a photon irradiance of ca. 20 mmol photons
m2 s1. Some replicate cultures were grown in
15-ml petridishes in which the medium was changed
weekly; others were maintained for long-term storage
in 60-ml screw-cap bottles at 158C under a photon
irradiance of ca. 5 mmol photons m2 s1. Cultures
were grown until they had sufficient biomass for
DNA extraction and karyological studies.
Karyological studies
Algae were fixed in 1:1 glacial acetic acid:absolute
ethanol, stained with Wittmann’s aceto-iron hematoxylin and mounted in 45% acetic acid (Maggs
1998). Stained dividing nuclei were photographed
using Kodak Technical Pan film and processed for
high contrast with Kodak HC 110 developer.
Chromosomes in different planes of focus were
traced in different colored pencils to build up a 3D
picture of the nucleus.
DNA extraction, PCR amplification, and sequencing
DNA was extracted from 50 mg fresh weight of
cultured algae, using the DNeasy Plant Mini Kit
(Quiagen, UK) according to the manufacturer’s instructions. Primers for amplifying the rbcL gene were
designed using Genbank sequence X54532 for
Antithamnionella spirographidis (as Antithamnion
sp.). Anti1 (50 -CAC AAC CAG GTG TTG ATC
CAA TTG AAG C-30 ) was used as the forward external primer and Anti4 (50 CTA CGA AAG TCA
GCT GTA TCT GTA GAA GTA TA 30 ) as the reverse
external primer. PCR amplifications were carried out
using either a Perkin Elmer DNA Thermal Cycler
480 (Perkin Elmer Biosystems) or a PTC-100TM
Programmable Thermal Cycler (MJ Research). The
cycle was 5 min denaturing at 948C, 30 cycles of
1 min at 948C and 3 min at 608C, followed by a
final extension phase at 608C for 10 min. Reactions
497
Speciation in red algae
Table 2 Antithamnion and outgroup Antithamnionella species sequenced for rbcL indicating source of isolate and GenBank numbers for
rbcS, 18S, and ITS sequences published by Lee et al. (2005)
Species
Genes sequenced (rbcL only) or obtained from GenBank, with collection or
isolate information
Antithamnion defectum Kylin
rbcL
JN089390: Isolate CAM 220 ¼ UTEX LB2261 (female), LB2262 (male) Friday Harbor, isolated
by John West, Sept 1963 (originally UWCC 240, also termed JAW 240 female and JAW 241
male)
JN089391: Isolate CAM 403, La Jolla, California, July 27, 1996
rbcS/18S/ITS
AY168256/ AY168239/AY168250 (UTEX LB2262)
Antithamnion sparsum Tokida
rbcL
JN089392: Isolate CAM 213 ¼ Daechon, Korea, April 23, 1992, collected by HG Choi (IK Lee
isolate 3010)
rbcS/18S/ITS
AF346221/AY168248/AY168238 (isolated from Bangpo, Korea, April 27, 1998; Lee et al. 2005)
Antithamnion kylinii Gardner
rbcL
JN089393: Isolate CAM 218 ¼ UTEX LB801
rbcS/18S/ITS
AF346223/ AY168240/AY168251 (UTEX LB801)
Antithamnion cruciatum (C. Agardh) Nägeli
rbcL
JN089394: Isolate CAM 216, Mulroy Bay, Co. Donegal, Ireland, February 16, 1993
Antithamnion densum (Suhr) Howe
rbcL
JN089395: Isolate CAM 214, Skellig Rocks, Co. Kerry, Ireland, July 1992
JN089396: Isolate CAM 302, Ranolien, Brittany, France, May 25, 1990 (MT L’Hardy-Halos
R3758).
rbcS/18S/ITS
AY168257/AY168241/AY168252 (isolate ¼ CAM 214)
Antithamnion aglandum Kim et Lee
rbcL
AY594700 (Wando, Korea, January 29, 1999)
rbcS/18S/ITS
AF346212/AY168234/AY168244 (Dokdo island, Korea, March 21, 1993)
Antithamnion nipponicum Yamada et Inagaki
rbcL
AY594699
rbcS/18S/ITS
AY168255/AY168235/AY168245 (Wando, Korea, January 20, 1996)
Antithamnionella sp.
rbcL
DQ787564 (isolate A31)
rbcS/18S/ITS
AF346225/AY168243/AY168254 (isolate ATN, Korea; Lee et al. 2005)
contained 200 ng each primer, 20 mM dATP, dCTP,
dGTP, dTTP (Ultrapure dNTP set, Amersham
Pharmacia Biotech), 2.5 mM MgCl2, and 5 U Taq
polymerase
(Biogene
Ltd).
