SYSTEMATICS OF THE COMPSOPOGONALES (RHODOPHYTA) WITH
EMPHASIS ON THE FRESHWATER FAMJLES IN NORTH AMERICA.
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
TARA RINTOUL
In phal fulfilment of requirements
for the degree of
Master of Science
August, 1998
0Tara Rintoul, 1998 National Library Bibliothèque nationale du Canada Acquisitions and Acquisitions et 8ibliographic Services services bibliographiques 395 Wellington Street 395, rue Wellingtori OttawaON KIAW Ottawa ON K1A ON4 Canacla Canada
The author has granted a non- L'auteur a accordé une licence non exclusive licence dowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seil reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique.
The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substmtial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT
The Compsopogonaies is supported as a valid taxonomic entity through phylogenetic analysis of gene sequence data (18s rRNA and rbcL genes). A well separated freshwater lineage was identified through these analyses and a close relationship was recognized between the Boldiaceae and the Cornpsopogonaceae.
Biogeographical distribution of the Boldiaceae and Compsopogonaceae in North America was assessed through gene sequence data of the 5.8s rRNA gene and internally transcribed spacer regions ITS 1 and ITS2 of the rRNA gene. Analysis of the Boldiaceae indicated that introductions occurred fiom southem populations into northem areas. No obvious geographic trend was evident in the Compsopogonaceae. hdividuals in both families are thought to be dispersed through vector-assisted transport. The
Compsopogonaceae is shown to be monotypic in North America, containhg the single species Compsopogon coenrletlr. Observations of developing monosporangia demonstrated that the fieshwater families share ultrastructurai characteristics with the marine Erythropeltidaceae, and that the method of monosporangia formation is a usefid taxonomic character to distinguish the order Compsopogonales. TABLE OF CONTENTS
TABLE OF CONTENTS
ACKNOWLEDGMENTS
LIST OF TABLES
LIST OF FIGURES AND PLATES
CHAPTER 1 : INTRODUCTION AND LITERATURE REVIEW
1.1 Systematics of the Order Compsopogonales
1.2 Distribution and biology of the freshwater families Boldiaceae and
Compsopogonaceae
i) Boldiaceae
ii) Compsopogonaceae
1.3 Taxonomy of the Cornpsopogonaceae
1.4 Ultrastructure of monospore production in the Compsopogonales
CHAPTER 2: MATERIALS AND METHODS
2.1 DNA Extraction, Polymerase Chain Reaction and DNA Sequencing
2.2 DNA Sequence analysis
2.3 Microscopy
i) Transmission electron microscopy
ii) Scanning electron microscopy
iii) Light microscopy
CHAPTER 3: RESULTS
3.1 Phylogeny of the Compsopogonales i) Phylogeny of the Compsopogonales based on rbcL gene sequences.
ii) 18s rRNA gene phylogeny of the Compsopogonales
iii) Combined 18s rRNA and rbcL gene phylogeny of the
Compsopogonales
3.2 Biogeography of the Boldiaceae
3.3 Biogeography of the Compsopogonaceae
3.4 Taxonomy of the Compsopogonaceae
3.5 Ultrastructure of monosporangia in the Boldiaceae and Compsopogonaceae
i) Boldiaceae
ü) Compsopogonaceae
CHAPTER 4:DISCUSSION
4.1 Phylogeny of the Compsopogonales
4.2 Biogeography of the Boldiaceae and Compsopogonaceae in North Arnerica
4.3 Taxonomy of the Compsopogonaceae
4.4 Ultrastructure of monosporangia in the Boldiaceae and Compsopogonaceae
SUMMARY
LITElRATURE CITED ACKNOWEDGMENTS
1 thank Dr. Robert Sheath for the opportunity, the support and the ideas which were essential in the completion of this research. 1 thank members of my advisory codtteeDr. Joe Gerrath and Dr. Bnan Husband for insight and enthusiasm. 1 appreciate the training and energy that Morgan Vis-Chiasson shared with me at the beginning of my project. 1 am gratefbi to Angela Holliss for assistance in DNA sequence analysis and Joseline Beaulieu, P.G. Davison, Ray Holton and Bnan Oates for collecting
and providing samples for this research.
To the people 1 have shared space and experience with, Lesley Campbell, Kirsten
Miiller and Alison Sherwood, 1 extend heartfelt thanks for times filled with ideas and mirth, and sympathetic support. Gord Lemon, my traveling cornpanion, translater and
good fnend, shared my joumey through this process and 1 was glad to have him dong.
1 am also greatly indebted to the people who surround me in the world away fiom
scientifïc endeavor, who maintain a delightful, enteriahhg and loving atmosphere for me
to corne home to. 1 thank my family for ali of their support and belief in my abilities. And
as the most kind, generous and understanding force in my life, I thank Robert Wouda for being with me and consta;ritly reminding me that I was capable of everything. LIST OF TABLES
Table Title Pages number Collection and gene sequence information for simples 20-22
included in phylogenetic dysisof the order
Compsopogonales. Collection information for samples included in North
Amencan biogeographic shidy of the Boldiaceae and
Compsopogonaceae. Primer sequences for phylogenetic and biogeographic
analyses of the Compsopogonales. rbcL gene percent sequence divergence and Kimura 2
parameter distances of the Compsopogonales. 18s rWA gene percent sequence divergence and Kimura 2
parameter distances of the Compsopogonales. 5.8s rRNA gene percent sequence divergence and Kirnura 2
parameter distances of the Boldiaceae. ITS 1 percent sequence divergence and Kirnura 2 parameter
distances of the Boldiaceae, ITS2 percent sequence divergence and Kimura 2 parameter
distances of the Boldiaceae. 5.8s rRNA gene percent sequence divergence and Kimura 2
parameter distances of the Compsopogonaceae. 10 ITS 1 percent sequence divergence and Kirnura 2 parameter
distances of the Compsopogonaceae. 11 ITS2 percent sequence divergence and Kimura 2 parameter
distances of the Compsopogonaceae. 12 rbcL gene percent sequence divergence and Kimura 2
parameter distances of the Compsopogonaceae. 13 18s rRNA percent gene sequence divergence and Kimura 2
parameter distances of the Compsopogonaceae. 14 Summary of important characters reported for the 97-99
Compsopogonales. LIST OF FIGURES AND PLATES
Figure Title Pages
Number Distribution of Boldiaceae in North America as represented by
reported collection sites. Distribution of Compsopogonaceae in North America as
represented by reported collection sites. Parsimony analysis of rbcL gene sequence data of the
Compsopogonaies and Bangiales. Neighbour joining distance tree of Kimura 2 Parameter distances
of rbcL gene sequence data of the Compsopogonales and
Bangiales. Parsimony analysis of 18s rRNA gene sequence data of the
Compsopogonaies and Bangiales. Neighbour joining distance tree of Kimura 2 parameter distances
fiom 18s rRNA gene sequence data of the Compsopogonales and
Bangiales. Parsimony analysis of the combined 18s rRNA and rbcL gene
sequence data of the Compsopogonales and Bangiales. Neighbour joinùig distance analysis of combined 18s rRNA and
rbcL gene sequence data of the Compsopogonales and Bangides. Distribution of sequenced collections of Boldicl erythrosiphon
fiom North Arnerica Parsimony analysis of combined sequence data of 5.8s rRNA 58-59 gene, ITS I and ITSZ of the Boldiaceae of North America Neighbour joining distance trees of 5.8s rRNA gene, ITS 1 and 60-6 1
ITS2 combined sequence data of the Boldiaceae of North
America. Distribution of sequenced collections of Cornpsopogonaceae in 63-64
North America, Parsimony analysis of combined sequence data of 5.8s rRNA 69-70 gene, ITS 1 and ITS2 of the Compsopogonaceae in North America. Neighbour joining distance analysis of 5.8s rRNA gene, ITSl and 72-73
ITSZ combined sequence data of Compsopogonaceae in North
America, Parsimony (A) and neighbour joining distance (B) anaiysis of 74-75 combined 5.8s rRNA gene, ITSl and ITS2 sequence data of the
Compsopogonaceae in North America with the collections ALTR and SL2A removed. Parsimony analysis (A) and neighbour joining distance (B) 78-79 analysis of 5.8s rRNA gene, ITSl and ITSZ combined sequence data of Compsopogonaceae. Mature thalli of Boldia erythrosiphon Transverse sections of thalli of Boldia erythrosiphon Transverse sections of thalli and monosporangia of Boldia
vii 20 Germlings, young plants and monosporaflgia of the 9 1-92
Compsopogonaceae. 21 Ce11 division and monosporangia of the Compsopogonaceae. 93-94 22 Gene tree of combined 18s rRNA and rbcL gene data of 100-
Compsopogonales with morphological characters mapped onto 101
branches. CHAPTER 1: INTRODUC'MON AND LITERATURE REWW
1.1: Systematics of the Order Compsopogonales
The order Compsopogonales currentiy consists of three families: the
Compsopogonaceae, Boldiaceae and Erythropeltidaceae (Garbary et al. 198Oa). The first two families are typically fkshwater in their occurrence, though Compsopogon coeruleus has ken collected in brackish envhnments (Tomas et al. 1980).In contrast, the third family is composed of only marine representatives. The Compsopogonales was erected in
1939 by Skuja and included only the family Compsopogonaceae and the single genus
Compsopogon at that time (Skuja 1939, Kylin 1956, Melchoir 1954, Das 1963). In
Krishnamurthy's (1962) review of the genus Cumpsopogon, a new genus
Cornpsopogonopsis was described based on his conclusions that the formation of rhizoidal filaments throughout the thaltus was a taxonornically significant morphological atûibute.
When the genus Boldia was established, Hemdon (1964) placed it in its own family, the Boldiaceae. This new family was then classined in the order Bangiaies with some resenations by Hemdon. As part of a study on the developmentai morphology and cytology of Boldia, Nichols (1964b) reported that "further consideration conceming the fdyBoldiaceae as a member of the Compsopogonales rather than the Bangides should be made." This statement resulted fiom his observations that chromosome number and spore formation were similar to Compsopogon. Nonetheless, the genus and fhlyhave been retained in the Bangides (Chapman 1974, Bourrelly 1985, Stock et al. 1987).
Bourrelly (1985) stated that " le type d'organisation de Boldia peut fort bien entrer dans le cadre des Bangiales et trés près de la famille des Erythropeltidacées au voisinage des
Porphyiopsis. " He thus suggested a strong relationship between the families
Erythope1tidace.eand Boldiaceae.
The family Erythropeltidaceae is the large* in the Compsopogonales (Abbott and
Hollenberg 1976, Garbary et al. 198Oa). It contains seven genera: Erylhrocladia,
Membranella and Srnithora. The familial name was erected by Skuja in 1939 and the gmup was originally placed in the Bangiales.
Garbary et al. (1980a) proposed the establishment of a new order, the
Erythropeltidales, to contain three families: Erythropeltidaceae, Boldiaceae, and
Compsopogonaceae. The diagnosis for the order fiom Garbary et al. (1980a) is as follows:
"f lants consisting of mutose, foliose, saccate or filamentous thdi, the latter with or without a cortical layer; chloroplasts one or more per cell, axial or parietal; pyrenoids present or absent; differentiated basal systems occurring in al1 families although rhizoids not produced; dlrnembers show a similar pattern of monosporangium formation in which undifferentiated cells divide with a curved wall, one cell of the two becoming the monospormgium; sexdreproduction reported but doubthl; pseudochonchoceIis phases present in some genera."
The intention of Garbary et al. (1980a) was to folIow the suggestion made by
Krishnamurtby (1 962) of placing the families Compsopogonaceae and Erythropeltidaceae together in the order Erythropeltidales, based on their similar mauner of monospore formation. As the family Boldiaceae pst-dated Krishnamurthy's evaluation it was not included in his aaalysis. Garbary et al. (1980a) included the Boldiaceae in the new order because of similar monospore formaton among the families. In his analysis
Krishnarriurthy (1 962) had incorrectiy attributed the ordinal name Erythropeltidales to
Skuja (1939), thus failing to provide a proper diagnosis for the order (Garbary et al.
1980a). This diagnosis was subsequently provided by Garbary et al. (1980a) and was generally accepted (Garbary et al. 1%Ob, Entwisle and Kraft 1984, van den Hoek et al.
1995). However, the proposal of the order Erythropeltidales by Garbary et al. (198Oa) was flawed taxonomically. Principle III of the International Code of Botanical
Nomenclature states that "The nomenclature of a taxonomie group is based upon priority of publication." Wynne (1985) corrected the proposed order by placing the three families in the order Compsopogooales, since "this order (Compsopogonales) was described by
Skuja (1939) and has priority over the Erythropeltidales of Garbary et al. (1980a)." The description of the order of Garbary et ai. (1980a), as maintained under the name
Compsopogonales of Skuja (1939), is taxonomically upheld today (e-g. Bold and Wynne
1985, Bourrelly 1985, Garbary and Gabrielson 1990, Freshwater et al. 1994).
