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SYSTEMATKS AND BIOGEOGRAPHY OF THE RED ALGAL ORDER HLLDENBRANDIALES (RHODOPHYTA)
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
ALISON RUTH SHERWOOD
In partial fulfihent of requirements
for the degree of
Doctor of Philosophy
December, 2000
O Aiison R. Sherwood, 2000 National Library Bibliothèque nationale du Canada
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SYSTEMATICS AND BIOGEOGRAPHY OF THE RED ALGAL ORDER
HILDENBRANDMES (RHODOPHYTA)
Alison Ruth Shercvood Advisor: University of Guelph, 2000 Professor R-G, Sheath
The genetic variability of the genus Hildenbrandia throughout its distributional range and the taxonomie implications of this variation were examined using a combination of DNA sequence analyses (rbcL and 18s rRNA genes, and ITS regions), othzr rno lecular marker techniques (ISSR analyses) and morpliometric analyses. The phylogenetic relationship of Hildenbrandia to the second genus of the Hildenbrandiales,
Apophlaea, was also exarnined using these techniques. Analyses of North American
Hildenbrandia demonstrated high sequence divergence values within and arnong marine and fieshwater forms (225.8% [rbcL]; ~9.7%[18S rRNA]); although these levels were comparable in marine European rnembers of the genus (524.9% [rbcL];55.8% [18S
&NA]), they were much lower for fieshwater European members (r 1.9% [rbcL]; ~3.6%
[18S &NA]). The rbcL gene was transitionally saturated within the North Amencan and global level comparisons, but the phylogenetic signal for the 18s rRNA gene was strong even among global collections. Biogeographic comparisons of marine and fkeshwater
Hildenbrandia collections fiom a small geographical region (southem Sweden) revealed different relationships among the samples, but comparisons to other samples fiom Europe indiczted a close phylogenetic relatiooship among al1 European freshwater
Hildenbrandia. Some trends revealed by morphometric analysis correlated with those yielded by phylogenetic analyses, especiaily at a global level. Except for the common marine species, rubra, samples with similar morphologies fonned clades in phylogenetic trees. Based on these analyses, as well as morphornetric examinations of the qpe specimens and global representatives, taxonomie revision is recommended to reduce the number of currently accepted marine Hildenbuandia taxa fiom 12 to seven, with no synonymies recommended for the fieshwater taxa Inclusion of Apophlaea in the analyses indicated that although Apophlaea is a monophy Ietic genus, Hildenbrundia is not. A rnicroscopic examination of the freshwater species, H. angolemis, and its unique form of asemal reproduction demonstrated that gemrnae develop within the algal thallus and are released from the thallus surface. Histochemical cornparisons between gemma and thallus cells revealed large amounts of starch in the gemmae, but not in thallus cells, Completing this degree would not have been possible without help and assistance fkom a nuniber of people and organizations. Most importantly, 1 would like to thank my advisor, Dr. Robert Sheath, for his guidance and support since my undergraduate days. 1 am gratefül for the multitude of research, travel and teaching opportunities that he has provided over the years. The other members of my advisory cornmittee (Dr. Joe Gerrath,
Dr. Denis Lynn and Dr. Larry Peterson) have also helped in the design of my projects and provided advice in their areas of expertise.
1 have shared the lab with a number of people at various points during my degree, and I would like to thank them al1 for making the thne fun and interestùig: Lesley
Campbell, Dana Couture, Laura Kline, Kirsten Müller, Tara Rintoul, Troina Shea and
Stacey Thompson. Thanks aiso to my fiends in both the botany and zoology departments.
1have received a large amount of help with my thesis projects fiom other people.
Many phycologists around the world have provided me with material for this thesis, and to them 1 owe a large "thank you" for giving me the opportunity to examine specimens with such a wide geographical scope. Troina Shea did rnuch of the PCR work for
Chapter 4 and Stacey Thompson helped with the light microscopy sectioning for Chapter
5. Dr. Paul Silva (UC Berkeley Herbarium) has kindly sorted through my taxonomie and nomenclatural quenes and Dr. Ten Crease (Dept. of Zoology, Guelph) hm spent a good deal of time helping me with my phylogenetic analyses. Angela Holiiss (Guelph
Molecular Supercentre) has sequenced innumerab2e templates for me over the past four years-
Many thanks to Ron Deckert, who has been a wonderfil field assistant, source of conimon sense, general botany guru, listener and friend. Also to my parents, John and
Glenda Shenvood, who have always been supportive of my academic endeavors.
1would like to acknowledge financial assistance fkom NSERC in the form of two post-graduate schola. awards. Research costs have been covered through NSERC grant
OGP 0 183 503 to Dr. Robert Sheath, the University of Guelph and a PSA grant-in-aid of research. *.. 111
TABLE OF CONTENTS
Page Acknowledgments i-ii Table of Contents z-vii List of Tables viii-ix List of Figures x-xiv Abbreviations xv-XVi Chapter 1: General Introduction 1-21 1.1. Morphology and distribution of the Hildenbrandides 1.2, Taxonomy of the Hildenbrandides 1-3. Phylogenetic positioning of the Hildenbrandiales 1.4. Biogeographic study of the Hiidenbrandiales 1S. The origins of fieshwater Hildenbrandia 1.6. Asexual propagation by gernmae in Hildenbrandia 1-7. Research objectives 1-8. Literature cited Chapter 2: Biogeography and systematics of Hildenbrandia in North America: inferences from morphometrics and sequence analysis of the rbcL and 18s rRNA genes 2.1. Introduction 2.2, Materials and Methods 2.2.1. Collection and identification of materials and DNA extraction 2.2.2. Morphometric analysis 2.2.3. Amplification and sequencing of the rbcL and 18s rRNA genes 2.2.4. rbcL and 185 rRNA gene analyses TABLE OF CONTENTS cont.
2.3. Results 2.3.1. Morphometric analysis 34 2.3 -2. Analysis of transitional saturation of the rbcL and 18s rRNA 39 genes 2.3 -3. rbcL gene distance, parsimony and quartet puzzIing analyses 47 2.3 -4. 18s rRNA gene distance and parsimony analyses 52 2.4. Discussion 57 2-5. Literature Cited 62 Chapter 3: Biogeography and systematics of Hildenbrandia in Europe: 67-101 inferences from morphometrics and sequence analysis of the rbcL and 18s rRNA genes 3.1. Introduction 3 -2. Materials and Methods 3.2.1. Sample collection and DNA extraction 3 -2.2. Morphometric analysis 3-2.3. Amplification and sequencing of the rbcL and 18s rRNA genes 3.2.4. rbcL and 18s rRNA gene sequence analyses 3 -3. Results 3 -3.1. Morphometric analyses 3 -3-2. Analysis of transitional saturation of the rbcL and 18S rRNA genes 3 -3-3. Parsirnony, distance and quartet puzzling analyses of the rbcL gene 3 -3-4. Parsimony, distance and quartet puzzling analyses of the 18s rRNA gene TABLE OF CONTENTS cont.
3 -4. Discussion 3 -5. Literature Cited Chapter 4: The relationship between marine and freshwater Hiïdenbrandia along an historical salinity gradient 4.1. Introduction 4.2. Materials and Methods 4.2.1. Collection of materials and morphologicd examination 4.2.2. DNA extraction, rbcL and 18s rRNA gene amplification, sequencing and gene sequence analyses 4.2.3. ITS 1 and ITS2 amplification and analyses 4.2.4. ISSR amplification and analyses 4.2.5. Determination of time since isolation f7om the Baltic Sea 4.3. Results 4.3.1. Morphological examination of collections 113 4.3 -2. Analyses of the ITS 1 and ITS2 regions 114 4.3 -3. ISSR-PCR results 221 4.3.4. rbcL and 18s rRNA analyses of representative collections 125 4-4. Discussion 131 4.5. Literature Cited 135 Chapter 5: Microscopic analysis and seasonality of gemma production 140-164 in Hilden brandia angolensis 5.1. Introduction 5.2. Materials and Methods 5.2.1. Collection of HiZdenbrundia angolensis 5.2.2. Microscopical and histochemical techniques 5.2.3. Seasonality of gemma production TABLE OF CONTlENTS cont.
5-3. Results 5-3.1. Gemma morphology and anatomy 144 5.3 -2. Histochemistry and X-ray microanalysis 145 5-3 -3. Gernma development 149 5 -3-4. Seasonality of gemma production 154 5.4. Discussion 157 5.5. Literature Cited 260 Chapter 6: Analysis of global collections and the type specimens of 165-140 Hildenbrandia 6.1. Introduction 6.2. Materiais and Methods 6.2.1. Type specimens, historicaliy signifTcant specimens and global collections analyzed 6.2.2. Morphometric analyses 6.2.3. rbcL and 18s rRNA gene sequence analyses 6.3. Results 6.3.1. Morphometric analyses 179 6.3-2. Analysis of transitional saturation of the rbcL and 18s rRNA 203 genes 6.3.3. 18s rRNA gene sequence analyses - parsimony analysis 203 6.3-4. 18 S rFWA gene sequence analyses - distance analy sis 209 6.3.5. 18s rRNA gene sequence analyses - quartet puzzling analysis 214 6.3.6. Sequence divergence values of the rbcL and 18s rRNA 214 genes 6.4. Discussion 215 6.5. Taxonomie proposais and revised descriptions 220 vii
TABLE OF CONTENTS cont,
6.6. Key to the Hildenbrandiales 233 6.7. Literature Cited 234 Chapter 7:General Conclusions 24 1-245 Appendix 1: Primers 246-248 Appendix 2: Means of morphometric measurements for al1 sarnples 249-25 1 --- VIl1
LIST OF TABLES
Page Table 1.1 Current classification of the Hildenbrandiales 6 Table 2.1 Collection information for specimens of marine and 25-27 fkeshwater Hildenbuandia used in North American study Table 2.2 Means and ranges of characters used in morphometric analyses of marine and fieshwater Hildenbrandia fiom North Amenca Table 3-1 Collection orm mat ion for specimens of marine and fkeshwater Hildenbrandia used in European study Table 3 -2 Means and ranges of characters used in morphometric analyses of marine and fieshwater HiZdenbrandia fkom Europe Table 4.1 Collection information, elevation, age of sampied water bodies and GenBank accession numbers for Swedish samples of marine and freshwater Hildenbrandia Table 4.2 Percent sllnilarity among marine and fieshwater Hildenbrandia samples fkom IS SR analyses based on the Dice coefficient and the Jaccard coefficient Table 4.3 The number of base pair changes in gene sequences among five representative samples of Hildenbrandia fiom Sweden for the rbcL and 185 rRNA genes Table 5.1 Results of histochernical reûctions for gemmae and thalli of Hildenbrandia angolensis Table 6.1 Collection or source information for specimens of marine and fkeshwater Hildenbrandia and ApophZaea used in global analyses LIST OF TABLES cont.
