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Dissertations, Theses, and Masters Projects Theses, Dissertations, & Master Projects
1992
Cytophotometric Estimation of Nuclear DNA Content Variation in Ten Species of Geniculate Coralline Algae (Corallinaceae, Rhodophyta)
Jeffrey Craig Bailey College of William & Mary - Arts & Sciences
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Recommended Citation Bailey, Jeffrey Craig, "Cytophotometric Estimation of Nuclear DNA Content Variation in Ten Species of Geniculate Coralline Algae (Corallinaceae, Rhodophyta)" (1992). Dissertations, Theses, and Masters Projects. Paper 1539625729. https://dx.doi.org/doi:10.21220/s2-cdzt-sp73
This Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. CYTOPHOTOHETRIC ESTIMATION OF NUCLEAR DNA CONTENT VARIATION IN TEN SPECIES OF GENICULATE CORALLINE
ALGAE (CORALLINACEAE, RHODOPHYTA)
A Thesis Presented to
The Faculty of the Department of Biology
The College of William and Mary in Virginia
In Partial Fulfillment
Of Requirements for the Degree of
Master of Arts
by
Jeffrey Craig Bailey
1992 APPROVAL SHEET
This thesis is submitted in partial fulfillment
the requirements for the degree of
Master of Arts
Approved, May 1992
^p^seph^L. Scott
. Broadwater
Stanton F. Hoegerman TABLE OP CONTENTS
Page
ACKNOWLEDGEMENTS...... iv
LIST OF TABLES...... V
LIST OF FIGURES...... vi
ABSTRACT...... vii
INTRODUCTION...... 2
MATERIALS AND METHODS...... 6
RESULTS AND DISCUSSION...... 10
REFERENCES...... 2 5
TABLES ...... 3 3
FIGURES...... 3 6
VITA...... 42
i i i ACKNOWLEDGEMENTS
The author would like to express his thanks to the
following group of persons for their professional guidance,
technical assistance and generosity: Dr. Joseph L. Scott,
Dr. Donald F. Kapraun, Dr. Richard M. DiHainan, Dr. James F.
Merritt, Cheryl Morrison, Jewel Thomas and Bill Saunders.
Special thanks are awarded Dr. Sharon T. Broadwater and Dr.
Stanton F. Hoegerman for greatly improving upon an original
draft of the present manuscript. The author also wishes to
gratefully acknowledge financial support, in the form of a
summer graduate assistantship, provided by the Center for
Marine Science Research, Wilmington, North Carolina.
iv LIST OF TABLES
Table Page
1. Estimated 2C genome sizes...... 33
2. One-way analysis of variance of estimated 2C genome sizes...... 3 4
3. Nuclear volume estimates...... 3 5
v LIST OF FIGURES
FIGURE PAGE
1. Photomicrograph of chicken erythrocytes and vegetative cell nuclei of Cheilosoorum saqittatum and Lithothrix aspergillum after DAPI staining...... 3 6
2. Photomicrograph of vegetative cell nuclei and cellular (nuclear) fusion in Bossiella orbigniana after DAPI staining...... 37
3. Plot of fluorescent fading vs. time for five species of geniculate coralline algae and chicken erythrocytes after DAPI staining...... 38
4. Frequency distributions of relative 2C DNA levels in epithallial cells of three species of geniculate coralline algae and chicken eryth rocytes after DAPI staining...... 3 9
5. Frequency distribution of relative DNA levels in three different vegetative cell types in Jania adhaerens...... 40
6. Logarithmic plot of 2C genome size vs. a theoretical doubling sequence...... 41
vi ABSTRACT
Cytofluorometric analysis of DAPI stained nuclei was used to examine intrathallial differences in relative ploidy levels associated with each of three different cell types and interspecific variation in 2C nuclear DNA content among ten species of geniculate coralline algae in the red algal order Corallinales. Relative DNA values gave evidence of substantial intrathallial variation of ploidy level in epithallial, cortical and medullary cell nuclei in all genera investigated. Epithallial cells of tetrasporophytic specimens were shown to invariably contain the 2C level of DNA, approximately half the value of cortical cell nuclei. Relative fluorescence values indicate that ploidy levels of medullary cells range from 8 - 3 2C. Nuclear volume estimates were found to be highly correlated to 2C DNA content. Estimates of 2C genome size from epithallial cell nuclei in the tetrasporophytic phase range from 0.6 picograms in Amphiroa zonata to 1.3 pg for Calliarthron tuberculosum and exhibit a large-scale discontinuous pattern of variation. Analysis of variance of 2C DNA amounts in epithallial cell nuclei using a conservative Sheffe test recognized two significantly different groups: (a) a low-DNA content group, and (b) a high-DNA content group with mean values approximately twice that of the first group. Despite apparent taxonomic inconsistencies in the low-DNA content group, composed of members of the Amphiroideae, Corallinoideae and Metagoniolithoideae, results indicate that nuclear DNA contents may have potential use as a taxonomic criterion. Interspecific variation in nuclear DNA quantity is discussed in terms of increases or decreases in the amount of repetitive DNA within the basic genome or polyploidization. It is possible that each of these phenomena could account for the distribution of DNA values determined in the present study and it is likely that such processes have played significant roles in evolution and speciation within the three subfamilies of geniculate coralline algae. A relationship amoung 2C nuclear DNA content, yearly reproductive cycles, spore dimensions, pattern of spore germination and characterisitc presence or absence of secondary pit connections and lateral cell fusion between medullary cells of adjacent vegetative filaments is suggested, lending support to an infraordinal classification of the Corallinales not based on the presence or absence of genicula. CYTOPHOTOMETRIC ESTIMATION OF NUCLEAR DNA CONTENT
VARIATION IN TEN SPECIES OF GENICULATE CORALLINE
ALGAE (CORALLINACEAE, RHODOPHYTA) INTRODUCTION
The order Corallinales (Rhodophyta) sensu Silva and
Johansen (1986) circumscribes a distinct assemblage of
Rhodophyceae containing a large number of species all of which deposit calcium carbonate (as calcite) in their
cell walls (Johansen 1976, 1981, Woelkerling 1988).