The morphological basis for the erection of the Compsopogonales was the single feature of monosporangium formation. The sporangia have been descnbed as developing fkom undifferentiated vegetative cells which undergo oblique divisions, by a curved ce11 wall, producing two cells the smaller of which develops into a monosporangium. From this single character Garbary et al. (1980a) concluded that the families Erythropeltidaceae and Boldiaceae (previously of the Bangides) and the Cornpsopogonaceae should be included in the single order Compsopogonales (=Erythopeltidales semu Garbary et al.
1980a). The placement of these families in this order has been widely accepted in the literature but the postulated relationships of these families have not been fiirther investigated.
In a cladistical analysis of red algal orders using morpholopical and life history amibutes, Gabrielson et al. (1985) suggested that Merdata are needed for ali taxa in the families Erythropeltidaceae and Compsopogonaceae in order to sort out relationships arnong these groups. However, they contend that the Compsopogonales appear well defined and represent a rnonophyletic group.
Molecula. data, especidy DNA sequences, have become a fkquently used tool for investigating relationships Ui the Rhodophyta at al1 taxonomic levels (e.g. Ragan et al.
1994, Freshwater et al. 1994, Vis et al. 1998). The order Compsopogonales has been included in two molecular phylogenetic analyses of the Rhodophyta. Freshwater et al.
(1994) analysed sequences of the chloroplast gene coding for the large sub-unit of ribulose-1 d-bisphosphate carboxylaseloxygenase enzyme (rbcL) of representative members of the division. Ragan et al. (1994) produced a phylogeny based on the nuclear small sub-unit rWA gene (SSU = 18s rRNA). Both of these analyses suggest that the order Compsopogonales, as represented by Compsopogon (rbcL), and Erythrotrichia and
Erythrocladia (1 8s rRNA), occupies a basal or position of ancient divergence within the
Rhodophyta.
The Compsopogonales have been descnbed as an ancient lineage in the
Rhodophyta because of its simplistic reproduction, cellular organization and molecular studies on gene sequence data (Gabrielson et al. 1985, Gabrielson and Garbary 1986,
Freshwater et al. 1994, Ragan et al. 1994). As the taxonomic organization of the Compsopogonales has been disputed, and no phylogenetic assessrnent has been carried out among a number of representatives in the order, this study was undertaken to assess whether this group is a valid taxonomie entity and better detennine its phylogenetic status. Parsimony and genetic distance analyses of sequences of the rbcL gene and the
18s rRNA gene fiom representative genera of the order assess whether the
Compsopogonales represents a monophyletic group.
1.2 Distribution and biology of the freshwater families Boldiaceae and
Compsopogonaceae i) Boldiaceae
Boldia erythrosiphon was first described in 1964 by Hemdon fiom Big Walker
Creek, Giles Co., Virginia, USA. Its range is presently fiom the hardwood forest regions of the southeastern United States through southem Ontario and into West central Quebec
(Figure 1 Howard and Parker 1980, Sheath and Hymes 1980, Sheath and Cole 1992).
Streams which contain B. erythrosiphon are permanently flowing, saturated with dissolved oxygen, have the universal presence of Na, Ni, Sn, Al, Mn, Be and Cu, are widely variable in CaC03 alkalinity, and have pH ranges of 7.0-8.5 (Howard and Parker
1980, Howard and Parker 198 1, Sheath et al. 1989). Boldia may occur attached to cobble in riffle or pool areas of fast moving streams and also has fiequently been reported
growing on the shells of pleurocend mails sometimes in large abundance (Hemdon 1964,
Howard and Parker 1980). The seasonal production of erect fionds has been observed to
commence between March and June. Fronds have been observed to degenerate by July
when water temperatures exceed 25 OC (Howard and Parker1980, Stock et al. 1987). Figure 1 : Distribution of Boldiaceae in North Amenca as represented by reported collection sites. I I v- 100 80 @ Reported collection sites of Boldia erythrosiphon Sites heavily sampled for stream macroalgae (Sheath and Cole 1992) TU tundra GR grassland CP coastal plain BF boreal forest DC desert-chappanil DE dccidous forest WC western TR tropical minforest HH hemlock- coniferous hardwood forest 7 forest Boldia erythrosiphon is peremial and consists of a heterotrichous thallus. Mature plants, with fionds producing monospores, range in length hm1 to 20 cm, occasiondly up to 75 cm and £kom 0.1 to 1.5 cm in diameter (Herndon 1964, Howard and Parker
1980). The species epithet erythrosiphon is translated as red tube and is reflective of the mature fom of the thallus (Flint 1970). Thalli are brownish red or olivaceous hollow, saccate fronds which grow singly or in clusters nom a multicellular base (Hemdon 1964).
The mature thallus is most often monostromatic in vegetative regions and is composed of four identifiable ce11 types: vegetative cells, monosporangia, granular cells, and intercalary filaments or rhizoidal cells. Vegetative cells contain a single nucleus and several, parietal, unbranched or irregularly lobed nbbon-like chloroplasts without pyrenoids (Howard 1977).
Asexual propagation takes place via monospores which have been described as king produced f7om cells which are denved fkom intercalary filaments (Nichols 1964b,
Howard and Parker 1980). Monospores are released from the monosporangia as naked protoplasts through a parietal pore and germinate in two different ways. In the fmt instance the monospore becomes surrounded by a gelatinous sheath and through several divisions forms a moud or disc of cells which initiates the erect portion of the plant
(Nichols 1964b). Each disc can give rise to a number of erect thalli. Ln the second mode of germination, the monospore divides to form a filament which gmws, potentially branching, dong the surface of the substrate. Cells of this filamentous stage may be released as monospores or may continue to develop foxming a mound of ceus that develops into a disc which produces upright thalli (Nichols 1964b, Howard and Parker 1980).
A second species Boldia angustata, was proposed by Deason and Nichok (1970) based on differences in thailus development and vegetative morphology. However, this taxonomie entity was disputed by Howard and Parker (1 980) through culturai studies of monospore germination and phenology. Al1 of the morphological variation attributed to
B. angustata was documented as present during the development of populations of B. erythrosiphon.
Cytologicai studies have demonstrated that metaphase chromosomes of Boldia are
arranged in a ring with one chromosome always outside of the ring (Nichols 1964b).
Counts made fiom both erect thalli and filamentous stages of development determined a
chromosome number of 8 * I.
The relatively Limited geographic distribution of Boldia was atîributed by Howard
(1977) to a lack of an efficient mechanisrn for dispening viable spores to suitable streams
which provide the substrats requirements for spore germination Boldia erythrosiphon
represents a good example of a geographically restricted algal species. It would,
therefore, be of great interest to ascertain the historical events that led to its curent
distribution. This study attempts to do this through analysis of DNA sequence data of the
internally transcribed spacer regions, ITSl and ITS2, and of the 5.8s rRNA gene.
ii) Compsopogonaceae
Compsopogonaceae occur in tropical and subtropical to temperate regions
worldwide (Krishnamurthy 1962, Sheath and Hambrook 1990,Entwisle and Price 1992).
The distribution of this family in North America is illustrateci in Figure 2 as summarized Figure 2: Distribution of Compsopogonaceae in North Amerka as represented by reported collection sites. I 1 O0 80 @D Reported Stream collection sites of Compsopogonaceae @ Ephemeral collections of Compsopogonaceae (Aquaria and garden ponds) 9 Sites heavily sarnpled for strearn rnacroalgae (Sheath and Cole 1992) TU tundra GR grassland CP costal plain BF boreal forest DC desert-chapparal DE decidous forest WC western coniferous TR tro ical rainforest HH hemlock-hardwood forest Pi forest hmthe following references: Vis et al. 1992, Manny et al. 1991, Starniach 1987, Rivers
1978, Masters 1940, Collins 19 16. Compsopogon and Compsopogunopsis grow in moderateiy fast streams and stiil waters such as ponds and ditches in hard, fresh to brackish water with high levels of alkalinity (Sheath and Hambrook 1990, Enhvisle and
Price 1992). Compsopogon has been described as a weedy or nuisance species in garden ponds, aquaria and irrigation ditches (Collins 1916, Smith 1950, Heynig 1971,Battiato et al. 1979, Entwisle and Price 1992, Komobis 1993). It has been suggested that the movement of Compsopogon into temperate regions of North America and Europe is due to their presence on tropical angiosperms which are imported for use in garden ponds and warm water aquaria (Collins 19 16, Zaneveld et al. 1976, Entwisle and Price 1992). It also has been postdated that shipping and boating have acted as sources of Compsopogon into brackish and fieshwater environments. Filaments or spores may have been transferred to new areas on the feet of migratory birds over both long and short distances (Chapman and
Cameron 1967, Entwisle and Price 1992). Warm water influxes fiom sources such as power plants (Entwisle and Pnce 1992) and Cotton mills (Weiss and Murray L 909) have been hypothesized to explain the presence and persistence of Compsopogon in temperate regions of Europe and New Zealand (Fritsch 1945, Enhnsle 1989), whereas populations in other temperate regions have proven to be ephemed, not surviving fiom year to year
(Collins 19 16, Masters 1940, Smith 1950).
The erect pomon of the Compsopogon thallus consists of branched filaments with diffuse growth ranging in colou.from gray-green to purple to bluish (Nichols 1964a).
Young filaments and apical regions are uniseriate and as they mature become corticated through divisions of the axial cells. As the filaments develop multiseriate organization the axial cells enlarge and sometimes collapse (Krishnamurthy 1962). Young vegetative cells contain a single plastid which separates into strips or bands of irregular size as it matures.
These cells also contain a large nucleus with chromosome numbers of 7 I 1 (Nichols
1964a).
Members of the Compsopogonaceae are attached to substrats by means of a prostrate portion of the plant, either a branched uniseriate filament or a basal disk. The lowermost portions of erect filaments will also produce tubula. outgrowths that grow down the axis fomùng rhizoids, which may supplement the basal structure or completely cover it (Knshnarnurthy 1962, Nichols l964a).
Sexual reproduction in this family has never been convincingly documented, so the only known method of reproduction is asexual propagation through the production of monospores (Nichols 1964a). Monosporangia are fomed by unequal divisions of cortical cells that result in the production of one small ce11 and one large ce11 separated by an oblique ce11 wall. The smaller of these ceils develops into a monosporangiurn. The cell contents are rounded up and released as a naked protoplast with a parietal sheet-like chloroplast (Nichols l964a). Monospores germinate in either a bi-polar or tri-polar manner. In bi-polar germination, a rhizoidal or filamentous base is produced, and in tri- polar germination, a basal disk or holdfast is produced (Nichols 1964a).
A large number of studies have documented the seasonal and geographic-based morphologid variability in members of this family. This has led to a number of taxonomie proposals which have both increased and decreased the numbers of species described for the group (Shyam and Sarma 1980, Vis et al. 1992, Necchi and Dip 1992,
Necchi and Pascoaloto 1995). This taxonomie controversy is described in detail in section
1.3. It is clear that the Cornpsopogonaceae are organisms which are quite capable of establishing themselves in new habitats provided that physicd conditions are suitable
(Entwisle and Price 1992). This leads one to question what may have been the major conditions that have resulted in the current distribution of the family in Noxth America.
This study attempts to define potential source populations or geogmphical trends of this
family through gene sequence data of the internally transcribed spacer regions, ITS l and
ITS2, and of the 5.8s gene of nuclear encoded ribosomal DNA.
13 Taxonomy of the Compsopogonaceae
The Compsopogonaceae contain two genera, Compsopogon and
Compsopogonopsis (Knshnamurthy 1962, Sheath and Hambrook 1990, Vis et al. 1992 ).
These genera are distinguished by the occurrence of rhizoidal filaments in the cortex,
which occur throughout the cortex of the plant in Compsopogonopsis but are restricted to
the base of the thallus in Compsopogon. Krishnamurthy (1962) concluded that there were
six valid species of Compsopogon and one species of Compsopogonopsis. Since 1962
five new species of Compsopogon (Pujals 1967, Reis 1977, Tracanna 1980, Chihara and
Nakamura 1980, Yadava and Kumano 1985) and one new species of Compsopogonopsis
(Chihara 1976) have been descnbed (Necchi and Dip 1992).
Vis et al. (1 992) used mdtivariate rnorphometncs of morphological characters to
determine the number of genera and kfhgeneric taxa in the Compsopogonaceae in
North America through cornparison of type and collected specimens. From this andysis two species of Compropogon, Compsopogon coeruleus and Compsopogunpproljkms
Yadara et Kumano, and one miesof Compsopogonopsis, Compsopogonopsis leptoclados (Mont.) Krishn. were detemiined to be well distinguished. Thus, the taxonomie revisions of Vis et al. resolved that "only three infîageneric taxa of
Compsopogonaceae should be recognised worldwide." Necchi and Dip (1 992) undertook a taxonornic revision of the Compsopogonaceae in Brazil. Because of the great variation of morphological characteristics of Compsopogon, which Necchi et al. (1990) had illustrated through cultural studies, herbarium specimens and collected plants, the only character which Necchi and Dip (1992) accepted as diagnostic was method of cortex formation. On the basis of this criterion they recognised two species "one with rhizoidal filaments in the cortex and another without." This character is currently used to separate the genera Compsopogon and Cornpsopogonopsis. Necchi and Dip (1 992) concluded that this single character was not sufficient for a separation of genera. Rather, it should be diagnostic at the species level distinguishing Compsopogon leptoclados and C. coemleus.
