Annals of Botany 95/1 © Annals of Botany Company 2005; all rights reserved
Nuclear DNA Content Estimates in Multicellular Green, Red and Brown Algae: Phylogenetic Considerations
Department of Biological Sciences, University of North Carolina-Wilmington, 601 South College Road, Wilmington, NC 28403-3915, USA
* E-mail kapraund{at}uncw.edu
Received: 22 October 2003 Returned for revision: 23 December 2003 Accepted: 11 February 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSION...PHAEOPHYTARHODOPHYTAGENERAL SUMMARYNOTES ON APPENDIXES I-III....APPENDIX I. CHROMOSOME NUMBER...KEY TO REFERENCESAPPENDIX II. CHROMOSOME NUMBER...KEY TO REFERENCESAPPENDIX III. CHROMOSOME NUMBER...KEY TO REFERENCESACKNOWLEDGEMENTSLITERATURE CITED |
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• Background and Aims Multicellular eukaryotic algae are phylogenetically disparate. Nuclear DNA content estimates have been published for fewer than 1 % of the described species of Chlorophyta, Phaeophyta and Rhodophyta. The present investigation aims to summarize the state of our knowledge and to add substantially to our database of C-values for theses algae.
• Methods The DNA-localizing fluorochrome DAPI (4', 6-diamidino-2-phenylindole) and RBC (chicken erythrocyte) standard were used to estimate 2C values with static microspectrophotometry.
• Key Results 2C DNA contents for 85 species of Chlorophyta range from 0·2–6·1 pg, excluding the highly polyploidy Charales and Desmidiales with DNA contents of up to 39·2 and 20·7 pg, respectively. 2C DNA contents for 111 species of Rhodophyta range from 0·1–2·8 pg, and for 44 species of Phaeophyta range from 0·2–1·8 pg.
• Conclusions New availability of consensus higher-level molecular phylogenies provides a framework for viewing C-value data in a phylogenetic context. Both DNA content ranges and mean values are greater in taxa considered to be basal. It is proposed that the basal, ancestral genome in each algal group was quite small. Both mechanistic and ecological processes are discussed that could have produced the observed C-value ranges.
Key words: C-value enigma, Chlorophyta, DNA C-values, eukaryotic algae, nuclear genome size, Phaeophyta, Rhodophyta
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSION...PHAEOPHYTARHODOPHYTAGENERAL SUMMARYNOTES ON APPENDIXES I-III....APPENDIX I. CHROMOSOME NUMBER...KEY TO REFERENCESAPPENDIX II. CHROMOSOME NUMBER...KEY TO REFERENCESAPPENDIX III. CHROMOSOME NUMBER...KEY TO REFERENCESACKNOWLEDGEMENTSLITERATURE CITED |
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About 20 years ago, publications began to appear encouraging the development of technologies for the genetic transformation of commercially important seaweeds into domesticated ‘sea crops’ (Zhao and Zhang, 1981
; Tang, 1982; Zhang, 1983; Saga et al., 1986; Polne-Fuller and Gibor, 1987; Cheney, 1988a, b, 1990). For the most part, these investigations were based on the adoption of biotechnology procedures employed with flowering plants (Evans et al., 1981; Harms, 1983; Torrey, 1985). In seaweeds, initial problems in confirming heterokaryon formation (Fujita and Migita, 1987; Kapraun, 1989, 1990) and in defining parameters for protoplast and heterokaryon regeneration (Cheney et al., 1987; Polne-Fuller and Gibor, 1987; Xue-wu and Gordon, 1987; Cheney, 1988a, b; Kapraun and Sherman, 1989) seemed to be surmountable. It appeared that development of new genotypes for seaweed mariculture would inevitably track the progress reported by crop scientists. However, this was not to be, as the more or less routine genetic manipulations in crop plants, including production of somatic hybrids, were made possible by an extensive body of knowledge from decades of genetic research. In brief, applied phycology could mimic the technology of crop science, but it could not deliver somatic hybrids with stable, desired genomes. Some of us suspected that the successful application of biotechnology procedures to the domestication of seaweeds would require criteria for pre-screening candidate species (Dutcher et al., 1990; Kapraun and Dutcher, 1991). For most target taxa, only chromosome complement data were available, usually without any reference to their karyotype (Cole, 1990). The extent of polyploidy in target taxa and related species was generally unknown. No published data existed for nuclear DNA contents, nuclear G+C molecular percentages and genome complexity. Random manipulations based on chance were more likely to result in unstable constructs than in fortuitous combinations (van der Meer and Patwary, 1983; van der Meer, 1987, 1990; Zhang and van der Meer, 1988). Consequently, the most comprehensive program to date was initiated at the Center for Marine Science Research, University of North Carolina—Wilmington to provide the basic genetic data that were otherwise unavailable for seaweeds (Kapraun, 1999). Specifically, it was our intent to pre-screen target taxa for genetic manipulation by identifying potentially significant nucleotype parameters, including nuclear genome size, organization and complexity (extent of unique and repetitive nucleotide sequences), chromosome number, karyotype pattern and presence of polyploidy in closely related taxa.