Table 6.2 Mean tetrasporangial dimensions for Apophlaea and 180 Hildenbrandia type and historicaiiy signincant specimens LIST OF FIGURES
Page Figure 1-1 Light micrograph of the thallus construction of 3 Hilden brandia Figure 1.2 Light micrograph of a herbarium sheet of Apophlaea Figure 1.3 Transverse section through a conceptacle of H. rubra Figure 1.4 Transverse section through the thallus and a gemma of H angolensis Figure 2.1 Locations of North Amencan marine and fieshwater collection sites of Hildenbrandia specimens Figure 2.2 Cluster dendrogram of North Arnerican specimens (excluding reproductive measurements) Figure 2.3 Principal CO-ordinatesbiplot of North Amencan specirnens (excluding reproductive measurements) Figure 2.4 Cluster dendrogram of North American specimens (including reproductive measurements) Figure 2.5 Principal CO-ordinatesbiplot of North American specimens (including reproductive measurements) Figure 2.6 a, b Graphs of number of transitions versus p-distances for rbcL and 18s rRNA genes of North Amencan specimens Figure 2.7 Parsimony tree for rbcL data of North American Hildenbrandia specimens Figure 2.8 Neighbor-joining tree for rbcL data of North Amencan Hildenbrandia specimens Figure 2.9 Parsimony tree for 185 rRNA data of North American Hildenbrandia specimens xi
LIST OF FllGURES cont.
Figure 2.1 0 Neighbor-joining tree for 18 S rRNA data of North American NiIdenbrandia specimens Figure 3.1 Locations of European marine and fieshwater collection sites of HiZden brandia specimens Figure 3.2 Cluster deadrogram of European Hildenbrandia specimens Figure 3.3 Principal CO-ordinatesbipIot of European Hildenbrandia specimens Figure 3.4 a, b Graphs of number of transitions versus p-distances for rbcL and 18s rRNA genes of European specimens Figure 3 -5 Parsimony tree of rbcL data for European Hildenbrandia specimens Figure 3 -6 Neighbor-joining tree of rbcL data for European HiZdenbrandia specimens Figure 3.7 Parsimony tree of 18s rRNA data for European HiZdenbrandia specimens Figure 3.8 Neighbor-joining tree of 18s rRNA data for European Hilden brandia specimens Figure 4-1 Map of sampling locations for brackish and ficeshwater Hildenbrandia in Sweden Figure 4.2 a, b Unrooted neighbor-joining and parsimony trees for ITS 1 sequences of Hildenbrandia samples fiom Sweden. Figure 4.3 Unrooted neighbor-joining tree for ITS2 sequences of Hildenbrandia samples Tom Sweden xii
LIST OF FIGURES cont.
Figure 4.4 a, b Unrooted neighbor-j oinuig and parsimony trees for combined ITS Z and ITS2 çequences of Hildenbrandia samples &om Sweden. Figure 4.5 a, b Unrooted neighbor-joining trees of Hildenbrandia samples fiom Sweden based on ISSR analyses using the Dice and Jaccard coefficients Figure 4.6 a, b Unrooted neighbor-joining and parsimony trees of Swedish and other European Hildenbrandia samples based on the rbcL gene Figure 4.7 a, b Unrooted neighbor-joining and parsimony trees of Swedish and other European Hilaenbrundia samples based on the 18s rRNA gene Figure 5.1 Scanning electron micrograph of the surface of H angolensis Figure 5.2 Light rnicrograph of a released gemma in H angolensis Figure 5.3 Gemma producing rhizoids in H. angolensis Figure 5.4 PAS-stained section of H. ungolensis thailus Figures 5.5-5.10 Developmental sequence of gernmae in the thallus of H. angdensis illustrated using light microscopy- sectioned material Figures 5.1 1-5.16 Transmission electron rnicrographs of vegetative thallus organization and gemma development and release in H. angolensis Figure 5.1 7 Graphs of the number of gemmae and the number of released gemmae produced by H angdensis in two Texas spring-fed streams LIST OF FIGURES cont.
Figure 6.1 Collection locations of Hddenbrandia and Apophlaea 176 specimens used in globd analyses Figure 6.2 Cluster dendrogram of type and histoncalIy significant 182 specimens (excluding reproductive measurements) Figure 6.3 Principal CO-ordinatesbiplot of type and historically 184 significant specimens (excluding reproductive measurements) Figure 6.4 Cluster dendrograrn of type and historically signifïcant 187 specimens (including reproductive measurements) Figure 6.5 Principal CO-ordinatesbiplot of type and histoncally 189 significant specimens (including reproductive measurernents) Figure 6.6 Cluster dendrogram of al1 Hildenbrundia global specimens (excluding reproductive measurements) Figure 6.7 Principal CO-ordinatesbiplot of al1 Hildenbrandia global collections (excluding reproductive measurements) Figure 6.8 Cluster dendrograrn of ail Hildenbrandia global marine specimens (includïng reproductive measurements) Figure 6.9 Principal CO-ordinatesbiplot of al1 Nildenbrandia global marine specimens (including reproductive measurements) Figure 6.10 Cluster dendrograrn of al1 Hildenbrandia global fkeshwater specimens Figure 6.1 1 Principal components analysis biplot of al1 Hildenbrundia global freshwater specimens xiv
LIST OF FIGURES cont.
Figure 6-12 Graphs of number of transitions versus p-distances for rbcL and 18s rRNA genes of global specimens Figure 6.13 Parsimony tree of global Hildenbrandiales specimens based on the 18s rRNA gene Figure 6-14 Neighbor-joining tree of global Hildenbrandiales specimens based on the 185 rRNA gene Figure 6.15 Quartet puzzling maximum likelihood tree of global Hildenbrandiales specimens based on the 18s rRNA gene Figures 6.16 a-b Tetrasporangial morphologies of marine species of Hildenbrandia 18s rRNA gene coding for the small subunit of ribosomal RNA ANOVA anaiysis of variance bp base pairs BP bootstrap proportion BSA bovine semm albumin CT consistency index DNA deoxyribonucleic acid ISSR intersimple sequence repeat ITS intemal transcribed spacer mas1 metres above sea level MP maximum parsimony NJ neighbor-joining OTU operationai taxonomie unit PAS periodic acid - Schiffs reaction PCA principal components analysis PCO principal CO-ordinatesanalysis PCR polymerase chah reaction QP quartet piizzling &%PD random amplified polymorphic DNA rbcL gene coding for the large subunit of the ribulose-l,5-bisphosphate carboxylase/oxygenase enzyme RFLP restriction fiagrnent length polymorphism RuBisCO ribulose- l,5-bisphosphate carboxylase/oxygenase enzyme SEM scanning electron microscopy TBE tris / boric acid / EDTA baer TBR tree bisection-recomection TEM transmission electron microscopy ABBREVIATIONS CONT.
UPGMA unweighted pair group method using arithrnetic means UV ultra violet Y years Ya years ago CHAPTER 1: General Introduction
1.1. Morphology and distribution of the Hildenbrandiales
The red algal order Hildenbrandiales contains a single family, the
Hildenbrandiaceae, and two genera, Hildenbrandiu and Apophlaea, which differ in both
their morphology and distribution (Denizot, 1968). HiZdenbrandia is an exclusively
cmstose alga, consisting of vertical files of cells that arise from a prostrate basal layer of
filaments @ig. 1.1 a), and includes both marine and fieshwater forms (Denizot, L 968;
Wornersley, 1994). In contrast, Apophlaea is strictly a marine algal genus which is
constructed of an adherent crust, similar to Hildenbrandia, but which gives rise to
upright, branched pseudoparenchymatous protuberances (Fig. 1.1 b) (Hawkes, 1983).
Although sexual reproduction has not been documented for either Hildenbranaicr or
ApophZaea, marine representatives of both genera reproduce through the production of
tetrasporangia in uniporate conceptacles (Fig. 1.1 c) (Inrine & Pueschel, 1994). The
fieshwater forms of Hildenbrundia do not form tetrasporangia, but spread through the
production of asemal propagules known as gemmae (Fig. 1.1 d) (Sheath et al.,1993)-
Hildenbrandia is one of the most widespread genera of macroalgae along rocky
coastlines, and has been reported from al1 continents. In North America its range extends
fi-om the Canadian Arctic (Lee, 1980) along the Pacific coast of the continent (Abbott &
Hollenberg, 1976; Scagel et al., 1986) including the Baja Peninsda and Centrd America
(Dawson, 1952; Dawson, 1957), along the Atlantic coast fiom Newfoundland (South &
Hooper, 1980) to the New England United States (Taylor, 1966; Villalard-Bohnsack,
1995) and the Caribbean (Taylor, 1960). Globally, Hildenbrandia has been collected 2
Figure 1.1. a) Light micrograph of the fieshwater species, Hildenbrandia ~.ivularis,
showing the thallus composed of vertical files of cells.
b) Photograph of a herbarium sheet of Apophlaea sinclairii showing the
branched, upright portion of the thailus.
c) A transverse section through a conceptacle of the marine species, H.
rubru (LR White embedded material, light microscopy section, stained
cvith Toluidine Blue O), containhg tetrasporangia (arrow) at various
stages of maturity.
d) A transverse section through the thaIIus of the fi-eshwater species, H
angolemis (preparation as for Fig. 1 c), illustrating the release of a gernma
(arrow).
4
Eorn such diverse locations as Chile (this thesis, chapter 6), Brazil (Deoliveira, 1977),
Peru (Dawson et al., 1964), Argentins (Vallarino & Elias, 1997), the British Ides (Irvine
& Pueschel, 1994), the Baltic Sea (Rosenvinge, 19 17; Nielsen, 1994), the Atlantic coast of western Europe (Ardré, 1959; Freshwater et al., 1994), the Canary Islands (Afonso-
Carrillo er al,, l992), the Mediterranean (Ballesteros, 1988), South Afnca and India
(Silva et al., 1996), Vietnam (Dawson, 1954), Korea (Lee & Kang, 1986), Japan
(Umezaki, 1969), Fiji (N7Yeurtet al., 1W6), Australia (Womersley, 1994) and Antarctica
(R. Moe, personal communication). The genus also contains several fieshwater forms, which are comrnon in lakes and streams of continental Europe and the British Isles (e.g
West & Fritsch, 1932; Bourrelly, 1955), but are more localized in their distribution on other continents, including North Amerka (e.g. Flint, 1955; Nichols, 1965), where they are usually collected fiom hard water streams of moderate current velocity (Sheath et al.,
1993). Ln contrast to the widespread distribution of Hildenbrandia, Apophlaea is known only from the coastlines of New Zealand, and has been comparatively little studied
(Hawkes: 1983). Both genera are found in the rnid to lower intertidal regions of rocky shorelines, and HiZdenbrandia can extend well into the subtidal (Rosenvinge, 19 17).
Freshwater forms of Hildenbrandia have also been reported fkom depths of over 50 m
(Skuja, 1938). Besides rock substrats, Hildenbrandia has also been reported growing on glass, brick, plastic, and shells, but not on wood or epiphytically on other plants
(Israelson, 1942).