PCR
products
(1242 bp) were reamplified if necessary as described
by Nam et al. (2000).
The fragments for sequencing reactions were purified using the High Pure PCR Product Purification
Kit (Boehringer Mannheim) according to the manufacturer’s instructions. The PCR-amplified products
were directly sequenced using dideoxy chain termination methodology as described in Nam et al.
(2000), except for the primers, which were Anti1,
Anti4, and two additional primers, Anti2 (50 -CGT
GAG CGT ATG GAT AAA TTT GGT CGT TC-30 )
and Anti3 (50 -TTA CTT TAC GTA AAG CAG CCC
AAT CTT GTT C-30 ) used in various combinations
to ensure that the entire 1242 bp region was
sequenced.
Phylogenetic analyses
In addition to our own sequences, some rbcL sequences were obtained from GenBank (Table 2).
rbcL sequences were aligned by eye using BioEdit
7.0.4.1 (Hall 1999). The rbcL dataset, trimmed to a
1242 bp alignment (84% of the 1473 bp rbcL gene),
was analyzed with Bayesian inference (BI) and maximum likelihood (ML) using MrBayes v3.1.2
(Ronquist and Huelsenbeck 2003) and PhyML
v2.4.4 (Guindon and Gascuel 2003) respectively. BI
and ML trees were computed under a general
time-reversible model with a proportion of invariable
498
sites and gamma distribution (GTRþIþG), as determined by the Akaike Information Criterion in
PAUP/Modeltest 3.6 (Posada and Crandall 1998;
Swofford 2002). BI analyses consisted of two parallel
runs of each of four incrementally heated chains, and
3 million generations with sampling every 1000 generations. A burn-in sample of 2000 trees was removed before constructing the majority-rule
consensus tree. For the ML trees, the reliability of
each internal branch was evaluated on the basis of
1000 bootstrap replicates.
Sequences of rbcS, 18 S and ITS1 were also obtained from GenBank (Table 2), and used to prepare
a concatenated alignment of rbcL þ rbcS spacer
(1791 bp); partial 18 S (810 bp); and ITS1 (161 bp)
giving a total alignment of 2762 bp.
Results
Success of fertilization in Neosiphonia harveyi
Success of fertilization for females was correlated
with the biomass of males per unit area of pool
(Table 1; rs ¼ 0.52, P50.05). As the average male
biomass in pools did not differ between
Crawfordsburn Country Park, Co. Down (November
2006) and Fanad, Co. Donegal (December 2006), the
pattern is unlikely to have occurred through
confounding differences in biomass between the
sites with other broad-scale environmental variables
(ANOVA for male biomass between locations,
F1,10 ¼ 0.13, P40.05). A nonlinear regression described the relationship between fertilization success
C. A. Maggs et al.
and male biomass in pools (Fig. 2, r2adj ¼ 84%,
P50.05). The intercept of the fitted equation is at
21%, giving an estimate of the background level of
fertilization success.
Many fertile male plants were identified in the
collection from Sandy Point Marina (February
2007), so it was established that there was no shortage of fertile males available for supplying fertilization in the area at this time. However, fertilization
success was equivalent to the background level in
tide pools lacking males (Table 1). The average fertilization success rate in tide pools with male gametophytes was 82% (SE 1.3). In the absence of males,
the fertilization success in pools was 21% (SE 8.6),
and not significantly different from the 14% fertilization success of the females collected from the
marina (ANOVA F3,8 ¼ 2.12, P40.05).
Cystocarps contained a minimum of 30 carpospores, ranging up to 80–100 carpospores in large
cystocarps. Minimum spore production per pool
was calculated, based on the assumption that at
least 30 spores are produced in each cystocarp.
Minimum spore production per tide pool ranged
from 2745 in a pool without males up to 305 700
in a pool with plentiful males.
