Annals of Botany

Annals of Botany 2005 95(1):7-44; doi:10.1093/aob/mci002

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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

DONALD F. KAPRAUN^*

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   

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 

• 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

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 
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.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Evolutionary tree constructed from a distance matrix of eukaryotic SSU rRNA sequences and based on ‘substitution rate calibration’. Redrawn from Van der Peer et al. (1996).

 

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 
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 (I_f) 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 I_f 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 I_f 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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 
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).

View larger version (16K): [in this window] [in a new window]   FIG. 2. Summary results of combined analysis using morphological, ultrastructural and large and small subunit rRNA gene sequences for the five classes of green algae and four lineages of embryophytes (liverworts, hornworts, mosses and tracheophytes). Redrawn from McCourt (1995).

 
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).

View larger version (14K): [in this window] [in a new window]   FIG. 3. Phylogram of conjugating green algae based on MP analysis of rbcL sequences. Redrawn from McCourt et al. (2000).

 
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.

View larger version (13K): [in this window] [in a new window]   FIG. 4. Comparison of 2n chromosome complements and estimated 2C nuclear DNA contents in Desmids. See Appendix I for data sets.

 
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).

View larger version (16K): [in this window] [in a new window]   FIG. 5. Comparison of 2n chromosome complements and estimated 2C nuclear DNA contents in charophycean and embryophyte lineages. Data for hornworts, liverworts and mosses from Renzaglia et al. (1995). Data for Charales in Appendix I.

 
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).

View larger version (14K): [in this window] [in a new window]   FIG. 6. Phylogenetic tree of the Ulvales and Ulotrichales inferred from 18S rDNA and rbcL sequence analysis. Redrawn from Hayden and Waaland (2002).

 
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.

View larger version (15K): [in this window] [in a new window]   FIG. 7. (A) Consensus phylogeny for the Dasycladales from analyses of rbcL (Zechman, 2003), and (B) 18S rDNA (Berger et al., 2003) gene sequence data.

 
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.

View larger version (23K): [in this window] [in a new window]   FIG. 8. (A) Phylogram based on 18S rRNA gene sequence analysis, and (B) nuclear DNA contents in members of the Cladophorales/Siphonocladales complex. Numbering (1 and 2) indicates major clades. Redrawn from Hanyuda et al. (2002).

 

View larger version (13K): [in this window] [in a new window]   FIG. 9. Comparison of 2n chromosome complements and 2C nuclear DNA contents in members of the Cladophorales/Siphonocladales complex. See Appendix I for data sets.

 
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.

View larger version (10K): [in this window] [in a new window]   FIG. 10. Mean and range of 2C nuclear DNA contents for species representing eight orders of Chlorophyta included in Appendix I.

 
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:

1. 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.
   
2. Estimated 2C nuclear DNA contents range from 0·2–4·9 pg.
   
3. G + C ranges from 35–56 mol %.
   
4. 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).
   

View larger version (16K): [in this window] [in a new window]   FIG. 11. Comparison of 2n chromosome complements and 2C nuclear DNA contents in species of Chlorophyta included in Appendix I.

 

	PHAEOPHYTA

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 
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’ (Müller, 1967, 1969, 1970, 1975, 1986). In the present study, the 2C nuclear genome size estimate of 0·50 pg for Ectocarpus siliculosus closely approximates previous estimates of 0·54 pg (as 524 Mb; Stache, 1990, 1991) and of 0·52 pg (as 500 Mb) for Pilayella littoralis (L.) Kjellman (Le Gall et al., 1993).

In the present study, 2C genome size estimates resulting from static microspectrophotometry generally approximate previously published estimates based on flow cytometry (Le Gall et al., 1993) for Laminaria saccharina and L. digitata (Appendix II). Both of these techniques appear to result in larger estimates than obtained by reassociation kinetics (Stam et al., 1988). In the present study, large nuclei were observed in older medullary cells of L. saccharina. However, these nuclei were too large to be accommodated by the aperture on the microspectrophotometry system, and their I_f could not be measured. Endopolyploid nuclei with DNA levels of 8C or greater have been reported in vegetative tissue of Laminaria saccharina and Alaria esculenta (Garbary and Clarke, 2002).

Our understanding of the classification and phylogeny of the Phaeophyta has undergone a marked change in the last decade (Peters and Müller, 1986; Peters, 1998; Peters and Clayton, 1998; Rousseau et al., 2001; Draisma et al, 2001). Traditional phylogenetic interpretations of classifications take progressive complexity and increasingly fixed or obligate life histories as evidence of evolutionary advancement (Siemer et al., 1998). In the brown algae, traditional phylogenetic schemes assigned an ancestral or basal position to the Ectocarpales (Papenfuss, 1951; van den Hoek et al., 1995) and assumed the Fucales to be the most recent, derived group. Contemporary DNA sequence data reveal a more complex pattern of phylogenetic relationships in the brown algae (Lee et al., 2003). Morphological grades of organization, modes of growth and type of life history have evolved and/or have been lost independently and repeatedly. Apparently, the Dictyotales and Sphacelariales are basal while the Ectocarpales (including the Scytosiphonales in Kogame et al., 1999) and the morphologically complex Laminariales are the most recent/derived group (Kawai and Sasaki, 2000; Stefano et al., 2001). Some refer to the Ectocarpales sensu lato as ‘simple brown algae’, thus avoiding the phylogenetic connotation of ‘primitive’ (Peters and Burkhardt, 1998). Interestingly, the Fucales occupy a phylogenetic position in the middle of the tree despite their suite of supposedly advanced characteristics including an oogamous, monophasic, diploid life history. It seems noteworthy that taxa included in the expanded circumscription of the order Ectocarpales (Kornmann and Sahling, 1977; Tan and Druehl, 1993; Druehl et al., 1997; Siemer et al., 1998) are characterized by having both smaller genome sizes and chromosome complements while the Dictyotales, Fucales and Sphacelariales have some of the largest nuclear genome sizes (Fig. 12) and chromosome complements. It has been suggested that algae with a large volume (plant size) at maturity usually display anisogamy or oogamy, as expected if larger zygotes permit more rapid growth to these adult sizes (Madsen and Waller, 1983). Assuming a positive correlation between nuclear genome size and cell size, especially of female gametes and eggs, brown algae with larger plants at maturity would tend to have larger nuclear genomes. Present data are consistent with this analysis. Orders that are characterized by oogamy and are reported to have large female gametes (eggs), have the largest nuclear genomes observed regardless of their phylogenetic position (Fig. 12).

View larger version (12K): [in this window] [in a new window]   FIG. 12. (A) Molecular phylogeny, and (B) range of 2C nuclear DNA contents in the Phaeophyta. Redrawn from Draisma et al. (2001) and Rousseau et al. (2001).

