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(American Journal of Botany. 2001;88:1209-1213.)
© 2001 Botanical Society of America, Inc.

Phycology

cpDNA-RFLP in Ceramium (Rhodophyta): intraspecific polymorphism and species-level phylogeny1

Rémi A. Wattier, Alistair L. Davidson, Barbara A. Ward2 and Christine A. Maggs3

School of Biology and Biochemistry, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland

Received for publication July 5, 2000. Accepted for publication November 30, 2000.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Restriction fragment length polymorphism (RFLP) analysis of chloroplast (cp) DNA is a powerful tool for the study of microevolutionary processes in land plants, yet has not previously been applied to seaweed populations. We used cpDNA-RFLP, detected on Southern blots using labeled total plastid DNA, to search for intraspecific and intrapopulational cpDNA RFLP polymorphism in two species of the common red algal genus Ceramium in Ireland and Britain. In C. botryocarpum one polymorphism was detected in one individual among 18 from two populations. Twenty-six individuals of C. virgatum from five populations at three locations exhibited a total of four haplotypes. One was frequent (80.8% of individuals); the others were rare (7.7, 7.7, and 4.2%) and were private to particular populations. Polymorphism was observed in two populations. The corrected mean was 2.26 ± 0.36 haplotypes per population, which was within the typical range determined for higher plants using similar techniques. The spatial distribution of haplotypes was heterogeneous, with highly significant population differentiation (P = 0.00018; Fisher's exact test). Intraspecific polymorphism in C. virgatum had no impact on species-level phylogenetic reconstruction. This is the first unequivocal report of both intraspecific and intrapopulational cpDNA-RFLP polymorphism in algae.

Key Words: Ceramium • intraspecific variation • plastid DNA • population genetics • red algae • RFLPs • systematics


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Restriction fragment length polymorphism (RFLP) analysis of chloroplast (cp) DNA is now recognized as a powerful tool for the study of land-plant evolutionary processes, both at the intraspecific level and in molecular systematics (reviewed by Soltis et al., 1997 ; Schaal et al., 1998 ; Ennos et al., 1999 ; Wiens, 1999 ). Until relatively recently, however, although cpDNA-RFLPs were used extensively for taxonomy and phylogeny, they were regarded as unsuitable genetic markers for intraspecific analysis. This view was based on the low level of polymorphism in Lupinus texensis reported in the first extensive analysis of intraspecific cpDNA-RFLPs (Banks and Birky, 1985 ) and on the slower rate of evolution of cpDNA than of nuclear DNA (Soltis, Soltis, and Milligan, 1992 ). It was generally assumed that intraspecific polymorphism would not interfere with phylogenetic reconstruction using cpDNA-RFLP. Likewise, when cpDNA-RFLP was used as a taxonomic identification tool, any difference, even minor, in banding patterns between two individuals was often interpreted as indicating specific status. The possibility that these differences could instead represent intraspecific diversity was ignored, and most phylogenetic reconstructions using cpDNA-RFLP were based on one sample per taxon (see Wiens, 1999 ).

Since 1985, however, many more examples of intraspecific polymorphism have been discovered: Soltis, Soltis, and Milligan (1992) listed nearly 60 cases in ferns, conifers, and angiosperms from 15 families. During the last 5 yr, at least 20 studies have employed cpDNA-RFLP to study microevolutionary processes, including the genetic structure of angiosperm species such as Lambertia orbifolia (Byrne, Macdonald, and Coates, 1999 ), hybrid zones (e.g., in Salix; Harding et al., 2000 ), and phylogeography (e.g., Quercus spp.; Ferris et al., 1998 ).

Several recent phylogenetic analyses of cpDNA diversity at the species-to-genus level have included an intraspecific component, with widely varying results. Some studies, e.g., of Syringa (Kim and Jansen, 1998 ) and Limonium (Palacios, Rosselo, and Gonzalez-Candelas, 2000 ), found strong support for species monophyly. In other genera, e.g., Phacelia (Levy et al., 1996 ) and Senecio (Bain and Jansen, 1996 ), cpDNA polymorphism does not conform to species boundaries: many haplotypes are shared extensively between taxa, and species-level phylogenies are deeply affected by intraspecific diversity.