1.2. Taxonomic study of the Hildenbrandiales
Taxonomic studies of Hildenbrandia are hindered by a paucw of morphological 5 characters. For comparative purposes the cmstose thallus construction of the genus has relatively few distinctive features compared to more complex erect red algal thalli. The taxonomy of Hildenbrandia currently relies on the following rnorphological characters: thallus thickness, filament height, basal layer height, cell Iength and diameter, conceptacle dimensions, reproduction by tetrasporangia or gemmae, tetrasporangial size and division pattern, presence or absence of paraphyses in the conceptacles, and to some extent, thallus color (Denizot, 1968;Sheath et al., 1993; Irvine & Pueschel, 1994;
Womersley, 1994). Many of these features are of dubious taxonomic validity since they vary with the age of the plant and the environment in which the dga occurs (e-g.thallus thickness, color and conceptacle dimensions)- The genus currently includes 12 marine and five freshwater species and subspecific taxa (Table 1.1). A taxonomic investigation of the genus, including an examination of the type specimens of the genus, is warranted given the lack of consensus on the validity of characters used to delimit species.
The genus Apophlaea is considered to be a member of the order Hildenbrandiales based on sirnilar pit plug morphology to Hilaenbrundia (both have a core with a single cap layer) and presence of zonate tetrasporangia in conceptacles (Hawkes, 1983;
Pueschel, 1989). PIants of Apophlaea fimdamentally differ fkom those of HiZdenbrandiu in that Apopldaea has a branched, upright portion of the thallus in addition to a crustose base. The limited distribution of Apophlaea contrasts strongly with the worldwide distribution of Hildenbrandia. ApophZaeu currently contains three species and subspecific taxa (Table 1.1). Table 1.1. Current classification of the Hïldenbrandiales.
Genus Specific or subspecific taxon Apophlaea A. lyallii Hoo kX et Harv. A. lyallii var. gigartinoides Hook-f. et Ham A. sinclairii Harv. ex Hook-f. et Harv. Hilden brmdia W. angolensis" Welw. ex W. West et GS-West H. arracana Zeller H. canariensis Bsrgesen
H, crouanii (J. Agardh) J. Agardh H dmvsonii (Ardré) Hollenb. EL expansa Dickie H kerguelensis (Askenasy) Y.M. Chamb. H lecannellieri Har. H. occidentalis Setch. ex N.L. Gardner H. occidentulis var. lusitanka Ardré H occidentalis var. yessoensis (Yendo) Ardré H. parula Womersley H. ramanaginairu M. Khan H. rivularis" (Liebm.) J. Agardh H rivularis ssp. chalikophild Palik H. rivularis var. drescherza LingeIsh. H. rubra (Somrnerf.) Menegh.
Yreshwater taxa 7
A Mertaxonomic issue within the Hildenbrandiales is the apparent Iack of female
garnetangia and post-fertilization events, which are typically used for taxonomic purposes
in other orders of the Rhodophyta. Culture studies attempting to elucidate the life history
of marine Hildenbrandia have only succeeded in producing tetrasporophyte plants
@eCew & West, 1977), and thus tetrasporogenesis in Hildenbrandia is believed to be mitotic, which is an unusual phenomenon among the red algae (Guiry, 1990).
Few taxonomic treatments of the Hildenbrandiales have been published, the rnost comprehensive being that by Denizot (1968). Several authors have presented treatments
for the marine species of specific geographical regions, including Australia (Womersley,
1994), the British Isles (Trvine & Pueschel, 1994) and the Indian Ocean (Silva et al,,
1996). No studies have systematically exarnined both marine and fieshwater forms of
Hildenbrandia worldwide.
Given the small nurnber of morphological characters available for classification within the Hildenbrandiales, molecular analyses of the order may provide insight into the relationships between the genera and among the species. Phylogenetic reconstruction using molecular markers, in particular, has become a comrnon tool in systematic phycology over the past decade, and many studies have been published that attempt to dari@ the taxonorny of algai groups using these techniques (e.g Hommersand et al.,
1994; Ragan et al., 1994; Oliveira et al., 1995). Molecular techniques have been
instrumental in determinhg relationships within and arnong algal groups; however, they
seem to be best used in combination with other forrns of data, such as morphometrics,
rnicroscopical or biochernical/physiological data. Two of the most comrnon molecular 8
markers that have been employed in recent years are the rbcL gene, a chloroplast gene that codes for the large subunit of the ribdose-1,s-bisphosphatecarboxyIase/oxygenase enzyme, and the nuclear gene coding for the small subuoit of ribosomal RNA (1 8s rRNA gene). The rbcL gene appears to be relatively well conserved at the specific and generic levels, and has been successfùlly used for phylogenetic reconstruction at these ievels withïn the red aigae (e-g. Freshwater & Rueness, 1994; Woolcott & King, 1998).
However, its utiIity is reduced at higher taxonornic levels for the red algae (cg.
Freshwater et al., 1994) as the DNA sequences Vary considerably among red algal orders.
The 18s rRNA gene is commonly used for phylogenetic reconstruction since it is universally present in eukaryotes, and is well conserved because it is constrained by fûnctional requirements (Van de Peer et al., 1993). The gene has been used at a number of levels for phylogenetic cornparisons (e-g.among orders - Ragan et al., 1994; arnong genera - Shunada et al-, 1999; among species - Bird et al., 1994).
1.3. P hylogenetic positioning of the HiIdenbrandiales
The phylogenetic positioning of the Hildenbrandiales with respect to other red algal orders has been exarnined by severai authors through phylogenetic reconstruction using molecular techniques. The earliest comprehensive studies of red algal molecular phylogeny, based on the rbcL (Freshwater et al., 1994) and the 18s rRNA genes (Ragan et al., 1994), both demonstrated the Hildenbrandiales to be basal or near the base of the monophyletic Flondeophyte lineage, although only a single sequence of Hildenbrnndia was used in each study. Subsequent sîudy based on the 18s rRNA gene demonstrated
Apophlaea to be closely allied to Hildenbrandia, supporting its placement within the 9
Hildenbrandiales (Saunders & Bailey, 1999). However, this study suggested that
Hildenbrandia rnay be paraphyletic, since one sequence of Hildenbrandia was positioned closer to Apophlaea in their phylogenetic tree than to the other Hildenbrandia sequence
(Saunders & Bailey, 1999). Thus, although the positionhg of the order widi respect to other red algal orders appears to be well established, the relationships within the order require merstudy. The addition of molecular sequence data for the second species of
Apophhea, A. sinclnirii, will also help determine whether Hildenbrandia is a monophyletic lineage.
1.4. Biogeographic study of the Hildenbrandiales
Acquiring data for the testing of biogeographic hypotheses has become simpler with the advent of molecular techniques to study evoLutionary and distributional patterns within the algae. Ngal distributions are dynamic; some of the factors contributing to geographic shifts include continental drift and plate tectonics, physiologicd tolerance ranges of individual taxa and dispersal events (Hommersand, 1990; Lüning, 1990). Thus, present-day distributions must be analyzed in relation to events in the past, on several scales. For example, the cold temperate and polar marine benthic floras of the Northern
Hemisphere differ substantially from those of the Southern Hemisphere, which can be explained by the presence of a warm-water barrier in the tropical regions for long evolutionary periods (Lüning, 1990). The distribution of individual taxa may also be a result, in part, of the dispersal mechanisïns employed by the alga, which impact on the range of an alga on a much smaller time scale (Pielou, 1979).
The cmstose nature of the Hildenbrandiales (excepting the upright portion of 10
Apophlaea) has interesting implications for biogeographic study of the order. Maggs
(1990) listed several reasons why cnistose dgae are of particuiar biogeographic interest: many grow slowly and require months to several years to reach reproductive maturity, spores released fiom crusts are less likely to enter the water column and be widely dispersed than for non-crustose algae, and many crusts do not grow epiphytically or on artificial substrats, so they are Iess likely to be transported long distances by vectors. In spite of these points, Nildenbrandia is widely distributed worldwide, which is interesting fiom a biogeographical standpoint, and it has been hypothesized that the Hildenbrandides is one of the most ancient red Aga1 orders based on its anatomy and distribution (Maggs,
1990).
CIadistic techniques have been used recently to test biogeographic hypotheses for several algal groups. For exarnple, van Oppen et al. (1994) used molecular markers to investigate the dispersal routes of the Arctic-Antarctic disjunct seaweed Acrosiphonia crrcta (Chlorophyta). Among other trends, they observed that Arctic Greenland populations were most likely established independently of other North Atlantic populations, based on restriction fiagment length polymorphisms (FWLP' s) and random amplified polymorphic DNA (EbVD's) techniques. Kooistra et al. (1992) used nuclear ribosomal interna1 transcribed spacer (ITS) sequences to gain support for the hypothesis that the green alga Cludophoropsis rnembranacea is of Tethyan origins, and that dispersal of the alga had occ~u-redbetween the Caribbean and the tropical eastem Atlantic. Similar investigations using rnolecular data to examine the biogeographic patterns of
HiZdenbrandia are warranted given the potentially great age of the genus, based on the hypothesis of Maggs (1 990).
1.5. The origins of freshwater Hildenbrandr'a
The fieshwater red algae can be divided broadly into two categories: those resembling their marine counterparts (Le. underwent early evolution in marine habitats) and those which have no obvious marine counterparts (i-e. underwent early evolution in streams). On this basis, Skuja (1938) proposed that this first group of eeshwater red algae may have arisen through invasion(s) by marine red algae into fieshwater habitats.
Examples of this first group include aigae that have both marine and freshwater representatives classified within the same genus, usually because they are morphologically similar, such as Bangia, Bostg~chiu,CaZogZossa and Nildenbrandia
(Sheath, 1984). Freshwater members of this f~stgroup are often red colored due to a predominance of the pigment phycoerythrin over phycocyanin (Sheatk, 1984). Examples of the second group include the genera Batrachospermum, Compsopogon, Lemanea,
Tuomeya and Sirodotia, and these are usually more bluish coloured than red due to a predominance of phycocyanin over phycoeryhin (Sheath, 1984).
Several mechanisms exist whereby a marine alga, such as HiZdenbrmdia, codd invade into a freshwater system, including long-terrn spread up estuaries, vector-assisted transport and successful maintenance after continental uplift or embayment enclosure
(Sheath, 1984). From examining the distribution of fieshwater Hilden6randia in North
America and Europe it appears that different mechanisms may be responsible for the curent distributions on the two continents. In many regions of Europe, HiZdenbrandîa is widespread in both stream and lake systems (e.g. Budde, 1926; Israelson, 1942; 12
Bourrelly, 1955; Starmach, 1969). In North America, however, reports of fieshwater
Nildenbrandia have been concentrated fiom the tropical and subtropical regions of the continent (e.g. Flint, 1955; Nichols, 1965; Boon et al., 1986; Sheath et al. 1993; although see unconfirmed reports fiom Ohio Pillick & Lee, 19341 and Pennsylvania Wolle,
18871). Many of these reports are fiom isolated springs, streams originating fkom springs, or fiom mountain streams in the Caribbean islands. The disjunct distribution of fieshwzter Hildenbrandia in North Arnerica thus differs strongly fkom the almost ubiquitous distribution of this alga in Europe, This pattern suggests that the European populations may have undergone a large amount of vector-assisted transport among inland locations, perhaps afier initial invasions by marine populations via long-term spread up estuaries or embayment enclosure. In contrast, the North American populations have probably achieved their disjunct status through multiple vector-assisted invasion events to their isolated habitats.