 
The Fucales constitute a large monophyletic order (Rousseau and Reviers, 1999a, b), and include about 40 of the approximately 265 genera reported in the Phaeophyta (Rousseau et al., 1997). The Fucales reportedly evolved and diversified in southern Australia (Clayton, 1988), but are now widely distributed throughout the world (Serrão et al., 1999). The order includes six families in two large, well-supported groups: Group I includes the Fucaceae and Hormosieraceae, among others, and Group II the Sargassaceae and Cystoseiraceae, among others (Rousseau and Reviers, 1999a, b; Rousseau et al., 2001). The Fucaceae in Group I have a bipolar distribution, with Fucus, Ascophylluum and Pelvetia being restricted to the North Atlantic and Hormosira and Xiphophora restricted to the southern hemisphere. Molecular data support a large divergence time between these northern and southern hemisphere taxa (Serrão et al., 1999). Unfortunately, there are no published C-value data for any southern hemisphere representatives, and data for only two species from the North Atlantic (Appendix II). It would be a matter of great interest to compare nucleotype data for these two well-circumscribed groups to determine what DNA content transformations may have followed their ancient divergence some 40 million years ago (Serrão et al., 1999).

Although most Fucales are restricted to cold-water environments, members of the Group II families, the Sargassaceae and Cystoseiraceae, have primarily tropical and warm temperate distributions (Bold and Wynne, 1985; Saunders and Kraft, 1995). Present data are insufficient to support any conclusions, but there is some indication that cold-water genera Ascophyllum and Fucus may have larger nuclear genomes than do the warm water genera Sargassum and Turbinaria (Fig. 12). Unfortunately, no data are available for any species of the Cystoseiraceae, which are other important Group II members.

Most orders of brown algae are reported to have basic chromosome numbers between 8–13 (Cole, 1967) with higher numbers for 1n chromosome complements resulting from polyploidy (whole-number multiples of a basic genome) (Lewis, 1996). If polyploidy has played a significant role in the evolution of the brown algae, then ancestral taxa could be expected to share a genome characterized by an ancestral chromosome complement and a 2C genome size. Published chromosome counts are available for 21 species of the brown algae (Lewis, 1996) included in the present study (Appendix II). Comparison of these 1n chromosome complements and estimated 2C genome sizes indicates a low correlation (r² = 0·2037; data not shown) indicative of significant aneuploidy processes (Kapraun, 1993c).

Conclusions and directions for further study
Phaeophyta that warrant further investigation include the Fucales as discussed above, and the Sphacelariales, which have the largest 2C nuclear genomes of all the brown algae investigated. Although this order is cosmopolitan in the world's oceans (Draisma et al., 2002), it is of particular interest because of the many species endemic to the Southern Hemisphere. As with the Fucales, geographic disjunction (northern vs. southern taxa) and habitat restriction (cold-water vs. temperate/tropical) almost certainly have resulted in nucleotype transformations.

Present and previous invesitigations permit some generalizations concerning nuclear genomes in the Phaeophyta:

1. Chromosome numbers range from 1n = 4–64, with 93 % of the species in the range of n = 8–32 (Lewis, 1996), and both polyploidy and aneuploidy events appear to have accompanied speciation in some taxonomic groups.
   
2. Estimated 2C nuclear DNA contents range from 0·2–1·8 pg.
   
3. G + C ranges from 28·6–49·7 mol % (Le Gall et al., 1993).
   
4. Reassociation kinetics indentified the presence of highly repetitive, mid-repetitive and unique sequences in species of Laminaria (Stam et al., 1988).
   
5. All of the brown algal orders investigated exhibit considerable variation in both chromosome numbers and nuclear genome sizes. Nuclear genome size and phylogenetic advancement are poorly correlated. However, orders that are characterized by oogamy (or pronounced anisogamy) and are reported to have large female gametes (eggs) have the largest nuclear genomes observed regardless of their phylogenetic position.
   

	RHODOPHYTA

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 
The red algae are predominantly marine organisms with more than 700 genera and 6000 species described in about two dozen orders (Chapman et al., 1998). Current classification schemes for the red algae based on molecular data (Freshwater et al., 1994; Saunders and Bailey, 1997; Harper and Saunders, 2001a, b) as well as organelle ultrastructure (Pueschel, 1989; Scott and Broadwater, 1990) recognize two subclasses: Bangiophycidae (which are generally uninucleate) with 3 or 4 orders, and Florideophycidae (which are typically multinucleate) with 14 orders (Woelkerling, 1990). The Bangiophycidae appears to be polyphyletic (Freshwater et al., 1994; Müller et al., 2001). The Florideophycidae form a monophyletic clade with most orders falling within two clades (Fig. 13) that terminate long branches of basal position and having specific synapomorphic pit plug characteristics. Group I orders have two pit plug cap layers and include the Acrochaetiales, Balbianiales, Balliales, Batrachospermales, Colaconematales, Corallinales, Nemaliales, Palmariales, Rhodogorgonales and Thoreales (Saunders and Bailey, 1997; Harper and Saunders, 2001a). Group II orders lack cap layers but possess a cap membrane and include the Bonnemaisoniales, Ceramiales, Gelidiales, Gigartinales, Gracilariales, Halymeniales, Plocamiales and Rhodymeniales (Freshwater et al., 1994; Ragan et al., 1994; Saunders and Bailey, 1997; Harper and Saunders, 2001b). The widely held traditional view that the Acrochaetiales are the most primitive and the Ceramiales the most highly derived of the florideophycidean red algal orders (Kylin, 1956; Dixon, 1973) is not supported by molecular data (Chapman et al., 1998). A more complex phylogenetic model is emerging for red algae characterized by ancient lineages often terminating in modern radiations (Saunders and Bailey, 1997).

View larger version (17K): [in this window] [in a new window]   FIG. 13. (A) Combined analysis phylogenetic tree using morphological, ultrastructural and gene sequence data, and (B) range of 2C nuclear DNA contents in the Rhodohyta. Numbering (1 and 2) indicates major clades in the Florideophycidae. Redrawn from Freshwater et al. (1994), Saunders and Bailey (1997), de Jong et al. (1998), and Harper and Saunders (2001a, b, 2002).