Other distinguishing features of the group include: apical and intercalary meristems, production of reproductive organs in roofed conceptacles, tetrasporangia undergoing simultaneous zonate division, two celled carpogonial branch on a supporting cell which
serves as the auxiliary cell, many supporting cells
fusing after fertilization to produce a large fusion cell
from which gonimoblast filaments arise, and pit connections with two-layered caps lacking cap membranes
(Bold & Wynne 1985, Johansen 1981, 1976, Woelkerling
1988, Pueschel 1987). Parenchymatous thalli are composed of joined filaments which are differentiated into three cell types: epithallial, cortical and medullary.
Coralline algae are essentially cosmopolitan, occurring
in all oceans, with various species often dominating upper subtidal areas.
Historically, coralline algae have been divided into two groups (1) non-geniculate (non-articulated) in the
2 3 form of crusts, often smooth or with conspicuous protuberances and (2) geniculate (articulated), with plants attached, having a branched frond composed of calcified segments (intergenicula) and uncalcified nodes
(genicula) (Johansen 1976, 1981, Woelkerling 1988) .
However, the validity of the basic dichotomy of the group, based on the presence or absence of genicula, has been questioned. Established subfamilies within each group appear to share characters, including yearly reproductive cycles, spore dimensions, pattern of spore germination and the presence or absence of secondary pit connections and lateral cell fusions between medullary cells of adjacent vegetative filaments, which suggest that some of the geniculate subfamilies may be more closely related to some of the non-geniculate subfamilies than to other geniculate subfamilies (Cabioch 1971, 1972,
Chihara 1973, 1974, Adey & Johansen 1972, Johansen 1976,
Woelkerling 1988). On the basis of these features,
Cabioch (1972) and Chihara (1973, 1974) consider the present taxonomic circumscription of the order
Corallinales unnatural. Differences in the two schemes, which are discussed by Adey and Johansen (1972), reflect the greater taxonomic and phylogenetic significance accorded to genicula by Johansen (Johansen 1969a, 1969b,
Johansen & Silva 1978, Johansen 1981).
Within the geniculate coralline algae, three strikingly different types of genicula are present and 4 these, along with several other notable characters, form the distinguishing characteristics of the three subfamilies; Amphiroideae, Corallinoideae, and
Metagoniolithoideae (Johansen 1969a, 1969b, Garbary &
Johansen 1987). Previous taxonomic treatments of the coralline algae were confined to morphological and reproductive data sets gathered at the level of the light microscope (Johansen 1981, Woelkerling 1988) and scanning electron microscopic studies of surface features of the group (Garbary 1978, Garbary & Scagel 1979, Garbary &
Veltkamp 1980, Garbary et al. 1981, Garbary & Johansen
1982) .
Recently, investigations of the karyology, genome size and genomic complexity among various taxa of marine
Rhodophyta (Goff & Coleman 1984a, 1986, 1990, Gonzalez &
Goff 1989, Kapraun & Bailey 1989) and Chlorophyta
(Kapraun & Shipley 1990) have proven useful in the elucidation of relative ploidy level changes associated with presumptive life history stages. These and other studies have provided a clearer understanding of the systematics of some algal taxa (Kapraun & Gargiulo
1987a, 1987b, Kapraun & Shipley 1990, Dutcher et al.
1990, Kapraun & Dutcher 1991, Bot et al. 1991, Kapraun &
Bailey 1992, Freshwater, in press.).
Thus far, genomic evolution in the marine red and green algae appears to have similarities to phenomena observed in higher land plants including, intra- and 5 interspecific variation in nuclear DNA content, mechanisms of karyotypic change, and nucleotide sequence complexity and evolution (Van't Hof 1965, Stebbins 1971,
Price 1976, 1988, Rees & Jones 1972, Sparrow et al. 1972,
Cavallini & Natali 1991). Speciation in chlorophycean and rhodophycean algal taxa may be accompanied by interspecific variation in DNA content which often exhibits a discontinuous pattern. Much of this variation is attributed to variability in the amount of repeated sequences. Repetitive genetic elements have been shown to constitute 10 - 95 % of the nuclear genome in some red and green algae (Bot et al. 1989a, Dutcher et al. 1990,
Kapraun et al. in press). Repetitive DNA is also a predominant feature of higher plant genomes, often comprising 40 - 9 0% of their nuclear genomes (Hake &
Walbot 1980, Flavel 1980, Smyth 1991). Various karyotype rearrangements apparently involving fusion or fission events (Kapraun & Bailey 1992), polyploidy (e.g. Kapraun
& Gargiulo 1987a, 1987b, Kapraun & Bailey 1989) and cryptopolyploidy (Kapraun et al. 1988) have been reported in several chlorophycean and rhodophycean taxa.