Following the taxonomy of the Compsopogonaceae as set forth by Vis et al.
(1992). Compsopogon coeruleus and Compsopogonopsis leptoclados have been included in the phylogenetic study of the order Compsopogonales. The sequence data of these two genera were compared through parsirnony, divergence and distance cnteria to determine if two distinct genera do exist. In addition, sequences of ITS 1, ITS2 and 5.8s gene of the rDNA were compared for two populations of Compsopogonopssis and 8 populations of
Compsopogon. 1.4 Ultrastructure of monospore production in the Compsopogondes
Ultrastructural characteristics are very important in red algai classification and in the detennination of phylogenetic relationships among various taxa (Pueschel 1990,
Sheath et al. 1996). As the method of monospore formation is diagnostic for the order
Compsopogonales, an ultrastructural study of monospore production was undertaken for the two fkshwater families of the Compsopogonales, Compsopogonaceae and
Boldiaceae.
Withi.the order Compsopogonales, ultrasbnictural studies have been camied out on Porphyropsis (McDonald 1972), Compsopogon (Nichols et al. 1966, Gantt et al. 1986,
Scott and Broadwater 1989), Erythrotrichia ( McBnde and Cole 1971 b, McDonald
1Wî), Srnithm (McBnde and Cole 1969,197 1a, McDonald 1972) and Erythrocladia
(Garpuiio et al. 1987). However, the majority of these studies focussed on vegetative ulmchne. The ultrastructure of monospore production has been investigated ody in
Srnithora (McBnde and Cole 1971 a, McDonald 1972) ,Porphyropsis (McDonald 1972) and Erythrotrichia (McBnde and Cole 1971 b).
Monosporangia have been typifïed as producing a single unuiucleate spore on either haploid or diploid plants which are usdymitotic (Gw1990). McBride and
Cole (1971b) described monospore production as "the differentiation of whole vegetative celis into spores" and noted that this is a common means of asexual reproduction in the
Bangiophycidae. A cfassification of types of spore formation observed in the
Bangiophycidae placed members of the Compsopogonales in a group called "Type 1:
Formation of monospores fiom differentiated sporangia" @rew 1956). In the
16 Compsopogonales, monosporangia are formed by the division of undifférentiated celis and formation of a cmed wall, with one of the redting cells developing as the monospomgium.
Srnithora naiadum possesses a monosporangeous region easily recognisable as a band of deeply pigrnented, round cells (McBride and Cole 197 1a). The cells in this area appear to undergo a greater rate of rnitotic division than the vegetative cells (McBride and
Cole 197 1a). In a developing monosporangium of S. naiadum there is a loss of vacuolation in the cell, more pores are visible in the nuclear envelope, many floridean starch granules are contained witbin the cell, which are larger than those found in the vegetative cells, and pnor to the release of the spore the ce11 wail is considerably thinner than that of the vegetative cells (McBride and Cole 1971a). The most active organelle in a developing monospore of S. naiadum is the dictyosome. It produces large numbers of two types of specialised vesicles, a "fibrous vacuole-like structure" and a "coalescent electron tramparent vesicle" (McBride and Cole 1971). The fibrous structure is released fkom the monospore prior to the spore's release, and is hypothesised to act as a lubncant for spore release. The îransparent structure is released after the spore has left the parent plant and is hypothesized to act as an adhesive for spore attachent to substrates
(McBride and Cole 1971 a, McDonald 1972). The release of monospores iÏom the S. naiadum thailus occurs by a break in the ce11 wall on either side of the thallus (McBride and Cole 1971a).
The monospores in Erythroirichia possess "ultrastructural details identical to those described for Srnithora" (McBride and Cole 1971b). The process of monospore
17 formation is initiated by an asyxnmetrical division of a vegetative cell. The larger daughter ce11 then develops into a "spherical, naked monospore which in some cases may gerrninate in the thallus but is usually released" (Dixon and Weiss 1967).
Porphyopsis coccinea produces monospores by oblique ceiI divisions
(McDonald 1972) these monospores contain several vesicles and numemus invaginations of the plasmalemma, with no observable distinct ce11 wail layer but surrounded by a fibdar-appearùig material (McDonaid 1972).
UItrastructural details of monospores in the freshwater families
Compsopogonaceae and Boldiaceae were investigated in the present study in order to provide Mershared characteristics of this important, potentially synapomorphic, character of the order Compsopogonales. Chapter 2: MATERIALS AM)METHODS
2.1: DNA extraction, Poiymerrue Chain Reaction and DNA sequencing.
Field-collected samples or cultured isolates were either kept alive in culture or hzenat -20 OC. Specirnens were rnaintained in Bold's Basal Media (freshwater taxa) or
Alga-Gro Seawater Medium (marine taxa) at room temperature with diurnal light cycles or at 10 OC with a 12 hou. light/dark cycle (Stein 1973). Collection and sequence information is listed in Table 1 (rbcL and 18s rRNA gene phylogeny samples) and Table
2 (i3oldiaceae and Compsopogonaceae Biogeography samples). Epiphytes were removed hmfield collected samples by examination in a dissecting microscope. These fiesh and hzen specimens were ground with a mortar and pestle in liquid nitrogen. DNA was exûacted fkom this ground material following the protocol of Saunders (1993) as descnbed beiow. Approximately 50 to 100 mg of the ground tissue were subjected to proteinase K digestion. The aqueous material obtained kmthis process was separated fiom the solid cellular debris through centrifugation and put through a phenoVchloroform extraction. The aqueous material was centrifüged producing a pellet, and was drawn off.
The pellet was washed with 70% ethanol and allowed to air dry. The pellet was then resuspended in 50 pL ultrapure water and the DNA extract was stored at -20°C.
DNA fkagments containhg the rbcL gene, the 18s rRNA gene and the ITSI -5.8s-
ITS2 region wereamplined fiom each DNA The primers used for amplincation are listed in Tabie 3. PCR reactions were carried out in a Perkin-Elmer Cetus 480 DNA Thermal
Cycler or a Perkin Elmer Gene Amp PCR System 2400. For the large mentrbcL and
18s rRNA gene sequences, an initial denahuaton penod at 95 OC for 2 minutes Table 1: Collection and gene sequence information for the samples included ir. phylogenetic analysis of the order Compsopogonales
Taxon CoiIection Site Date of Collection / Sequence Sequence GenBank Reference Data Accession Type Num ber Rhodophyta Cornpsopogonales Boldiaceae Boldia erythrosiphon Big South Fork, Tennessee, coll. R,Holton Hemdon USA (TNBSF) June 22,1993 Boldia erythrosiphon Champlain Rapids, Ottawa coll. J. Beaulieu River, Ontario, Canada August 18,1993 (ONOR) Hiawassee River, Tennessee, coll. R. Holton USA (TNHR) June 22,1993 Holton et al. (1998)
Compsopogonaceae Compsopogon coeruleus Route 17,0.4 km South coll. M.L. Vis (Balbis) Montagne of Brownville Line, Florida, June 5,1993 USA (FL27) Compsopogon coeruleus Whiskey Chitto, Louisiana, coll. R.G.Sheath and M.L. Vis rbcLIl8S AF087116, USA (LAWC) December 3,1993 MO87128 Compsopogon coeruleus Juncture Monier, Brelotte coll. R. Sheath River and Grand Riviere, St. March 1997 Lucia (SL2A) Compsopogonopsis Kamo'oloa Stream, Lïhu'e coll. R.G. Sheath leptoclados (Montagne) District, Kaua'i, Hawaii, USA August 15, 1992 Knshnamurthy (HI 14) Compsopogonopsis Blue Hole, New Mexico, USA coll. A. Sherwood rbcL/18S AF087120, leptoclados (NMBH) December 29, 1996 AF087123 Erythropeltidaceae Eryrhrocladia sp. UTEX1 1637, La Jolla, coll, L. A. Loeblich rbcL118S AFO87117, California, USA Spring 1966 L26189 Ragan et a1 (1 994) C4 Erythrotrichia carnea UTEX' 1690, Pebble Beach, coll. J. Ramus rbcLll8S AF087118, (Dillw.) J. Ag. California, USA May 1965 L26188 Ragan et al. (1994) Smithora naiadum Whiffen Spit, Sooke, Bntish coll. B. Oates Hollenberg Columbia, Canada (BCWS) May 5,1996 Smithora naiadum Moss Beach, California, USA coll. R. Sheath (CAMB) July 1996
Table 2: Collection information for samples included in North American biogeographic study of the Boldiaceae and Compsopogonaceae.
Taxon Collection Site Date of GenBank Collection Accession Number Boldiaceae Boldia Hiawassee River, cou. R Holton MO871 11 erythrosiphon Tennessee June 22,1993 USA (ml Big South Fork, cou. R. Holton Al70871 10 Tennessee, USA June 22,1993 (TNBSF) Greenbrier River, coll. R. Holton AF087112 West Virginia, USA July 5,1993 (WVGW Craig Creek, coll. R. Holton AFO87 11 1 Virginia, USA July 5, 1993 (VACC1 Passage Creek, coll. R. Holton MO87 113 Vkginia, USA December 7,1993 (VApc1 Champlain Rapids, coll. J. Beaulieu AF087108 Ottawa River, August 18,1993 Ontario, Canada (ONOR) Black River, Ontario, cou. J. BeauIieu MO87 IO9 Canada (ONBR) August 23,1993 Compsopogonaceae Compsopogon Tennessee River COU, P.G. Davison MO87098 coeruleus below Wheeler Dam, November 16, Lawrence Co., 1997 Alabama, USA (ALTRI Tom Amado, Costa coll. RG. Sheath Rica (CRI 3) and K. Müller March 1998 Route 17,0.4 KM coll. M.L.Vis South of Brownde June 5,1993 Line, Florida, USA (FL 27) Whiskey Chitto coll. R.G. Sheath River, Louisiana, and M.L. Vis USA (LAWC) December 3, 1993 RT 85, Sibrio coll. G. Lemon Lnternado Indigena, and T. Rintoul Queretaro, Mexico April24,1997 (Mex 3B) Juncture Monier, col1 R.G. Sheath Brelotte River and March 21, 1997 Grand Riviere, St. Lucia (SL2A) Tributary of coll. R.G.Sheath Soufiiere River , St. March 1997 Lucia (SL6A) Compsopogon Rt. 120, Jalpan, coll. G. Lemon cf. proiificur Queretaro, Mexico and T. Rintoul -7) May 1997 Compsopogonopsis Blue Hole, New coll. A. Sherwood leptoclados Mexico, USA December 29, (NMW 1996 Kamo 'oloa Stream, coll. R.G. Sheath Lïhu' e District, August 15 1992 Kaua'i, Hawaii, USA ml41 was followed by 35 cycles of denaturation at 93 OC for 1 minute, primer armealhg at 47
OC for 1 minute and extension for 4 minutes at 72OC. This process was then completed by a final extension time of 3 minutes at 72 OC. For the ITS 1-5.8s-ITS2 region, the PCR was executed with the foUowing amplification conditions: an initial denaturation period at 95 OC for 2 minutes was followed by 35 cycles of denaturation at 93 OC for 1 minute? primer annealing at 48 OC for I minute and extension for 2 minutes at 72 OC. The reaction was completed by a final extension time of 72 OC for 3 minutes.
PCR products were either purified directiy or gel pmified using the WizardM
PCR Preps DNA Purification Systems (Promega) following the manufacturer's protocol.
The products were then quaatified using a Pharmacia Genequant RNAfDNA
Spectraphotometer. PCR products were sequenced using the AB1 PrismTMCycle
Sequencing Ready Reaction Kit and the Applied Biosystems 377 Automated DNA
Sequencer. Sequencing reactions were performed using each of the PCR primers as well
as intemal primers so that complete forward and reverse sequences were defined (Table
3)-
2.2 DNA SEQUENCE ANALYSIS
SeqED was used to construct consensus sequences fkom the data obtained for both
strands of each PCR product. Primary alignment of the consensus sequences was done
using ClustaI X (Thompson, J. et al. 1997). Further dignment of 18s rRNA gene
sequences, based on the secondary structure models of Van de Peer et al. (1997), was
done using DCSE (Dedicated Comparative Sequence Editor, De Rijk, P. 1993).