 
The Bangiales and Compsopogonales are sister groups in the polyphyletic Bangiophycidae and are basal to, and more ancient than, any of the Florideophycidae (Freshwater et al., 1994; Oliveira et al., 1995). Compsopogon coerulus (Compsopoganales) has a 2C DNA content of 0·25 pg and a reported chromosome complement of 1n = 7 ± 1 (Nichols, 1964). Estimates of 2C nuclear DNA contents range from 0·6–1·2 pg for the seven isolates of Bangia and Porphyra (Bangiales) investigated (Fig. 13). Published chromosome complements for these isolates range from 1n = 3–5 (Appendix III). These data are consistent with a basal (ancestral) red algal nucleotype characterized both by small genome sizes and small chromosome complements. Comparison of 2n chromosome complements with 2C nuclear geonome size estimates for species in the Bangiales indicates a poor correlation consistent with an aneuploid sequence (Kapraun et al., 1991). Regional isolates of Porphyra leucosticta and P. spiralis var. amplifolia exhibited different chromosome complements and/or estimates of nuclear DNA contents, underscoring the inherent difficulty in delineating species when substantial genotypic divergence (Lindstrom and Cole, 1992; Dutcher and Kapraun, 1994; Oliveira et al., 1995) is masked by narrow phenotypic expression (Kapraun et al., 1991; Chapman et al., 1998).

DNA amount data are available from five of the ten orders in Group I of the Florideophycidae, but four of these five orders are represented by only one to a few species (Fig. 13). The newly recognized order Colaconematales is apparently one of the more derived in this group (Harper and Saunders, 2002). In the present study, uninucleate vegetative cells of Colaconema daviesii were found to have 2C DNA contents of 0·6 pg. The Corallinales appear to be sister to other orders in Group I (Saunders and Bailey, 1997). Members of this order have 2C DNA contents of 0·1–1·3 pg (Appendix III). Coralline red algae can be divided into two types: geniculate (with alternating calcified internodes and uncalcified nodes) and non-geniculate (which usually grow as crusts) (Woelkerling et al., 1993). Recently, molecular studies demonstrated that genicula are non-homologous structures that evolved independently in several families (Bailey and Chapman, 1996, 1998). When DNA content data are superimposed on this molecular phylogeny (Fig. 14), it becomes apparent that geniculate clades are represented by species with larger nuclear genomes (0·6–1·3 pg) while non-geniculate clades contain species with relatively small nuclear genomes (0·1–c.0·4 pg). Neogoniolithon spectabile and Titanoderma pustulatum, non-geniculate species with 2C DNA contents of 0·8 and 1·0 pg, respectively, appear to be the sole exceptions to this generalization among the species investigated. The strong correlation between the geniculate/nongeniculate morphotype and a ‘polyploid’ nucleotype is remarkable as it implies a significant role for the nucleotype (in addition to a substantial genotype role) in the expression of this morphotype.

View larger version (17K): [in this window] [in a new window]   FIG. 14. (A) Molecular phylogeny, and (B) estimated 2C nuclear DNA contents and of the coralline red algae. Geniculate genera are indicated by open vertical bars. Note that genicula are non-homologous structures that evolved independently in multiple clades. Geniculate taxa are characterized by larger nuclear genomes (>0·6 pg) and crustose taxa are characterized by smaller (<0·6 pg) with the exception of Neogoniolithon and Titanoderma. Phylogeny redrawn from Bailey and Chapman (1998). DNA content data from J. C. Bailey and D. F. Kapraun (unpubl. res.).

 
Since coralline red algae deposit calcite in their cell walls, they are represented by an extensive fossil record (Wray, 1977). Crustose (non-geniculate) taxa, with a fossil record extending to the mid-Mesozoic and beyond, appear to be more ancient than articulate (geniculate) taxa, which experienced rapid and substantial radiation following the Cretaceous/Tertiary extinction (K/T event) (Adey and Johansen, 1972; Adey and Macintyre, 1973). If available molecular data have been correctly interpreted and geniculate taxa are polyphyletic and arose independently in several families (Bailey and Chapman, 1996, 1998), then multiple polyploidy events must have occurred independently in these several lineages following the K/T event (Fig. 15).

View larger version (15K): [in this window] [in a new window]   FIG. 15. Phylogeny for some Corallinales based on the fossil record (redrawn from Wray, 1977) and inferred from 18S rRNA gene sequence analysis (Bailey and Chapman, 1998; Bailey, 1999). Nuclear genome size estimates from J. C. Bailey and D. F. Kapraun (unpubl. res.). Proposed polyploidy events are indicated by [P]. Vertical dashed lines indicate 70 my (million year) intervals. Note proposed polyploidy events and subsequent radiation at the K/T boundary.

 
In a previous investigation of coralline green algae (i.e. Dasycladales), which also have an extensive fossil representation, it was noted that some genera experienced similar rapid and expansive speciation following the K/T event some 65 million years ago. Extant species are characterized by nuclear DNA contents that are 2 times the values found in taxa assumed to be ancestral or basal (Kapraun and Buratti, 1998). For the most part, genera with elevated nuclear DNA content values are more species-rich than are genera with smaller (ancestral) genome sizes. Again, we wonder at the constellation of environmental circumstances that appears to have rewarded nuclear genome size increase in a diversity of genotypes in the post-K/T marine environment. The classical explanation for success of diploidy and polyploidy relies on the protection it offers against expression of deleterious mutations, especially those that are particularly harmful (Perrot et al., 1991). It is tempting to speculate that the post-K/T marine environment may have included elevated UV radiation levels leading to increased DNA hazards.

Group II of the Florideophycidae is characterized by a nuclear 2C DNA content range of 0·2–2·8 pg (Appendix III). Five of these Orders (Gelidiales, Gigartinales, Gracilariales, Halymeniales and Rhodymeniales) have particularly narrow ranges of DNA contents (data not shown). In the Gelidiales, the relatively narrow range of small DNA content values but substantial range of chromosome numbers (Appendix III), and the absence of a correlation between nuclear genome size and chromosome number suggests a significant role of aneuploidy in their evolution (Kapraun and Dunwoody, 2002). Analyses of rbcL and LSU gene sequence data have resulted in a molecular phylogeny for the Gelidiales (Freshwater and Bailey, 1998; Thomas and Freshwater, 2001). This well-circumscribed order includes only a handful of genera, but is particularly species-rich (Thomas and Freshwater, 2001). It would be a matter of great interest to obtain nucleotype data for additional representative species of this economically important group of agarophytes to determine the possible role of aneuploidy in their evolution.

The order Gracilariales, like the Gelidiales, includes just a handful of genera, but some of them, e.g. Gracilaria, are species-rich (Fredericq and Hommersand, 1990). Unlike the Gelidiales, the Gracilariales are noted for nucleotype constancy, with all species of Gracilaria investigated having identical 2C DNA contents of 0·4 pg and chromosome complements of 2n = 48 (Kapraun and Dutcher, 1991; Kapraun, 1993a). Species of the closely related Gracilariopsis (Bird et al., 1994: Bellorin et al., 2002; Gurgel et al., 2003) similarly have constant 2C DNA contents (0·4 pg) and 2n chromosome complements (2n = 64).