Until recently, meaningful testing of this invasion hypothesis for individual genera was difficult, due to the lack of fossil data. However, with the advent of the use of molecular tooIs, which allow researchers to address questions of phylogeny or evolutionary origins, this proposal can be addressed. By examining patterns of DNA sequence variation among marine and fieshwater sarnples within the genus, it is possible to determine whether the two groups have been isolated fiom one another for a long petiod of time, and if the fieshwater samples represent single or multiple invasion events.
Addressing Skuja's (1938) invasion hypothesis for the genus Hildenbrandia is important since our present understanding of the phylogenetic relationships between marine and 13
fieshwater species is based solely on a Iimited suite of morphological characters,
discussed in Section 1.2 (although in addition to this list, Pueschel cl9891 reported a
similar pit plug morphology for both marine and fieshwater Hildenbrandia). Any taxonomie revision within the genus involving both the marine and freshwater species shodd incorporate data that address the evolutionary relationship between the two groups. Previous studies have examined recent invasion events using molecular markers, for exarnple in the green algal genus Codiurn, and have determined that the intemal transcribed spacer regions of the nuclear ribosomal genes (ITS 1 & ITSî), as well as some fiagment analysis techniques, are often appropnate markers at this level (Coleman, 1996).
1.6. Asexual propagation by gemmae in Hikienbrandia
The reproductive structures of the marine forms of Hildenbrandia (tetrasporangia) are common in the florideophyte lineage, but the propagules of the fieshwater forms
(gemrnae) have few parallels among the red algae. Examples of the few red algae that produce propagules include Batrachospermurn breutelii, Deucalion levringii and
Anisosclzizuspropagdi (J3uisma.n & Kraft, 1982; Sheath & Whittick, 1995), but the morphology of their propagules differs substantially from the gemrnae of Kildenbrandia.
Early culture studies of freshwater Hildenbrandia failed to produce any life history stage other than the gemma producing plants (NichoIs, 1965; Seto, 1977), and it is still believed that these are the sole reproductive structures. Gemmae have been reported for the two most comrnody reported fieshwater species, H. angolensis and H. rivularis (Starmach,
1952; Sheath et al., 1993).
Gemmae are tight clusters of cells that are produced within the thallus and are 14 released from the surface (Starmach, 1952), after which they settle and divide to fom a new thallus. Although severai studies have examined their morphology and development
(eg NichoIs, 1965; Seto, 1977; Necchi, 1987), details of their development at the ultrastructural level and histochemical differences between gemma and thallus cells have not been elucidated. This contrasts to the large nurnber of studies which have examined tetrasporogenesis in marine red aigae, including marine Hildenbrandia (e-g. Guiry, 1978;
Pueschel, 1983; Guiry, 1990).
Given the wide distribution of freshwater Hildenbrandia, at least on some continents, and thus the likelihood that gemmae are heavily involved in the spread and maintenance of these populations, more microscopical studies are required to understand the nature of their composition and the sequence of events leading to their release fiom the thallus. Details of gemma structure may also shed light on the origins of the fieshwater Hildenbrandia forrns; if the freshwater forrns are derived fio m marine fonns they may display homology in reproductive structures.
1.7. Research objectives
The following research objectives of this thesis aim to clarZy the phylogenetic and morphological relationships arnong members of the red algal genus Hildenbrandia in order to re-evaluate the taxonomy of the genus, and to examine the currently accepted phylogenetic relationships within the Hildenbrandiales:
1. To analyze in detail the biogeographic and systematic patterns evident from
HiEdenbrandia collections fkom the North Arnerican and European continents, and
on a smaller scale fiom other regions of the world, using a combination of 15
morphological, morphometric and molecular analyses.
2. To examine the possibility of a marine ongin for the freshwater forms of
Hilden brandin through comparisons of marine and keshwater collections fiom
several continents based on the rbcL gene, 18s rRNA gene and ITS regions of the
rRNA genes.
-l 3. To study the process of gemma development and release in the freshwater species
of Hildenbrandia, H. angolemis, using several foms of microscopy and
histochemistry.
4. To re-evaluate the systematics of the genus NiZdenbrandia through rnorphometric
analyses of the type specimens and worldwide collections of the genus, as well as
comparisons through molecular analyses.
5. To re-examine whether Hildenbrandia and Apophlaea have a close phy 10 genetic
relationship based on molecular analyses.
1.8. Literature cited
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Ardré, F. 1959. Un intéressant Hildenbrandfiadu Portugal. Revue Algologique 4: 227- 237.
Ballesteros, E. 1988, Estnichira y dinhica de la cornunidad de Cystoseira rnedirerranea Sauvageau en el Mediterrkeo noroccidental. lnvestigacidn Pesquera 52: 3 13- 334. Bird, C.J., Ragan, M.A., Critchley, A.T., Rice, E.L. & Gutell, R.R. 1994, Molecular relationships arnong the Gracilariaceae (Rhodophyta): merobservations on some undetermined species. European Journal of Phycology 29: 195-20?.
Boon, P.J., Jupp, B.P. & Lee, D.G. 1986. The benthic ecology of rivers in the Blue Mountains (Jamaica) prior to construction of a water regulation scheme. Archiv fur Hydrobiologie 3: 3 15-355.
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Budde, H, 1926. Zweiter Beitrag ZLU Entwicklungsgeschichte von Hildenbrandia rivularis (Liebmann). Deutsche Botanische Gesellschaft Berichte 44: 367-3 72.
Coleman, A. W, 1996. DNA analysis methods for recognizing species invasion: the exarnple of Codium, and generally applicable methods for algae. Fifieenth International Seaweed Symposium (Eds. S.C. Lindstrom & D.J. Chapman). Hydrobiologia 326/327:29-34.
Dawson, E,Y, 1952. Marine Red Algue of Pacrjic Mexico Parr I. Bangiales to Corallinaceae Subf: Corallinoideae. Allan Hancock Pacific Expeditions, Volume 17, Number 1, The University of Southem California Press, Los Angeles, California, 239 pp.
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Freshwater, D.W., Fredencq, S., Butler, B.S., Hommersand, M.H. & Chase, M.W. 1994. A gene phylogeny of the red aigae (Rhodophyta) based on plastid rbcL. Proceedings of the National Academy of Science USA 91: 728 1-7285.
Freshwater, D. W. & Rueness, J. 1994. Phylogenetic relationships of some European GeZidiurn (Gelidiales, Rhodophyta) species, based on rbcL nucleotide sequence analysis. Phycologia 33: 187- 194.
Guiry, M.D. 1978. The importance of sporangia in the classification of the Florideophyceae. In: Modern Approaches to the Tmonomy of Red and Brown Algae. (Eds. D.E.G. Irvine & J.H. Pnce). Academic Press, London, pp. 1I 1-144.
Guiry, M.D. 1990. Sporangia and spores. In: Biology of the Red Algae (Eds. K.M. Cole and R.G. Sheath). Cambridge Universiq Press, Cambridge, pp. 347-376.
Hawkes, M. W. 1983. Anatomy of ApophZaea sinclairii - An enigrnatic red alga endemic to New Zealand. Japanese Journal of Phycology 31: 55-64.
Hommersand, M.H. 1990. Biogeography of the marine red algae of the North Atlantic Ocean. In: Evolutionary Biogeography of the Marine Algae of the North Atlantic. (Eds. D.J. Garbary & G.R. South). NATO AS1 Series, Vol. G 22, pp. 349-4 10.
Hommersand, M.H., Fredericq, S. & Freshwater, D .W. 1994. Phylogenetic systematics and biogeography of the Gigartinaceae (Gigartinales, Rhodophyta) based on sequence analysis of rbcL. Botanica Marina 37: 193-203.
Huisman, J.M. & Kraft, G.T. 1982. Deucalion gen. nov. and Anisoschizus gen. nov. (Ceramiaceae, Cerarniales), two propagule-formïng red algae fiom southern Australia. Journal of PhycoZogy 18: 177-192.
Irvine, L.M. & Pueschel, C.M. 1994. Hildenbrandiales. In: Seaweeds of rhe British Ides, Volume 1, Rhodophyta, Part 2B, Corallinales, Hildenbrandiales. (Ed. L .M. hine & Y.M. Chamberlain). The Natural History Museum, London, pp. 235-241.
Israelson, G. 1942. The Freshwater Florideae of Sweden: Studies on their taxonomy, ecology and distribution. Sym bolae Botanicae Upsalienses 4: 1 - 13 5.
Kooistra, W.H.C.F., Stam, W.T., Olsen, J.L. & van den Hoek, C. 1992. Biogeography of Cladophoropsis mem bwnacea (Chloro phyta) based on cornparisons of nuclear rDNA ITS sequences. Journal of Phycology 28: 660-668. Lee, I.K. & Kang, J. W. 1986. A Check List of the Marine Algae in Korea, 272e Korean Journal of PhycoZogy 1: 3 11-325-
Lee, R.K.S. 1980. A Catalogue of the Marine Algae of the Canadian Arctic. National lMuseums of Canada Pzrb!icatfons in Botany 9: 1-82.
Lillick, L.C. & Lee, LM. 1934. A check-list of Ohio aigae with additions Çom the Cincinnati region. Arnerican MidZand NaturaZist 15: 7 13 -75 1.
Lüning, K. 1990. Seaweeds: Their Environment, Biogeography, and Ecophysiology. John Wiley & Sons, New York, 527 pp.
Maggs, C.A. 1990. Distribution and evolution of non-coralline crustose red algae in the North Atlantic. Ln: Evolzttionav Biogeography of the Marine Algae of the North Atlantic. (Eds. D.J. Garbary & G.R. South). NATO AS1 Series, Vol. G 22, pp. 24 1-264.
Necchi, O., Jr. 1987. Estudos sobre as Rodophyta de 6gua.s continentais do Brasil - 6. Ocorrência de Hildebrmdia rivularis (Liebmann) J. Agardh nos Estados do Rio de Janeiro e Sao Paulo. Neritica 2 (suppl.): liC'LlS2.
Nichols, H.W. 1965. Culture and development of Hildenbrandia rivularis ftom Denmark and North Amerka. Americun Journal of Botany 52: 9-1 5.