Polyploidy in the Antithamnion densum/sparsum
complex
Karyological studies
Chromosome counts were made successfully for
A. densum from Ireland and Antithamnion sparsum
Fig. 2 Non-linear regression of female fertilization success by male biomass in tidepools for N. harveyi showed an excellent fit
(r2adj ¼ 84%; P50.05). Fertilization success ¼ 20.5 þ 62.9 male biomass/(0.001 þ male biomass). The equation has an intercept at 20.5%,
which can be taken as a background level of fertilization success.
Speciation in red algae
499
Fig. 3 Antithamnion densum with tetrasporocytes (A), showing 33 chromosomes in a germinating haploid tetraspore (B and C) and a
dividing meiotic tetrasporocyte with 33 paired chromosomes (D and E ). Male A. defectum (F). Antithamnion sparsum male (G) and
chromosomes in a dividing meiotic tetrasporocyte at two planes of focus and an integrated diagram (H–J) showing 61 paired
chromosomes.
from Korea (Fig. 3). Unfortunately, the cultures of A.
defectum from Washington State did not grow well
(the UTEX LB2261 female isolate appeared to have
developed a mutant morphology after several decades
in culture so it could not be crossed to produce
tetrasporophytes). A. densum had n ¼ ca. 33 chromosomes, seen in a germinating haploid tetraspore, and
in numerous dividing meiotic tetrasporocytes with
33 paired chromosomes. The diploid number of
63-67 chromosomes was observed in dividing apical
500
C. A. Maggs et al.
Fig. 4 Phylogenetic analysis of rbcL sequences for Antithamnion species, rooted with Pterothamnion villosum (A), and a concatenated
alignment of rbcL-rbcS, partial 18S and ITS1 (B). Upper values at nodes are Bayesian probabilities; lower values are Maximum Likelihood
bootstrap values.
cells of tetrasporophytes. In A. sparsum initial observations on meiotic tetrasporocytes indicated
40 chromosome pairs (as reported by Kim et al.
2008) but further examination of better preparations
revealed 57 4 (probably 61) chromosome bodies in
tetrasporocyte nuclei and 51 4 chromosomes in
germinating tetraspores.
Karyological data for Antithamnion cruciatum isolate CAM 216 from Ireland (Table 2) were obtained
for comparison. The tetrasporophyte culture produced only non-viable tetraspores. Mitotic apical
cells had 85 10 chromosomes, and dividing meiotic
tetrasporocytes contained 40 2 paired chromosomes (not shown).
Phylogenetic analysis
In the rbcL tree (Fig. 4A) Pterothamnion villosum was
designated as outgroup based on its position in analyses of larger sets of taxa. Two main clades of
Antithamnion species were resolved, as seen with
the concatenated alignment (Fig. 4B). In the clade
containing A. sparsum and its relatives, one of the
two European isolates of A. densum (Ireland and
Brittany) grouped with a GenBank sequence clearly
misidentified as A. cruciatum. This was sequenced in
our laboratory, and we assume there was contaminant DNA from A. densum; DNA samples from
A. cruciatum repeatedly degraded, presumably due
to a constituent of the alga. The relative positions
of several taxa are not resolved, but A. defectum is
not monophyletic with regard to Korean A. sparsum,
which branches within the A. defectum clade.
Analysis of the concatenated alignment (Fig. 4B)
likewise groups A. sparsum and A. defectum in a
robust clade, but the position of A. densum in this
clade is not resolved.
Discussion
Fertilization success in Neosiphonia harveyi
In sperm casting species, unlike broadcast spawners,
the influence of gametes’ traits on fertilization success is largely limited to the sperm (Serrão and
Havenhand 2009). Fertilization success of N. harveyi
female gametes in tide pools was generally very high,
80%. This is probably the most accurate estimate
of female fertilization rates in a marine red alga. The
closest comparable study is that of Polysiphonia
lanosa in the Bay of Fundy by Kaczmarska and
Dowe (1997). Female fertilization success ranged
from 26% to 54% in different months for all age
classes combined, and up to 91% for young thalli,
but the study suffered from some difficulties caused
by loss of older cystocarps and ambiguous enlarged
501
Speciation in red algae
procarps. As pointed out by Serrão and Havenhand
(2009) high values of fertilization success are in conflict with the hypothesis (Searles 1980) that sperm
limitation was the selective pressure for mitotic cloning of the zygote into carpospores and the origin
of the distinctive florideophyte triphasic life history
(Santelices 2002). Instead, it may be that a high production of carpospores simply minimizes the effects
of variation in fertilization success on population
structure and dynamics (Fierst et al. 2005).