The Gigartinales is a large and diverse order (Fredericq et al., 1996; Hommersand et al., 1999; Tai et al., 2001) including commercially important carrageenophytes such as Eucheuma and Kappaphycus (Craigie, 1990). Members of this order are characterized by a wide range of chromosome complements (2n = 10–70) and a narrow range of small nuclear DNA contents (2C = 0·2–0·9 pg) (Appendix III).

The Ceramiales is the largest red algal order, with more than 325 genera and 1500 species described (Kraft and Woelkerling, 1990). Nucleotype data are available for fewer than 2 % of these species (Appendix III). Members of this order have both the largest DNA contents and the greatest range of DNA content values (0·5–2·8 pg). Recent molecular systematics investigations indicate that three families (Dasyaceae, Delesseriaceae and Rhodomelaceae) have evolved from the paraphyletic Ceramiaceae (de Jong et al., 1998; Phillips, 2000; Lin et al., 2001; Choi et al., 2002). Consistent with the molecular phylogeny, the smallest DNA value (2C = 0·5 pg) was found in Ceramium (Fig. 16). When nuclear DNA content data are superimposed on a consensus molecular phylogeny for the order, each family is seen to have at least one (ancestral?) species with a 2C DNA content of 0·8–1·2 pg as well as species with elevated (polyploid?) DNA contents (Fig. 16). The simplest explanation is that polyploidy, characterized by even-number multiple increase in chromosome complements as well as increase in nuclear genome size, accompanied speciation in each of these lineages. A strong correlation between chromosome complements and nuclear genome size in many Ceramiales investigated is consistent with this explanation. Conspicuous exceptions include Acanthophora spicifera with 2n = 64 and 2C = 1·1 pg, and Antithamnion villosum with 2n = 48 and 2C = 2·0 pg. Clearly, in some genera, polyploidy events were followed by chromosome reorganization, including fission/fusion processes ultimately resulting in aneuploidy (Fig. 17) as described for species of Polysiphonia (Kapraun, 1993a).

View larger version (10K): [in this window] [in a new window]   FIG. 16. (A) Molecular phylogeny, (B) and range of 2C nuclear DNA contents in the Ceramiales. Redrawn from de Jong et al. (1998), Phillips (2000) and Zuccarello et al. (2002). The figure in square brackets [ ] represents smallest DNA content (pg) observed in each family. n = number of species and isolates represented by data.

 

View larger version (9K): [in this window] [in a new window]   FIG. 17. Comparison of 2C DNA contents and 2n chromosome numbers for seven species and isolates of Polysiphonia (Ceramiales). Data from Kapraun (1979, 1993b) and Kapraun and Dunwoody (2002).

 
The Ceramiales appear to be a basal and ancient lineage relative to other Group II Florideophicidae (Saunders and Bailey, 1997), yet on average have larger nuclear genome contents (2C = 3·5 pg) than do most of the taxa that are believed to have diverged after them (Fig. 13). Unless an assumption is made that the other taxa in the Florideophycean lineage have experienced nuclear genome size decrease, an explanation is required to account for the larger genome sizes in the Ceramiales. Two explanations seem worth considering: (1) a mechanistic model; and (2) an ecological model.

Although the existence of mechanisms for decreasing DNA amounts has been proposed (Wendel et al., 2002), it is more probable that polyploidy and transposable element amplification will result in genome size increase through time (Bennetzen, 2002), ultimately resulting in genomic ‘obesity’ (Bennetzen and Kellogg, 1997). Since the Ceramiales are arguably the oldest members of the Group II Florideophycean lineage, they would have accumulated the largest genomes and may have been subject to a predictable genomic expansion. Although data are severely limited, there appears to be a correlation between antiquity of these red algal lineages and their mean nuclear DNA contents (Fig. 13).

An ecological model suggests that the role of selective forces can be a significant factor in effecting genome size transformations. It can be argued that the single-cell stage is the most vulnerable period in any multicellular organism's life history. This is especially applicable for red algae, which uniquely lack flagellated (motile) cells in their life history. If the non-motile tetraspore and carpospore do not survive, the life history is not completed (Searles, 1980). The survivability of the single cell may depend, in part, on nuclear genome size (DNA content) (Destombe et al., 1992) because of its correlation with cell size (Swanson et al., 1991), nuclear volume, and cell cycle length (Price, 1988). The implication is that cell (spore) size may be indirectly adaptive. For example, larger spore size could reduce predation by zooplankton, promote rapid settlement, and accommodate greater energy reserves for increased initial growth after germination. But selective forces may be more directly related to genome size, such that cellular DNA content results from a compromise between two conflicting forces: smaller genomes increasing cellular growth rates and larger genomes increasing cell size (Parker et al., 1972).

Where there is a high degree of competition and fewer resources, larger cell sizes and slower rates of development are favoured. These parameters coincide with larger and smaller genomes, respectively, in both plants and animals (Grime and Mowforth, 1982; Price, 1988). The corresponding selection types have been designated K and r, where K-selection favours slower development, delayed reproduction, larger body size, and longer life span, and r-selection favours rapid development, high population growth rate, early reproduction, small body size, and short life span (Begon et al., 1990). On the basis of developmental rates, body size, and life span (cell longevity), K-selected species would tend to have larger reproductive cells in smaller numbers and r-selected species would tend to have smaller reproductive cells produced in large quantities (Madsen and Waller, 1983).

In a previous investigation of the relationship of nuclear genome size to reproductive cell parameters in the Rhodophyta (Kapraun and Dunwoody, 2002), three general trends regarding carpospore production were noted: (1) increase in genome size is positively correlated with increase in carpospore volume; (2) species with larger genome sizes produce fewer carpospores; and (3) species that produce larger carpospores produce fewer carpospores. Members of the Ceramiales, with their larger genome sizes, typically produce fewer, but larger carpospores and generally behave as predicted in a K-selection model (Fig. 18). In contrast, members of the Gelidiales, Gigartinales and Gracilariales, with their smaller genome sizes, typically produce large numbers of small carpospores as predicted in an r-selected model (Kapraun and Dunwoody, 2002). The conspicuous limitation of this ecological model is that the Ceramiales generally produce small, structurally simple, short-lived plants (associated with r-selection), while the other orders generally produce large, structurally complex, long-lived plants (associated with K-selection).

View larger version (16K): [in this window] [in a new window]   FIG. 18. Negative correlation of carpospore volume with the number of carpospores per cystocarp for four orders of Florideophycidae. Regression analysis of data for all orders, r2 = –0·512. Without data for the Ceramiales, r2 = 0·719.

 
Conclusions and directions for further study
Members of Group I red algae that warrant further investigation include the Nemaliales, Acrochaetiales and Colaconematales. These three orders are among the oldest of the florideophycean algae, are widely distributed, and contain many genera that are species-rich (Saunders et al., 1995; Harper and Saunders, 1998), yet published information for their nucleotypes is very limited. It is to be wondered if the relatively large DNA content of 2·3 pg in Galaxaura is representative of the Nemaliales.