Nielsen, R. 1994. Danske Hmalger, Udbredelse og Danske Navne. Mil.@-og Energiministeriet / Skov- og NaturstyreIsen, Copenhagen, 123 pp.
N'Yeurt, A.D.R., South, G.R. & Keats, D.W. 1996. A revised checklist of the benthic marine algae of the Fiji Islands, South Pacific (Including the Island of Rotuma). Micronesia 29: 49-98.
Oliviers, MC., Kumiawan, J., Bird, C .J., Rice, E.L., Muphy, C.A., Singh, R.K., Gutell, R.R. & Ragan, M.A. 1995. A preliminary investigation of the order Bangides (Bangiophycidae, Rhodophyta) bmed on sequences of the nuclear small-subunit ribosomaI RNA genes. PhycoIogical Research 43 : 7 1-79.
Pielou, E.C. 1979. Biogeography. John Wiley & Sons, New York, 3 5 1 pp.
Pueschel, C.M. 1982. Ultrastructural ~bservationsof tetrasporangia and conceptacles in HiZdenbrandia (Rhodophyta: Hildenbrandiales). British Phycological Journal 17: 333-341.
Pueschel, C.M. 1989. An expanded survey of the ultrastructure of red algal pit plugs. Journal of Phycology 25: 625-636.
Ragan, MA., Bird, C.J., Rice, E.L., Gutell, R.R., Murphy, C.A. & Singh, R.K. 1994. A molecular phylogeny of the marine red algae (Rhodophyta) based on the nuclear small-subunit rRNA gene. Proceedings of the National Academy of Science USA 91: 7276-7280.
Rosenvinge, L.K. 191 7. The Marine Algue of Denmark: Contributions to their Natural HrStory. Vol. 1. Rhodophyceae. Andr. Fred. Host & Son, Copenhagen, 486 pp.
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Seto, R- 1977. On the vegetative propagation of a fiesh water red alga, Kildenbrandia rivularis (Liebm.) J. Ag. Bzrlletin of the Japanese Society of Phycology 25: 12% 136.
Sheath, R.G. 1984. The biology of fieshwater red algae. Progress in PhycoIogical Research 3: 89-157.
Sheath, R-G., Kaczmarczyk, D. & Cole, K.M. 1993. Distribution and systematics of fkeshwater Hildenbrandia (Rhodophyta, Hildenbrandiales) in North America. European Jozirnal of Phycology 28: 115-1 2 1.
Sheath, R.G. & Whittick, A. 1995. The unique gonimoblast propagules of Batrachospermum brezltelii (Babachospermales, Rhodophyta). PhycoZogia 34: 33-38.
Shimada, S., Horiguchi, T. & Masuda, M. 1999. Phylogenetic affrnities of genera Acanthopeltis and YatabeZZa (Gelidides, Rhodophyta) inferred fiom molecular analyses. Phycologia 38: 528-540.
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Skuja, H. 1938. Comments on the fkesh-water Rhodophyceae. Botunical Review 4: 665- 676. South, G.R. & Hooper, R.G. 1980. A Catalogue and Atlas of the Benthic Marine Algue of the Island of Newfoundland. MemonaI University of Newfoundland Occasional Papers in Biology, Number 3, 136 pp.
Stmach, K- 1952. O rozmnazaniu sis krasnorosta Hildenbrandia rivularis (Liebm.) J. Ag. (The reproduction of the fiesh water Rhodophyceae Hildenbrandia rivularis). Acta Societatis Botanicorurn Poloniae 2 1: 447-474-
Starmach, K. 1969. Growth of thalli and reproduction of the red dga Hildenbrandia rivularis (Liebm.) J. Ag. Acta Societatis Botanicorurn Poloniae 38: 523-533.
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Taylor, W.R. 1966. Marine Algae of the Northeastern Coast of North America. University of Michigan Press, Ann Arbor, 509 pp.
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Villalard-Bohnsack, M. 1995. L?lus~atedKey to the Seaweeds of New England The Rhode Island Natural History Survey, Kingston, 144 pp.
West, G.S. & Fritsch. F.E. 1932. A Treatise on the British Fresh-Water Algae. Cambridge University Press, Cambridge, 534 pp.
Wo lle, F. 18 87. Fresh- Water Algae of the United States (Exclusive of the Dintornaceae) . Comenius Press, Pennsylvania, 364 pp.
Womersley, H.B.S. 1994. The Marine Benthic Flora of Southern Australia, Part IIL4. Flora of Australia Supplementary Senes Number 1, 508 pp. Woolcott, G.W. & King, R.J. 1998. Porphyra and Bangia (Bangiaceae, Rhodophyta) in warm temperate waters of eastem Austrdia: Morphological and molecular analyses. Phycological Research 46: I 11 - 123. 22
CHAPTER 2: Biogeography and systematics of Hildenbrandia in North America:
inferences from morphometrics and sequence analyses of the rbcL and 18s rRNA genes
2.1. Introduction
The red algal genus Hildenbrandia Nardo is characterized by a thin crustose thallus that is composed of a unistratose basal layer that gives rise to vertical files of cells
(Womersley, 1994). The files are connected by secondary pit plugs (Pueschel, 1988), which may contribute to the toughness of the thallus. Tetrasporangia and gemmae are the only reproductive structures known; the former are produced in conceptacles that develop erosively (Rosenvinge, 1917; Pueschel, 1982). Within the genus, species are delineated based on habitat (marine or fieshwater), mode of reproduction (marine species by tetraspores, fieshwater species by gemrnae), ce11 size, thallus thickness, conceptacle size and shape. and tetrasporangial division pattern (Nichols, 1965; Denizot, 1968; Irvine &
Pueschel, 1994). The tetrasporangia of Hildenbrandia are al1 considered to be zonately divided, but two types of basic division pattern are recognizable mine & Pueschel,
1994). Parallel divisions are seen in such species as H. occidentalis Setch. ex N.L.
Gardner and H crouanii (J. Agardh) I. Agardh (Denizot, 1968; Irvine & Pueschel, 1994), while non-pardel divisions, which result fiom one transverse division followed by two oblique divisions, are characteristic of H. rubra (Sommerf.) Menegh. (Denizot, 1968;
Irvine & Pueschel, 1994).
It has been argued that conceptacle size and thallus thickness are not taxonomically useful characters for Xildenbrandia because they Vary with age and development (Denizot, 1968; lrvine & Pueschel, 1994). In addition, the presence or
absence of paraphyses in the conceptacles has been debated (Ardré, 1959; Pueschel,
1982), although some species have been descnbed based on this character (e-g.
dawsonii [Ardré] Hollenb. has paraphyses and H. canariensis Bmgensen does not).
Thus, relatively few reliable morphological characters are available for taxonomie
purposes and other data need to be incorporated into the systematic analyses of this
genus. Currently 12 marine species of HiZdenbrandia and five fieshwater species are
recognized (Denizot, 1968; Abbott & Hollenberg, 1976; Sheath et al-, 1993a; Irvine &
Pueschel, 1994). The fieshwater Xildenbrandia species are morphoiogically similar to
marine representatives, but relationships among these groups are unciear.
Hildenbrandia is widespread in marine habitats in North Arnerica, but is more
restricted in its range in fieshwaters. Marine Hildenbrandia species have been reported
from Alaska (Lindstrom, 1977) to Costa Rica (this study) in Pacific North Arnerica, and range fiom the Arctic (Zee, 1980) as far south as the Caribbean on the Atlantic coast
(Taylor, 1960). HiZdenbrandia occurs in fieshwaters fiom Texas to Costa Rica and is also cornmon in the Caribbean islands (Sheath et al-, 1993a). Freshwater Hildenbrandia has also been reported fiom Ohio (Lillick & Lee, 1934) and Pennsylvania (Wolle, 1887).
In marine habitats Hildenbrandia usually OCCLUS in the intertidal and subtidal regions and
often in the crevices of rocks, on the shady sides of boulders, or in tidepools (Bird &
McLachlan, 1992). Freshwater Hilden brandia species are generally found in tropical to warm temperate streams with high specific conductance values (Sheath et al., 1993a).
Moiecular studies have proven valuable in resolving phylogenetic relationships 24 among groups of red algae. Both the chloropiast rbcL gene and the nuclear 18s rRNA gene have been used extensively at various taxonomic levels within the Rhodophyta (e.g.
Freshwater et al., 1994; Ragan et al., 1994; Müller et al., 1998; Vis et al., 1998).
Previous studies employing sequence analysis have shown Hildenbranrlia to occupy a basal position in the Florideophyceae (Freshwater et al., 1994; Ragan et al., 1994;
Saunders & Bailey, 1997, 1999). A phylogenetic and biogeographic analysis of marine and freshwater Hildenbrandia fkom North America based on rnorphometric and DNA sequence analyses was carried out in order to cl- the relationship among the marine and fieshwater species and to examine patterns of biogeographic variation within the genus.
2.2. Materials and Methods
2.2.1. Collection and identification of materials and DNA extraction
Collection information and identifications based on morphological characters for specimens included in these analyses are shown in Table 2.1 and collection locations are illustrated in Fig. 2.1. The following taxonomic references were used for these identifications: Denizot (1968), Abbott & Hollenberg (1974), Bird & McLachlan (1992) and Sheath et al. (1993a). Freshwater species identification was based on the cnterion of thallus cell dimensions (Sheath et al., 1993a and references therein). Marine species were examined for tetrasporangial division pattern, tetrasporangial appearance, conceptacle size and shape, thallus and ce11 dimensions and the presence or absence of fungal hyphae or paraphyses in the conceptacles. Marine specimens with shallow conceptacles and tetrasporangia with some non-parallel divisions were designated H. rubra (Taylor, 1960). Table 2.1. Collection information for North American specimens of marine and freshwater Hildenbrandia. Collection numbers refer to sarnple codes on map.
Collection Sample number Collection information GenBank Accession Number number and identification rbcL 18s rRNA
AKSW 1 Berner's Bay, AK, USA. Coll, S. Lindstrom, 30 April 1998, AF107811 (H.r~tbra) BCSWl Half Moon Bay, BC, Canada, Coll. R. Sheath and M, Koske, 09 June 1997. (H. rtrbra) BCSW2 Sechelt, BC, Canada. Coll, R. Sheath and M. Koske. 22 March 1998, AF107813 (H. rtrbra)
BCSW3 French Beach, Vancouver Island, BC, Canada. Coll. R. Sheath. 09 May rl (H.rirbra) 1998, BCSW4 Parkesville Bay, Vancouver Island, BC, Canada. Coll, S. Thompson and C, to submit submit (H. occidenfalis) Sarzyzick. 05 October 1 999.
WASWl Beaverton Cove, San Juan Island, WA, USA, Coll. D. Garbary. 30 March 4 (H. rtrbra) 1998. ORSW2 Port Orford, OR, USA. Coll, A, Sherwood and R. Deckert. 10 June 1998, (H. rribra) ORSWl Battlerock, OR, USA. Coll, A, Sherwood and R. Deckert. 09 June 1998. (H,rrrbra) CASW3 Trinidad Harbor, CA, USA. Coll, A Sherwood, R, Deckert and F. (H. occidentalis) Shaughnessey .O9 June 1998.