The present investigation was initiated to provide basic information on the nuclear genomes of selected members of geniculate coralline algae. These results and previously published information are used to discuss interspecific and subfamilial genetic relationships among geniculate coralline algae. 6
MATERIALS AMD METHODS
SOURCE OF SPECIMENS
Mature tetrasporocytes of Lithothrix aspergillum
Gray, Amphiroa zonata Yendo, Corallina Vancouveriensis
Yendo, Bossiella orbigniana ssp. dichotoma (Manza)
Johansen, Jania adhaerens Lamour. and Calliarthron tuberculosum (Post. & Rupr.) Dawson were periodically collected from shallow subtidal habitats in the vicinity of Laguna Beach, Orange County, California from December
1990 to October 1991. Collections of Amphiroa beauvoisii
Lamour. and Corallina officinalis L. were made at South
Masonboro Inlet Jetty, Wrightsville Beach, North Carolina in June 1989 through August 1991 (see Abbott & Hollenberg
1976 and Kapraun 1980, 1984 for location map and habitat descriptions, respectively). Specimens of
Metagoniolithon radiatum (Lamarck) Ducker and
Cheilosporum sagittatum (Lamour.) Aresch. were obtained from Rye, Victoria, Australia in October 1991. Material was either field-fixed in 3 absolute ethanol : 1 glacial acetic acid or was returned to the laboratory and cleaned of debris and epiphytes prior to fixation for 24h.
Tetrasporophytic plants were microscopically identified 7 and transferred to 70% ethanol for storage (Kapraun &
Martin 1987) .
DETERMINATION OF NUCLEAR DNA CONTENT
Fixed material for measurement of nuclear DNA was treated as follows: branch tips (including the first 2 -
3 intergenicula) were placed in a 2 - 5% solution of
sodium ethylenediaminetetraacetic acid (EDTA) for 24 -
72h to remove all calcium carbonate. Decalcified branch tips, along with a drop of EDTA solution, were then placed on the rough ground glass end of a frosted slide and cells were liberated by maceration with a second slide. The resultant aqueous cell suspension was applied to glass coverslips treated with Subbing solution (0.01 g chromium potassium sulfate and 0.1 g gelatin in 100 ml of distilled water). Larger fragments were removed from the suspension, leaving a film of cells which were allowed to air dry (10 - 15 min).
Chicken erythrocytes (RBCs), used as the internal standard, were collected by pipette from decapitated 18- day embryos and placed in a solution of 19 mg sodium citrate in 0.5 ml of distilled water. RBCs were subsequently centrifuged, fixed in 9 methanol : 1 formaldehyde (Gold & Price 1985) for 20 min and then re centrifuged. The resulting pellet was rinsed in distilled water, centrifuged three times to remove 8 fixative, and transferred to 70% ethanol for storage at
4° C (Gold & Price 1985). Fixed RBCs were smeared on glass coverslips and air dried (10 - 15 min).
After drying, coverslips with algal material and
RBCs were rehydrated in 0.2 M KC1 for 15 min (Goff &
Coleman 1984a, 1985), and stained in the dark for 45 min with 0.5 ug/ml 4',6 - diamidino-2-phenylindole dihydrochloride (DAPI; Sigma Chemical Co., St. Louis, MO) in Mcllvain's buffer, adjusted to pH 4.1. Coverslips were then mounted in DAPI on glass slides, sealed with clear nail polish, and refrigerated overnight prior to use.
Microspectrophotometric data for RBCs with a known
DNA content of 2.5 pg per nucleus (Rasch et al. 1971) were used to quantify mean fluorescence intensity (If) values for epithallial cells of geniculate coralline algae. Proportionality of DAPI-staining between the RBC standard and algal cell nuclei has been demonstrated previously by comparison of If values obtained with red algae having known DNA contents (Kapraun & Dutcher 1991,
Kapraun et slI. 1991, Freshwater unpubl.). Frequency distributions for If values corresponding to 2C contents for each species were determined following the methodology of Goff & Coleman (1986). Relative fluorescence intensity values for epithallial cell nuclei were transformed into estimates of 2C nuclear DNA content for each species by solving the equation: RBC If / sample
If = 2.5 pg / X pg (Johnson et al. 1987, Tiersch et al. 9 1989) . The number of epithallial cell nuclei examined in each sample and the number of samples (each containing 2
- 4 plants) are indicated in Table 1.
Observations and photomicrographic records were made with brightfield and (incident) epi-UV illumination using an Olympus BH2-RFK microscope equipped with a high pressure mercury vapor lamp (HBO, 100W) in combination with exciter filter UG-1 and dichroic mirror DM-400, which are specific for DAPI emissions (Goff & Coleman
1990). A microphotometer (Kintek Photometry Systems,
OPELCO, Washington, DC, USA), equipped with a rotating array of perforated diaphragms, permitted selection of pinhole aperatures corresponding to the diameter of the nucleus being viewed (Kapraun & Shipley 199 0).