Initially, efforts were focused on commercially important red seaweeds, including Porphyra (Kapraun et al., 1991; Dutcher and Kapraun, 1994); agarophytes including taxa of the Gracilariales (Dutcher et al., 1990; Kapraun and Dutcher, 1991; Kapraun, 1993b; Kapraun et al., 1993a, b, 1996; Lopez-Bautista and Kapraun, 1995) and the Gelidiales (Freshwater, 1993; Kapraun et al., 1993a, 1994) and selected carrageenophytes including Eucheuma and Kappaphycus (Kapraun and Lopez-Bautista, 1997), Agardhiella (Kapraun et al., 1992), Gymnogongrus (Kapraun et al., 1993b) and Hypnea (Kapraun et al., 1994). Eventually, these investigations were expanded beyond strictly applied research to include opportunistic studies of other available taxa (Kapraun, 1993a; Kapraun and Dunwoody, 2002).
The significance of nuclear genome size variation in seaweeds is best appreciated in the larger context of our emerging understanding of the role of the nucleotype on phenotypic expression (Wenzel and Hemleben, 1982; Bennett, 1985). Specifically, an up to 200 000-fold variation in nuclear DNA content (C-value) has been reported in eukaryotes (Gregory, 2001). Although little correlation generally exists between nuclear genome size and an organism's complexity (the C-value paradox; Thomas, 1971), there is substantial evidence that the nucleotype affects the phenotype in a non-genic manner in response to environmental demands (Bennett, 1972; Cavalier-Smith, 1978, 1985a, b; 2005; Ohri and Khoshoo, 1986). In both plants and animals (Bachmann et al., 1972; Grime and Mowforth, 1982; Price, 1988) genome size and cell size extend their influence to ecological selection types. Larger genome size is associated with K-selection that favours slower development, delayed reproduction and larger body size. Smaller genome size is associated with r-selection that favours rapid development, high population growth rate, early reproduction and small body size (Bennett, 1972, 1987; Cavalier-Smith, 1978, 1985a; Begon et al., 1990).
An appreciation began to develop that nuclear genome profile data acquired for target species associated with the commercial seaweed industry might have an equally valuable basic research application in promoting our understanding of nucleotype transformations that have accompanied evolution in the major groups of marine algae. For example, in multinucleate coenocytic green algae, very large nuclear genomes (2C DNA contents = 2·6–4·9 pg) have a role in maintaining nucleus/cytoplasm ‘domains’ (Kapraun and Nguyen, 1994). In the Dasycladales (e.g. Acetabularia), nuclear genome content data superimposed on a phylogeny of the group suggest that ancient polyploidy events accompanied major radiations in extant families (Kapraun and Buratti, 1998). In red algae, nuclear genome size was found to be positively correlated with both size and number of reproductive spores and with ecological considerations, including K- and r-selection (Kapraun and Dunwoody, 2002). In addition, ‘basal’ or ancestral groups of red algae appear to have somewhat larger nuclear genomes than do more recently derived taxonomic groups (Kapraun and Dunwoody, 2002).
There are no published nucleotype data for representatives of many major groups of the Chlorophyta (Kapraun, 1993c) and the Rhodophyta (Kapraun and Dunwoody, 2002). The present investigation expands our knowledge of both groups with numerous original DNA content estimates. Nucleotype data for brown algae appear to be restricted to three investigations treating a handful of species (Dalmon and Loiseaux, 1981; Stam et al., 1988; Le Gall et al., 1993). Consequently, we initiated a significant effort to obtain nuclear genome size data for representatives of the major orders of brown algae. The present paper includes DNA content values for 44 species and varieties of Phaeophyta, only five of which had been previously investigated.
Certainly, one of the greatest challenges of this paper is to discuss nuclear genome size variation and trends that apply to all of the major groups of multicellular eukaryotic algae. These photosynthetic organisms have little more in common than the name ‘algae’, which has greater ecological implications (aquatic habitat) than taxonomic significance (Fig. 1) as algae are only distantly related to each other, and to photosynthetic land plants (Van de Peer et al., 1996). Red and brown algae have plastids surrounded by four membranes and contain chlorophyll a and c (or phycobillins; Chapman et al., 1998). The Chlorophyta, including the Zygnematales, Desmidiales and the Charales in the charophycean lineage, are characterized by plastids with two membranes and contain chlorophyll a and b as in land plants (McFadden et al., 1994a, b). Although classical taxonomic schemes implied that morphologically simple green algae where probably ancestral to land plants (Bold and Wynne, 1985), it is now understood that they are sister clades, and probably share a common ancestor (Mishler et al., 1994; Kenrick and Crane, 1997). This paper will discuss each of the three major groups of multicellular algae separately, elaborating their distinctive features and summarizing their similarities. Nuclear DNA content data from the present investigation and from the literature are summarized in three Appendices. Excluded are the numerous groups of mostly unicellular, microalgae such as the familiar diatoms (Chrysophytes), green microalgae and Prasinophytes, and eukaryotic Cyanidiophyceae (red algae). For some of these groups, limited anecdotal information is included in the text to support discussions on nuclear genome sizes in ancestral and basal algal lineages.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSION...PHAEOPHYTARHODOPHYTAGENERAL SUMMARYNOTES ON APPENDIXES I-III....APPENDIX I. CHROMOSOME NUMBER...KEY TO REFERENCESAPPENDIX II. CHROMOSOME NUMBER...KEY TO REFERENCESAPPENDIX III. CHROMOSOME NUMBER...KEY TO REFERENCESACKNOWLEDGEMENTSLITERATURE CITED |