CASW2 Samoa Boat Launch, CA, USA. Coll. A, Sherwood, R, Deckert and F. (H,rirbra) Shaughnessey, 09 June 1998, CASWl Brook's Island, San Francisco Bay, CA, USA, Coll, R, Moe, 03 February Table 2.1. Continued.
Collection Sample number Collection information GenBank Accession Number number and identification rbcL 18s rRNA
MEXSWl La Bufadora, Baja California, Mexico, Coll, A Sherwood and R, Deckert, - AF108410 (H, rwbra) 04 Juiie 1998. MEXSW2 Bahla de Los Angeles, Baja California, Mexico. Coll. A, Shenvood and R. AFI07823 (H. riibra) Deckert. 05 June 1998. MEXSW3 Todos Santos, Baja California, Mexico. Coll. S. Fredericq, 24 October to submit to submit (H. dmvsonii) 1999.
CRSWl Doininical, Costa Rica. Coll, R, Sheath and K. MUller. 17 February 1998. - " (H,rubra) CRSW2 Stream outflow at Dominical, Costa Rica. Coll, R, Sheath and K. Muller, 20 AF107819 (H. rubra) February 1998. BLZSW 1 water taxi dock at Belize City, Belize. Coll, R, Sheath and M. Koske, 3 1 II - (H. rirbra) December 1999. NFSWl Cape Broyle, NF, Canada. Coll. K. Muller and B. Brace. 22 June 1998, AF 107824 AF108411 (H. rubra)
NSSWl Herring Cove Lookoff, Halifax, NS, Canada, Coll, A. Shenvood and J, AF 107825 AFIO8412 (H, ritbra) Shenvood. 08 February 1998.
MASW 1 Wood's Hole, MA, USA. Coll. R,Searlcs. 29 April 1997, AF 10782 1 AF 1 08409 (H, r~rbra)
RlSW 1 Fort Wetherill, RI, USA. Coll. M, Harlin, 22 June 1998, I (H, nrbra)
CTSW 1 Seaside Point, Waterford, CT, USA. Coll, J, Foertch and R,Wilkes, 27 AF 107820 AFlO8408 (H, rrlbra) January 1998.
Fig. 2.1. Locations of North American marine and freshwater collection sites of Hildenbrandia specimens. Numbers refer to collection numbers in Table 2.1.
30
Specimens with deep conceptacles containing fungai hyphae and bead-like tetrasporangia
with only parallel -divisionswere designated H. occidenfalis (Gardner, 19 17). Although
samples of H: crouanii were not collected during this study, they are reported fiom the
Atlantic coast of North America (South et al-, 1988) and would have been distinguishable fiom H. occidentalis by their shallower conceptacIes and thinner thalli. Specimens with thin thalli, shallower conceptacles containing filaments, but tetrasporangia simïlar to occidentalis were designated H: dawsonii (Hollenberg, 197 1). Eight fkeshwater
Hildenbrandia collections were obtained, ranging fiom Texas and Florida to Costa Rica, and hcluded samples fiom both the Greater and Lesser Antilles of the Caribbean islands.
Twenty-two marine collections were studied, extending fiom Berner's Bay, Alaska, to centra! Costa Rica on the Pacific coast, and fkom Newfoundland to Connecticut on the
Atlantic coast. Field-collected samples were scraped from their substratum with a razor blade. In order to avoid collection of more than one species in a single sample, the material for morp hometrk and molecular analysis was taken fiom a clearly-delineated patch of Kildenbrandin. Samples were later verificd to be of a single species by observation using a compound microscope. For morphometrïc analysis collections were fixed in the field in 2.5% CaC0,-buffered glutaraldehyde. For molecular analyses samples were transported to the laboratory chilled or dried in silica gel. Samples were cleaned of visible epiphytes to prevent contamination, blotted, and frozen at -30°C. DNA extraction procedures followed those of Saunders (1993) with modifications given in Vis
& Sheath (1997).
2.2.2. Morphometric analysis 3 1
Specimens were examined for the following characters in replicates of 10-30
(S heath et al., 1993a) : filament height, basal layer height, diameter and height of erect filament cells, fieshwater or marine habitat, and if reproduction is by tetraspores, the division pattern of tetrasporangia Marine or fieshwater habitat and tetrasporangial division pattern were binary coded as two separate characters. All measwements were made on an Olympus BH- light microscope using either a Cohu 2222-1000/0000 color video carnera with a SnappyTMframe grabber and the SigrnaScan Pro 3.0 software (Jandel
Scientific), or a CoolSnapmf digital carnera with the software package MetaView
(Universal haging). The Gower sirnilarity coefficient was used since it allows cornparisons including both the quantitative and binary-coded data (Dm& Everitt,
1982). Data were ranged according to Gower (1 97 1). Both cluster analysis (UPGMA algorithm) and principal CO-ordinatesanalysis (PCO) were carried out using MVSP
(Multi-Variate Statistical Package; Kovach Computing Services, 1986-88). Means of the resulting clusters were compared with one-way ANOVA using the Minitab statistical package (Ryan er aL, 1985) with a significance level of p c 0.05. Reproductive characters of marine samples, including conceptacle and tetrasporangial dimensions, were measured but included only in a separate andysis of marine samples for which these data were available.
2.2.3. Amplification and sequencing of the rbcL and 18s rRNA genes
Multiple primer combinations were used to ampli@ 1O 1O bp of the rbcL gene of
H'enbrandia, corresponding to positions 394-1404 of the cornplete rbcL sequence for
Anrithamnion sp. (GenBank). The fonvard primers HILFl and HILF2 wexe designed for 32
Hildenbrandio based on conserved regions near the beginning of the rbcL gene
(Appendur 1). The prïmers F 160, rbcLR, COMP 1, and COMP2 were used for some samples (Appendix 2). Gene amplification was performed in a Perkin Elmer Gene Amp
2400 Thermal Cycler as follows: initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation for 1 min at 93 OC,primer annealing for 1 min at 47"C, extension for 4 min at 72°C and a final extension of 6 min at 72" C. The total reaction volume was
97yL, which contained 2yL genomic DNA, 20 rnM each of dATP, dTTP, dCTP and dGTP, 0.4mM of each primer, 2mM MgCl?, 10pL 1OX reaction buffer (Perkin Elmer) and 2.0 units of Taq polymerase. PCR products were visualized on 2% agarose gels stained with ethidium bromide. PCR products were purified for sequencing using the
QIAquick PCR Purification Kit (Qiagen) or the Wizard PCR Prep DNA Purification
System (Prornega), according to the manufacturer's specifications- The double-stranded
PCR products were sequenced in one direction using the AB1 PRISM Taq FS Cycle
Sequencing Ready Reaction Kit (Applied Biosystems) and an AB1 3 77 Automated DNA
Sequencer.
Partial sequences (ca. 1600 bp) of the 18s rRNA gene were amplified for as many sarnples as possible (Table 2.1). The region was arnplified in two overlapping pieces, employing the primer pairs HF 18S.2 and G08.1, and G04.1 and GO7 (Appendix 1).
Seven of the marine Hildenbrandia specirnens analysed were determined to contain a group ICI intron at position 1506 of the 18s rRNA gene, as previously reported by Ragan et al. (1993). These data were excised from the gene sequences before Meranalysis.
Amplification procedures for the 18 S rRNA gene were the same as for the rbcL gene, 33 except that an annealing temperature of 55 OC was employed.
Sequence data of the rbcL and 18s rRNA genes were submitted to GenBank, and the accession nurnbers are listed in Table 2.1 - The coding of sarnplcs on the phylogenetic trees and morphometric dendrograms indicates both species designation by morphology and collection location. Standard state and provincial abbreviations were used, with a few exceptions: CR = Costa Rica, MJ3X = Mexico, PR = Puerto Rico and SL = St. Lucia.
"SW" indicates sait water or marine collections. Nurnbers following the letter abbreviations refer to the collection location within the geographical area.
2.2.4. rbcL and 18s rRNA gene analyses
DNA sequencing software (ABI) was used to assemble the sequences for both genes- Since the rbcL gene in red algae contains no insertions or deletions these sequences were easily aligned. Sequences of the 18s rRNA gene were aligned according to secondary structure models using DCSE (Dedicated Comparative Sequence Editor; De
Rijk, 1993). Regions of the 18s rRNA gene that could not be aligned unarnbiguously were eliminated fiom the analysis. Parsirnony trees for both the rbcL gene and the 18s rRNA gene were generated in PAUP *4.0 (Phylogenetic Analysis Using Parsimony;
Swofford, 2000) using the heuristic search option and random addition (100 replicates), steepest descent and tree bisection-recomection (TBR) branch-swapping, Each base was equally weighted in both the rbcL and 18s rRNA gene parsimony analyses. Neighbor- joining trees were constructed using PHYLIP (Phylogeny Inference Package; Felsenstein,
1993) from a Kirnura two-parameter distance rnatrix (Kimura, 1980). A transition / transversion ratio of 2.0 and a single category substitution rate were selected. Quartet 34
puPling maximum likelihood reconstruction was carried out using the puzzle function in
PAUP *4.0 (Swofford, 2000) as described by Stnmmer & von Haeseler (1996) with 1000
puplhg steps. Sequence statistics were estimated fiom the aliments using the
program MEGA (Molecular Evolutionary Genetics Analysis; Kumar et al-, 1993).
Bootstrap resarnpling (2000 replicates) was run for both the parsimony and distance data.
Decay analysis for the parsimony tree was carried out using AutoDecay (Eriksson, 1997),
following the protocol of Bremer (1988).
Phylogenetic analyses were performed using several outgroup taxa as described by
Swofford et al. (1996) (Bangia sp. CAF043366 rbcL; AF043365 18s rm,
Eryrhrotrichia camen (Dillwyn) J. Agardh [MO87 118 rbcL; L26 189 18s rRNA],
Porphyra carolinensis Coli et Cox PO4041 rbcL],Porphyra kuniedai Kurogi
[Al?12305 1 Z 8s rRNA], Srnithora naiad2tm (Anderson) Hollenb. [AF087119 rbcL;
MO87126 18s rRNA] and Porphyridiunz aerugineum Geitler D(17597 rbcL; L27635
18s rRNA]).
In consideration of the fiequent presence of fungal endophytes in the thalii of
Hildenbrundia species and the possible problems of contamination due to the crustose
habit of the aiga, al1 consensus sequences for both the rbcL and 18s rRNA genes were
checked by a "BLAST" search to the GenBank database to confirm that they were indeed
red algal sequences. In addition, the amplification of both the rbcL and 18s rRNA genes
in several discrete fragments using red algal-specific prirners helped to ensure that
contamination was not a factor.