Sequence statistics, number of basepair changes between sequences and the
25 Table 3: Primer sequences for phylogenetic and biogeographic analyses of the Compsopogonales.
Region PRIMER PRIMER SEQUENCE GENERA NAME 5' 3' rbcL Compl GAA TCT TCT ACA GCA ACT TGG AC Boldia, Compsopogon, Compsopogonopsis, Erythrocladia, Erythrotrichia, Smithora Comp2 GCA TCT C?T ATT ATT TGA GGA CC Boldia, Compsopogon, Compsopogonopsis, Erythrocladia, Erythrotrichia FI60 CCT CAA CCA GGA GTA GAT CC Bangia F65O ATT AAC TCT CAA CCA TTT ATG CG Bangia, Boldia, Compsopogon, Compsopogonopsis, Erythrocladia, Erythrotrichia, Smithora CTG TAA GTG GAT GCG TAT GGC A Bangia, Compsopogonopsis, Erythrocladia, Erythrotrichia, Sm ithora ACG GTT AAC ACC TTC CAT TGG AG Smithora GCA CGT TCG TAC ATA TCT TCC Boldia, Compsopogon, Compsopogonopsis, Erythrocladia, Erythrotrichia CGA GAA TAA GTT GAG TTA CCT GC Bangia ACA TTT GCT GTT GGA GTC TC Bangia, Smithora, GO1 CAC CTG GTT GAT CCT GCC AG Boldia, Compsopogon, Srnithora G02.1 CGA TTC CGG GAG AGG GAG CCT G Compsopogon, Compsopogonopsis, Smithora G04.1 GTC AGA GGT GAA ATT CTT GG Boldia, Compsopogon, Compsopogonopsis, Smithora GO7 TCC TTC TGC AGG TTC ACC TAC Compsopogon, Compsopogonopsis G10.1 GCG CGC CTG CTG CTT CCT TGG Boldia, Compsopogon, Compsopogonopsis, Smithora G12.1 CAA CGA GGA ATT CCT TGT AGG Compsopogonopsis G15.1 CTT GTT ACG ACTT CTC CTT CC Boldia, Srnithora ITSl & ITSS GGA AGT AAA AGT CGT AAC AAG G Boldia, Compsopogon, 5.8s Compsopogonopsis ITSIFSA GTT TCC GTA GGT GAA CCT GGG Compsopogon ITSlO GCT GCG TTC TTC ATC GAT Boldia, Compsopogon, Compsopogonopsis ITS3 ACA TCG ATG AAG AAC GTA GC Boldia, Compsopogon, Compsopogonopsis AB 28 ATC CAT ATG CRAAC TTC AGC GGG T Boldia, Cornpsopogon, Compsopogonopsis identification of variable or phylogenetically informative cbaracters were determined using MEGA ( Molecular Evolutionary Genetics Analysis Version 1.O 1; Kumar et al.
1993).
Phylogenetic trees based on cladistic critena were constnicted using PAUP 3.1.1
(Phylogenetic Analysis Using Parsimony, Swofford and Begle 1993). Search algorithms used were heuristic, branch and bound and exhaustive. As al1 algorithms produced trees of consistent topologies it was heuristic searches which underwent bootstrap resampling and branch decay analysis. Heuristic searches were cmied out on variable characters under the constraints of random (1000 replicates) stepwise sequence addition, steepest descent and tree bisection recomection (TBR)branch swapping. Analyses were completed on sequence data using equal weights for al1 characters. Bootstrap resampling of 1000 replicates was applied to the data set to obtain bootstrap support values for each branch. Decay values were calculated with the program AutoDecay ver 2.9.7 (Eriksson,
T. 1997) based on the protocol of Bremer (1 988).
Distance analyses were carried out in PHYLIP ver. 3 .Sc ( Phylogeny uiference
Package Felsenstein, J. 1993). From the complete data matrix pairwise Kimura "2
Parameter" distauce values were calculated for al1 samples with a transition/transversion
ratio of 2.0 and a single category of substitution rate. These distance analyses were
bootstrap resampled 1000 times to provide braoch support values and were used to
constmct neighbour joining 50% majority consensus trees. Maximum likelihood
analyses were Camed out using DNAml in the PHnIP program package. 23 MICROSCOPY
i) Transmission Electron Microscopy
Spimens of Compsopogon coenrleus, Compsopogonopsis leptoclados and
Boldia eryîhrosiphon were visually inspected and cleaned of epiphytes. Primary fixation
was with Karnovsky's fixative (Kamovsky, 1965) in Sorensen's phosphate beer (0.1 M,
Clark 1981) at 4 O C. Samples were rinsed with buffet and a secondary fixation was carried out over a one hour period in 2% 0s04in Sorensen's buffer. Samples were rinsed
again with Sorensen's bufTer and then dehydrated by a graded ethanol senes: 0.5 hours in
5%,10%,25%,35%,50%,70% and then one hour in 80%, and 95% ethanol. Specimens
were then lefi overnight in 100% ethanol. Two more changes of 100% ethanol followed
at one hour each. Specimem were infiltrated with Spurr's resin (Spurr 1968) according to
the following ratios and times: 2: 1 ethanol:Spurr's for one hour, 1 :1 ethano1:Spurr's for
one hour and 1:2 ethano1:Spurr's for one hou. Specimens were left overnight in 100%
Spurr's resin. They were then positioned in embedding blocks, covered with Spurr's resin
and polyrnerized for 12-1 8 hours at 60°C. Blocks were thin sectioned using a Reichert
Om U3 Ultramicrotome, mounted on carbon-coated copper grids and stained with uranyl
acetate and lead citrate. Prepared sections were viewed üsing a JEOL 100 CX
transmission electron microscope at 60 kV.
5) Scanning Electron Microscopy
Samples for analysis using the SEM were prepared following the protocol
described for the TEM with the exclusion of the osmium post fixation. The dehydration
series was followed by critical point drymg and sputter coating with AuPb doy.
29 Specimens were then viewed using a JEOL 35-C scanning electron microscope.
üi) Light Microscopy
Thin sections fiom the matenal prepared for the TEM were heat fixed to glas slides and stained with Toluidene Blue O in 0.5% sodium borate for 1 minute. These sections were viewed with an Olympus BH2 compound microscope equipped with an
Olympus PM 10 AK camera system. CHAPTER 3 :RESULTS
3.1 Phylogeny of the Compsopogonales i) Phylogeny of the CompsopogonaIes based on rbcL gene sequences.
Sequences of 17 taxa were obtained representing mernbers of the three families of the Compsopogonales (Boldiaceae, Compsopogonaceae and Erythropeltidaceae) as weii as taxa of the Bangiaceae and Cryptomonadaceae as listed in Table 1. Sequences of the rbcL gene were 1050 base pairs (bp) in length in the finai alignment. It was not possible to sequence the entire gene as it was necessary to design forward and reverse primers based on previously pubiished sequences (Freshwater et al. 1994) and these were positioned within the gene. There were no insertions and deletions in the rbcL gene which led to certain alignment of al1 sequences. For nine taxa, consensus sequences of the rbcL gene were constructed using cornparisons of complete forward and reverse sequences. Sequences of Boldia erythrosiphon from Ontario and Compsopogon coemleus fkom Louisiana were only sequenced in the fonvard direction in order to provide verification of the sequences of those species fiom other collections. Sequences have been deposited in Gen Bank with the accession numbers listed in Table 1.
Intraspecific variation was tested for Bangïa sp., Compsopogon coerulew,
Compsopogonopsis leptoclados and Boldia erythrosiphon (Table 4). Variation was found to be very low between collections of C. coeruleus fiom Florida and Louisiana (0.38%) and Cs. leptoclados from New Mexico and Hawaii (0%) but was an order of magnitude higher between B. erythrosiphon fiom Ontario and Tennessee (4.6%) and Bangia sp. from kshwater (Lake Michigan) and marine envkonments (Rhode Island)(7. 1%).
31 Interspecific sequence divergence ranged fiom O (C. coeruleur and Cs. ZeptocZudos) to
1 6.5% (Boldaand Erythrotrichia).
Cornparisons of all sequences revealed 440 informative characters distributed evenly throughout the data set. The sequences of C. coerzdeus fiom Louisiaoa and Cs. leptoclados hmNew Mexico were identical to the sequence of Cs. leptoclados fiom
Hawaii and were included in the parsimony analyses as a single entity. Parsimony analysis carried out on the variable characters of the rbcL data resulted in one most parsimonious tree of a minimal length of 1023 steps, a consistency index (CI) of OS68 and a retention index (RI) of 0.624 (Figure 3). Outgroup rooting was based on
Ciyptornonas Q strain as it has been demoastrated that this taxon provides an acceptable outgroup when investigating red algae ( Ragan et al. 1994).
The parsimony analysis showed support for a monophyletic compsopogonalean clade containing al1 three families with a bootstrap support of 53% and decay value of 8 steps. Within this clade the two fieshwater families, Boldiaceae and Compsopogonaceae, fuxm a distinct lheage with strong bootstrap and decay support (83%. 12 steps) separate
£iom the marine family, Erythropeltidaceae. Smithora naiadum is not positioned within the erythropeltidacean clade in the parsimony analysis but occupies a separate branch witfiin the Compsopogonales.
Maximum iikelihood analysis carried out on the data set produced a tree of similar topology to the parsimony analysis and is not show. In this cladogram Smithora naiadm is positioned on a branch which diverges early in the fkshwater compsopogonalean clade and these together are depicted as derived fiom a clade
32 containing Evhrohichia and Erythrockadia.
Distance analysis was carrïed out on al1 1050 characters of the 17 samples
(Table 4) and were the basis of the neighbour joining distance consensus tree illustrated in Figure 4. It produced a very similar topology to that produced in the parsimony analysis except that S. naiadum sits within the erythropeltidacean ciuster with weak bootstrap support (56%). The distances within the compsopogonaiean clade are much larger than those observed within the bangidean clade. This suggests that there has been a longer period of divergence among members of the Compsopogonales, specifically among families than in the Bangides. Sequence divergence is much higher in the families
Boldiaceae and Erythropeltidaceae than in the Compsopogonaceae. u) 18s rRNA gene phylogeny of the Compsopogonales
The finai data matrix of the 18s rRNA gene sequence data was constructed based on alignment of primary sequence structure and secondary structural elements of each sequence, fiom which regions of uncertainalignment were removed. The data rnatrix was
1645 bp in length and contained 469 informative characters. Sequences have been deposited in GenBank with the accession nurnbers Listed in Table 1. Sequence divergence percentages of included taxa were calculated and are iilustrated in Table 5 (upper nght matrix). Within the rhodophyte representatives sequence divergence ranged fiom O to
14%. Intraspecific variation of geographically distant collections was measwd in the
Bangiaceae (Bangia 7.2%), Erythropeltidaceae (S. naiadum 0.49%) and
Compsopogonaceae (derailed below). Within the Compsopogonaceae al1 collections of Figure 3: Parsimony analysis of rbcL gene sequence data of the Compsopogonales and
Bangides. Length = 1023 steps, CI = 0.568, RI = 0.624. Numbers above intemal branches indicate bootstrap resampling results (% of 1000 replicates). Branches lacking values had less than 50% bootstrap support. Numbers below intemal branches indicate decay values and represent the number of steps which would be added to the tree for those branches to coiiapse. Porphyra amplissima 1 100r Porphyra amplissima 2 Porphyra miniata
lPl lPl Bangin sp. LM Bangia sp. RI -
-Compsopogon coentleus op O Cxoerulezu LA t CD Cs. leptocladus HI,NM CD Boldia erythrosiphon TN ]i Boldia erythrosiphon ON CD %
Figure 4: Neighbour joining distance tree of Kimura 2 Parameter distances of r6cL gene sequence data of the Compsopogonales and Bangiales. Numbers above intemal branches indicate bootsûap resampling results (% of 1O00 replicates). Branches lacking values had
Iess than 50% bootstrap support. Porphyra yezoensis Porphyra carolinensis 100 Porphyra amplissima Porphyra amplissima ] L- Porphyra miniata b- b- Bangia sp. LM -Bangia sp. RI Cs. Ieptocladus NM Cs. leptocladus HI
B. erythrosiphon ON B. erythrosiphon TN 1 Erythrocladia sp. - Erythrotrichia carnea 1"3 L. I a Srnithora naiadum BCWS O CD 91 (D
C. coeruleuî (FL27, SLU, LAWC) and one collection of Cs. leptoclados (NMBH)had identical sequences, and were included as a single entity in the phylogenetic analyses. Cs. leptuclados (IfI14) in cornparison to this group demonstrated a divergence of 0.06%. Due to the exclusion of some regions of the sequence of the 18s rRNA gene, Eiythrocladia sp. and Erythrotrichia carnea were aiso identical, but were included as two separate entities.
All search afgorithms based on parsùnony criteria produced single most parsimonious trees of identical topology. Consideration of aiignment gaps as fifth characten, single events or missing data did not affect the topology of the tree. Coding gaps as missing characters or single events simply reduced the number of autapomorphic characters in the analysis, which decreased the length of the most parsimonious tree. As an example, analyses of the 18s rRNA gene data with gaps as a fifth character produced a tree 794 steps in length, gaps as single events produced a tree of 774 steps in length, and gaps coded as missing produced a tree of 732 steps. The consistency index (CI) was in al1 cases 0.820 and the topology of the tree remained unchanged. Based on these consistencies parsimony analyses were then carried out under heuristic search criteria with gaps coded as missing data.