Therefore, extranuclear (cytoplasmic) fluorescence was greatly reduced, eliminating the need for subtraction of background readings (Goff & Coleman 1984a).
DETERMINATION OF NUCLEAR VOLUME
Nuclear volume (NV) estimates for nine species of geniculate coralline algae and RBCs were made by photographing interphase nuclei stained with DAPI, viewing the negatives on a microfiche reader and tracing the projected images (Kapraun & Freshwater 1987). Since the majority of all nuclei observed appear oval, rather than spherical, average diameter was computed to be: d = 10 length + 0.5 width / 2 for use in the formula NV = 3.14 d 3 / 6
(Sparrow & Nauman 1973).
RESULTS AND DISCUSSION
Microspectrophotometry with DNA-localizing fluorochromes has been used previously for karyological studies of marine green and red algae (Goff & Coleman
1984a, 1984b, 1985, 1986, Kapraun et al. 1988, Kapraun &
Shipley 1990, Kapraun & Bailey 1989, 1992, Kapraun &
Dutcher 1991). In the present study, DAPI staining (Goff
& Coleman 1984a, 1985, 1990, Gonzalez & Goff 1989) for 45 min followed by overnight storage at 4° C prior to examination resulted in reproducible, intense nuclear fluorescence with negligible cytoplasmic interference
(Figs 1, 2). Relative fluorescence intensity values for
DAPI stained nuclei were found to be markedly stable over time (Fig. 3). Similar experiments performed with a second DNA-localizing fluorochrome, hydroethidine
(Kapraun 1989), were characterized by spurious extranuclear staining and extreme variability in observed relative fluorescence values within and between experiments. Similarly, estimates of 2C nuclear DNA contents for geniculate coralline algae using hydroethidine staining procedures varied two to three fold in some cases (unpubl. data). Species or taxon specific properties of fluoro- chromes (Goff & Coleman 11 1990) , potentially resulting in unacceptable levels of experimental variation, urges that controlled pilot
studies be initiated based on experimental design and objectives.
Variations in relative ploidy levels associated with gametophytic and tetrasporophytic phases of some red algae have been demonstrated by microspectrophotometry
(Kapraun & Bailey 1989, Kapraun & Shipley 1990, Goff &
Coleman 1990, Kapraun & Dutcher 1991). In the absence of gametophytic specimens in this study, relative 2C DNA
levels were established by examining from 2 to 4 pairs of unreplicated early-telophase division figures in cortical-epithallial cells. Frequency distributions of relative DNA values for interphase epithallial cell nuclei indicate sharp 2C level profiles in all ten species of geniculate coralline algae used in the present study (see Figs 4 & 5 for selected species). A survey of relative fluorescence values for each of three cell types
(epithallial, cortical and medullary) is presented for
Jania adhaerens (Fig. 5). From these studies it is apparent that geniculate coralline algae exhibit a distribution of nuclear DNA levels which is correlated with cell type. Such patterns of intrathallial DNA level variation were similarly observed in each species
included in the investigation.
To date, nearly all red algae examined have cell cycles in which DNA replication occurs very soon after 12 mitosis, and nuclei predominantly reside in the G 2 stage of the cell cycle (Goff & Coleman 1990). In contrast, mature epithallial cell nuclei of tetrasporophytic specimens stay in G^ and invariably contain a 2C level of
DNA while cortical and medullary cells' DNA levels may, taken together, commonly range from approximately 4 -
32C. Relative fluorescence values, and therefore DNA level, in meristematic apical cells were not determined in this study. Due to the large size (diameter) of these cells' nuclei, they could not be accommodated by even the largest aperture in our photometry system. Apical cell nuclei excepted, extrapolated ploidy levels are greatest in medullary cell nuclei. Exceptionally high ploidy levels demonstrated in this study for some medullary cell nuclei are postulated to be the result of nuclear migration and subsequent nuclear fusion (Borowitzka &
Vesk 1978, Johansen 1981) (Figs 2 & 5). In general, estimated ploidy levels for cortical cell nuclei exhibit a 4C peak, approximately twice the nuclear DNA level observed in unreplicated G1 phase epithallial cells (Fig.
5). These results suggest a minimal investment of genetic material and energy in the production of epithallial cells. It is interesting to note that nonmeristematic epithallial cells, which are at times periodically sloughed off and replaced by meristematic activities of the underlying cortical layer (Borowitzka &
Vesk 1978) , reach G q in the G^ phase of the cell cycle 13 and are replicatively inactive following cell division and remain so indefinitely. Further, it would appear that replication in these cells is selected against due to their ephemeral nature.
In addition to allowing determination of relative
DNA contents, microspectrophotometry can be used to quantify mean fluorescence (X If) values, provided that standards with known DNA contents are used and equivalent staining is achieved in both standard and experimental nuclei (Ruch 1970, Bennett & Smith 1976). Recently, quantitative estimates of nuclear DNA content have been provided for various taxa of marine green and red algae via cytofluorometric techniques (Kapraun & Shipley 1990,
Hinson & Kapraun 1991, Kapraun & Dutcher 1991, Kapraun &
Bailey 1992, Freshwater in press, Kapraun et al. in press). In the present study, chicken RBCs were used to quantify mean If values for 10 species of geniculate coralline algae.