2.3. Results Fig. 2.2. CIuster dendrogram of North American Hildenbranaia collections based on vegetative characters, habitat (marine versus freshwater) and tetrasporangial division pattern. The numericai scale indicates similarity among clusters according to the Gower similarity coefficient. Group A includes al1 freshwater specimens and Group B includes al1 marine specimens. The freshwater group is denoted by a gemma symbol (cluster of cells) and marine specimens are denoted by a tetrasporangial illustration with the corresponding division pattern.
Fig. 2.3. Principal CO-ordinatesbiplot of North Amencan HiZdenbrandia specimens, using the same characters as in Fig. 2.2. Group A includes all fieshwater specimens and Group B includes al1 marine specimens. The fieshwater group is denoted by a gemma symbol (cluster of cells) and marine specimens are denoted by a tetrasporangial illustration with the corresponding division pattern.
2.3.1. Morphometnc Analysis
The cluster and PCO analysis based on vegetative characters, coding for marine versus fieshwater habitat and tetrasporangial division pattern distùiguished two groups of
Hildenbrundia specimens among the North Amencan coliections (Figs. 2.2 & 2.3). One- way analysis of variance indicates that these groups are signincaotly different: Group A contains al1 of the freshwater samples, which are separated firom the remaining samples by producing gemmae as their only reproductive structures. In addition, the fieshwater samples have significantly shorter filaments @ < 0.003) and smaller basal layer height @
< 0.00 1) than the marine samples (freshwater filament 1engt.h = 26.0 - 61.7pm, marine filament height = 50.6 - 335pm; fieshwater basal layer height = 3.6 - 6.1pm, marine basal layer height = 7.0 - 15.9pm) (Table 2.2). Group B contains al1 the marine Hildenbrandia specimens (H rtrbra, H. occidentalis and H. cimvsonii)-
The second set of rnorphometnc analyses, based on vegetative and reproductive characters of the marine collections, distinguished two groups (A & B) of specimens
(Figs. 2.4 & 2.5). Group A contains the marine specimens with tetrasporangia that have parallel cleavage (H. dmvsonii and H. occidentalis), while Group B contains the marine specimens with tetrasporangial divisions that are not al1 parallel (H. rzrbrn). These two groups are significantly different based on filament height and conceptacle dimensions.
Group A has signifïcantly longer filaments (Z = 241pm versus 109pm), wider conceptacles (x = 93.5pm versus 72.3pm) and deeper conceptacles (n = l32pm versus
72.3pm) than Group B.
2.3.2. Analysis of transitional saturation of the rbcL and 18s rRNA genes Table 2.2. Means and ranges (in parentheses) of characters used in morphometric analyses of marine and freshwater Hildenbrandia from North Amer ka,
Morphomettic cell ceII length filament basal layer conceptacle conceptacle tet, tet, reproductive tet, grOUP diameter (pm) tieight height (pm) diameter depih diameter length body division (l-4 Olm) (pm) (w.0 (PO pattern - - Group A 4.4 - gemmae NIA (H (3,6-6,1) angoleru is)
Group B.i. 8,9 93.5 132 6.7 23,3 tetrasporangia "crouanii (marine (7,9- 10.5) (73.4- 1 13) (106- 15 1) (4.9-7,7) (20.7- -likeH gtoup 1) 258)
Group B.ii. 10.4 72.3 72.2 8,5 24.2 tetrasporangia "rubra- 8 (marine (7.0- 15.9) (5 1.9- 102) (47.4- 1 12) (5.7- (1 7.6- 1i ke" group II) 12.1) 32.4) Fig. 2.4. Cluster dendrogram of North American marine Hihienbrundia samples, based on both vegetative and reproductive characters. Numerical scale indicates similarity according to the Gower similarïty coefficient. Group A includes aiI marine specimens with paralle: teîrasporangial cleavage and Group B includes al1 marine specimens with some non-parallel divisions. The marine specimens are denoted by a teîrasporangial illustration with the corresponding division pattern.
Fig. 2.5. Principal CO-ordinatesbiplot of North Amencm marine Hdldnbrandia samples, based on both vegetztive and reproductive cliaracters. Group A inc ludes aI1 marine specimens with parallei tetrasporangiaI cleavage and Group B includes d marine specimens with some non-paraIlel divisions. The marine specirnens are denoted by a tetrasporangial illustration with the corresponding division pattern.
Fig. 2.6. a) Graph of the number of transitions versusp-distances for al1 pairs of North American Hildenbrandia samples for the rbcL gene- Transitional saturation appears to occur at approxhately 15% sequence divergence.
b) Graph of the nurnber of transitions versus p-distances for dl pairs of North Amencan Nildenbrandia samples for the 18s rRNA gene. Transitional saturation does not occur in these samples. 46 Hildenbrandia rbcL gene (North America)
0.00 0.05 0.10 0.15 0.20 0.25 p-distances
Hildenbrandia 18s rRNA gene (North America)
0.00 0.02 0.04 0.06 0.08 0.10 p-distances 47
The painvise p-distances (uncorrected sequence divergences) were plotted against
the corresponding number of transitions between pairs of sequences for both the rbcL
(Fig. 2-6 a) and 18s rRNA (Fig. 2.6 b) genes of North American collections of
Hildenbrandia. Although sorne flattening of the curve for the rbcL gene occurs at
approximately 15% sequence divergence, no flattening of the curve was apparent for the
data based on the 18s rRNA gene. Thus, the rbcL gene is transitionally saturated for the
North American Hildenbrandia data, Phylogenetic reconstmction was nonetheless
attempted for the rbcL data set, with interpretations limited to the least divergent sets of
samples.
2.3.3. rbcL distance, parsimony and quartet puzzling analyses
A final alignment of 10 10 bp was generated for the rbcL gene of the
Hildenbrandia collections. Parsimony analysis of 3 03 phylogenetically informative
characters generated two most parsimonious trees with a consistency index (CI) of 0.46
and length of 1237 steps, one of which is shown here (Fig. 2.7). The two trees differ only
in the positioning of the outgroups with respect to one another (ie. positioning of Bangia
+ Porphyra and Erythrotrichiu + Srnithora). This tree strongly resembles the neighbor- joining tree generated by distance anaiysis (Fig. 2.8). In general, terminal clades are well
supported in the analyses; however, the support is much lower for intemal nodes of the
tree. The quartet puzzling maximum likelihood tree had the same topology as the
parsirnony tree and thus is not show, These analyses indicate that the fieshwater
samples of Hildenbrandia (K. angolensis) are paraphyletic with respect to the marine
samples. For the marine samples, clades represent biogeographic groupings for the most Fig. 2.7. One of two most-parsimonious trees generated fiom North American Hildenbrandia data for the rbcL gene (CP0.46; Iength= 123 7 steps). Group A includes Atlantic marine samples and Group B inchdes Pacific marine samples. BCS W4 and CASW3 correspond to K- occidenialis and the remaining ingroup samples are fkom fieshwaters. AKSWl & NFSWl
Bangia
Pophyra Fig. 2.8. Neighbor-joining tree based on distance analysis of North American samples of Hildenbrandia for the rbcL gene. Group A includes Atlantic marine samples and Group B includes Pacific marine samples. BCS W4 and CAS W3 correspond to H occidentalis and the remaining ingroup samples are fkom fkeshwaters. I 64 66 AKSWI & NFSWI
C MASWI IO0 ,
1- 1- Bangia 1 û0 I Po rphyra
Srnithora 99- - 1 Erythrotrichia 52 part. One clade (A) (97-1 00% bootstrap proportion PP]; decay 13 steps) contains ail of the H rubra samples fiom the North Atlantic, and also uicludes the sample of this species collected eom Berner's Bay, Alaska. Within this clade NFS Wl and AKS W 1 are identicai. Clade B contains samples of H. rubra from the Pacific coast of North America.
The two collections of H occidentalis (BCSW4 & CASW3) are identical in sequence for the rbcL gene and thus are collapsed in the analyses. The two fieshwater sarnples fiom
Costa Rica also group together strongly (100% BP; decay 39 steps). Bootstrap suppoa was generally higher for the neighbor-joining tree than for the parsimony tree.
Percent sequence divergence values for the rbcL gene among samples of
Hildenbrandia were extrernely high. For example, the sarnple from Alaska differed by
18.4% from the CRSW2 sample. Divergence ranged from O - 25.8% within the genus, as compared to 10.2 - 24.7% among the outgroups and 21 -4 - 3 1.3% among the outgroups and the samples of Hildenbrandiu.
2.3.3. 18s rRNA gene distance, parsimony and quartet puzzling analyses
A fmal alignment of 1440 bp was generated for the 18s rRNA gene regions of the
HiZdenbrandia collections. The Group IC 1 intron previously reported by Ragan et al.
(1993) for H. rubra was detected in approximately half of the H. rubra sarnples examined, but was not present in any of the fieshwater collections.
Parsimony analysis of 41 1 phylogenetically informative cl~aractersgenerated five most-parsimonious trees for the 18s rRNA gene of HiZdenbrundiu (CI = 0.65, length =
1016 steps) (Fig. 2.9), of which a strict consensus is shown. Intemal nodes are weakly Fig. 2.9. Strict consensus of five most-parsimonious trees generated fkom North American Hildenbrandia samples for the 18S rRNA gene (CI=O-65; lengtkl0 16 steps). Group A indudes fieshwater collections (H. angolensis) as well as two marine H. occidentalis samples (BCSW4 & CAS W3) and one marine H dawsanii sample (MEXSW3). Group B includes Atlantic marine samples (H. rubra), and Group C includes Pacific marine samples (H rubra). AKSWI NFSWI
MASWl IB
Bangia
Erythrotnchia
Srnithora
Porphyndium Fig. 2.10. Neighbor-jolliing tree based on distance analysis of North Amencan Hildenbrandia samples for the 18s rRNA gene. Group A includes fieshwater collections (H angolensis) as well as two marine H. occidentalis samples (B C SW4 & CAS W3) and one marine dawsonii sample WXSW3). Group B includes Atlantic marine samples (H rubru), and Group C includes Pacific marine samples (H rubra). Erythro trichia Srnithora 57 supported; however, relationships among terminal taxa are much better supported. The neighbor-joining tree (Fig. 2.10) closely resembles the parsimony tree, at least with respect to parts of the parsimony tree that are well resolved. Quartet puzzling araalysis yielded a tree with the same topology as the parsimony tree, and thus is not shown-
The topologies of the 18s rRNA gene eees differ considerably from those of the rbcL gene trees. The fkeshwater samples of H. arzgolensis are grouped much closer together in the 18s rRNA gene analyses and would form a monophyletic group except for the unresolved position of the H. occidentalis sarnpIes (BCSW4 & CASW3) and the H. dwsonii sample (MEXSW3) within clade A; albeit with little support (< 50% BP in parsimony; 79% BP in distance analysis). An Atlantic coast group of H rubra samples, containing NSSwl, MAS Wl and CTS W 1, is delineated here in the distance analysis (B), as was observed in the rbcL gene analyses, but is unresolved in the parsirnony ariia1ysis.