Parsimony analysis of the 18s rRNA gene resulted in a single most parsimonious tree of 732 steps with a CI = 0.820 and a retention index ('= 0.875 (Figure 5). The rhodophyte representatives are separated into two distinct clades with strong bootstrap and decay value support which are representative of the red aigal orders Bangiales
(100%, 55 steps) and Compsopogonales (98%, 14 steps). The genera Bangia and
40 Porphyra are paraphyletic within this m. Within the Compsopogonales each family is strongly associated into its own clade: Erythropeltidaceae (100%*26 steps),
Compsopogonaceae (100%, 26 steps) and Boldiaceae. The two fkeshwater families
Boldiaceae and Cornpsopogonaceae share a more ment comon ancestor with each other than with the Erythropeltidaceae (97%, 1 1 steps). The Boldiaceae is only represented by a single collection so no support values for the family cm be obtained-
The Compsopogonaceae represent a strong clade with bootstrap support of 100% and decay value of 26 steps. Cmpsopogon and Compsopogonopsis are paraphyletic. Within the Erythropeltidaceae, Erythrotrchio and Erythrocladia are more closely related to each other (100%, 7 steps) than to Srnithora. The two populations of Srnithora fiom California and BC are positioned on a well supported branch (96%. 4 steps). Maximum likelihood analysis carried out on the data set produced a tree of identical topology to the parsimony andysis and is not show.
Kirnura 2 "parameter" distances for sequences of the 18s rRNA gene of 17 samples are listed in Table 5 (lower left matrix) and were used to construct the neighbour joining 50% majority consensus tree illustrated in Figure 6. This adysis produced a tree topology identical to that of the parsimony analysis. Bootstrap support values are robust with strong support for the Bangiales (100%) adCompsopogonales (94%). The clades representing the Compsopogonalean families are quite distinct with strong bootstrap support: Erythropeltidaceae (1 00%), Compsopogonaceae (100%) and Boldiaceae. The relationship between the two fkeshwater families is also strongly supported (100%)).It is important to note the length of the branch for Boldia erythrosphon This indicates that
41 Figure 5: Parsimony analysis of 18s rRNA gene sequence data of the Compsopogonales and Bangides. Length = 732, CI = 0.820, RI = 0.875. Nurnbers above internai branches indicate bootstrap resampling results (% of 1000 replicates). Branches lacking values had leu than 50% bootstrap support. Nmbers below interna1 branches indicate decay values and represent the number of steps which wodd be added to the tree for those branches to coilapse. 100 Porphyra yezoensis 88 9 Porphyra tenera 1 3 73 y Ban@ sp. RI 2 00. 1 O0 r 22 1O0 Porphyra miniata 55 1 Ban@ sp. LM
100 Erythrocladia sp. 7 100 1 Erythrotr ichia carnea 26 Srnithora naiudum CA 96 98 4 Smithora naiadum BC 14 1Boldiaceae IBoldia erythrosiphon 7 Cs. leptocladus HI 14 C. coeruleus FL, SL, LA OQ & Cs. leptocludus NM 3
CD Figure 6: Neighbou.joining distance tree of Kimuni 2 parameter distances from 1 8s rRNA gene sequence data of the Compsopogonales and Bangides. Numbers above
intemal branches indicate bootstrap resampling results (Y0 of 1000 replicates). Branches
lacking values had less than 50% bootstrap support. IPorphyra miniata Porphyra amplissimu Bangia sp. RI
-~an~ia sp. LMA Erythrocladia sp. 100
Erythrotrichia carnea O 1 O0 - Srnithora naiadum CA
-I 96 Srnithoru naiadum BC CP z Boldia erythrosiphon-1 BO (P % Cs. leptocladus HI C. coeruleus SL, FL, a0 LA & Cs. leptocladm NM g there is potentially a great amount of thesince the divergence of the Boldiaceae and the
Compsopogonaceae. The 18s rRNA gene sequence data show agaiu the high level of genetic divergence within the Compsopogonales particuiarly among families. The
Bangiales also exhibits a high level of divergence fiom the Compsopogonaies (Kimura 2 parameter distances = 0.123 - 0.1582).
üi) Combined 18s rRNA and rbcL gene phylogeny of the Compsopogonales
The combined data set of 18s rRNA and rbcL gene sequence data included 14 samples for which complete data were available. Sequence data were identical for the collections of Compsopogon coenileur fiom Louisiana and Compsopogonopsis leptocludos fiom New Mexico, so these were included as a single entity in the analyses.
Sequence data for Boldia erythrosiphun were not available for both genes from a single collection so the representative entity in the analysis is fkom two collections of Boldio fkom Tennessee (rbcL gene = Big South Fork River, 18s rRNA gene = Hiawassee River
Holton et al. 1998.).
Parsimony analysis was carried out on the 888 informative characters fkom the
2695 base pairs of the combined 18s and rbcL gene sequence data of the
Compsopogonales. This resulted in a single most parsimonious tree 1664 steps in length with a CI of 0.690 and RI of 0.734 (Figure 7). This dysisaiso lends strong support to taxonomie divisions as they currently stand at the ordinal and familial level. Of the three parsimony analyses this combined data set gives the strongest bootstrap and decay value support for the bangidean (100%, 95 steps) and compsopogondean clades (99%, 18 steF's)- Figure 7: Pmimony analysis of the cornbined 18s rE2NA and rbcL gene sequence data of the Compsopogonales and Bangides. Length = 1664 steps, CI = 0.690, RI =
0.734.Nurnbers above intemal branches indîcate bootstrap resampling redts (Y0 of 1 O00 replicates). Branches lacking values had less than 50% bootstrap support. Numbers below intemal branches indicate decay values and represent the number of steps which would be added to the tree for those branches to coiiapse. - r Porphyra yezoensis Porphyra miniata
Porphyra amplissima
-Bangia sp. LM -
10 100 Erythrocladia sp. 19 Smithora naiadum BC -99 Boldia erythrosiphon Compsopogon coenrleus LA & Cs. leptocladus NM Cs. leptocludus HI14 Figure 8: Neighbour joining distance tree of cornbined 18s rRNA and rbcL gene sequence data of the Compsopogonales and Bangiales. Numbers above intemal branches indicate bootstrap resampling results (% of 1 O00 replicates). Branches lacking values had less than 50% bootstrap support. 100 IPorphyra miniata 1
~angiasp. RI
I m -94
1O0 .' Compsopogon meruleus FL 3 100 C. coenrleus LA & 1 Cd O Compsopogonopsis m O leptocladw NM 3 E Cs. leptocludus HI 1p,CD0- Cgptornonas Q>
0.05 Within the Compsopogonales al1 three fdesare supported by 100% bootstrap values and high decay values (19-79 steps). Within the Erythropeltidaceae a close relationship between Erythmîrichia and Etytrhrocladia is again evident in cornparison to
Srnithora. The Boldiaceae and Compsopogonaceae are allied into a clade with bootstrap support of 100% and 23 step decay vaiue. The species within Compsopogonaceae are paraphyletic.
Kimwa 2 parameter distances derived fiom the combined sequence data were used to construct a neighbour joining distance tree (Figure 8). Tree topology is identical to that produced by the parsimony anaiysis and bootstrap support values are comparably robust to those in the parsimony tree.
The trees derived from the rbcL, 18s rRNA gene sequences and combined sequence data are congruent, with stronger resolution observed in the 18s rRNA gene and combined trees with regards to the taxonornic relationships within the Compsopogonales.
These data support the current higher level taxonomic organization of these groups. The
Compsopogonales is strongly supported by the 18s rRNA gene sequences and combined data as being a monophyletic group.
The fieshwater families Compsopogonaceae and Boldiaceae are grouped more close1y together than with the marine Erythropeltidaceae. Within the Erythropeltidaceae the level of genetic divergence is higher (rbcL gene = 9.2-12.7%, 18s rRNA gene = 0-
1.1%) than that observed in the other two families. In contrast, within the
Compsopogonaceae there is a dehite homogeneity of the gene sequence data across the group. This can also be compared to the rbcL sequence divergence (4.0%) observed in
51 the monotypic Boldiaceae which is much higher than in the Compsopogonaceae (rbcL gene = O-0.038%, 18s rRNA gene = O-0.06%).
33 Biogeography of the Boldiaceae
For six collections of Boldia erythrosiphon, the single species of the Boldiaceae, sequence data were obtained for the 5.8s rRNA gene and the two intedly transaibed spacer regions which Bank it, ITS 1 and ITS2. These collections represent much of the entire geographic range of Boldia as illustrated in Figure 9. Collection information for the sequenced sarnples is given in Table 2.
The 5.8s rRNA gene ranged in length fiom 107 - 160 base pairs, the final alignment of al1 sequences was 167 characters. Sequence divergence percentages ranged from O - 33.5% (Table 6 upper nght matrix) with identicai gene sequence shared between the collections fiom Hiawassee River, TN (TNHR) and Craig Creek, VA (VACC).
Kimura 2 parameter distances ranged fiom O - 1.374 (Table 6 lower left matrix).
Sequence data of ITS 1 ninged in length fiom 385- 462 base pairs and the final data matrix alignment was 468 characters. Sequence divergence percentages ranged fiom
O - 19% (Table 7 upper nght matrix) with identical ITS 1 sequences shared between coilecîions TMIR and VACC. Kimura 2 parameter distances ranged fiom O - 1.O465
(Table 7 lower left matrix).
Sequence data of ITS2 ranged in length fiom 279 - 588 base pairs with the ha1 alignment of 620 characters. Sequence divergence ranged fiom O - 20.9% (Table 8 upper right matrix) with identical sequences once again shared by the collections TNHR and
VACC. Kirnuni 2 parameter distances ranged fiom O - 1.6195.
52 Figure 9: Distribution of sequenced collections of Boldia erythrosiphon fiom North
America.
Table 6: 5.8s rRNA gene sequence divergence percentages and Kimura 2 parameter distances of the Boldiaceae.
------TNHR VACC WGR VAPC ONBR ONOR TNBSF TNWR - O 7.8 7.2 13.2 19.2 29.3 VACC O - 7.8 7.2 13.2 19.2 29.3
WGR 0.1583 0.1583 d 4.2 10.2 19.8 29.3 VAPC 0.1444 O. 1444 0.0804 - 5.9 20.4 29.9 ONBR 0.292 0.292 0.2122 0.1 178 - 19.8 28.7 ONOR 0.4974 0.4974 0.5304 0.5328 0.5 12 1 - 33.5 TNBSF 0.942 0.942 O -9292 0.9678 0.8931 1.374 -
Table 7: ITS 1 sequence divergence percentages and Kimura 2 parameter distances of the
Boldiaceae.
VGPC ONBR TNHR VACC WVGR ONOR TNBSF VAPC - 0.2 1 15.4 15.4 16.9 17.5 19 ONBR 0.006 - 15.2 15.2 16.7 17.3 18.8 TNHR 0.7016 0.6857 O 13-7 16.4 17.5 VACC 0.7016 0.6857 O - f 3.7 16.4 17.5 WVGR 0.824 0.8052 0.5833 0.5833 - 16.7 19 ONOR 0.885 0,8642 0.7869 0.7869 0.8062 - 16.7 TNBSF 1.0443 1,0187 0.8841 0.8841 1.0465 0.8063 - Table 8: ITSZ sequence divergence percentages and Kimura 2 parameter distances of the
Boldiaceae.
VACC VAPC WGR ONBR ONOR TNBSF VACC - O 0.32 0.16 14.2 20 20.9 TNHR O - 0.32 O. 16 14.2 20 20.9 VAPC 0.009 0.009 - 0.16 14 20.1 20.8 WGR 0.004 0.004 0.004 - 14 20 20.8 ONBR 0.5254 0.5254 0.5167 0.5169 - 21.4 23.1 ONOR 0.9276 0.9276 0.9435 0.9282 1.0738 - 24.7 TNE3SF 1.O229 1.0229 1.007 1.O059 1.2835 1.6 195 - The colIections TMIR and VACC were included as a single entity in following analyses due to identical sequence data. The collection of Boldia fiom Big South Fork
River, Tennessee (TNBSF)was unaiignable with the other collections due to very high levels of sequence divergence and the Kimura 2 parameter distance values greater than one. This collection was excluded from al1 Meranalyses.
Al1 of the Boldia erythrosiphon collections had similar nucleotide composition of the combined ITSl and ITS2 sequences. The mean composition was 27.2%A, 26.5% T,
22.6% C and 23.7% G.
From the 957 bp of the combined data matrk of ITS1, ITS2 and 5.8s rRNA gene sequence data there were 462 variable sites. Heuristic searches carried out on these variable characters produced a single most parsimonious tree 735 steps in length (Figure
10). It was not possible to obtain an outgroup for this data due to alignment problems so al1 trees were produced unrooted. Parsirnony analysis supports two distinct clades, one consisting of the southemmost populations (TNHR,VACC & WVGR) and the other of the northernrnost populations (ONBR &VAPC). The collection made at the Ottawa
River, Ontario, Canada does not ally itself closely with any of the other collections and its relationship with either clade is unresolved.
Maximum likelihood analysis of this data set produced a tree of comparable topology to that produced in parsimony analyses and is not shuwn.