A second group of red algae that warrant our attention is the Ceramiales. Continuing molecular phylogenetic investigations provide us with evolutionary schemes (de Jong et al., 1998; Phillips, 2000; Lin et al., 2001; Choi et al., 2002) upon which nucleotype data can be superimposed to reveal the extent that speciation was accompanied by nuclear transformations.

Present and previous invesitigations permit some generalizations concerning nuclear genomes in the Rhodophyta:

1. Chromosome numbers range from 1n = 2–68 (–72) (Cole, 1990), and both polyploidy and aneuploidy events appear to have accompanied speciation in some taxonomic groups.
   
2. Estimated 2C nuclear DNA contents range from 0·22–2·85 pg.
   
3. G + C ranges from 28·6–49·7 mol % (Kapraun et al., 1993b; Le Gall et al., 1993).
   
4. Reassociation kinetics indentified the presence of highly repetitive, mid-repetitive and unique sequences in species of Gracilariales and Gelidiales investigated (Kapraun et al., 1993a).
   
5. Some red algal taxa such as the Gracilariales were found to have remarkably constant chromosome numbers and nuclear genome sizes, while other taxa such as species of the Gelidiales have considerable variation in both chromosome complements and karyotype patterns, and in nuclear genome sizes. Members of the Ceramiales are characterized by having both the largest nuclear genomes and the largest chromosome complements (Fig. 19).
   

View larger version (14K): [in this window] [in a new window]   FIG. 19. Comparison of 2n chromosome complements and 2C nuclear DNA contents in the Rhodophyta. Data taken from Appendix III.

 

	GENERAL SUMMARY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 
Nuclear DNA content estimates for the Rhodophyta (2C = 0·2–2·8 pg), Chlorophyta (0·2–6·1 pg ) and the Phaeophyta (2C = 0·2–1·8 pg) approximate an order of magnitude. DNA contents in the freshwater charophycean orders Charales and Zygnematales are significantly larger (39·2 and 20·7 pg, respectively). The size of these algal genomes is best appreciated when compared with the minimum amount of DNA estimated for specifying the mRNA sequences required for angiosperm development. Specifically, the genome of Arabidopsis thaliana (L.) Heynhold, with 0·16 pg = 157 Mb (Bennett et al., 2003) is one of the smallest found in angiosperms (Bennett and Smith, 1976) but still has 30000 or twice the estimated 15000 genes per haploid genome required for development (Flavell, 1980). Correlation between genome size and phylogeny is indicated in some of the algal groups investigated. Genome size appears to correspond more closely to specific reproductive and developmental parameters in all three algal groups.

	NOTES ON APPENDIXES I–III. CHROMOSOME NUMBERS AND NUCLEAR DNA CONTENT ESTIMATES IN SPECIES OF MACROSCOPIC ALGAE

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 
(a) Orders are listed alphabetically. In all three major groups of algae, insights from continuing molecular phylogeny investigations impact on our understanding of the delineation and composition of taxa at all levels: orders, family and genus. An attempt has been made to assign genera to currently recognized families, but on-going molecular investigations have demonstrated that many of these families are not natural assemblages. Synonyms are provided in cases where chromosome complements and/or nuclear DNA content estimates were originally published under different genus and/or species epithets. Footnotes are provided in the Appendixes for some of these examples. References within these footnotes are included in the general Literature Cited. References within the tables themselves are listed in a key below each individual Appendix.

(b) Most comprehensive lists of chromosome numbers have been published as haploid (1n) values for the Chlorophyta (Kapraun, 1993), Phaeophyta (Lewis, 1996) and the Rhodophyta (Cole, 1990). In the Appendixes, chromosome numbers are extrapolated from 1n numbers (and ranges of probable 1n numbers).

(c) Since most DNA amounts in the literature are given in picograms (pg), unless otherwise indicated Mbp values in the Appendixes are derived, using the expression 1 pg = 980 Mbp (Cavalier-Smith, 1985a; Bennett et al., 2000). DNA amounts originally published as megabase pairs (Mbp) are indicated with a dagger (). These values were derived from reassociation kinetics (Olsen et al., 1987; Stam et al., 1988; Bot et al., 1989a, b, 1990, 1991; Kooistra et al., 1992;), with the sole exception of LeGall et al. (1993) who used ethidium bromide (Hoechst 33342) and mithramycin A, two fluorochromes specific for the bases A–T and G–C, respectively, with RBC standard and flow cytometry.

(d) Algal life histories typically are characterized by an alternation of haploid gametophyte and diploid sporophyte generations (Kapraun, 1993c; Kapraun and Dunwoody, 2002). Thus, DNA content (pg) measurements could be based on either or both 2C replicated haploid nuclei or 4C replicated diploid nuclei. In practice, most published DNA content (pg) values are for 2C diploid nuclei and most 1C and 4C values are extrapolated. In the Appendixes, the original published DNA content (pg) value for each species is indicated with an asterisk (*). In some samples, available specimens were not reproductive and ploidy level could not be determined with certainty. Assignment of DNA content to specific C-level for these isolates is speculative (¹).

(e) Previously unpublished data are indicated as (unp). 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. Nuclear DNA content estimates for members of the Desmidiales and Zygnematales are taken from Honors investigations by William Purvis and Mickie Marlowe.

(f) Standard species. The vast majority of nuclear DNA estimates for algae have used chicken red blood cells or erythrocytes (RBC) for a DNA standard and the published value of 2·4 pg accepted for the 4C DNA content of Gallus gallus (Clowes et al., 1983; Riechmann et al., 2000). Limitations of RBC as a standard for plant material has been discussed elsewhere (Johnston et al., 1999; Bennett et al., 2000). Mouse (Mus) sperm was used as a standard by Hamada et al. (1985), the fish Betta splendens was used as a standard by Spring et al. (1978) and Allium cepa was used by Maszewski and Kolodziejczyk (1991). Initial investigations in our laboratory utilized a standard line based on the fluorescence intensity of an alga with a known DNA content and an angiosperm: Antirrhinum majus L. (e.g. Kapraun and Shipley, 1990; Hinson and Kapraun, 1992; Kapraun and Bailey, 1992) or Impatiens balsamina L. (e.g. Kapraun and Shipley, 1990). Species used as a calibration standard for published algal nuclear DNA content estimates are listed in Table 1.

View this table: [in this window] [in a new window]   TABLE 1. Species used as a callibration standard in Appendixes I, II and III

 
(g) Methods. Both flow cytometry (FC) (Le Gall et al., 1993) and static cytometry or microspectrophotometry (MI) (Kapraun 1994; Kapraun and Buratti, 1998) have been shown to be reliable methods for quantification of nuclear DNA contents in green algae. Feulgen microdensitometry (Fe) was used by Maszewski and Kolodziejczyk (1991). Reassociation kinetics (RK) has been used successfully as well (Bot et al., 1989a, b, 1990, 1991; Kooistra et al., 1992; Olsen et al., 1987).