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Algal material was fixed in Carnoy's solution and stored in 70 % ethanol at 4 °C. Preserved material was rehydrated in water and softened in 5 % w/v EDTA (Goff and Coleman, 1990
) for between 30 min and 3 h. Algal specimens were transferred to cover slips treated with subbing solution, air-dried and stained with DAPI (0·5 µg mL–1 4', 6-diamidodino-2-phenylindole; Sigma Chemical Co., St. Louis, MO 63178) as previously described (Goff and Coleman, 1990; Kapraun and Nguyen, 1990). Detailed procedures for microspectrophotometry with DAPI and requirements for reproducible staining have been specified previously (Kapraun and Nguyen, 1990; Kapraun, 1994) using a protocol modified after Goff and Coleman (1990). Microspectrophotometric data for Gallus (chicken erythrocytes or RBC) with a DNA content of 2·4 pg (Clowes et al., 1983) were used to quantify mean fluorescence intensity (If) values for algal specimens (Kapraun, 1994). DAPI binds by a non-intercalative mechanism to adenine and thymine rich regions of DNA that contain at least four A–T base pairs (Portugal and Waring, 1988). Consequently, RBC are best used as a standard for estimating amounts of DNA when the A–T contents of both standard and experimental DNA are equivalent (Coleman et al., 1981). Gallus has a nuclear DNA base composition of 42–43 mol % (molecular percent) G + C (Marmur and Doty, 1962). Limited published data for algae indicate mean values of 43·5 mol % G + C (n = 9, range = 40–47 mol %) for the Phaeophyta (Olsen et al., 1987; Stam et al., 1988; Le Gall et al., 1993), 41·6 mol % G + C (n = 22, range = 28–49 mol %) for the Rhodophyta (Kapraun et al., 1993b, c; Le Gall et al., 1993), and 46·2 mol % (n = 22, range = 35–56 mol %) for the Chlorophyta (Olsen et al., 1987; Freshwater et al., 1990; Kooistra et al., 1992; Le Gall et al., 1993). Algae investigated in this study are assumed to have a similar range of base pair compositions, and linearity is accepted between DAPI-DNA binding in both RBC and algal samples (Le Gall et al., 1993). Nuclear DNA contents were estimated by comparing the If values of the RBC standard and algal sample (Kapraun, 1994). All three algal groups contain taxa with some or all of their cells being multinucleate and often endopolyploid (Goff et al., 1992; Kapraun and Nguyen, 1994; Garbary and Clarke, 2002; Kapraun and Dunwoody, 2002). In addition, both red algae (Goff and Coleman, 1986) and green algae (Kapraun, 1994) have taxa that exhibit a nuclear ‘incremental size decrease associated with a cascading down of DNA contents’. Consequently, methodologies were developed specific for specimens of each algal group to permit assignment of C-level and interpretation of If data. Materials and methods, as well as information for collection locations, and data for number of algal nuclei examined in each sample and estimates of nuclear genome size (pg ± s.d.) are available at http://www.uncw.edu/people/kapraund/DNA. |
RESULTS AND DISCUSSION CHLOROPHYTA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSION...PHAEOPHYTARHODOPHYTAGENERAL SUMMARYNOTES ON APPENDIXES I-III....APPENDIX I. CHROMOSOME NUMBER...KEY TO REFERENCESAPPENDIX II. CHROMOSOME NUMBER...KEY TO REFERENCESAPPENDIX III. CHROMOSOME NUMBER...KEY TO REFERENCESACKNOWLEDGEMENTSLITERATURE CITED |
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The Division Chlorophyta contains the eukaryotic green algae, which possess chlorophylls a and b, as well as starch stored inside plastids with stacks of two to six thylakoids per band (Bold and Wynne, 1985
). Approximately 425 genera and 6500 species have been described (Alexopoulos and Bold, 1967). Simplicity and antiquity of green algae have long been accepted as evidence of their apparent ancestry to land plants (McCourt, 1995). Recently, parsimony analysis of sequence data for the RuBisCO large subunit (rbcL) (McCourt, 1995; McCourt et al., 1996) and small-subunit (SSU) rRNA group I interons (Bhattacharya et al., 1994) contradict this view and present a compelling case that an ancient divergence separates green plants into two major monophyletic lineages: the Chlorophyta and the Streptophyta (McCourt, 1995; Karol et al., 2001). The Chlorophyta contain the classical ‘green algae’, primarily the Chlorophyceae and Ulvophyceae (Watanabe et al., 2001; Fig. 2). The Streptophyta includes the charophycean lineage comprised of five orders, along with bryophytes and tracheophytes (Mishler et al., 1994). Identification of the charophycean lineage as the sister group of land plants suggests that their common ancestor was a branched, filamentous organism with a haplontic life cycle and oogamous reproduction (Karol et al., 2001).
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A third polyphyletic green plant lineage, at the base of the split of the Chlorophyta and the Streptophyta, includes the green alga Mesostigma viride (Turmel et al., 2002a
). A significant body of research centered around Mesostigma is emerging that provides insights into the timing of events that restructured both mitochondrial (mtDNA) and chloroplast (cpDNA) genomes during the evolution of green algae (Turmel et al., 2002b) and the transition from charophytes to land plants (Turmel et al., 2002a). The exact placement of Mesostigma remains controversial as some phylogenetic analyses include this species with the Prasinophyceae (Lemieux et al., 2000; Turmel et al., 2002b) while others consider it to be basal in the charophycean lineage (Karol et al., 2001). Whatever the exact position of Mesostigma, there is no doubt that this alga belongs to a deeply diverging lineage because it represents the most basal branch in trees inferred from sequences of land plants and all five orders of charophytes (Karol et al., 2001).