Distance analyses produced trees of similar topologies exhibiting the same relationships between southem and northem populations of the species (Figure 11 A &
B). The distance trees show the high level of divergence between the Ontario collections
57 Figure 10: Parsimony analysis of combined sequence &ta of 5.8s rRNA gene, ITS l and
ITS2 of the Boldiaceae of North Amerka. Length=735 steps. TNHR & VACC Figure 1 1: Neighbour joining distance analysis of 5.8s rRNA gene, ITS 1 and ITS2
combined sequence data of the Boldiaceae of North Amerka ONBR
HRTN & VACC WVGR in cornparison to the lower levels of divergence observed in the US collections. Even though the northernmost populations group together, these high levels of sequence divergence may represent multiple pst-glacial introductions of Boldia evthrosiphon into the northem areas of its range.
33 Biogeography of the Compsopogonaceae
Sequence data was obtained for the 5.8s rRNA gene and the two intemaily transcribed spacer regions, ITS 1 and ITSZ, fiom seven collections of Compsopogon coenrleus (ALTR, CR1 3,FL27,LAWC, MEX3B, SL2A, SL6A), two collections of
Compsopogonopsis leptoclados (HI 14, WH)and one probable collection of
Compsopogon cf. prolificus (MEX7). The collection of Cs. leptocladus from Hawaii
(HI 14) was not a part of the biogeographic analysis of the family but was included in the taxonomie analysis so this collection is it is presented in some of the results of this section. The site MEX7 is the collection site from which Vis et al. (1992) collected and identifiai C. proZz~cz(s.However, the plants collected for this study were immahire, without copious bmching, and the species character of branches curling around the main axis was not observed. The geographic distribution of these collections is illustrated in
Figure 12 and collection information is outlined in Table 2.
The length of the 5.8s rRNA gene ranged fiom 122 to 176 base pairs with the nnal alignment containing 192 chamcters. Within the 5.8s rRNA gene sequence divergence ranged fiom O - 16.1 % (Table 9 upper right rnatrix) with identical sequences shed among Compsopogon coeruleus (CR13,LAWC, SLoA), Compsopogon cf prolificus (MEX7) and Compsopogonopsis leptoclados (HI14). The most divergent
62 Figure 12: Distribution of sequenced collections of Compsopogonaceae in North
America. sequences were between two populations of C. coeruleuî (AL= SLZA). Kimura 2 parameter distance values ranged hmO - 1.268.
The ITS l region ranged in length fiom 3 19 - 345 base pairs and upon final alignment the data matrix contained 372 characters. Sequence divergence percentages of
ITS 1 ranged fiom 0.27 to 37.4 % (Table 10 upper nght matrix) and Kimura 2 Parameter distance values ranged fiom O - 1.12455 (Table 10 lower lefi matrk) for the ITS 1 data.
The ITS2 region ranged in length fiom 322 - 567 base pairs, the final length of the aligned &ta matrk was 843 base pairs. Sequence divergence percentages ranged fiom O to 17.5 % (Table 1 1 upper right matrix) and Kimura 2 parameter distances ranged nom O to 1.192 (Table 11 lower lefi matrix).
Nucleotide composition of combined ITS 1 and ITS2 data showed a moderate G bias in al1 col1ections averaging 34.2% and ranging nom 27.6 (SL2A) - 36.2% (CR13). In contrast, A's accounted for 20.9%' T's for 21.9% and C's for 23%.
Combined sequence data of the intemally transcribed spacer regions ITS 1 and
ITS2 and the 5.8s rRNA gene gave a final alignment of 1215 base pairs of which 674 characters were observed to be variable. Heuristic searches carried out on this data set with gaps coded as missing characters provided a single most parsimonious tree of 1505 steps (Figure 13). Since it was impossible to align another taxon for outgroup purposes to the sequence data al1 trees were unrooted. This analysis produced two well resolved clades, one was separated into two smaller groups: NMBH associated with LAWC and
MEX7 with SL6A and the second contained SL2A and ALTR . The collection MEX38 Table 9: 5.8s rRNA percent gene sequence divergence and Kimura 2 parameter distances of the Compsopogonaceae.
CR13 MEX3B HI14 LAWC MEX7 SL6A NMBH FL27 SL2A ALTR CR13 MEX3B HI 14 LAWC m O\ MEX7 SL6A NMBH FL27 SL2A ALTR Table 10: ITS 1 percent sequence divergence and Kimura 2 parameter distances of the Compsopogonaceae.
FL27 HI14 ALTR SL6A SL2A CR13 MEX3B LAWC NMBH MEX7
ALTR 0,205
WCLA 0.02 NMBH 0.85 Table 1 1 : ITS2 percent seauence divergence and Kimura 2 ~ararneterdistances of the Com~so~ononaceae. HI14 LAWC SL6A CR13 NMBH MEX7 MEX3B FL27 SL2A ALTR HI14 LAWC SL6A CR13 NMBH MEX7 MEX3B FL27 SL2A ALTR Figure 13: Parsimony analysis of combined sequence data of 5.8s rRNA gene, ITS1 and
ITS2 of the Compsopogonaceae in North America. Length = 1505 steps. NMBH
ALTR and the FL27KR13 entity remained uoassociated with other collections.
Maximum likelihood dysisproduced a tree of identical topology to that produced in the parsimony analysis and is not shown.
Neighbour joining analyses of Kimura 2 parameter distances provided a tree of similar topology to those produced in the parsimony analysis (Figure 14). In this case the collections MEX3B, FL27KR13, LAWC and NMBH are unassociated into distinct clades. The clades formed by ALTR and SL2A and SL6A and MEX7 are again supported.
The distance analysis demonstrates the high level of divergence of ALTR and
SL2A fiom the other collections. These samples were removed fiom the analysis to assess whether long branch attraction was affecthg the topology of the trees when they were included (Figure 15). With SL2A and ALTR removed the data matrix contained
1058 charactee of which 300 were variable. The length of the most parsimonious tree was reduced by more than 50% to 41 1 steps (Figure 15A). Sorne topological changes observed in the parsimony tree were changes in anagernents of the two clades which contain only C. coenrlezis; ikst the support of a close relationship between the two collections fiom Mexico (MEX3B and MEX7) and another with SL6A in a clade with the
CR13EL27 entity. In this data set the Cs. ZeptocZidos collection (NMBH) remains in a group with C. coeruleus (LAWC). Relationships among collections in the distance adysis of the reduced data set were consistent with those descnbed in the parsimony analysis except that SL6a did not associate with the FL27\CR13 entity (Figure 15B). Figure 14: Neighbour joining distance anaiysis of 5.8s rRNA gene, ITS 1 and ITS2
combined sequence data of Compsopogonaceae in North Arnerïca NMBH LAWC Figure 15: Parsimony (A) and neighbour joining distance (B) analysis of combined 5.8s rRNA gene, ITS 1 and ITS2 sequence data of the Compsopogonaceae in North Amenca with the collections ALTR and SLZA removed. A:Length = 41 1 steps.
3.4 Taxonomy of the Compsopogonaceae
Sequence data were obtained for three coding regions (&CL, 18s and 5.8s rRNA gene) and two non-coding spacer regions (ITS 1 and ITSZ) for a geographic and taxonomie diversity of representatives of the Compsopogonaceae. For the rbcL gene, sequences were obtained hmthree collections of Compsopogon meruleus (FL27,
LAWC, UTEX Freshwater et al. 1994) and two collections of Compsopogonopsis leptoclados (HI 14, NMBH). Sequence divergence in these collections ranged fiom O to
0.76% within the 1050 bp compared (Table 12). In parsimony and distance analyses of the rbcL gene sequence data, the genera of the Compsopogonaceae were represented as paraphyletic with branches containhg both Compsopogon and Compsopogonopsis as a single entity due to identical sequence data (See Figures 3 & 4).
18s rRNA gene sequence data were obtained fiom three collections of
Compsopogon coenrleus (FL27, LAWC, SL2A) and two collections of
Compsopogonopsis leptocludos (HI14, NMBH). From the conservative data matrix of
1645 base pairs sequence divergence ranged fiom O - 0.12% (Table 13). Identical sequences were obtained from four collections (FL27, LAWC, SL2A and NMBH) which represented both C. coeruleuî and Cs. leptoclados. Parsirnony and distance analyses of the 18s rRNA gene sequence data represented these taxa as paraphyletic with one another. The genetic distance between these taxa is negligible (See Figiiles 5 L 6).
Combination of the 18s rRNA and rbcL gene sequence data produced trees depicting the same trends as the trees based on separate data analysis. C. coenrlew
LAWC and Cs. leptocladus NMBH yielded identical sequence data. This entity is located
76 in a clade with Cs. leptoclados HI14 which has bootstrap support of 77% and a low decay value of 1 step. In the distance analysis of the combined data these two entities again fa11 into the same topology but the distance between that clade and the collection of
C. coerulew FL27 is negligible (Figure 8).
Sequenced data for the 5.8s rRNA gene and the two non-coding spacer regions
ITSl and ITS2 were obtained from seven collections of Compsopogon coeruleus (ALTR,
CR13, FL27, LAWC, MEX3B, SL2A, SL6A), two collections of Compsopogonopsis leptoclados (HI 14, NMBH) and one collection of Compsopogon cf. pro1ifim.s (MEX7).
Sequence lengths, divergence values and Kimura 2 parameter distances are discussed in detail in section 3.3 and the values for 5.8s rRNA gene, ITSl and ITS2 sequence data are listed in Tables 9, 10 and 1 1.
In analyses of the combined sequence data fiom the 5.8s rRNA gene and the internally transcribed spacer regions ITSl and ITS2, there are no clear trends which follow the curent taxonomie scheme of the Compsopogonaceae (Figures 16). The species Compsopogon cf. prolz~cusis well supported as a member of a clade containing
Compsopogon coeruleus and the genera Compsopogon and Compsopogonopsis are shown to be paraphyletic in both parsimony (Figure 16Aa nd distance analysis (Figure
16B).
As described above, the genus Compsopogonopsis was observed to be paraphyletic in al1 of the gene sequence data analyses of rbcL, 18s rRNA gene, 5.8s rRNA gene, ITSl and ITS2. There was also extremely low levels of sequence divergence between Compsopogonopsis and Compsopogon in al1 gene sequence data, which fa11
77 Figure 16: Parsimony (A) and neighbour joining distance (B) analysis of 5.8s rRNA gene, ITSl and ITS2 combined sequence data of Compsopogonaceae. A: Length=l332 steps. within previously reported intmspecific ranges for rhodophyte taxa. Molecuiar sequence data provided an increased number of characters for phylogenetic analyses and has shown through cladistic analyses that the feature of cortical formation does not represent a synapomorphic character because Compsopogonopsis is not a well-separated, monophyletic taxon. Table 12: rbcL gene percent sequence divergence and Kimura 2 parameter distances of the Compsopogonaceae
FL27 UTEX LAWC NMBH Hl4 FL27 - 8 4 8 4 UTEX 0.0077 - 4 8 4 LAWC 0.0038 0.0038 - 4 O NMBH 0.0077 0.0077 0.0038 - 4 HI14 0.0038 0.0038 O 0.0038 -
Table 13: 18s rRNA gene percent sequence divergence and Kimm 2 panimeter distances of the Compsopogonaceae
FL27 SL2A LAWC NMBH HI14 FL27 - O O O 2 SL2A O - O O 2 LAWC O O - O 2 NMBH O O O - 2 HI14 0.0012 0.00 12 0.00 12 0.0012 - 3.5 Ultrastructure of Monosporangia in the Boldiaceae and Compsopogonaceae i) Boldiaceae
Mature saccate thalli of Boldio erythrosiphon (Figure 17a) are composed of four types of cells; vegetative cells, granula cells, rhizoidal cells or intercalary filaments and monosporangia (Figure 17 bd). Widalcells are localized mainly at the base of the plant (Figure 17b) but are also observed growing between cells on the surface of the thallus (Figure 17c-e). Rhizoidal cells are longer (30 - 65 pm) and narrower (2 - 5 pm) than vegetative cells (4 - 40 pm) and extend downwards as rhizoids at the base of the plant (Figure 17 b). Rhizoidal cells, which also have been called intercaiary filaments
(Howard and Parker 1980) may also be blunt ended cells growing among vegetative cells throughout the thallus (Figure 17 ce). The majority of the volume of rhizoidal cells were filied by densely staining chloroplasts and a large peripheral nucleus with a large darkly staining nucleolus (Figure 18%b). Centrally positioned vacuoles were not observed in these cells.
Vegetative cells make up the bulk of the plant, and in surface view are square- shaped to rounded and contain one to a few large parietal chloroplasts (Figure 17 c,d,e,f).
Transverse sections of these cells show a peripheral protoplast and a large central vacuole
(Figure 18a4 19 ad). Granula cells occur scattered throughout the thallus, and were not observed above the light microscope level (Figure 17c).