For algae, in contrast, the great majority of studies have used cpDNA-RFLP, based on restriction patterns of purified cpDNA, strictly as a molecular taxonomic tool (e.g., Parsons, Maggs, and Douglas, 1990 ; Goff et al., 1992 ; Chopin et al., 1996 ). The largest number of conspecific samples investigated to date is nine (Goff and Coleman, 1988 ), mainly due to the lengthy procedures involved in isolating plastid DNA. Furthermore, several cpDNA-RFLP studies included species of ambiguous taxonomic status, so observed polymorphism could not be assigned to either intraspecific or supraspecific levels (e.g., Rice and Bird, 1990 ; Gonzalez et al., 1996 ). Recently, limited phylogenetic analyses have included conspecific samples (Maggs and Ward, 1996 ; Blomster, Maggs, and Stanhope, 1998 ), but there have been no intrapopulational cpDNA-RFLP analyses in algae.

The aims of the present study were to determine whether intraspecific and intrapopulational cpDNA-RFLP polymorphism could be detected and used for population genetics in two closely related Ceramium species (Rhodophyta) and to assess the possible impact of intraspecific polymorphism on species-level phylogenetic reconstruction. We also evaluated the use of cpDNA-RFLP, detected on Southern blots using labeled total plastid DNA, in comparison with other reported tools for molecular taxonomy and population genetics of red algae.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Sampling
The four Ceramium species included in this study are C. botryocarpum Griffiths ex Harvey, C. virgatum Roth [synonym C. nodulosum (Lightfoot) De Candolle (Maggs et al., 2001)], C. secundatum Lyngbye, and C. pallidum (Nägeli ex Kützing) Maggs et Hommersand. The first three species are known to be closely related from analyses of a large cpDNA-RFLP data set (Ward, 2000 ) and unpublished rbcL sequence data, and C. pallidum is a member of a sister clade. Ceramium pallidum can be readily distinguished from the other species by its morphology (branching pattern). Ceramium virgatum differs from C. botryocarpum and C. secundatum by its irregular branching and the last two species can be separated anatomically by the number of periaxial cells (6–7 vs. 7–8 cells; Maggs and Hommersand, 1993 ).

Populations of C. botryocarpum and C. virgatum were sampled by removing all thalli growing in randomly selected 5 x 5 cm squares of a subdivided quadrat and sorting the algae carefully to remove epiphytes and to separate individual thalli. A total of 18 thalli of C. botryocarpum was sampled from two locations: Annalong, County Down, Ireland, 9 July 1994 (N = 12) and Lyme Regis, Dorset, southern England, 16 September 1993 (N = 6). Both sites were similarly sheltered fucoid-dominated shores with a turf of small red algae on shaded vertical faces. For C. virgatum, a total of 26 individuals was sampled from three locations. Two of these sites were ~80 km apart in County Down, on the southeast coast of Northern Ireland, at Annalong (20 individuals, 27 January 1994) and Crawfordsburn, Belfast Lough (four thalli, 25 January 1994). At the Annalong site samples were taken from three different populations, all within 100 m, located in rockpool 1 (N = 6), rockpool 2 (N = 5), and lowershore channels (N = 9). All C. virgatum thalli were epiphytic in permanent pools. At the third location, Martinshaven, Pembrokeshire, Wales, two thalli (25 July 1992, epiphytic on Fucus serratus) were analyzed. Single individuals of C. pallidum (Doorin Point, west Donegal, Ireland, epiphytic on Fucus spp. in upper mid-shore pools on 20 March 1992) and C. secundatum (Marble Hill, Sheephaven Bay, north Donegal, epiphytic in deep intertidal lagoons on 5 April 1992) were used for phylogenetic analysis only.

Molecular techniques
Plastid DNA-RFLPs were detected using Southern hybridization of genomic DNA probed with nonradioactively labeled (Digoxigenin, Boehringer Mannheim, now Roche Molecular Biochemicals, Lewes, Sussex, UK) purified plastid DNA, according to Maggs and Ward (1996) . Two restriction enzymes, EcoRI and PstI, were used for all samples, and four additional hexanucleases (KpnI, HindIII, BglII, and PvuII) were used only on a subset of 12 C. botryocarpum individuals from one population.