Several DNA-localizing fluorochromes have been used in published investigations. DAPI (4', 6-diamidino-2-phenylindole) is certainly the most popular, especially in recent studies (Kapraun 1994; Kapraun and Buratti, 1998). Hydroethidine (H) (Kapraun and Bailey, 1992), ethidium bromide (EB) and mithramycin (Kapraun et al., 1988; Le Gall et al., 1993) and propidium iodide (PI) (Spring et al., 1978) were used in selected green algal investigations.

Recently, the Angiosperm Genome Size Workshop (Bennett et al., 2000) identified ‘best practice’ methodology for nuclear genome size estimation in plant tissues (for details and recommendations, see http://www.rbgkew.org.uk/cval/conference.html under Angiosperm Genome Size Discussion Meeting). Virtually none of the published genome size data for algae resulted from investigations adhering to all of the best practice recommendations. Even in cases where the preferred methodology of Feulgen microdensitometry was employed, researchers typically used animal (RBC) rather than plant (Allium or Pisum) standards. Consequently, all present and previously published data included in these Appendices should be considered accurate only to ±0·1 pg (Kapraun and Shipley, 1990; Hinson and Kapraun, 1991; Kapraun and Dutcher, 1991; Kapraun and Bailey, 1992).

	APPENDIX I. CHROMOSOME NUMBER AND NUCLEAR DNA CONTENT IN SPECIES OF CHLOROPHYTA

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 
A key to the references appears at the end of this Appendix.

View this table: [in this window] [in a new window]

 

	KEY TO REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 
1. Beutlich A, Borstelmann B, Reddemann R, Seckenbach K, Schnetter R. 1990. Notes on the life histories of Boergesenia and Valonia (Siphonocladales, Chlorophyta). Hydrobiologia 204/205: 425–434.[CrossRef]

2. Bodenbender S, Krause UR, Schnetter R. 1988. Notes on life cycles in Colombian isolates of Ernodesmis and Boodlea (Siphonocladales, Chlorophyta). Cryptogamie, Algologique 9: 279–287.

3. Bot PVM, Holton RW, Stam WT, van den Hoek C. 1989a. Molecular divergence between north Atlantic and indo-West Pacific Cladophora albida (Cladophorales, Chlorophyta) isolates as indicated by DNA–DNA hybridization. Marine Biology, Berlin 102: 307–313.[CrossRef]

4. Bot PVM, Stam WT, van den Hoek C. 1989b. Biogeographic and phylogenetic studies in three North Atlantic species of Cladohora (Cladophorales, Chlorophyta) using DNA–DNA hybridization. Phycologia 28: 159–168.

5. Bot PVM, Stam WT, van den Hoek C. 1990. Genotypic relations between geographic isolates of Cladophora laetevirens and C. vagabunda. Botanica Marina 33: 441–446.

6. Bot PVM, Brussaard CPD, Stam WT, van den Hoek C. 1991. Evolutionary relationships between four species of Cladophora (Cladophorales, Chlorophyta) based on DNA–DNA hybridization. Journal of Phycology 22: 617–623.[CrossRef]

7. Brandham PE. 1965. Some new chromosome counts in the desmids. British Phycolgical Bulletin 2: 451–455.

8. Chowdary Y. 1963. On the cytology and systematic position of Physolinum monilia Printz. Nucleus 6: 44–48.

9. Chowdhury TK, Sarma YSRK. 1984. Nuclear cytology of desmids from India. The Nucleus 27: 179–198.

10. Enomoto S, Hirose H. 1970. On the life history of Anadyomene wrightii with special reference to the reproduction, development and cytological sequences. The Botanical Magazine, Tokyo 83: 270–280.

11. Hamada J, Saito M, Ishida MR. 1985. Nuclear phase in vegetative and gamete cells of Closterium ehrenbergii: fluorescence microspectrophotometry of DNA content. Annual Reports of the Research Reactor Institute 18: 56–61.

12. Hinson TK, Kapraun DF. 1991. Karyology and nuclear DNA quantification of four species of Chaetomorpha (Cladophorales, Chlorophyta) from the western Atlantic. Helgoländer Meeresuntersuchungen 45: 273–285.[CrossRef]

13. Hoshaw RW, Wang J-C, McCourt RM, Hull HM. 1985. Ploidal changes in clonal cultures of Spriogyra communis and implications for species definition. American Journal of Botany 72: 1005–1011.[CrossRef]

14. Jose G, Chowdary Y. 1977. Karyological studies on Cephaleuros Kunze. Acta Botanica Indica 5: 114–122.

15. Kapraun DF. 1970. Field and cultural studies of Ulva and Enteromorpha in the vicinity of Port Aransas, Texas. Contributions in Marine Science 15: 205–285.

16. Kapraun DF. 1993. Karyology of marine green algae. Phycologia 32: 1–21.

17. Kapraun DF. 1994. Cytophotometric estimation of nuclear DNA contents in thirteen species of the Caulerpales (Chlorophyta). Cryptogamic Botany 4: 410–418.

18. Kapraun DF, Bailey JC. 1992. Karyology and cytophotometric estimation of nuclear DNA variation in seven species of Ulvales (Chlorophyta). Japanese Journal of Phycology 40: 13–24.

19. Kapraun DF, Buratti JR. 1998. Evolution of genome size in the Dasycladales (Chlorophyta) as determined by DAPI cytophotometry. Phycologia 37: 176–183.

20. Kapraun DF, Flynn EH. 1973. Culture studies of Enteromorpha linza (L.) J. Agardh and Ulvaria oxysperma (Kuetz.) Bliding (Chlorophyceae, Ulvales) from Central America. Phycologia 12: 145–152.

21. Kapraun DF, Gargiulo GM. 1987a. Karyological studies of three species of Cladophora (Cladophorales, Chlorophyta) from Bermuda. Giornale Botanico Italiano 121: 165–176.

22. Kapraun DF, Gargiulo GM. 1987b. Karyological studies of four Cladophora (Cladophorales, chlorophyta) species from coastal North Carolina. Giornale Botanico Italiano 121: 1–26.

23. Kapraun DF, Martin DJ. 1987. Karyological studies of three species of Codium (Codiales, Chlorophyta) from coastal North Carolina. Phycologia 26: 228–234.[Medline]

24. Kapraun DF, Nguyen MN. 1994. Karyology, nuclear DNA quantification and Nucleus-cytoplasmic domain variations in some nultinucleate green algae (Siphonocladales, Chlorophyta). Phycologia 33: 42–52.