DAPI stained interphase epithallial cell nuclei examined with episcopic UV illumination revealed interspecific variation in amounts of 2C nuclear DNA content ranging from 0.6 picograms in Amphiroa zonata to
1.3 pg in Calliarthron tuberculosum (Table 1). Although published data for other Rhodophyta are limited, values range from 0.4 to 2.1 pg for Porphvra. Gelidium.
Pterocladia. Aqardhiella. Gracilaria and Polvsiphonia
(Kapraun & Dutcher 1991, Kapraun et al. in press, 14 Freshwater in press, D.F. Kapraun pers. comm.)• A one way analysis of variance (ANOVA) using a conservative
Scheffe test (Table 2) revealed that two significantly different groups could be distinguished on the basis of estimated 2C nuclear genome size. In the present investigation, those members of the subfamily
Amphiroideae fall into one discrete group and are joined by the species Cheilosnorum saaittatum and
Metagoniolithon radiatum of the subfamilies
Corallinoideae and Metagoniolithoideae, respectively.
The second group is comprised solely of those remaining species in the subfamily Corallinoideae. It is noted that 2C nuclear DNA estimates in the second group, with a mean value of 1.2 pg, are approximately twice the mean value of the first group (0.68 pg). Despite apparent taxonomic inconsistencies in the first group, comprised of members of each of the three currently recognized subfamilies of geniculate coralline algae, these data indicate that nuclear DNA quantity may be helpful as an exclusionary taxonomic criterion in delimiting problematic species within the family Corallinaceae.
Nuclear volume (NV) values have been shown to be highly correlated with the amount of DNA per nucleus
(e.g. Sparrow & Miksche 1961, Price 1976, Cavilier-Smith
1978) . Estimates of nuclear volume for at least one cell type in each of 10 species of geniculate coralline algae were made from interphase nuclei visualized after DAPI 15 staining (Figs 1,2 and Table 3). Estimates of NV were found to be highly correlated (r = 0.94, p < 0.05) with quantitative 2C genome sizes determined from epithallial cell nuclei for each of 9 species used in the investigation and chicken RBCs used as the internal standard.
In the present study, nuclear DNA determinations based on microspectrophotometric analysis with DAPI
(Table 1) and NV estimates (Table 3) gave evidence of a bimodal, large-scale discontinuous distribution of DNA content, with the mean value of one group representing approximately twice the value of the other. This pattern of DNA variation observed in geniculate coralline algae, characterized by discontinuity and nearly regular progression of values (Fig. 6) is also known for vascular plants and for the coenocytic green marine algal genus
Codium (Kapraun et al. 1988). Hypothetically, there are three ways to account for variation in nuclear DNA levels: 1) differential polynemy, or "strandedness" of chromosomes, 2) polyploidy, resulting in a doubling, or near doubling, of the basic genome size, and 3) an increase or decrease in the amount of repetitive DNA within the genome.
Since most biochemical and ultrastructural studies support a general uninemic chromatid model (see Rees et al. 1966 and Rees 1972 for reviews), differential polynemy has been rejected as a likely mechanism for 16 groupings of species DNA values (Callan 1967, Laird 1971,
DuPraw 1972, Price & Bachmann 1975). Alternatively, discontinuous patterns have been explained in terms of lengthwise addition or loss of DNA (Price 197 6, Narayan &
Rees 1976, Narayan 1983). Current molecular evidence and genetic models suggest that DNA (gene) duplication along the length of a chromosome (Callan 1967, Rees & Jones
1972, Fieldhouse & Golding 1991, Smyth 1991) would apparently result in symmetrical distribution throughout the genome of large-scale (saltatorial) DNA addition and deletion events (Britten & Kohne 1968, Narayan 1983). It has been suggested that duplicated sequences that are dispersed at many scattered locations within the genome, or on chromosomes, are likely to be mobile genetic elements (Smyth 1991).
There is considerable evidence that both increase and decrease in DNA content have accompanied speciation and phylogenetic advancement in vascular plants (Rees et al. 1966, Rees & Jones 1972, Price & Bachmann 1975, Price
1976, 1988). Historically, cytogenetic studies of the
Rhodophyta have been handicapped by a number of features, including complexities of florideophycidean life histories, smallness of nuclei and chromosomes, and periodicity and rapidity of cell division (Cole 1990).
Karyological investigations of several taxa of geniculate coralline algae used in this study, Amphiroa zonata,
Amphiroa beauvoisii. Lithothrix aspergillum. Bossiella 17 orbigniana ssp. dichotoma and Calliarthron tuberculosum, met with minimal success. Squash preparations of apical cell regions stained with 2% aceto-orcein (Kapraun &
Martin 1987) resulted in poor, uneven staining of most cell nuclei and permitted little resolution of nuclear features. Squash preparations stained with DAPI were clearly superior to those produced by the former method but a lack of mitotic activity precluded accurate chromosome counts. Branch tips treated in the field with either of two mitotic inhibitors, 4 x 10”5 M griseofulvin or 3 x 10”3 M 8 - hydroxyquinoline, for up to 14 hr prior to fixation yielded numerous early-late prophase nuclei, but few clear metaphase figures. Although precise chromosome counts were not obtained, it is likely that the chromosome complements of Bossiella orbigniana ssp. dichotoma and Ca11iarthron tuberculosum are characterized by pronounced karyotype asymmetry (pers. observ.). At present, karyological information is available for only 4 species of geniculate coralline algae, all within the subfamily Corallinoideae (Cole 1990). Magne (1964) reported a diploid chromosome complement of 2N = 48 for tetrasporophytic specimens of Jania rubens and Corallina officinalis var. mediterranea. Similarly, Suneson (1937,
1950) reported a haploid chromosome complement of N = 2 4
for gametophytic specimens of Corallina officinalis and
Marainosporum aberrans. Due to the paucity of such data, any speculation concerning the relationship among 18 chromosome number, genome size, phylogenetic position, and systematics within and between the subfamilies of geniculate coralline algae must be considered tentative.