Data analysis
Banding patterns were analyzed visually. Restriction fragments were scored as present or absent to generate a data matrix. Phylogenetic trees were constructed using Dollo parsimony and neighbor-joining options in PAUP* 4 (Swofford, 1999 ), rooted with C. pallidum as outgroup, bootstrapped with 1000 replications, and edited using TREEVIEW 1.6.1 (Page, 1996 ). Heterogeneity of distribution of absolute haplotype frequencies between populations of C. virgatum was tested with Fisher's exact test for population differentiation (Raymond and Rousset, 1995a ), using the STRUC program included in GENEPOP version 3.2 (Raymond and Rousset, 1995b ). The mean number of haplotypes per population for reported studies of higher plants was corrected for small sample sizes by multiplying the number of haplotypes by n/(n – 1), where n = number of samples ± 1 SD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Of the two species assayed for intraspecific polymorphism of cpDNA-RFLPs, C. botryocarpum exhibited only one polymorphism, detected using two restriction enzymes on DNA from 18 individuals sampled at two localities 500 km apart (Fig. 1). One individual differed from the others by the loss/gain of a PstI restriction site (not shown). Screening 12 samples from one of the populations with four additional enzymes detected no further polymorphisms. In contrast, a total of four haplotypes was observed for C. virgatum (Table 1). Haplotype A was very frequent (80.8%). Haplotype B, which differed by the gain of one EcoRI and one PstI restriction site, was rare (7.7%). Haplotypes C and D, which both differed from A by the gain of one EcoRI restriction site each, were also rare (3.8 and 7.7%, respectively). Polymorphism was observed within two of the populations (Crawfordsburn and Annalong rockpool 2; Fig. 2). The corrected mean number of haplotypes per population was 2.26 ± 0.36.



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  		Fig. 1. Chloroplast DNA Southern-RFLP, digested with BglII, for 12 individuals of Ceramium botryocarpum from one population (Annalong, Northern Ireland), with sizes of labeled marker (kb). No polymorphisms were detected with this enzyme

 

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  		Table 1. Infraspecific cpDNA diversity in Ceramium virgatum in Ireland and Wales

 


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  		Fig. 2. Chloroplast DNA Southern-RFLP, digested with EcoRI, for 15 individuals of Ceramium virgatum from three populations, with marker sizes (kb). Crawfordsburn individuals 2 and 3 are haplotype A; individuals 1 and 4 are haplotype B, in which a gain of a restriction site in an 11.0-kb fragment (dot; one of a doublet) has generated 8.3 and 2.6 kb fragments (arrows). At Annalong, all six individuals from pool 1 are haplotype A. Of the five individuals shown from pool 2, 7–9 and 11 are haplotype A, and 10 is haplotype C, in which a restriction site gained in the 4.0-kb fragment (dot) has generated a 2.4-kb fragment (arrow); the expected 1.6-kb fragment is below the size detection limit for this blot

 
The spatial distribution of haplotypes was heterogeneous, with significant population differentiation (P = 0.00018; Fisher's exact test). The frequent A haplotype was shared by all populations, but each rare haplotype was present in only one population: B at Crawfordsburn, C in Annalong rockpool 2, and D at Martinshaven, Wales (Table 1).

The intraspecific polymorphism observed in C. virgatum had no impact on species-level phylogenetic reconstruction (Figs. 3, 4). All four haplotypes of C. virgatum form a clade with 100% bootstrap support in both parsimony and neighbor-joining analyses, and the tree topology is identical to that in analyses using only the frequent C. virgatum haplotype A.



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  		Fig. 3. Chloroplast DNA Southern-RFLP, digested with PstI, for samples of Ceramium pallidum (P), C. virgatum (V), C. botryocarpum (B), and C. secundatum (S), with sizes (kb) for unlabeled marker. Bands shared by C. botryocarpum and C. secundatum, the two most closely related species, but not by C. virgatum, are indicated by dots. Fragments unique to C. virgatum are indicated by filled triangles

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
This is the first unequivocal report of both intraspecific and intrapopulational cpDNA-RFLP polymorphism in algae. As noted above, very few previous investigations of seaweeds have included any tests for intraspecific polymorphism. In the most extensive of these, Goff and Coleman (1988) found "almost identical" banding patterns, using eight hexanucleases on purified cpDNA, for nine pooled samples of Gracilariopsis (as Gracilaria) lemaneiformis (Rhodophyta) from separate locations along a 3500-km stretch of Pacific North America. In the invasive green seaweed Codium fragile ssp. tomentosoides, Goff et al. (1992) reported no definite cpDNA-RFLP variation using four to six enzymes on samples from six geographic locations on Atlantic and Pacific coasts of North America. However, among seven samples of Chondrus crispus from both sides of the North Atlantic Chopin et al. (1996) found an unspecified low level of restriction site polymorphism with two of nine enzymes.