A residium of related unicellular micromonadophytes (= Prasinophytes; Kantz et al., 1990; Steinkötter et al., 1994; Karol et al., 2001) is, likewise, associated with the Chlorophyta–Streptophyta divergence (Fig. 2). Nuclear genome size and organization remain largely unknown in the Prasinophytes. Pulse field gel electrophoresis of Ostreococcus tauri (Prasinophyceae) resulted in a nuclear genome size estimate of 10·20 mbp (Courties et al., 1998) or 0·1 pg using the expression 1 pg = 980 Mbp (Bennett et al., 2000). The minute size of this genome, one of the smallest among free-living eukaryotic organisms, is best appreciated by comparison with the chloroplast (cpDNA) genome size of 118360 bp or 0·012 pg (Lemieux et al., 2000) reported in the closely related Mesostigma viride. It is assumed that this small nuclear genome size is evolutionarily derived rather than ancestral (Courties et al., 1998) as other members of the Mamiellaceae represent secondarily reduced forms (Daugbjerg et al., 1995). If such extreme reduction of nuclear genome size is typical of the micromonads, it may not be possible to reconstruct a hypothetical ancestral nuclear genome from extant species.
Charophycean algae
The charophycean lineage includes the Chlorokybales (Qiu and Palmer, 1999), Klebsormidiales (Karol et al., 2001), Conjugophyta (Zygnematales; Hoshaw et al., 1990), the Coleochaetales (Bhattacharya et al., 1994; McCourt, 1995) and the Charophyta (Surek et al., 1994; McCourt et al., 1996; Fig. 3).
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Coleochaetales. Members of this small and obscure group are minute epiphytes on aquatic angiosperms and aquatic algae (Bold and Wynne, 1985
). Feulgen microspectrophotometry was used to elucidate the life history of Coleochaete scutata (Hopkins and McBride, 1976). However, data were given as relative fluorescence units (rfu) without reference to a DNA standard. Apparently, there are no published estimates for nuclear DNA contents in any member of this order. Karyological studies of three Coleochaete speces have been published (Sarma, 1982). Reported chromosome complements of 1n = 24, 36 and 42 suggest a polyploid sequence derived from a basic complement of x = 12. The reported chromosome complement of n = 22 in Klebsormidium (Chaudhary and Sarma, 1978) likewise is consistent with polyploidy.
Desmidiales and Zygnematales. The conjugating green algae or Zygnematales make up a widely distributed group of freshwater algae characterized by the lack of flagellated cells and reproduction by conjugation (Hoshaw et al., 1990). Most biologists are familiar with Spirogyra and its strikingly prominent ribbon-shaped spiral chloroplast (Bold and Wynne, 1985). The Zygnematales are among the most investigated green algae cytologically (Sarma, 1982). The lowest chromosome number of n = 2 is recorded in several species of Spirogyra. The group is known for extensive polyploidy with chromosome complements of n = 30, 60, 90 to 592 reported (Sarma, 1982). Presence of polycentric chromosomes in both filamentous and unicellular (desmid) forms is a unique feature of the group (King, 1960; Hoshaw and McCourt, 1988). Karyotype analyses indicate an extraordinary range in chromosome lengths as well, from 1–20 µm (King, 1960). DAPI microspectrophotometry was used to investigate a species complex in Spirogyra (Wang et al., 1986). Specimens identified as three separate species, based primarily on filament diameter and cell size, were determined to be polyploid races of a single species. Ploidal changes observed in both culture and field material was described as autopolyploidy, characterized by spontaneous even-number multiplication of the genome (Wang et al., 1986). Data were given in rfu and nuclear DNA contents were not quantified. In the present study, these same isolates (UTEX 2465 and 2466) were re-investigated and found to have essentially equivalent nuclear DNA amounts (Appendix I). Apparently, autopolyploid forms in these algae are unstable and can spontaneously revert to lower ploidy levels in culture.
The sole published estimate of nuclear DNA contents in the true desmids (Desmidiales) is for Closterium (2C = 2·7 pg; Hamada et al., 1985). Unpublished investigations in our laboratory of the filamentous Zygnematales (Purvis, 1998) and unicellular Desmidiales (Marlowe, 1998) are summarized in Appendix I. The 2C nuclear DNA contents in the Zygnematales ranged from 0·5–4·2 pg, and from 1·1–20·7 pg in the Desmidiales. Several desmids investigated had nuclei too large to be accommodated by the photometer aperture system and could easily have had nuclear DNA contents in excess of 4x specimens that were measured. Thus, nuclear DNA contents approaching 100 pg may occur in some desmids. In the desmids that permitted quantification, reported chromosome complements and nuclear DNA contents are highly correlated (r2 = 0·7897), providing circumstantial evidence of polyploidy in the group (Fig. 4). In contrast, no correlation was observed between nuclear DNA contents and reported chromosome complements for several filamentous Zygnemataceae (data not shown) as would be expected if higher chromosome numbers resulted primarily from duplication of chromosome fragments resulting from fusion and/or fission events associated with their polycentric centromeres.
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Both the filamentous Zygnemataceae and the true desmids have undergone explosive speciation, resulting in thousands of described species (for example, see Prescott et al., 1972
, 1977, 1981; Hoshaw and McCourt, 1988) on every continent except Antarctica. Now that a basic understanding of phylogenetic relationships is emerging for the Zygnematales (Surek et al., 1994; McCourt et al., 2000) and the Desmidiales (Denboh et al., 2001), it would be a matter of great interest to identify possible nucleotype transformations that have accompanied their speciation. Many of the Zygnemataceae appear to be characterized by polyploid ‘species complexes’ (Hoshaw and McCourt, 1988) and reported large cell sizes in many of the Desmidiales suggest that polyploidy in these uninucleate, unicellular organisms has produced some of the largest nuclear genome sizes known in plants.