However, it seems significant that estimated 2C nuclear genome sizes within the group exhibit an essentially bimodal distribution, with one value approximately twice the other (Tables 1, 2 and Fig. 6).
In the absence of detailed karyological information for nearly all species of geniculate corallines, it seems likely that the large-scale discontinuous patterns of nuclear DNA content observed in this study, with modal values varying approximately two-fold, may be explained by doubling of genome size by polyploidy. One of the most direct effects of polyploidy is the abrupt quantitative increase in chromosome number and nuclear
DNA content (Ohri & Khoshoo 1986). Empirical evidence for such concomitant changes in chromosome number, ploidy level and DNA content is widespread in vascular plants and well documented in the literature (e.g. Sparrow &
Nauman 1973, Price 1976) and has recently been observed in several taxa of multinucleate green marine algae (
Kapraun et al. 1988, Kapraun & Shipley 1990, Hinson &
Kapraun 1991). It is possible that those species forming a discrete group having significantly higher DNA values may be polyploid derivatives. Karyological studies of 4 geniculate and 4 non-geniculate species of coralline algae (Cole 1990) indicate that a haploid chromosome 19 complement of N = 24 has apparently been conserved within the order and polyploidy, perhaps derived from a basic genome of X = 12 or 6, may have played a significant role
in speciation and evolution of coralline algae. This hypothesis awaits further study.
Johansen (1969a) proposed a phylogenetic scheme for the genera of Corallinoideae based primarily on conceptacular origin and vegetative anatomy. Results
from the present investigation suggest phylogenetic advancement within the subfamily Corallinoideae may be associated with increasing nuclear DNA contents (Johansen
1969a, Table 1). The derived position of the genus
Cheilosporum from a Corallina-like ancestor, tentatively proposed by Johansen (1969a), is of special interest.
Estimated 2C genome sizes for Corallina officinalis (1.2 pg) and Corallina Vancouveriensis (1.3 pg) are approximately twice that of Cheilosporum saqittatum (0.7 pg). Assuming intergeneric differences in nuclear DNA content are attributable to the accumulation of repetitive DNA sequences or polyploidization, the derived position of Cheilosporum suggests a possible reduction of
DNA content in this species. A plausible alternative explanation for this curious taxonomic distribution of
DNA content is that the genus Cheilosporum diverged from the latest common ancestor it shared with the extant genus Corallina prior to the apparent accumulation of additional DNA which has effectively doubled DNA amounts 20 in the genus Corallina. This explanation, if correct,
suggests that genome size in the genus Cheilosporum may be considered primitive within the subfamily
Corallinoideae.
Johansen (1977), in a revision of his earlier hypothesis (Johansen 1969a), suggested that Cheilosporum
is more closely related to Jania and Haliptilon than to
Corallina. Arthrocardia and other genera in the subfamily
Corallinoideae on the basis of carpogonial and male
conceptacle anatomy. An estimated 2C genome size of 1.1 pg for Jania adhaerens determined in the present study places this species in the high-DNA content group with
other, members of the subfamily Corallinoideae, excepting
Cheilosporum. Despite the apparent confusion concerning the generic relationships within the Corallinoideae it is
conceivable that both increase and decrease in DNA amounts may have accompanied speciation and evolution in the subfamily. A strong trend toward low DNA values for evolutionarily advanced or specialized species has been postulated for some plant (Rees & Jones 1972, Price
1976) and animal groups (Hinegardner 19 68, Bachmann et al. 1972, Hinegardner & Rosen 1972). Alternatively, as a result of their karyological investigation of grasses,
Sparrow and Nauman (1973) contend that large-chromosome
species with primitive characteristics and presumably high DNA content are derived from ancestral small- chromosome species with low DNA contents by incremental 21 genome increases. At the same time, small-chromosome / low DNA content advanced forms may continue to evolve from the same basic ancestral types, but without the evolutionary slowing-down presumed to be imposed by the accumulation of redundant genetic information (Kapraun &
Gargiulo 1987b). It is obviously beyond the scope of the present study to determine if genome size in Cheilosporum saaittatum represents an ancestrally primitive character or autapomorphic condition.
Garbary and Johansen (1987) used light- and scanning electron microscopy of Lithothrix aspergillum and Amphiroa spp. to resolve homologies in vegetative anatomy and morphogenesis in the two genera, emphasizing genicular structure in determining phylogenetic trends in geniculate coralline algae. On the basis of these features, the authors place Lithothrix sp. in a basal position within the monophyletic subfamily Amphiroideae, concluding that any feature present in Amphiroa spp. that is also present in Lithothrix aspergillum, is presumed to be primitive (Garbary & Johansen 1987). Thus, Amphiroa spp. is considered phylogenetically advanced within the subfamily Amphiroideae. Results from the present investigation indicate that speciation and evolution within the subfamily have occurred independently of
large-scale changes in nuclear DNA content (Table 1).