Our observations in C. virgatum of four haplotypes in total and of a corrected mean of 2.26 ± 0.36 haplotypes per population represent a level of intraspecific and intrapopulational polymorphism in the center of the range determined for land plants using similar techniques (purified cpDNA, probes covering the majority of the chloroplast genome, or using complete cpDNA as a probe). Comparable mean values for five angiosperm species (Rhododendron flammeum/R. canescens, Kron, Gawen, and Chase, 1993 ; Thymus vulgaris, Tarayre et al., 1997 ; Beta vulgaris ssp. maritima, Forcioli et al., 1998 ; Penstemon haydenii, Caha, Lee, and Stubbendieck, 1998 ; Lambertia orbifolia, Byrne, Macdonald, and Coates, 1999 ) are 1.03–2.77. Trifolium pratense is exceptional, with 22 haplotypes found in 96 individuals, and 9.99 ± 1.13 haplotypes per population (Milligan, 1991 ).

Nevertheless, in view of the relatively small number of individuals of C. virgatum examined, the restricted area sampled, the fact that only two enzymes were employed, and the highly significant population differentiation demonstrated here, it might reasonably be expected that more haplotypes would be observed if sampling size, geographic area, and number of enzymes were increased. Recent studies using single-strand conformational polymorphism (SSCP) of the spacer region between the plastid-encoded genes for the small and large subunits of Rubisco have revealed intraspecific (3–9 haplotypes) and intrapopulational polymorphism in three tropical red algae in the Ceramiales, Bostrychia moritziana, B. tenuissima (Zuccarello, West, and King, 1999 ; Zuccarello et al., 1999 ), and Caloglossa leprieurii (Zuccarello, Bartlett, and Yeates, 2000 ). In one of these species, C. leprieurii, haplotype diversity was geographically structured.

The lack of impact of intraspecific polymorphism in C. virgatum on species-level phylogenetic reconstruction is similar to results for Limonium (Palacios, Rosselo, and Gonzalez-Candelas, 2000 ), for example, rather than for species such as Senecio (Bain and Jansen, 1996 ) in which cpDNA polymorphism does not reflect species boundaries. However, the difficulties of interpreting potential intraspecific variability are highlighted by Brodie et al.‘s (1996) suggestion that Porphyra (Rhodophyta) sequences that differed at a single position in the Rubisco spacer represent separate species.

Restriction digestion of purified cpDNA, because it is time consuming and not compatible with processing large number of samples, has largely been superseded by PCR-based methods. However, primer design for red algal plastids is problematic for several reasons. Of the available "universal" chloroplast primers (Taberlet et al., 1991 ; Dumolin-Lapegue, Pemonge, and Petit, 1997 ) none has been tested on red algae, which are not closely related to green plants (Ragan and Gutell, 1995 ), and the evolutionary diversity of the Rhodophyta is so great that the possibility of designing universal primers for them is very limited. In fact, only two complete red algal chloroplast genomes are available, and both of these are for members of the small subclass Bangiophycidae. In addition, marine algae, unlike most land plants, have abundant congeneric and conspecific epiphytes growing on them, which can cause serious difficulties for any PCR-based approach to genetic analysis of field-collected individuals (e.g., Blomster et al., 2000 ). Ceramium virgatum, for example, is almost ubiquitous as an epiphyte.

Our method of employing the complete chloroplast genome as a probe on digests of total genomic DNA maximizes the probability of finding polymorphisms, and, by avoiding PCR amplification, reduces the potential for generating spurious results. Both conserved and variable regions of the plastid genome are sampled simultaneously, providing information relevant to phylogenetic reconstruction at different levels. The present demonstration of intraspecific polymorphism in red algae shows the potential of this technique for the study of genetic differentiation at a geographic scale and probably down to within-population spatial structure.



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  		Fig. 4. Phylogenetic analysis by Dollo parsimony of Southern-RFLP EcoRI and PstI data for Ceramium virgatum (four haplotypes), C. botryocarpum, and C. secundatum, rooted with C. pallidum. The results of 1000 bootstrap replications for both parsimony and neighbor-joining analyses are shown at nodes. The intraspecific variation in C. virgatum has no effect on the inferred phylogeny

 

   FOOTNOTES
 
1 The authors thank John Taggart for his contribution to the methodology and Jim Provan for helpful discussions. This research was supported by the Natural Environment Research Council, grant GR3/7945. Back

2 Current address: Veterinary Sciences, Department of Agriculture and Rural Development, Dundonald, N. Ireland. Back

3 Author for correspondence (c.maggs{at}qub.ac.uk ). Back


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 DISCUSSION
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