Charales. The Charales, commonly known as stoneworts or brittleworts, flourish in fresh and brackish water habitats throughout the world (Bold and Wynne, 1985). Charophytes are prone to calcification and have left an abundant fossil record up to the Cretaceous, and perhaps beyond (Grambast, 1974; Feist et al., 2003). The order is well circumscribed and includes a mere handful of extant genera (McCourt et al., 1996), remnants of a once diverse, but now largely extinct group (Feist et al., 2003). The base chromosome number for Chara is n = 7 and in Nitella is n = 3. However, many species exhibit polyploidy, with chromosome complements up to n = 70 reported (Sarma, 1982). Published C-value data are limited to a single investigation of five species of Chara (Maszewski and Kolodziejczyk, 1991). Two of these species, with 2n = 28, have 2C DNA contents of about 14 pg. Interestingly, while one of the species with a polyploid 2n = 56 has the expected 2DNA content of 28 pg, the other two species with 2n = 56 have 2C DNA contents of about 19 pg or three times the lowest value (Appendix I).
Comparative molecular data indicate that the charophycean green algae are a sister group and paraphyletic to land plants (Mishler et al., 1994; McCourt, 1995; McCourt et al., 2000). It is perhaps informative to compare the C-values of these green algal groups with those of the oldest group of land plants, the bryophytes (Kenrick and Crane, 1997). Unfortunately, data for the basal groups in the charophycean lineage (Chlorokybales and Klebsormidiales) are limited to chromosome numbers for Klebsormidium (Sarma, 1963). Published information for members of the Zygnematales and the Charales indicate that they can be characterized either by chromosome complements of more than 2n = 30 or 2C nuclear DNA contents greater than 1 pg, or both (Fig. 5). Unfortunately, no DNA content estimates are available for any member of the Coleochaetales, but the smallest chromosome complements reported in the order, 2n = 44 and 48, are consistent with polyploidy and a larger nuclear genome. In contrast, hornworts, liverworts and mosses, in general, have chromosome complements less than 2n = 30 and/or 2C nuclear DNA contents less than 1 pg (Renzaglia et al., 1995; Voglmayr, 2000). Although greater values for both parameters are known in the bryophytes, they appear to be restricted to polyploid species and do not contradict the generalization. For example, more than 80 % of the nuclear DNA C-values in mosses were reported to occur in a narrow peak between 0·25–0·6 pg (Voglmayr, 2000). It has been suggested that the small DNA amounts and low C-value variation are linked to the biflagellate nature of bryophyte sperm cells (Renzaglia et al., 1995). As nuclear genome size and sperm cell size are tightly correlated, and sperm cells are thought to drastically lose their motility with increasing size, a strong selection pressure against larger sperm, and therefore also against larger DNA amounts, is hypothesized (Voglmayr, 2000).
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These observations gain additional significance in the context of the suggestion that the common ancestor of all angiosperms may have possessed a small genome (Leitch et al., 1998
, 2005). Small genome size appears to be correlated with phenotypic characteristics such as rapid seedling establishment, short minimum generation times, reduced cost of reproduction, and an increased reproductive rate (Bennett, 1987; Midgley and Bond, 1991). Consequently, small genome size may permit greater evolutionary flexibility (Leitch et al., 1998) whereas larger size and amplification may lead to ‘genomic obesity’ (Bennetzen and Kellogg, 1997). It is to be wondered if the relatively large nuclear genomes found in the charophycean algae, perhaps appropriate in an ancient atmosphere with low amounts of oxygen and UV-absorbing ozone, rendered them unsuitable contenders for the colonization of land as atmospheric conditions improved (Graham, 1993).
Ulvophycean algae
The other major monophyletic lineage related to the charophycean algae discussed above contains the classical ‘green algae’, primarily the Chlorophyceae and Ulvophyceae (Watanabe et al., 2001). The Chlorophyceae apparently arose during the later stages of green algal evolution and are not a basal lineage (Watanabe et al., 2001). This group includes many of the familiar flagellates such as Volvox and Chlamydomonas and is characterized by the predominance of freshwater taxa. There are few published DNA content estimates for members of the Chlorophyceae. The pioneering investigation of Holm-Hansen (1969) which used ‘fluorometric measurement’, reported 2C = 0·6 pg for Dunaliella tertiolecta. However, no calibration standard was specified. Higashiyama and Yamada (1991) used pulse field electrophoresis to estimate a 2C genome size in Chlorella of 40 Mbp (or 0·04 pg using the expression 1 pg = 980 Mbp (Bennett et al., 2000). The Ulvophyceae are primarily marine species, most with larger and more complex morphologies than typically found in the Chlorophyceae. Molecular data support a model for the Ulvophyceae sensu Mattox and Stewart (1984) with two separate lineages: a clade including the Ulotrichales and Ulvales (Hayden and Waaland, 2002) and a clade with the Caulerpales, Cladophorales/Siphonocladales complex, Dasycladales and the Trentepohliales (Zechman et al., 1990; Hanyuda et al., 2002). Published information is available for all of the major groups of the Ulvophyceae, and significant new data are included in this study (Appendix I).