Chihara (1973, 1974) divided both the geniculate and non-geniculate coralline algae into two groups on the 22 basis of their yearly reproductive cycles, spore dimensions, and spore germination characteristics. This alignment of certain members of the Corailinaceae into two groups, termed Amphiroa - Lithophvllum and Corallina
- Lithothamnium. coincides with a grouping based on the manner of connection between cells of adjacent filaments.
In the Amphiroa - Lithophvllum group secondary pit connections are common and lateral cell fusions within the medulla are absent. In the Corallina - Lithothamnium group secondary pit connections are absent and the medulla is characterized by conspicuous lateral cell
fusions (Cabioch 1972, Chihara 1974). Both characters have been considered useful features in the taxonomy of the order and lend support to an infraordinal classification of the Corallinales (sensu Cabioch 1972) not based on the presence or absence of an erect segmented system (Gabrielson & Garbary 1986).
In the present study relatively high DNA content values were shown to be characteristic of members of the subfamily Corallinoideae (Table 1, Fig. 5). A second,
low-DNA content group was established comprised of a heterogeneous assemblage of members of the subfamilies
Amphiroideae (Lithothrix aspergillum. Amphiroa zonata and
Amphiroa beauvoisii), Corallinoideae (Cheilosporum saaittatum) and Metagoniolithoideae (Metaaoniolithon radiatum) (Table 1, Fig. 5). Although species-by-species comparisons cannot be made, these results suggest that 23 nuclear DNA amounts may also support infraordinal
interrelationships indicated by both spore germination patterns and characteristic presence or absence of
secondary pit connections and lateral medullary cell
fusion in the Corallinaceae. It is unfortunate that
spore germination patterns in the genus Metaqoniolithon have not been investigated. If, indeed, DNA amounts are
correlated with patterns of spore germination, an
Amphiroa - Lithophvllum type pattern is predicted for this genus. However, it is of interest that
Cheilosporum. which exhibits an estimated low DNA content
(0.7 pg) characteristic of the Amphiroideae, shares the
Corallina - Lithothamnium type pattern and spore dimensions typical of the subfamily Corallinoideae.
Similarly, it is notable that Jania rubens is reported to be heterogeneous with regard to spore germination, exhibiting both Corallina - Lithothamnium and Amphiroa -
Lithophvllum patterns (Chemin 1937, Chihara 1974), although observation of the latter pattern may be considered questionable (Chemin 1937).
Reconciling apparent heterogeneity in spore germination patterns, spore dimensions (see Chihara 1974) and seemingly discordant DNA contents within the present taxonomic scheme for the Corallinales requires further study. Analysis of DNA amounts for members of the non- geniculate subfamilies of Corallinaceae are currently underway in our laboratory. It will be significant new 24 evidence if DNA quantities similarly support infraordinal relationships suggested by patterns of spore germination and vegetative anatomy. 25
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Table 1.2C genome size (pg) for epithallial cell nuclei after DAH staining in 10 species of geniculate coralline algae. Data standardized to nuclear DNA content o f diploid chicken erythrocytes (2.5 pg: Rasch gt al. 1971) . n = number of nuclei observed in each sample. Mean ± SD is given for each species.
Subfamily/Species n 2C Genome Size (pg)
Amphiroideae
Amphiroa zonata 40 0.58 50 0.74 60 0.60 97 0.46 50 0.45 90 0.69
X= 0.59 ±0.12
Amphiroa beauvoisii 221 0.55 180 0.76 120 0.76
X= 0.69 ± 0.12
Lithothrix aspergillum 50 0.68 120 0.74 40 0.66 110 0.71 110 0.68 120 0.91
X= 0.73 ±0.09
Corallinoideae
Corallina officinalis 70 1.17 100 1.20 44 1.19
X= 1.19 ±0.02
Corallina vancouveriensis 32 1.30 Table 1. continued. 61 1.24 90 1.25
X= 1.26 ± 0.03
Jania adhaerens 160 1.00 120 1.05 98 1.12 100 1.38
X= 1.14 ±0.17
Bossiella orbigniana 120 1.08 ssp. dichotoma 125 1.12 90 1.25 120 1.28
X= 1.18 ±0.10
Calliarthron tuberculosum 30 1.20 120 1.38
X= 1.29 ±0.13
Cheilosporum sagittatum 50 0.82 50 0.63 40 0.67 45 0.73
X= 0.71 ±0.08
Metagoniolithoideae
Metagoniolithon radiatum 50 0.80 41 0.75 30 0.41 50 0.78
X= 0.69 ± 0.18 3~4
Table 2. Results of one-way analysis of variance (ANOVA) with unequal sample sizes performed on quantitative estimates of 2C nuclear DNA contents in 10 species of geniculate coralline algae. (p<0.05).
One-way ANOVA Summary Table
Source of Variation SS df MS F P
Between groups 2.709124 9 0.3010137 22.00 0.000 Within groups 0.396793429 1.368253E-2
Total 3.105917 38
Scheffe Test: a difference between any two group means greater than 0.3748454 is significant a the 0.05 level or less.