Fertilization success in N. harveyi was positively
correlated with the biomass of male gametophytes
present within the pool. When no males existed in
a rock pool, fertilization success rates ranged from
5% to 35%, with a background level of fertilization
estimated by regression analysis to be 20% (Fig. 2).
These females depend on immigrant spermatia from
other pools to fertilize their gametes and they experience sperm limitation. These results are similar to
those of the only previous work addressing gene flow
by spermatia, which involved a painstaking paternity
analysis of cystocarps of the perennial red alga
Gracilaria gracilis in tidepools in northern France
(Engel et al. 1999, 2004; Engel and Destombe
2002). In this pioneering ‘‘seascape genetics’’ study
female fertilization success was estimated by cystocarp yield per unit female thallus (assuming an
even distribution of the sessile female gametes).
Male fertilization success, estimated by the individual
contribution of different males to zygotes, was assessed by paternity analyses of 350 cystocarps. They
showed that 9% of successful male gametes originate
from outside the female’s pool (Engel et al. 1999).
In the intertidal zone, the retreat and advance of
the tide allows immigration of spermatia to surrounding rock pools and some cross-fertilization of
populations in different rock pools occurs. In more
isolated pools, immigration of spermatia will be
extremely limited (Engel and Destombe 2002). In
G. gracilis fertilization success was higher in highshore pools than low-shore pools. The high shore
is isolated from the sea for longer periods of time
than is the low shore, which may allow a higher
concentration of spermatia to build up in high
shore pools, thereby increasing the chance of successful encounters between male and female gametes
(Engel and Destombe 2002). Higher genetic diversity
was observed in low-shore pools of Gracilaria gracilis
due to the directional gene flow from high-tide pools
to low-tide pools (Engel et al. 2004). It is clear that
restricted gene flow between tide pools, particularly
at different levels of the shore where there are contrasting physiological stresses (Engel et al. 1999),
could contribute to the processes of sympatric
speciation.
The presence of even small numbers of male
N. harveyi in a pool increased fertilization rates to
near saturation levels (Fig. 2). In Polysiphonia lanosa,
using the lower boundary assumptions for male fecundity, the spermatia: carpogonia ratio was calculated to be 3000–4700:1 (Kaczmarska and Dowe
1997). Theoretical considerations predict that in species in which sperm competition occurs selection
should favor the evolution of tiny sperm produced
in high numbers (Parker 1982). An extremely large
number of spermatia can therefore be released into
the rock pool from a small biomass of male gametophyte. Rock pools develop circulation currents that
should be able to transport spermatia around the
pool. At low tide spermatia will accumulate in the
rock pool and fertilization rates are expected to be a
function of spermatial concentration up to a certain
point; however it is possible that after a certain concentration is reached further increase will have little
effect on fertilization success as other factors such as
female fertility may take over. The genetic consequences of differences in numbers of successful fertilizations between pools are greatly amplified by the
mitotic production of carpospores. Even assuming
minimum values for production of carpospores,
numbers per pool differed by two orders of magnitude from less than 3000 when no males were present to over 300,000 in a pool with plentiful males.
Most populations of N. harveyi occur in tidepools,
so the results from tidepools are likely to be more
characteristic for this species, and possibly for other
tidepool species. The low fertilization success of
N. harveyi in the current-exposed marina, despite
abundant reproductive males, is presumed to be because released spermatia suffer from rapid dilution,
diffusion and dispersal. These conditions may be
similar to those experienced by algae in intertidal
pools during high tide. In Gracilaria gracilis only
6.3% of fertilizations occurred during high tides
(Engel and Destombe 2002). Motion of the water
and exposure of the shore both have a major effect
on fertilization rates. Lower fertilization rates occur
in areas with swifter or more turbulent flow of water
(Denny 1988). The marina at Sandy point, Hayling
Island is quite open and the collection area is exposed with strong water currents.
Polyploidy in the Antithamnion densum/sparsum
complex
Antithamnion sparsum is clearly polyploid (n ¼ c. 60)
by comparison with A. densum (n ¼ 33). Polyploidy
502
was previously reported by Kim et al. (2008) but
their estimates of chromosome number were of
n ¼ 44, made on dividing spermatangial cells.