Ulvales. Recent phylogenetic investigations using chloroplast and nuclear DNA sequences have redefined the boundary between the Ulotrichales and Ulvales (Hayden and Waaland, 2002). Species of Capsosiphon and Monostroma, included in the Ulvales by Bliding (1963, 1968) appear to be more closely related to the Ulotrichales (Fig. 6). The amended order Ulvales is monophyletic, but the chief characteristic used to separate the familiar genera Ulva and Enteromorpha, i.e. blade vs. tubular thallus, lacks taxonomic significance (Hayden and Waaland, 2002). The Ulva and Enteromorpha morphologies apparently arose independently several times throughout the evolutionary diversification of the group, making distinctions between these two genera problematic (Tan et al., 1999; Shimada et al., 2003). Estimates of nuclear DNA contents for species of the Ulvales range from 2C = 0·2–1·1 pg (Appendix I). The absence of a correlation between nuclear genome size and chromosome number in these species (data not shown) suggests a significant role of aneuploidy in their evolution (Kapraun and Bailey, 1992).
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Ulotrichales. The Ulotrichales as presently delimited has been expanded to include the Acrosiphoniaciae (sensu Kornmann and Sahling, 1977
). Nuclear DNA content data, available for only three species of this large and diverse order, suggest that it is characterized by small 2C values of 0·5–0·9 pg (Appendix I). These relatively small genome sizes are complemented by relatively small chromosome numbers of 2n = 8–24 (Kapraun, 1993c).
Trentepohliales. The order Trentepohliales includes more than 60 species of subaerial and terrestrial green algae (Lopez-Bautista et al., 2000). Molecular investigations place members of this order with the second lineage of the Ulvophyceae (Mishler et al., 1994; Chapman et al., 1995), which are otherwise almost exclusively marine. Nuclear DNA content estimates of 2C = 1·1–4·1 pg and reported chromosome complements of 2n = 22–36 (Appendix I) are indicative of polyploidy (Lopez-Bautista et al., 2000). However, there is no apparent correlation between chromosome number and nuclear DNA content.
Dasycladales. The order Dasycladales includes extant tropical and subtropical benthic marine green algae and existed as long ago as the Cambrian (approx. 570 mya; Berger and Kaever, 1992). Members of the Dasycladales are unicells characterized by a highly differentiated cell body with radially disposed branches and a persistent primary nucleus (Spring et al., 1978). Detailed investigations of evolution in the order have benefited from the abundance of fossilized morphotypes, which record periodic radiations and extinctions (Olsen et al., 1994). Only 11 of 175 known fossil genera are extant, representing 38 species in two families: Dasycladaceae and Polyphysaceae (= Acetabulariaceae). The small number of extant genera permits characterization of the Dasycladales as living fossils. Monophyly of the Dasycladales is unchallenged and supported by morphological, ultrastructural, biochemical and DNA sequence data (O'Kelly and Floyd, 1984; Mishler et al., 1994; Watanabe et al., 2001; Zechman, 2003).
In the Dasycladales, estimated 2C DNA contents range from 0·7–3·7 pg (Appendix I). The smallest 2C DNA values occur in the basal (and primitive) genera Bornetella and Cymopolia (Fig. 7). The relatively larger DNA contents found in more recently evolved taxa almost certainly reflect a sequence of multiple polylploidy events. It is noteworthy that although the dasyclads are an ancient lineage, most extant species are recent, resulting from dramatic radiation events within the last 65 million years. In most taxa investigated, cyst volume was found to be inversely related to genome size (Kapraun and Buratti, 1998). The adaptive significance seems to be that small genome size and large cyst size result in the production of increased numbers of gametes per cyst.
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Recent molecular investigations based on analyses of rbcL (Zechman, 2003
) and 18SrDNA (Berger et al., 2003) sequence data indicate that reproductive cap morphotypes characteristic of Polyphysa and Acetabularia (Sawitzky et al., 1998) are polyphyletic. The revised and expanded circumscription of Acetabularia now includes Polyphysa peniculus and Acicularia schenckii (Berger et al., 2003). Acetabularia species are characterized by an earlier cap ray initiation relative to the formation of corona superior hairs compared with development in other members of the Polyphysaceae (Berger et al., 2003). It is noted with great interest that nuclear genome size is highly correlated with these developmental patterns. Specifically, all species of the expanded genus Acetabularia have 2–3 times the 2C DNA contents found in Polyphysa clavata and Polyphysa parvula, which are basal to other Polyphysaceae (Fig. 7). The strong correlation between cap morphotypes (Sawitzky et al., 1998) and cap morphogenesis (Kratz et al., 1998) and ‘polyploid’ nucleotypes in the Dasycladales implies a significant but poorly understood role for the nucleotype in gene expression (Gregory, 2001).
Caulerpales. Members of the Caulerpales (Codiales sensu Taylor, 1960) are multinucleate and coenocytic. Preliminary molecular data seem to support classical taxonomic treatments that separate the order into two groups (Zechman et al., 1990), one generally characterized by diplobiontic life histories and non-holocarpic production of gametes (e.g. Bryopsis and Codium), the other generally characterized by haplobiontic and diploid life histories and holocarpic production of gametes (e.g. Caulerpa and Halimeda; Kapraun, 1994). Nuclei with endopolyploid DNA contents have been reported in several caulerpalean algae, and a remarkable regular, incremental size decrease (cascading) in DNA contents of vegetative nuclei corresponding to values of 8C to 2C was observed in Halimeda (Kapraun, 1994). Estimates of 2C nuclear DNA contents range from 0·2–6·1 pg (Appendix I). The largest nuclear genome (2C = 6·1 pg) was observed in Codium fragile subsp. tomentosoides isolates from North Carolina. Originally endemic to Japan or the northwest Pacific (Goff et al., 1992), this invasive seaweed spread throughout the North Atlantic during the 20th century and became a nuisance species in some localities. It reportedly reproduces exclusively by parthenogenetic female gametes (Searles et al., 1984) and fragmentation (Fralick and Mathieson, 1973). Because of its mode of reproduction and unusually large nuclear genome, it is speculated that its success as a weed could be attributed, in part, to its behaviour as an autopolyploid apomict (Kapraun and Martin, 1987; Kapraun et al., 1988).