Monosporangia of Boldia erythrosiphon are seatîered on the surface of mature portions of the thallus (Figure 17 c,e,t) laying in the intercellular spaces surrounding groups of vegetative ceus. In the light microscope they can be recognised by their small size and dark, dense appeaniace in cornpiinson to the vegetative cells surroundhg them
(Figure 17 c,e). In some monosporangia a large nucleus was observed (Figure 17e). When
viewed with the scanning electron microscope, the regular linear arrangement of
vegetative cells in young portions (Figure 17d) of the thdus becornes disrupted by the production of monospores which surround vegetative cells, sometimes covering them and protruding fiom the surface of the thallus (Figure 17 f). Empty monosporangia can be
identified by the pores fiom which monospores were released (Figure 18f) which are clearly visible in scanning electron micrographs. Empty monosporangia were cornpletely deteriorated in some portions of the material and obscured the surface of the thallus (Not
shown). Using the TEM, the most noticeable characteristic of monosporangia is the
absence of the large irregular vacuole which is positioned in the centre of vegetative cells
(Figure 19 c,d). Ultrastructural characteristics which contribute to the dense appearance
of the monospore also include the closely appressed plastids, and high number of
floridean starch granules which occupy a large proportion of the volume of the ceil
(Figure 19c,d).
Another prominent feature observed in monosporangia is large nuclei, sometimes two per ce11, with clearly defined nucleoii; often two per nucleus (Figure 19 c). This
indicates that the monospores are actively dividing or preparing for division upon release.
ii) Compsopogonaceae
Monospores of Compsopogon germinated to form multicellular basal disks
(Figure 20a), which enlarged and produced uniseriate filaments (Figure 20 b).
Once a filament became multisetiate the cortical cells began to form monosporangia Figure 17: Matme thalii of Boldia erythrosiphon; a) Light micrograph (LM) of plant bearing four saccate tballi (st), b) Scanning electron micrograph (SEM) of base of mature plant with rhizoidal cells (rc), c) LM of surface of thallus bearing rhizoidal cells (rc),
grandar cells (gc), monosporaugia (m) and vegetative cells (vc). d) SEM of surface of
immature portion of thallus bearing rhizoidal cells (rc) and vegetative cells (vc), e) LM of
daceof mahue portion of thallus bearing rhiwidal cells (rc), vegetative cells (vc) and
monosporangia (m) with prominent nucleus (arrow). f) SEM of surface of mature portion of thallus containing vegetative cells (vc), monosporangia (m) and empty monosporangia
(em) with pores @).
Figure 18: Transverse sections of thalli of Boldia erythrosiphon a) Light micrograph
(LM) of cross section of thallus stained with Toluidene Blue O showing vegetative cells
(vc) and rhizoidal cell (rc), b) Transmission electron micrograph of rhizoidal ce11 at extemal edge of thallus (et) and neighbouring vegetative celi (vc) showing chloroplast
(c), nucleus (n) with prominent nucleolus (nc) and ce11 wall (cw). c & d )LM of cross sections of thallus stained with Toluidene Blue O showing vegetative cells (vc), rnonosporangia (m) and empty monospomgia (em).
Cell division was observed in cortical cells (Figure 21a). Both mother and daughter celis showed prominent nuclei with darkly staining nucleoli. The mother ce11 was observed to retain the majority of the centrai vacuole, and each ceil contained chloroplasts and sorne floridean starch granules (Figure 21a). nie cell wall was fonned centripetaily fiom the outer and inner edge of the thallus (Figure 21a).
The monosporangia viewed with the light microscope are visible as cells which are smaller and darker than vegetative cells (Figure 20c). Monosporangia share an oblique ce11 wall with the neighbouring vegetztive ceii and appear rounded (Figure 20c,
Vis 1992). In surface view with the SEM,monosporangia are observed as smaller cells which protrude fiom the surface of the thallus (Figure 20d). Transverse sections of a mature thallus show the monosporangia as small densely staining cells which are oriented to the exterior of the thallus (Figure 21b). Vegetative cells contain a large central vacuole, which is lacking in monosporangia (Figure 2 1c,d). The buk of the monosporangiurn is filled by chloroplasts and floridean starch granules (Figure 21 c, d). The floridean starch granules obsemed in monosporangia were larger than those observed in vegetative cells and much more abundant (Figure 21c, d). Nuclei in rnonospomgia are large and positioned in the centre of the ceIl (Figure 2 lc), unless more than one is present (Figure
2 1d). Nuclei of monosporangia contain a large densely staining nucleolus (Figure 2 1c, d). Figure 19: Transverse sections of thalli and monosporangia of Boldia erythrosi'on. a & b) Light micrographs showing numerous monosporangia (m), empty monosporangia (em) and a monostromatic layer of vegetative cells (vc). c & d) Transmission electron micrograph of monosporaagia and neighbouring vegetative ceiis (vc) showing chloroplasts (c), £loridean starch granules (fs), large nuclei (n) with prominent nucleoli
(nc), vacuoles (v) and ce11 walls (cw).
Figure 20: Gemilings, young plants and monosporangia of the Compsopogonaceae. a)
Light micrograph (LM) of the base of germlings of Cumpsopogon coemleus genninated fiom monospores. b) LM of cushion-like base (b) of a young plant of C. coeruleus producing uniseriate filaments (uf). c) LM of cortical cells (cc), monosporangia (m) and an empty monosporangia (em) of C.coeruleus. d) Scanning electron micrograph of
surface of mature multisenate filament of Compsopogonopsis leptoclados showing cortical cells (cc) and monosporangia (m).
Figure 2 1: Cell division and monosporangia of the Compsopogonaceae. a) Transmission electron micrograph (TEM) of division of cortical ceils in Compsopogon coeruZeus showing formation of ce11 wall (cw), large central vacuole (v), large nuclei (n) with prominent nucleoli (nc), chloroplasts (c) and floridean starch granules (fs). b) L ight micrograph of cross section of thallus of C. coerilleus stained with toluidene blue O showing vegetative cells (vc) and monosporangia (ms).c & d) TEMs of monosporangia and neighbouring vegetative cells (vc) of C. coenrleus with chloroplasts (c), vacuoles (v), large nuclei (n) with prominent nucleoli (nc), ce11 walls (cw) and floridean starch granules (m.
CHAPTER 4: DISCUSSION
4.1 Phylogeny of the Compsopogoaales
This study has shown the order Compsopogonales to be a monophyletic group which is strongly supported by phylogenetic analysis of chloroplast (rbcL) and nuclear
(1 8s rRNA) gene data. Relationships within the Compsopogonales also have been Mer resolved by these analyses with a closer relationship between the freshwater families
Compsopogonaceae and Boldiaceae than between either of these taxa and the
Erythropeltidaceae. The existence of a well separated fieshwater clade is also supported by the ultrastructural characteristics outlined below. This analysis shows that these families share a more recent common ancestor than either does with the Erythropeltidaceae.
The use of these molecular data has provided characters which help to overcome the paucity of usefbi morphological characters avaiiable for cladistic analysis withi.this group
(Gabrielson et al. 1985). The Compsopogonales is not a taxonomically diverse group,
containing only 9 genera and 35 species (Nichols and Lissant 1967, Heerebout 1968,
Abbott and Hollenberg 1976, Howard and Parker 1980 Kornma~1984, Kornmann and
Sahling 1985, Kommann 1987, Konimann 1989, Schneider and Searles 1991, Vis et al.
1992). However, the group does represent a wide range of morphological form. The range
of these morphological characters is illustrated in Table 14 and has been mapped onto the
phylogenetic tree derived fiom the combined sequence data of the rbcL and 18s rRNA
genes. Chanicters are represented by a single line if the specific character occurs once, or is
Merent in form for each taxon represented by a temiinal branch. Characters which have
undergone multiple changes in the lineage are represented by two crossed lines. The
95 compsopogonalean lineage is supported by the following characters: mowsporangia
formation (Garbary et al. 1980a), spore germination patterns (Gabnelson et al. 1985), form of the immature thallus as a uniseriate filament (Nichols 1964%Nichols l964b, Nichols and Lissant 1967, Heerebout 1968, Murray et al. 1972, Nichols and Sommerfield 1971) chloroplast type (Nichols 1964%Nichols l964b, Abbott and Hollenberg 1976), presence of
B phycoerythrin (Gabrielson and Garbary 1986), and the absence of a dictyosome/mitochon~alassociation (Scott and Broadwater 1989). The ~eshwater
lineage, Compsopogonaceae and Boldiaceae, is supported by the following attributes: freshwater habitat, chloroplast ultrastructure (Scott and Broadwater f989), and characteristics of mitosis (Scott and Broadwater 1990). The erythropeltidaceaen lineage
shares the characters marine habitat, bi-polar germination of monospores (Gabnelson et al.
1985), and the presence of pyrenoids in the chloroplasts (Abbott and Hollenberg 1976).
Each terminal taxon is distinguished on the basis of mature thallus form. Unique characten
include the presence of pit connections in the Compsopogonaceae (Scott et ai. 1988) and
sexual reproduction in Smithnaiudum (Hawkes 1988). Charactes which undergo a
change in the lineage include the transition fiom parietal discoid to axial stellate
chloroplasts in Smithora naiadum and Erythrorrichio carmu, and the encnisting disk and
bulb form of the immature thailus in Smithora naiadum.
The Compsopogonales is well supported as being monophyletic based on the taxa
that are included in the present analysis. However, there is evidence that the
Rhodochaetaies, with its single species Rhodochaete pmla Thuret, may be closely
aligned with the Compsopogonales (Scott and Broadwater 1989, Garbary and Gabrielson
96 Table 14: Surnmary of important characters reported for the Compsopogonales. Nurnbers and bracketed short forms correlate with character node labels of Figure 16. 1 Erythropeltidaceae Compsopogonaceae Boldiaceae 1 Habitat Marine (MH) BrackishlFreshwater (FH) Freshwater (FH) 2 Monosporangia Oblique division of vegetative Oblique division of Oblique division of vegetative formation (MF) ce11 vegetative ce11 ce11 (Garbary et al. 1980a) 3 Spore germination Unipolar/bipolar (BG) Unipolar (UG) Unipolar (UG) (Gabrielson et al. 1985) 4 Immature thallus Uniseriate filament Uniseriate filament (UI) Uniseriate filament (UI) (Nichols and (UI)(Erythrocladia, Lissant 1967, Erythrotrichia, Porphyropsis ) Heerebout 1968, Encrusting disk and bulb (DI) Murray et al. (Srnithora Porphyrostromium) 1972,Nichols and Sommerfield 1972) 5 Mature thallus Crusts (CR), uncorticated Corticated filament (CF) Monostromatic tube or sac (ST) morphology filament (UF), foliose blade (FT)
1 1 Pit connections Absent Primitive Absent (PC) (Scott et al. 1988) 12 Sexual Present in Srnithoru and Absent Absent reproduction (SR) Porphyrostromium (as (Hawkes 1988 Erythrobichopel fis) Kommann 1984, 1987) 13 Sue of nuclear Normal Reduced Reduced Associated Organelle in rnitosis (NAO) (W u\O (Scott and Broadwater 1990) 14 Chromosome* 7 (Erythropeltis subinfegra ) 7+ 1 ,7 or 8 numbers (Nichols l964a, Nichols 1964b, Shyarn and Sarma 1980, Cole 1990) *Not mapped ont0 gene tree. Figure 22: Gene tree of combhed 18s rRNA and r6cL gene data of Compsopogondes with morphological characters mapped onto branches. Character node labels correspond to numbers and short forms outlined in Table 14. Single lines represent the first occurrence of that character on the tree, crossed lines represent derived characters which evolve more than once or reversais.
1990). The pit connection morphology and peripheral endopfasmic reticulum-dictyosome relationship of Rhodochaete (Scott and Broadwater 1989) are close to those described in the Compsopogonaceae. In addition its Me history (sexual and asexual reproduction) is very similar to Porpyhyrostromium of the Erythropeltidaceae (Pueschel and Magne 1987,
Garbary and Gabnelson 1990). Rhodochaete has also been described as lacking an association between mitochondria and the dictyosome (Scott and Broadwater 1989), comparable to that described in the Compsopogonaies. Rhodochaete possesses apical growth and a heteromorphic life history not fomd in the Compsopogonales. Once molecular data for Rhodochaete have been obtained it would be of great interest to include it in a phylogenetic analysis with the data presented in this study in order to assess evolutionary relationships between these groups.
4.2 Biogeography of the Boldiaceae and Compsopogonaceae in North America
The Compsopogonaceae and Boldiaceae are evolutionarily ancient lineages representing very early events in the diversification of the red algae (Freshwater et al.
1994, Ragan et al. 1994). This has been Mersupported by the high Ievel of sequence divergence observed in the internally transcnbed spacer regions (ITS 1 and ITS2) obtained in this study. Previously reported sequence divergence values of the internally transcnbed spacer regions, ITSl and ITS2, for representatives of the Rhodophyta are as follows:
Batruchospermum gelutinosum (L.)de Candolle, intraspecific = < 1% (Vis and Sheath
1997); Palmariaceae, intraspecific = 0.4 - 5% (Lindstrom et al. 1996); Phycodrys,
interspecific = 0.35 - 4.2 1 % (van Oppen et al. 1995); and Gracilmia and Gracilmiopsis,
intmspecific = 0.22 - 4.1 % (Goff et al. 1994). The sequence divergences of Boldiaceae
102 (O - 22%) and Compsopogonaceae (0.24%) exhibit much greater ranges than those reporteci in these flondeophyte species. In both families, there were collections which
were even undigoable to the sequence data of the rest of the collections (Boldiaceae; Big
South Fork, Tennessee, Compsopogonaceae; Cornpsopogon oishii, Okamura, Japan).
