Abstract

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 Antithamnionsparsum is closely related to the diploid species Antithamniondefectum, 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 Antithamniondensum 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 + 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 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 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 µm 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 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 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. Neosiphoniaharveyi was also collected from Sandy Point Marina, 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 (>2 knots).

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).

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 (m2Approx. volume (m3Male 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 1.2 0.0000 0.0367 0.0000 0:2 204 35 
C2 10 2.0 0.0750 0.0770 0.9740 1:4 10,190 79 
C3 0.9 0.2 0.1111 0.7144 0.1556 2:7 780 90 
C4 0.2 0.0500 0.5420 0.0925 1:4 814 79 
C5 0.6 0.3196 0.1300 2.4585 3:2 555 85 
C6 0.5 0.1 0.0800 1.0200 0.0784 1:2 1057 90 
C7 10 2.0 0.0075 0.0230 0.3261 1:3 353 76 
D1 1.5 0.5 0.0000 0.2027 0.0000 0:1 230 
D2 0.5 0.2 0.4620 0.4620 1.0000 3:2 143 80 
D3 1.0 0.0253 0.0223 1.1345 5:2 310 76 
D4 2.25 0.7 0.1244 0.0809 1.5377 1:4 261 74 
D5 1.5 0.5 0.0000 0.2587 0.000 0:1 92 
Marina – – – – – – – – 5–37 mean: 14.0 
Tide pool Area (m2Approx. volume (m3Male 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 1.2 0.0000 0.0367 0.0000 0:2 204 35 
C2 10 2.0 0.0750 0.0770 0.9740 1:4 10,190 79 
C3 0.9 0.2 0.1111 0.7144 0.1556 2:7 780 90 
C4 0.2 0.0500 0.5420 0.0925 1:4 814 79 
C5 0.6 0.3196 0.1300 2.4585 3:2 555 85 
C6 0.5 0.1 0.0800 1.0200 0.0784 1:2 1057 90 
C7 10 2.0 0.0075 0.0230 0.3261 1:3 353 76 
D1 1.5 0.5 0.0000 0.2027 0.0000 0:1 230 
D2 0.5 0.2 0.4620 0.4620 1.0000 3:2 143 80 
D3 1.0 0.0253 0.0223 1.1345 5:2 310 76 
D4 2.25 0.7 0.1244 0.0809 1.5377 1:4 261 74 
D5 1.5 0.5 0.0000 0.2587 0.000 0:1 92 
Marina – – – – – – – – 5–37 mean: 14.0 

Note. For a yacht marina in southern England, only fertilization success of N. harveyi females is provided.

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 rbc
JN089390: Isolate CAM 220 = 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 rbc
JN089392: Isolate CAM 213 = 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 rbc
JN089393: Isolate CAM 218 = UTEX LB801 
rbcS/18S/ITS 
AF346223/ AY168240/AY168251 (UTEX LB801) 
Antithamnion cruciatum (C. Agardh) Nägeli rbc
JN089394: Isolate CAM 216, Mulroy Bay, Co. Donegal, Ireland, February 16, 1993 
Antithamnion densum (Suhr) Howe rbc
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 = CAM 214) 
Antithamnion aglandum Kim et Lee rbc
AY594700 (Wando, Korea, January 29, 1999) 
 rbcS/18S/ITS 
AF346212/AY168234/AY168244 (Dokdo island, Korea, March 21, 1993) 
Antithamnion nipponicum Yamada et Inagaki rbc
AY594699 
rbcS/18S/ITS 
AY168255/AY168235/AY168245 (Wando, Korea, January 20, 1996) 
Antithamnionella sp. rbc
DQ787564 (isolate A31) 
rbcS/18S/ITS 
AF346225/AY168243/AY168254 (isolate ATN, Korea; Lee et al. 2005
Species Genes sequenced (rbcL only) or obtained from GenBank, with collection or isolate information 
Antithamnion defectum Kylin rbc
JN089390: Isolate CAM 220 = 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 rbc
JN089392: Isolate CAM 213 = 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 rbc
JN089393: Isolate CAM 218 = UTEX LB801 
rbcS/18S/ITS 
AF346223/ AY168240/AY168251 (UTEX LB801) 
Antithamnion cruciatum (C. Agardh) Nägeli rbc
JN089394: Isolate CAM 216, Mulroy Bay, Co. Donegal, Ireland, February 16, 1993 
Antithamnion densum (Suhr) Howe rbc
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 = CAM 214) 
Antithamnion aglandum Kim et Lee rbc
AY594700 (Wando, Korea, January 29, 1999) 
 rbcS/18S/ITS 
AF346212/AY168234/AY168244 (Dokdo island, Korea, March 21, 1993) 
Antithamnion nipponicum Yamada et Inagaki rbc
AY594699 
rbcS/18S/ITS 
AY168255/AY168235/AY168245 (Wando, Korea, January 20, 1996) 
Antithamnionella sp. rbc
DQ787564 (isolate A31) 
rbcS/18S/ITS 
AF346225/AY168243/AY168254 (isolate ATN, Korea; Lee et al. 2005

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  

(1)
formula
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 20°C, in a regime of 16:8 h light:dark, at a photon irradiance of ca. 20 µmol 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 15°C under a photon irradiance of ca. 5 µmol 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 (5′-CAC AAC CAG GTG TTG ATC CAA TTG AAG C-3′) was used as the forward external primer and Anti4 (5′ CTA CGA AAG TCA GCT GTA TCT GTA GAA GTA TA 3′) 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 94°C, 30 cycles of 1 min at 94°C and 3 min at 60°C, followed by a final extension phase at 60°C for 10 min. Reactions 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 (5′-CGT GAG CGT ATG GAT AAA TTT GGT CGT TC-3′) and Anti3 (5′-TTA CTT TAC GTA AAG CAG CCC AAT CTT GTT C-3′) 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 sites and gamma distribution (GTR+I+G), 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 + 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 = 0.52, P < 0.05). 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 = 0.13, P > 0.05). A nonlinear regression described the relationship between fertilization success and male biomass in pools (Fig. 2, r2adj = 84%, P < 0.05). The intercept of the fitted equation is at 21%, giving an estimate of the background level of fertilization success.

Fig. 2

Non-linear regression of female fertilization success by male biomass in tidepools for N. harveyi showed an excellent fit (r2adj = 84%; P < 0.05). Fertilization success = 20.5 + 62.9 male biomass/(0.001 + male biomass). The equation has an intercept at 20.5%, which can be taken as a background level of fertilization success.

Fig. 2

Non-linear regression of female fertilization success by male biomass in tidepools for N. harveyi showed an excellent fit (r2adj = 84%; P < 0.05). Fertilization success = 20.5 + 62.9 male biomass/(0.001 + male biomass). The equation has an intercept at 20.5%, which can be taken as a 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 = 2.12, P > 0.05).

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 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 = 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 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.

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 (HJ) showing ∼61 paired chromosomes.

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 (HJ) showing ∼61 paired chromosomes.

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.

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.

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 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 high-shore 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 = c. 60) by comparison with A. densum (n = 33). Polyploidy was previously reported by Kim et al. (2008) but their estimates of chromosome number were of n = 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 = 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 < 2 µm even in the Ceramiales), and karyology has rarely been applied to studies of breeding systems. An exception was the discovery that one population identified as Gracilaria gracilis (as G. verrucosa) from Cape Gris-Nez (northern France) has an anomalous chromosome number of n = 16–18, instead of the typical number of n = 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.

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.

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.

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Author notes

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.