The large and diverse genus Caulerpa includes more than 75 described species, mostly from tropical shallow marine habitats (Price et al., 1998). Nuclear DNA contents published for four of these species are essentially identical (2C 
0·2 pg). Now that a molecular phylogeny has been published (Famà et al., 2002), it would be a matter of great interest to determine if evolution in this group has been accompanied by transformations involving chromosome complements and nuclear DNA contents.
Recently, the genus Caulerpa attracted considerable media attention as species expanded their ranges into more temperate environments (Olsen et al., 1998). One of these, C. taxifolia, is especially aggressive (Meinesz et al., 1993; De Villèle and Verlaque, 1995). It has been variously labelled as a mutant or superstrain that may have resulted from autopolyploidy or hybridization. Although the mechanism of its origin remains speculative, gigantism, fast growth rates, low temperature tolerances and facultative apomixes make it a formidable competitor (Olsen, 1997). Based on previous experience with Codium fragile, it would be a matter of great interest to determine if invasive C. taxifolia likewise is characterized by an elevated nuclear DNA content and functions as a polyploid apomictic strain.
A recent molecular and morphological analysis of Bryopsis revealed the presence of four genetically distinct clades from the western Atlantic and Caribbean that appear to be either seasonally or geographically disjunct (Krellwitz et al., 2001). However, these genetic clades do not coincide with current morphological species concepts in the genus. It has been suggested that investigations based on mis-identification of these polymorphic, poorly delimited species might account for the considerable variation in reported chromosome numbers, including 1n = 7, 8, 10, 12 and 14 (Kapraun, 1993c). Nuclear DNA estimates are available for only three Bryopsis species (Appendix I). In light of the investigation by Krellwitz et al. (2001), species assignment of these specimens, based solely on morphological features (Kapraun and Shipley, 1990), requires reconfirmation. It would be a matter of great interest to obtain both chromosome complement and nuclear genome size data for these molecularly delimited clades.
Cladophorales/Siphonocladales complex. Since nuclear volume is strongly correlated with cell size and cell cycle lengths in higher plants (Shuter et al., 1983) it is not surprising that these algae with their large, multinucleate cells and relatively long cell generation times have relatively large genomes (Kapraun and Nguyen, 1994). Many algae are characterized by an alternation of haploid gametophyte and diploid sporophyte generations. If the phases are isomorphic, a mechanism must be present to equilibrate the ratio between nuclear volume and cytoplasmic area to maintain a constant area of cytoplasmic domain per standardized nuclear DNA unit (Goff and Coleman, 1987, 1990). In members of the Cladophorales/Siphonoclades complex investigated, isomorphy is maintained by both increasing the number of nuclei per cell and increasing the ploidy level of nuclei (Kapraun and Nguyen, 1994).
The Cladophorales and Siphonocladales are a related patristic lineage sharing a gradation of ‘architectural’ morphological types (van den Hoek et al., 1988). Immunological distance estimates (Olsen-Stojkovich et al., 1986; van den Hoek et al., 1988) and cladistic analyses of nuclear encoded rDNA sequences (Zechman et al., 1990; Hanyuda et al., 2002) support a close relationship between the Cladophorales and Siphonocladales. Contemporary molecular studies support a phylogeny consisting of three well-supported clades: (1) species belonging to the cladophoracean genera Chaetomorpha, Cladophora and Rhizoclonium; (2) species belonging primarily to the Siphonocladales sensu Børgesen (1913); and (3) mostly freshwater species of cladophoracean genera, including Pithophora and Wittrockiella (Hanyuda et al., 2002). Confusingly, the genera Chaetomorpha, Cladophora and Rhizoclonium are polyphyletic, and their characteristic morphologies appear to have evolved several times, independently, in all three clades
Karyological studies indicate that species in this first clade, without exception, share a unique constellation of karyotype features including: (1) six basic chromosomes, three of which have median centromeres and three with submedian ones; and (2) almost universal polyploidy, resulting in chromosome complements in most species of x = 12, 18, 24, 30, 36, etc. (Wik-Sjöstedt, 1970; Kapraun and Gargiulo, 1987a, b). Species in the second clade have (1) various combinations of both metacentric and acrocentric chromosomes (Kapraun and Breden, 1988; Bodenbender and Schnetter, 1990; Kapraun and Nguyen, 1994); and (2) chromosome complements consistent with an aneuploid origin: 1n = 8, 12, 14, 16, 18, and 20 (Kapraun, 1993c; Kapraun and Nguyen, 1994). Nuclear DNA content estimates indicate that members of clade II (Fig. 8) have relatively small genomes (2C = 0·2–0·7 pg) while members of clade I, including the bulk of the Siphonocladales, have much larger genomes of 2C = 2·0–5·7 pg (Appendix I). Although the cladophoracean morphotype appears to have evolved independently in all of the clades, the combination of karyotype pattern and nuclear genome size characteristic of the core clade of the Cladophorales appears to be unique and diagnostic (Fig. 9). It would be a matter of great interest to obtain karyotype and nuclear genome size estimates for representative members of all three clades to determine if these generalizations are universal in the Cladophorales/Siphonocladales complex.