Such high levels of sequence divergence, so that alignment becornes impossible, have
been previously observed among collections of another bangiophyte red alga, Bangia sp.
(Müller et al. 1998).
Within the Boldiaceae, even with the large sequence divergence values,
phylogenetic analyses of the combined ITS 1, ITSZ and 5.8s rRNA gene data provided
some insight into probable historical events which led to the current distribution of the
family in its limited range of Eastern North Amenca The Canadian populations couid not
have existed until after the last glaciation, the Laurentide ice Sheet, receded from
southern Ontario between 13,000 and 1O, 000 years ago (Pielou 199 1). In con-
populations as far north as Virginia, USA may not have been affected by the maximum
glaciation of 18,000 years ago and may have existed in that region for hundreds of
thousands of years (Pielou 1991). The parsimony and distance analyses have supported a
relationship between the most northem collections of Boldia eryihrosiphon and a similar
grouping of southern populations. The populations of Boldia erythrosiphon sampled in
Ontario are quite divergent which may indicate that more than one south to north
introduction has occurred from populations for which sequence data was not obtained. It
is possible that these populations were dispersed through vector-assisted transport, such
as the migration of waterfowl (Kristiansen 1996). There are a number of species of ducks and geese which follow a migratory route that parallels the distribution of Boldia
(Bellrose 1980). There are also a number of migratory routes (Bellrose 1980) which cross through the localities of Boldia populations and continue westward, yet the distribution of the Boldiaceae remains restricted to eastern North America.
Analysis of combined sequence data of ITS 1, ITS2 and the 5.8s rRNA gene did not identiQ any clear geographic trends in the Compsopogonaceae of North America-
Members of the Compsopogonaceae are easily dispersed by the large nurnbers of asexual spores, as well as the persistent cushion-like bases which are capable of producing adult plants (Weiss et al. 1909, Collins 1916, Chapman and Cameron 1967, Zaneveld et al.
1976, Entwisle and Price 1992). In recent geologic time (a0,000 years) no large scale environmental changes, such as glaciation, have afYected the Compsopogonaceae in its tropical to sub-tropical habitat. Therefore this family has not been restricted by available habitat in the same way as the Boldiaceae.
Compsopogon is a nuisance plant in aquaria around the world ( Heynig 1971,
Rivers 1978, Starmach 1987, Battiato et al. 1979, Komobis 1993 ). Nydrodjc~on
(Chlorophyta) was introduced into New Zealand from East Asia through a hatchery supplying fish and aquatic plants to aquarists (fistiamen 1996). It is quite likely that events such as this also have occurred with the Compsopogonaceae in North America tbrough the aquarium trade between South and Central Amenca and temperate to sub- tropical North Amena. This method of dispersal may especially account for the identical sequences shared between collections of Compsopogon fiom Costa Rica and Florida
The likelihood of shipping andlor boating and migratory waterfbwl as modes of distribution is also supported by the genetic distribution of this group. This is especiaily evident in the high sequence divergence values observed between the two collections made on St Lucia, a small island spanning less than 50 krn, in the Lesser Antilles. This level of cüfTerence must be representative of multiple introductions to the islaud. These modes of introduction would also provide an explanation for the presence of
Compsopogonopsis in Blue Hole, New Mexico, which is an isolated artesian sprïng-fed pool popular with divers and perchance ducks (Portales/Roosevelt County Chamber of
Commerce pers. comm.).
The lack of resolution arnong collections of the Compsopogonaceae in North
America indicates that this family needs to be examined on a worldwide basis.
Populations fiom South Arnenca and Asia need to be compared to those in North
Amerka, in order to resolve the biogeographic history of this group.
43Taxonomy of the Compsopogonaceae
This study has demonstrated that Cmpsopogon and Cornpsopogonopssis are paraphyletic taxa based on analysis of sequence data for chloroplast (rbcL) and nuclear
(18s rRNA, 5.8s rRNA) genes as weU as intemally transcribed spacer regions (ITS1 and
ITS2). More importantly, there are very low levels of sequence divergence observed in the rbcL and 18s rRNA genes which fall well withui previously reported interspecific values of the Rhodophyta Reported sequence divergences for the rbcL gene of rhodophyte taxa include the following: Batrachospermales, intwpecific = 0.1-0.4% (Vis et al. 1998); Gelidium, intraspecific = 0- 1.8% and interspecific 1.2 - 4.8% (Freshwater and Rueness 1994); Bangia, among collections = O - 16% (Müller et al. 1998). Sequence divergence values for the 18s rRNA gene of rhodophyte taxa are: Porphya, interspecific
= 0.8 - 13% (Stiller and Waaland 1993) and 0.3 - 15% (Oliveira et al. 1995), Bungia, among collections = O - 10.6% (Miiller et al. 1998).
Krishoamurthy (1 962) distinguished species of the Compsopogonaceae based on dimensions of the thaili, cells and monosporangia, as well as type of basal system and number of erect branches on the basal system. These characteristics were all shown to be extremely variable within and between populations, on a seasonal basis and in culturai studies ( Nichols l964a, Shyam and Sarma 1980, Necchi et al. 1990, Necchi and Dip
1992, Vis et al. 1992). Thus they are dubious for distinguishing hfk&milial taxa. The method of cortex formation which has been proposed as a character delimiting genera by some authors (Knshnamurthy 1962, Vis et al. 1992) and species by other authos (Necchi and Dip 1992) is not supported by these analyses of molecular data This study has demonstnited that Compsopogon and C~rnp~~pugonopsisdo not represent unique evolutionary lineages, but rather are positioned within the same branches with relatively little sequence divergence. It is therefore proposed that Cumpsopogonopsis leptocludos be placed in synonymy with Compsopogon coeruleus as outlined in the taxonomie proposais and revised descriptions noted below.
Taxonomie proposais and revised descriptions (Revised from Vis et al. 1992).
Compsopogonaceae Schmitz, in Engler und Prantl, Die nati&lichen Pfranzenfamilien
1:3 18-320, 1897.
Compsopogon Montagne in Bory et Durieaux, Flore d Algérie 1 :1 52, 1816.
TYPE SPECIES: Compsopogon coeruleus Balbis) Montagne. SPECIES:
(1) Compsopogon coeruleuî (Balbis in C. Agardh) Montagne Flore d Algérie 1: 152,
1846.
BASIONYM: Conferva coerulea Balbis in C. Agardh Systema Algamm: 122,1824.
HETEROTYPIC SYNONYMS: Compsopogon aenrginosus (J. Agardh) Kützing, Species
Algatum:433, 1849. Compsopogon chalybeus Kütnng, Species Algarum:43 3,1857.
Compsopogon corinaldii (Meneghini) Kiitzing, Tabulae Phycologicae 7: 35, 18 5 7.
Compsopogon corticrassus Cbihara et Nakamura, Journal of Jopanese Botany 55: 136,
1980. Compsopogon hoohri Montagne, Flore d 'Algéria 1: 1 57,1846. Compsopogon
lusitanieus Reis. Boletim da Sociedude Broteriana Série 2 5 1: 9 1, 1 977. Compsopogon
oishii Okamura, Icones of Japanese Algue 3 :128, 19 15. Compsopogonopsis leptoclados
(Montagne) Krishoarnurîhy, Journal of the Linnean Society (Botaqv) 58: 2 19, 1962,
Compsopogon leptocludos Montagne, Annales des Sciences naturelles, Série 3, 14 298,
1850, Compsopogon leptoclados Montagne ,Annules des Sciences Naturelles, Série 3,
14:298, 1850, Compsopogonopsisjaponica Chihara, Journal of Japanese Botany 5 1: 289,
1976..
DESCRIPTION: Rhizoids throughout thallus or restricted to thallus base, main axis
diameter 121 - 3000 p,cortical cell length 7.5 - 55 pm, monosporangium length 7.5 -
27.5 Pm, unisenate branch diameter 10 - 55 p,cortical layers 1-4.
A potential representative of the species Compsopogon prolz3m.s was included in
analyses of the combined gene sequence data of 5.8s rRNA, and ITSl and ITS2. This
collection was made at the site MEX7 which was the site nom which Vis et al. (1992) identifieci this species. However, the collection used in this study consisted of immature unbrancheci plants, so the species character of branches curling around the main axis was not observed. in the cladograms of the gene sequence data, C. cf. prof@çs~sgrouped with a collection of C. coeruleus from St. Lucia and exhibited low sequence divergence in the
5.8s rRNA (0.0.52%) and ITS2 (O - 2.8%) regions, but very high in the ITS 1 (34 - 37.4%) region in cornparison to the other collections of C. coerufeuî and Cs. leptoclados.
However, without a positive identification of this species it is not possible to mer specuiate on its validity.
4.4 Ultrastructure of monosporangia in the Boldiaceae and Compsopogonaceae
Molecular sequence data of this study confirm the production of monosporangïa through oblique ce11 divisions as a defining, synapomorphic chmter for the
CompsopogonaIes.
Cellular morphology of the vegetative cells of uniseriate portions of thalli of the
Compsopogonaceae has been investigated in detail (Nichols et al. 1966, Gantt et al. 1986,
Scott and Broadwater 1989). in this study cornparisons between cortical cells of multiseriate portions of the thalli and monosporangia were made. Features which were observed in this study and previously reprted for vegetative cells of the
Compsopogonaceae include: the presence of a single, large, central vacuole, peripheral cytoplasm, nuclei located at the edge of the cek and a complex parietal chloroplast
(Nichols et al. 1966, Gantt et al. 1986, Scott and Broadwater 1989).
Vegetative cells of the Boldiaceae have been previously investigated only at the level of the light microscope. They were described as con-g a centrally located nucleus with a prominent nucleolus (Nichols 1964b), chloroplasts single and central in young cells, mahiring to be several, parietai, ribbn-shaped chloroplasts, and a large central vacuole (Hemdon, 1964, Howard and Parker 1980), as observed in this study.
Howard and Parker (1988) observed the disruption of cellular arrangement of vegetative cells by the production of monosporangia scattered throughout the thallus, similar to this study .
Monosporangia of the Boldiaceae and Compsopogonaceae were described through observations with the Iight microscope as deeply pigmented, thick walled, smd cells (Krisburthy 1962, Hemdon 1964, Nichols 1964% l964b, Howard and Parker
1980). Based on transmission electron microscopie observations in this shidy, the pigmentation appears to be due to the absence of a large central vacuole and the aggregation of large amounts of floridean starch granules in this study. These ultrastnictural characteristics have also been described for monosporangia of the
Erythropeltidaceae (McBride and Cole 197 1 a, McDonald 1972, McBnde and Cole
1971 b). In this study developing monosporangia of the Compsopogonaceae and
Boldiaceae were observed to be mitotically active in preparation for germination upon release fiom the thallus of the parent plant, as has ken described as characteristic of monosporangia (Guiry 1990). This shidy has identified the Compsopogonales (Rhodophyta) as a natural evolutionacy grouping based upon analyses of chloroplast (rbcL) and nuclear (1 8s rRNA) gene sequence data The group is characterized by the method of monosporangium formation, the presence of the pigment B phycoerythnn and the lack of an association between dictyosomes and the mitochondria Hence, the order should be retained as a valid taxonomie entity.
High levels of sequence divergence in the ITS 1 and ITS2 spacer regions suggest that the kshwater families Boldiaceae and Compsopogo~l~fceaeare an ancient ance- assemblage. Biogeographic analysis of the Boldiaceae based on these data dernomte that within this family populations were probably introduced into more northem regions of North Amenca fiom southem populations, likely by vector-assisted transport. No large-scale directional, histo ric, biogeographic trends were uncovered by analysis of ITS 1 and ITS2 and the 5.8s rRNA gene of representatives of the Compsopogonaceae.
However, multiple introductions seem to be occunhg in some areas and there is a definite link among some populations based on high sequence similarity. The curent distribution of the Compsopogonaceae may not be assessed adequately without investigating populations on a world-wide scale. It has been demonstrated that likely vectors for the dispersal of this family in North Amenca include the migration of water fowl and anthropogenic means such shipping, boating and the fieshwater aquarium trade.
Systematic analysis of chloroplast (rbeL) and nuclear (18s and 5.8s rRNA) genes and intemally transcribed spacer regions (ITSl and ITS2) showed that the family Compsopogonaceae is monotypic in North America, containing the single species
Compsopogon coeruleus. Analyses of these molecular data for C. coenrleus and Cs.
leptocladus have shown that the presence of rhizoids throughout the thallus, as opposed
to king confinecl to the base, does not represent an evolutionary novelty worthy of the
separation of Compsopogon and Compsopogonopsis.
Observations of developing monosporangia showed that the fieshwater families
Boldiaceae and Compsopogonaceae share ultrastructurai characteristics comparable to
those reported for representatives of the Erythropedtidaceae, and that the mode of
monosporangia formation is a taxonomically useful characteristic. LITERATURE CITED
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