25. Kapraun DF, Shipley MJ. 1990. Karyology and nuclear DNA quantification in Bryopsis (Codiales, Chlorophyta) from North Carolina, USA. Phycologia 29: 43–453.

26. Kapraun DF, Gargiulo GM. Tripodi G. 1988. Nuclear DNA and karyotype variatrion in species of Codium (Codiales, Chlorophyta) from the North Atlantic. Phycologia 27: 273–282.

27. Kasprik W. 1973. Beitrage zur Karyologie der Desmidaceen Gattung Micrasterias. Nova Hedwegia 42: 115.

28. King GC. 1960. The cytology of the desmids: the chromosomes. New Phytologist 59: 65–72.[CrossRef]

29. Kooistra WHCF, Boele-Bos SA, Stam WT, van den Hoek C. 1992. Biogeography of Cladophoropsis membranacea (Siphonocladales, Chlorophyta) as revealed by single copy DNA distances. Botanica Marina 35: 329–336.

30. Le Gall Y, Brown S, Marie D, Jejjad M. Kloareg B. 1993. Quantification of nuclear DNA and G-C content in marine macroalgae by flow cytometry of isolated nuclei. Protoplasma 173: 123–132.[CrossRef]

31. Lopez-Bautista JM, Kapraun DF, Waters DA, Chapman RL. 2000. Nuclear DNA quantification and the life cycle in Cephaleuros parasiticus (Trentepohliales, Chlorophyta). American Journal of Botany 87(6, suppl.): 248.

32. Maszewski J, Kolodziejczyk P.1991. Cell cycle duration in antheridial filaments of Chara spp. (Characeae) with different genome size and heterochromatin content. Plant Systematics and Evolution 175: 23–38.

33. Neumann K. 1967. Der Ort der Meiosis und die sporenbildung bei der siphonalen Grünalge Derbesia marina. Naturwissenschaften 4: 121.[CrossRef]

34. Olsen JL, Stam WT, Bot PV M, van den Hoek C. 1987. scDNA–DNA hybridization studies in Pacific and Caribbean isolates of Dictyosphaeria cavernosa (Chlorophyta) indicate a long divergence. Helgoländer Meeresuntersuchungen 41: 377–383.[CrossRef]

35. Prasad BN, Godward MBE. 1966. Cytological studies in the genus Zygnema. Cytologia 31: 375–391.[Medline]

36. Puiseaux-Dao S. 1963. Les Acétabulaires, matériel de laboratoire. Les resultants obtenus avec ces Chlorophycées. L'Annee Biologie (II) 3/4: 99–154.

37. Singh SJ. 1973. Cytology of some marne siphonaceous green algae. Ph.D. Thesis. Banaras Hindu University.

38. Spring H, Grierson D, Hemleben, Stöhr M, Krohne G, Stadler J, Franke W. 1978. DNA contents and numbers of nuclei and pre-rRNA-genes in nuclei of gametes and vegetative cells of Acetabularia mediterranea. Experimental Cell Research 114: 203–215.[CrossRef][Web of Science][Medline]

39. Suematu S. 1960. The somatic nuclear division in Trentepohlia aurea, the aerial alga. Bulletin Liberal Art College Wakayama University Natural Sciences 10: 111.

40. Wells CV, Hoshaw RW. 1971. The nuclear cytology of Sirogonium. Journal of Phycology 7: 279–284.[CrossRef][Web of Science]

41. Werz G. 1953. Über die Kernverhältnisse der Dasycladaceen, besonders von Cymopolia barbata (L.) Harv. Archiv für Protistenkunde 99: 148–155.

42. Wik-Sjöstedt A. 1970. Cytogenetic investigations in Cladophora. Hereditas 66: 233–262.

	APPENDIX II. CHROMOSOME NUMBER AND NUCLEAR DNA CONTENT IN SPECIES OF PHAEOPHYTA

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 
A key to the references appears at the end of this Appendix.

View this table: [in this window] [in a new window]

 

	KEY TO REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 
1. Caram B. 1964. Sur la sexualité l'alternance de générations d'une Phéophycée: le Striaria attenuate. Compte rendu Habdomadaire de Séances del'Académie des Sciences, Paris 259: 2495–2497.

2. Evans LV. 1962. Cytological studies in the genus Fucus. Annals of Botany 26: 345–360.[Abstract/Free Full Text]

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	APPENDIX III. CHROMOSOME NUMBER AND NUCLEAR DNA CONTENT IN SPECIES OF RHODOPHYTA

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 
A key to the references appears at the end of this Appendix.

View this table: [in this window] [in a new window]

 

	KEY TO REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 
1. Austin AP 1956. Chromosome counts in the Rhodophyceae. Nature (London) 178: 370–371.

2. Bailey JC, Kapraun DF. UNC-W, Wilmington, NC, USA, unpubl. res.

3. Bird CJ, Rice EL. 1990. Recent approaches to the taxonomy of the Gracilariaceae (Gracilariales, Rhodophyta) and the Gracilaria verrucosa problem. Hydrobiologia 204/205: 111–118.[CrossRef]

4. Bird CJ, van der Meer JP, McLachlan J. 1982. A comment on Gracilaria verrucosa (Huds.) Papenf. (Rhodophyta: Gigartinales). Journal of the Marine Biological Association of the UK 62: 453–459.

5. Cole KM. 1990. Chromosomes. In: Cole KM, Sheath RG, eds. Biology of the red algae. Cambridge: Cambridge University Press, 71–101.

6. Cordeiro-Marino M, Yamaguishi-Tomita N, Yabu H. 1974. Nuclear divisions in the tetrasporangia of Acanthophora spicifera (Vahl) Boergesen and Laurencia papillosa (Forsk.) Greville. Bulletin of the Faculty of Fisheries, Hokkaido University 25: 79–81.

7. Freshwater DW. 1993. Cytophotometric estimation of inter- and intraspecific variation in nuclear DNA content in ten taxa of the Gelidiales (Rhodophyta). Experimental Marine Biology and Ecology 166: 231–239.[CrossRef]

8. Hanic LA. 1973. Cytology and genetics of Chondrus crispus Stackhouse. Proceedings of the Nova Scotia Institute of Science 27 (Suppl.): 23–52.

9. Harris RE. 1962. Contribution to the taxonomy of Callithamnion Lyngbye emend. Naegeli. Botanica Notiser 115: 18–28.

10. Kapraun DF 1978. A cytological study of varietal forms in Polysiphonia harveyi and P. ferulacea (Rhodophyta, Ceramiales). Phycologia 17: 152–156.

11. Kapraun DF. 1989. Karyological investigations of chromosome variation patterns associated with speciation in some Rhodophyta. In: George RY, Hulber AW. eds. Carolina Oceanography Symposium. Washington: National Undersea Research Program Research Report 892, 65–76.