Unfortunately, it was not possible to determine chromosome numbers for A. defectum, although Kim
et al. (2008) reported n ¼ 21. They also reported
a similar number in A. densum, so their counts may
be too low. Polyploidy in A. cruciatum sporophytes
from Ireland (85 10 chromosomes) was not unexpected because it was previously observed in A. cruciatum from Newfoundland (85–110 chromosomes:
Whittick and Hooper 1977). Their culture did not
produce sporangia, whereas our Irish isolate formed
tetraspores but these were not viable. It seems likely
that sporophytes of both strains were triploid so that
meiosis failed. Generally, in red algae, polyploidy
seems common only in the Ceramiales (Maggs
1988).
Antithamnion defectum was paraphyletic with
regard to A. sparsum in rbcL analyses. A. sparsum
appears to have emerged from a lineage of A. defectum, as reported by Kim et al. (2008) on the basis of
RAPD analyses. Taxonomically, in order to recognize
A. sparsum, as advocated by Kim et al. (2008), multiple species of A. defectum should be described.
Phylogeny of nuclear and plastid genes was congruent, suggesting that A. sparsum is likely to be an
autopolyploid. Furthermore, two Korean isolates
and one Japanese isolate of A. sparsum were identical
for all molecular markers used by Lee et al. (2005).
This is compatible with A. sparsum having spread
from a single event of polyploidization. Fully isolated
polyploid species of higher plants can arise in one or
two generations if they can establish clonal growth
(Rieseberg and Willis 2007). In the case of A. sparsum, cells are larger than in the diploid species A.
defectum (Kim et al. 2008), and isolates grew vigorously in culture, suggesting that A. sparsum could be
a strong competitor with the parent diploid species.
Boo and Lee (1983) found evidence of partial interfertility between Korean A. sparsum and the UTEX
isolate of A. defectum. A. defectum females could be
fertilized by A. sparsum males but the reciprocal
cross produced no mature cystocarps (possible
post-zygotic isolation). Kim et al. (2008) did not
observe A. sparsum–A. defectum interfertility using
A. defectum UTEX isolate LB2261 although this contrasting result could be related to this UTEX isolate
having then been in culture for over 40 years.
Studies of chromosomes in seaweeds are hampered by their very small sizes (typically 52 mm
even in the Ceramiales), and karyology has rarely
been applied to studies of breeding systems. An exception was the discovery that one population
C. A. Maggs et al.
identified as Gracilaria gracilis (as G. verrucosa)
from Cape Gris-Nez (northern France) has an anomalous chromosome number of n ¼ 16–18, instead of
the typical number of n ¼ 24 found in other populations of G. gracilis, which suggested that this population is genetically isolated (Godin et al. 1993).
The result was difficult to interpret at that time,
but now Destombe et al. (2010) have found that
‘‘G. gracilis’’ in northern France consists of two sibling species, G. gracilis and G. dura. Although these
entities are genetically distinct for nuclear, plastid
and mitochondrial markers, evidence of introgression
suggested that interspecific hybridization has occurred at some time between these sibling species.
General conclusions
The Ceramiales (Rhodophyta) offer enormous potential for studying speciation processes in the laboratory and in nature. Unusual life-history features such
as the lack of flagella may be related to the growing
evidence that that sperm limitation is uncommon in
the red algae, at least in intertidal (tidepool) species.
Ongoing studies of gamete recognition may help us
to understand one mechanism by which sympatric
speciation can occur. Polyploidy is apparently widespread in the Ceramiales, and has contributed to
genetic isolation of particular populations.
Acknowledgments
We thank the organizers of this symposium,
Anuschka Faucci, Maria Pia Miglietta, and
Francesco Santini, as well as the other participants,
for the opportunity to contribute to this symposium.
We are grateful to Prof. I. K. Lee (Korea) and
Dr L’Hardy-Halos (France) for cultured isolates.
Prof. J. A. Brodie (Natural History Museum,
London) is thanked for helpful discussions.
Funding
The Antithamnion study was supported through a
Natural Environment Research Council Advanced
Fellowship to C.A.M. Our research on non-native
species is funded by the AXA Research Fund.
The symposium was supported by the Society for
Integrative and Comparative Biology.
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