Group A: Amphiroa zonata Amphiroa beauvoisii Lithothrix aspergillum Cheilosporum sagittatum Metagoniolithon radiatum
Group B: Corallina officinalis Corallina vancouveriensis Jania adhaerens Bossiella orbigniana ssp. dichotoma Calliarthron tuberculosum 3 5
Table 3. Nuclear volume estimates for nine species of geniculate coralline algae and chicken erythrocytes used in this study. Estimates for different cell types (epithallial, cortical and medullary) are presented for selected species.
SPECIES NV (um3)
Epithallial Cortical Medullary
Amphiroa zonala 3.7 --
Amphiroa beauvoisii 1.6 3.5 9.4
Lithothrix aspergillum 3.3
Corallina vancouveriensis 6.6
Bossiella orbigiana 4.2 - -
Calliarthron tuberculosum 4.3 8.1 24.8
Jania adhaerens - 8.3 21.7
Cheilosporum sagittalum 2.5 5.1
Metagoniolithon radialum 2.3 4.4
Chicken erythrocytes 24.3 36
Fig. 1. Chicken erythrocytes (RBCs) and interphase vegetative cells of two species of geniculate coralline algae following DAPI staining. (A) RBCs, (B) Cheilosporum saaittatum. and (C) Lithothrix aspergillum. e = epithallial cell(s), c = cortical cell(s). Scale bar = 10 um. A
m f
0 Fig 2. Interphase vegetative nuclei in Bossiella orbigniana ssp. dichotoma after DAPI staining. (A) epithallial and cortical cell nuclei, (B) cortical cell (C) and (D) medullary cell and nuclear fusion. e = epithallial cell, c = cortical cell, m = medullary cell Scale bar = 10 urn. % © 38
Fig 3. A - E. Fluorescent extinction (fading) plotted against time for epithallial cell nuclei in four species of geniculate coralline algae and chicken erythrocytes (RBC) following DAPI staining. Note stability of fluorescence intensity values over time. A. Metagoniolithon radiatum. B. Amphiroa beauvoisii. C. Cheilosporum saqittatum. D. Lithothrix aspergillum. E. RBC. A. Metagoniolithon radiatum
B. Amphiroa beauvoisii
¥ " r ■ ■ 1 a
C. Cheilosporum sagittatum
• ■ k" i I 2 4 6 8 10
Time (sec.) D. Lithothrix aspergillum
, — ■■ — ■ i i.y i.m— ...... 1
E. RBC
2 4 6 8 10
Time (sec.) 39
Fig. 4. A - D. Frequency distributions of relative 2C level DNA values, after DAPI staining, for epithallial cell nuclei in three species of geniculate coralline algae: chicken erythrocytes (RBC) are used as an internal standard. A. Amohiroa zonata, B. Lithothrix aspergillum. C. Calliarthron tuberculosum. D. RBC. n = number of nuclei used to determine X If± SD values, If = fluorescence intensity. A
Amphiroa zonata
Relative Fluoresecence (If) B 60 i
Lithothrix aspergillum
Relative Fluorescence (If) Calliarthron tuberculosum
Relative Fluorescence (If) Number of Nuclei (n) D Relative Fluorescence (If) Fluorescence Relative 0 0 80 60 40 40
Fig. 5. Relative nuclear DNA profile for each of three cell types in Jania adhaerens following DAPI staining. Cross-hatched regions indicate extent of overlap in relative fluorescence values between different cell types. Indicated regions of the histogram correspond to relative DNA values for (A) epithallial cell nuclei indicating a sharp 2C peak, (B) cortical cell nuclei which exhibit a 4C peak, and (C) values obtained from large medullary cell nuclei whose mean value approximates an 8C peak. Intrathallial variation in relative DNA contents is evidenced by regular progression of mean intensity fluorescence values (X If) . n = number of nuclei examined for each cell type. X If= IB
n = 122 40
32 Jania adhaerens
•T—I 73 24 <+H X If = 43 O
16
8
30 50 70 90 110 130 150
Relative Fluorescence (If) 41
Fig. 6. Logarithmic plot of estimated 2C genome size against a theoretical doubling sequence. Data represent interphase 2C nuclear DNA contents determined from epithallial cell nuclei for ten species of geniculate coralline algae after DAPI staining (Table 1). Note that the number of similar DNA values converging on a single data point is indicated. Estimated 2C Genome Size (pg) 2 1 Theoretical Doubling Sequence Doubling Theoretical 42
VITA
Jeffrey Craig Bailey
Born in Bluefield, West Virginia, May 5, 1968.
Graduated from Princeton Senior High School, Princeton,
West Virginia, June 1986. Undergraduate studies were conducted at the University of North Carolina at
Wilmington, North Carolina, concluding with a Bachelor of
Science in Marine Biology, cum laude, with Honors in
Biology, awarded May 1990. In August 1990 entered graduate school with support from a teaching assistantship in the Department of Biology at the College of William and Mary, Williamsburg, Virginia. Thesis research and coursework was undertaken in fulfillment of the requirements for the Master of Arts degree in
Biology. Recently accepted in the graduate program at
Louisiana State University, Baton Rouge, Louisiana, to pursue the Doctor of Philosophy degree in molecular evolution and systematics.