12. Kapraun DF. 1993a. Karyology and cytophotometric estimation of nuclear DNA content variation in Gracilaria, Gracilariopsis and Hydropuntia (Gracilariales, Rhodophyta). European Journal of Phycology 28: 253–260.

13. Kapraun DF. 1993b. Karyology and cytophotometric estimation of nuclear DNA variation in several species of Polysiphonia (Rhodophyta, Ceramiales). Botanica Marina 36: 507–516.

14. Kapraun DF, Dunwoody JT. 2002. Relationship of nuclear genome size to some reproductive cell parameters in the Florideophycidae (Rhodophyta). Phycologia 41: 507–516.

15. Kapraun DF, Dutcher JA. 1991. Cytophotometric estimation of inter- and intraspecific nuclear DNA content variation in Gracilaria and Gracilariopsis (Gracilariales, Rhodophyta). Botanica Marina 34: 139–144.

16. Kapraun DF, Freshwater DW. 1987. Karyological studies of five species in the genus Porphyra (Bangiales, Rhodophyta) from the North Atlantic and Mediterranean. Phycologia 26: 82–87.

17. Kapraun DF, Lopez-Bautista J. 1997. Karyology, nuclear genome quantification and characterization of the carrageenophytes Eucheuma and Kappaphycus (Gigartinales). Journal of Applied Phycology 8: 465–471.[CrossRef]

18. Kapraun DF, Bailey JC, Dutcher JA. 1994a. Nuclear genome characterization of the carrageenophyte Hypnea musciformis (Rhodophyta). Journal of Applied Phycology 6: 712.

19. Kapraun DF, Dutcher JA, Freshwater DW. l993b. Quantification and characterization of nuclear genomes in commercial red seaweeds: Gracilariales and Gelidiales. Hydrobiologia 260/261: 679–688.[CrossRef]

20. Kapraun DF, Dutcher JA, Lopez-Bautista J. 1992. Nuclear genome characterization of the carrageenophyte Agardhiella subulata (Rhodophyta). Journal of Applied Phycology 4: 129–137.

21. Kapraun DF, Hinson TK, Lemus AJ. 1991. Karyology and cytophotometric estimation of inter- and intraspecific nuclear DNA variation in four species of Porphyra (Rhodophyta). Phycologia 30: 458–466.

22. Kapraun DF, Dutcher JA, Bird KT, Capecchi MF. l993a. Nuclear genome characterization and carrageenan analysis of Gymnogongrus griffithsiae (Rhodophyta) from North Carolina. Journal of Applied Phycology 5: 99–107.[CrossRef]

23. Kapraun DF, Ganzon-Fortes E., Bird K, Trono G, Breden C. 1994b. Karyology and agar analysis of the agarophyte Gelidiella acerosa (Forsskal) Feldmann et Hamel from the Philippines. Journal of Applied Phycology 6: 545–550.[CrossRef]

24. Kapraun DF, Lopez-Bautista J., Trono G, Bird KT. 1996. Quantification and characterization of nuclear genomes in commercial red seaweeds (Gracilariales) from the Philippines. Journal of Applied Phycology 8: 125.

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27. Lopez-Bautista J, Kapraun DF. 1995. Agar analysis, nuclear genome quantification and characterization of four agarophytes (Gracilaria) from the Mexican Gulf Coast. Journal of Applied Phycology 7: 351–357.[CrossRef]

28. Magne F. 1964. Recherches caryologiques chez les Floridées (Rhodophycées). Cahiers Biologique Marine 5: 461–671.

29. McLaughlin J, van der Meer JP, Bird NL. 1977. Chromosome numbers of Gracilaria foliifera and Gracilaria sp. (Rhodohyta) and attempted hybridizations. Journal of the Marine Biological Association UK 57: 1137–1141.

30. Nichols HW. 1964. Culture and developmental morphology of Compsopogon coeruleus. American Journal of Botany 51: 180–188.[CrossRef]

31. Patwary MV, van der Meer JP. 1984. Growth experiments on autopolyploids of Gracilaria tikvahiae (Rhodophyceae). Phycologia 23: 21–27.

32. Rao CSP, Gujarati AR. 1973. Second meiotic division in the tetrasporangium of Chondria dasyphylla (Woodw.) C. Ag. Current Science 42: 361–362.

33. Ravanko O. 1987. Preliminary studies on cells and chromosomes in Polysiphonia violacea. Societas pro Fauna et flora Fennica. Flora Fennica 63: 45–50.

34. Rosenberg T. 1933. Studien über Rhodomelaceen und Dasyaceen. Lund: Akadamie Abhandlung.

35. Sheath RG, Cole KM. 1980. Distribution and salinity adaptations of Bangia atropurpurea (Rhodophyta), a putative migrant into the laurentian Great Lakes. Journal of Phycology 16: 412–420.[CrossRef][Web of Science]

36. Suneson S. 1937. Studien über die Entwicklungsgeschichte der Corallinaceen. Kongliga Fysiografiska Soelskapets i Lund. Handlingar 48: 1–101.

37. Van der Meer JP. 1976. A contribution towards elucidating the life history of Palmaria palmata (= Rhodymenia palmata). Canadian Journal of Botany 54: 2903–2906.

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	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 
I thank Professor M. D. Bennett and Dr I. J. Leitch for their encouragement to compile published information on algal nuclear genomes and to continue these investigations to expand our database, and for providing an opportunity to present this information at the Second Plant Genome Size Workshop, 2003. Much of the data in Appendixes I–III resulted from student research at UNC-W. Consequently, I recognize contributions from the following students, both undergraduate and graduate, who conducted phycological investigations under my direction: Dr J. C. Bailey, Dr P. W. Boone, P. C. Breden, J. R. Buratti, M. F. Capecchi, R. A. Criswell, J. T. Dunwoody, J. A. Dutcher, Dr D. W. Freshwater, Dr G. M. Gargiulo, T. K. Hinson, Dr J. Lopez-Bautista, M. Marlowe, Dr D. J. Martin, M. N. Nguyen, W. Purvis, M. J. Shipley. I thank Drs J. C. Bailey, D. W. Freshwater, G. Saunders and J. West for providing algal specimens used in this study. I acknowledge Dr G. Chandler and my son, Dustin Kapraun, for technical assistance in producing the computer-generated graphics. Financial support is gratefully acknowledged from a UNC-W Cahill Award.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION... PHAEOPHYTA RHODOPHYTA GENERAL SUMMARY NOTES ON APPENDIXES I-III.... APPENDIX I. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX II. CHROMOSOME NUMBER... KEY TO REFERENCES APPENDIX III. CHROMOSOME NUMBER... KEY TO REFERENCES ACKNOWLEDGEMENTS LITERATURE CITED

 

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