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Conclusions and directions for further study
We are not aware of published investigations of G + C mol % or reassociation kinetics for any charophyceaen algae. Consequently, their nucleotype characterization is restricted to chromosome complement, karyotype pattern and nuclear DNA content estimates. In general, charophycean algae have larger genomes (2·0–20·7 pg; Fig. 10) and larger chromosome complements (1n = 2–90 up to 592) than do most ulvophycean algae. The two orders most studied, the Zygnematales and Charales, have unique karyotypes. The former is known for its large, polycentric chromosomes; the latter for long chromosomes (up to 12 µm) with a high heterochromatin content.
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The unicellular Desmidiales, characterized by thousands of morphotypes, should be a target group for investigations of nuclear DNA content variation. Specifically, (1) reported large cell size could be compared with nuclear genome size, and (2) coincidence of elevated (polyploid) genome sizes with the number of described species per genus could be evaluated to determine if morphotypes delimited as species have primarily a genotypic or a nucleotypic basis.
The exact relationship of the Prasinophytes to land plants remains unclear (Qiu and Palmer, 1999) and the apparent miniaturization of their nuclear genomes may defeat attempts to use them as a model in reconstruction of land plant ancestral genomes (Cunningham et al., 1998; Oakley and Cunningham, 2000). Consequently, the basal groups in the charophycean lineage (Soltis et al., 1999), including the Chlorokybales, Klebsormidiales and Coleochaetales, may provide the best opportunity for gaining these insights, yet there are no published estimates of DNA contents in any member of these orders. Species of both Coleochaete and Klebsormidium are commonly investigated and are readily available to researchers. It should be a priority to obtain nuclear DNA content values for these green algae. The present investigation has noted that charophycean algae appear to be characterized either by chromosome complements and/or nuclear DNA contents greater than typically encountered in primitive land plants. It should be a priority to obtain data for many additional charophycean algae to evaluate this suspected relationship.
Finally, no published data are available for the flagellated unicellular and colonial Chlorophyceae, including the familiar Chlamydomonas and Volvox. It should be a priority to obtain nucleotype data for comparison with speciation patterns resolved in emerging molecular phylogenetic studies for these algae (e.g. Nozaki et al., 1995).
The present and previous investigations (Olsen et al., 1987; Bot et al., 1989a, b, 1990, 1991; Kooistra et al., 1992) permit some generalizations concerning nuclear genomes in the predominantly marine species of the Ulvophyceae:
- Chromosome numbers range from 1n = 5–12 (excluding polyploid values), and both polyploidy and aneuploidy events appear to have accompanied speciation in specific groups. Comparison of 2n chromosome numbers and 2C nuclear DNA contents results in a low correlation of r2 = 0·3177 (Fig. 11), consistent with a high occurrence of aneuploidy, i.e. chromosomal fusion and/or fission events.
- Estimated 2C nuclear DNA contents range from 0·2–4·9 pg.
- G + C ranges from 35–56 mol %.
- Reassociation kinetics has identified the presence of highly repetitive, mid-repetitive and unique sequences in the few species investigated. These preliminary results indicate a predominance of unique and mid-repetitive sequences and a relatively small proportion of highly repetitive sequences. The findings are consistent with the suggestion that much of the reported variation in nuclear genome sizes may result from accumulation and/or deletion of non-genic, repetitive elements (Cavalier-Smith and Beaton, 1999).
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PHAEOPHYTA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSION...PHAEOPHYTARHODOPHYTAGENERAL SUMMARYNOTES ON APPENDIXES I-III....APPENDIX I. CHROMOSOME NUMBER...KEY TO REFERENCESAPPENDIX II. CHROMOSOME NUMBER...KEY TO REFERENCESAPPENDIX III. CHROMOSOME NUMBER...KEY TO REFERENCESACKNOWLEDGEMENTSLITERATURE CITED |
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The brown algae or Phaeophyta are an essentially marine assemblage of more than 265 genera and 1500 species (Bold and Wynne, 1985
). Nuclear genome size estimates in Appendix II include previously unpublished observations (Criswell, 1998) as well as data from the present study. The range of 2C nuclear genome sizes estimated for the Phaeophyta (0·2–1·8 pg) approximates one order of magnitude (Appendix II). The smallest mean 2C genome sizes were found in the Ectocarpales (0·2–0·9 pg) and the largest 2C genome sizes were found in the Sphacelariales (1·8 pg), Fucales (1·7 pg) and Laminariales (1·6 pg). Previous published information for genome sizes in the Phaeophyta based on data from reassociation kinetics (Stam et al., 1988) and quantitative staining with DAPI (Stache, 1991; Le Gall et al., 1993) for six species of Phaeophyta indicated haploid genome sizes range from 430–1550 Mb. These researchers published DNA content estimates of 0·45–1·6 pg using the expression 1 pg = 0·965 x 109 (Britten and Davidson, 1971). The currently accepted conversion factor of 1 pg = 980 Mb (Cavalier-Smith, 1985a) results in slightly smaller estimates of 0·44–1·58 pg. Members of the Ectocarpales are notorious for development of polyploid populations, with ‘haploid, diploid and tetraploid plants connected with each other in a complex system of meiosis, heteroblasty and spontaneous increase in chromosome numbers’ (Mü