The NFS Wl and AKS W Z H- mbra sarnples again are closely associated (100% BP: decay 9 steps), but they are not grouped with the remainder of the east coast collections.
A Pacific grouping cm be distinguished from these analyses (C), with the exceptiion of
ORSW2.
The neighbor-joining tree shows that the outgroups are much more distana &orn the major@ of the HiZdenbrandia samples than the Hildenbrandia sequences are fiom each other. Sequence divergence values of the 18s rRNA gene for samples of
Hildenbrandia were much lower than for the rbcL gene, ranging from O - 9.7%.
2.4. Discussion
Many authors have used DNA sequence analysis of the red algae to help 58
understand phylogenetic relationships among various groups at the ordinal, familial,
generic and specific level. Although some studies employing both morphological and
molecular data have dernonstrated agreement among data sets (e-g.Freshwater &
Rueness, 1994; Hommersand et al., 1994), incongruency has been also recently reported
in the phycological literanire (e-g. Saunders & Bailey, 1997; Müller et al., 19%). For
example, the relationship between Bangia and Porphy-a is cmentiy uncIear; the two
genera are easily distinguished based on vegetative morphology, but molecular studies thus far have indicated paraphyly of Porphyra within Bangia (Oliveira et aL, 1995;
Müller et a[., 1998). The patterns discerned among the three data sets used in the
analyses of Hildenbranaia (rbcL gene sequences, 18s rRNA gene sequences and
morphometric measurements) do not closely resemble one another, with few trends
appearing in more than one form of analysis. Although as many different collections
were made as possible in North Amenca, limitations of accessibility produce the possibility of missing some genotypic diversity. For example, no specimens attributable to H crouanii were collected during the study. However, the coIlections cover a large
geographical range of both marine and fkeshwater Hildenbrandia in North America-
The levels of sequence divergence observed for both the rbcL gene (O - 25.8%)
and the 18s rRNA gene (O - 9.7%) for the samples of Hildenbrandia analysed are high
within-genus values. These data imply that Hildenbrandia is an ancient red alga, as
speculated in previous molecular phylogenetic studies (Freshwater et al., 1994; Ragan et
al., 1994; Saunders & Bailey, 2997). For the rbcL gene, maximum idiageneric
divergence values of 7.2% were reported by Hommersand et al. (1994) for members of 59 the family Gigartinaceae, and 16% among populations of Bangia by Müller et al. (1998).
In terms of the 18s rRNA gene, maximum sequence divergences of 15% have been reported between species of Porphyra (Oliveira et al,, 1995) and 10.6% between populations of Ban* (Müller et al., 1998). Thus, it appears that the genus
Hildenbrandia is anomdous in its extremely high levels of ïnfragenerïc sequence divergence for the rbcL gene, but well within the reported range for the 185 rRNA gene.
Both the rbcL and the 18s rRNA gene sequence analyses indicated that clades containing Atlantic samples can be distinguished fkom those containing Pacific samples.
Al1 of the Atlantic specimens, which are identifiable as H. rubra, fa11 within a cold temperate group according to Lüning (1990). In contrast, the Pacific samples of this species and H. occidentalis were collected fiom botli the cold and the warm temperate coastal regions, as defined by Lüning (1990); they are separated to some degree fiom each other in our sequence analyses. One interesting biogeographic result is the positioning of the Alaskan sample in the Atlantic group of H rubra. This sarnple grouped with the H. rubra sample fiom Newfoundland in ail analyses with strong (100% BP; decay 9 steps) support. Trans-Arctic invasions by red algae have been hypothesized based on nuclear intemal transcribed spacer regions of the rRNA gene (ITS 1 and ITS2) for Phycodrys rubens (L,.) Batters (van Oppen et al. 1995) and PalmariapaZmata (L.) Kuntze
(Lindstrom et al.,1 W6), and for Bangia based on rbcL and 18s rRNA gene sequences by
Muller et al. (1 998). The low sequence divergence between the Alaskan and
Newfoundland samples may thus reflect a trans-Arctic interchange of H. rzrbra.
However, it would be necessary to study material fiom several Arctic locations to fùlly test this hypothesis.
The rbcL and 18s rRNA gene sequence analyses do not adequately clarify the relationship between marine and fieshwater Hildenbrandia. It is possible that the fieshwater forms represent repeated invasions by marine species into freshwater habitats
(Skuja, 1938). If various postulated mechanisms of invasion are considered (emg vector- assisted transport, long-term spread up estuaries, embayment after continental uplifting;
Sheath, 1984), the possible migration of Hildenbrandia inland by vector-assisted transport seems to be the most likely mechanism considering its distribution in isolated springs on continental North Arnerica, such as those in Texas. In contrast, long-term spread may account for fieshwater populations in subtropical areas and the Caribbean
Islands. Other potential invaders from brackish habitats into Central American and
Caribbean streams have been identified, including members of the rhodophyte order
Ceramiales (Sheath et al-, 1993b). Embayment of coastal areas to form inland seas or estuarine environments does not seem likely since fieshwater Hildenbrandia in North
Amerka is restricted to fast-flowing Stream systerns (Sheath et al., 1993a). The paraphyly of fieshwater Hi!denbrandia in the rbcL gene analyses supports the concept of multiple invasions by different populations. Although the fieshwater collections form a monophyletic group in some of the 18s rRNA gene analyses, the unresolved nature of most of the marine sarnples in these trees makes the group of freshwater sarnples diffrcult to cori-E~rm.These invasions could have occurred by the transport of Hildenbrandia by various animals, including waterfowl (Coleman, 1996) and insects (Kristiansen, 1996).
Interestingly, despite the large corrected sequence divergence values among collections of 61 fkeshwater Hildenbmdia (0.4 - 18.3% for rbcL and 0.6 - 6.8% for 18s rRNA), morphologicaily and morphometrïcaiiy the samples al1 correspond to a single species, H angolensis, as was determined by Sheath et al. (1993a).
The separation of the three marine species analysed, H. occidentalis, H. rubra and
H. dawsonii, is only partially supported by our analyses due to the poor resolution of the
18 S rRNA trees, where the H. dawsonii sample was included (this sample was not included in the rbcL analyses due to PCR amplification failure). Researchers have questioned the validity of many of the characters that have been used in the past to distinguish species of Hildenbrandia (cg. Ardré, 1959; Denizot, 1968; lnine & Pueschel,
1994). Our fmdings fiom the molecular analyses indicate that some of these characters
(e-g.tetrasporangial division pattern) may need to be examined in more detail to evaluate their utility in making taxonomie distinctions. Furthermore, additional samples of H occidentalis, H dawsonii and H crouanii need to be examhed to ciarifi the relationship of these species to H. rubra.
While the taxonomy of North Arnerican Hildenbrandia remains unresolved, this study has demonstrated several key trends: marine clades are correlated with geography
(ocean basin), at least to some degree, tram-Arctic invasion may have occurred, and multiple invasions into fieshwaters are consistent with the rbcL analyses.
2.5. Literature Cited
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Ardré, F. (1959). Un intéressant HiZdenbrandtia du Portugal. Revue AlgoIogrque 4: 227- 237, Bird, C.J. & McLachlan, J-L. Z 992. Semeed FZora of the Maritimes. 1. Rhodophyta - The Red Algue. Biopress Ltd., Bristol, 177 pp.
Bremer, K. 1988. The Iirnits of amino acid sequence data in angiospenn phylogenetic recons~ction.Evolution 42: 795-803.
Coleman, A. W, 2 996. Are the impacts of events in the earth' s history disceniable in the current distributions of fieshwater algae? In: Biogeography of Freshwater Algae (Ed- J. Kristiansen), Hydrobiologia 336, Kluwer Academic Publishers, Dordrecht, pp. 137-142,
Denizot, M. 1968. Les Algues Floridees Encroutantes (à Z 'excltaion des CoraZlinacées). Museum National d'Histoire Naturelle, Paris, 3 10 pp.
De Rijk, P. 1993. DCSE Dedicated Comparative Sequence Editor. An Interactive Tool for Sequence Alignmenr and Secondary Strz~ture Research. Universiw of Antwerp WA), Antwerp.
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Freshwater, D.W. & Rueness, J. 1994. Phylogenetic relationships of some European Gelidium (Gelidiales, Rhodophyta) species, based on rbcL nucleotide sequence analysis. Phycologia 33: 187- 194.
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CHAPTER 3: Biogeography and systematics of Hilden brandia in Europe: infereuces
from morphometrics and sequence analyses of the rbcL and 18s rRNA genes
3.1. Introduction
Since the crustose red algal genus Hildenbrandia was originally described fiom
Europe (Venice, Itaiy) by Nardo in 1834, a plethora of taxonornic names have been proposed, many of which (e.g. H- nardi ZanardÙii, N. paroliniana Zanardini, H. rosea
Kütz. and H sanguines Kütz.) were eventually placed in synonomy with the type species,
H. rubra (Sommerf.) Menegh. (Denizot, 1968; Silva et aL, 1996). Currently, 12 marine species of Hildenbrandia are recognized (summarized in Chapter l), of which three are reported firom European marine habitats: H. rubra, H crouanii (J. Agardh) J. Agardh and
H. occidentalis var. lusitanica Ardré (Ardré, 1 959; Inrine & Pueschel, 1994). These three species are distinguished based on their planes of tetrasporangial division, thallu thickness and conceptacle size and shape. H. rubra and H. crouanii are reported to be very similar, except that the tetrasporangia of H crouanii possess ody parallel divisions and those of H rzrbra do not. H occidentalis var. Zusitanica has tetrasporangia similar to
H. crouanii (divisions dl parallel), but deeper conceptacles and a thicker thallus (Ardré,
2959; Denizot, 1968). The most commonly reported fieshwater species in Europe is iY rivularis (Liebm.) J. Agardh, which also includes two subspecific taxa (H. rivuluris ssp. chalikophila Palk and H. rivularis var. drescheri Lingelsh.) (Lingelsheim, 1922; Palik,
1961). Recently, the common fieshwater species in North America, H. angolensis Welw. ex W. West et G.S. West, was reported for the first time fiom Europe in Spain (Ros et al.,
1997). 68
In European fieshwaters Hildenbrda has been reported fkom the British Ides
(e. g. West & Fritsch, 193 2; Raven et al., 1994), Scandinavia (e-g.Israelson, 1942;
Nichols, 1965) and in most regions of continental Europe (eSg Starmach, 1984;
Hofiam, 1987). Marine Hildenbrandia species have also been collected fiom most regions of Europe, including the British Isles @vine & Pueschel, 1994), Scandinavia
(Rosenvinge, 19 17), France (Agardh, 185 l), My(Nardo, 1834) and Portugal (Ardré,
1959). Although the fieshwater Hildenbrandia species are morphologically simila. to the marine species, the relationship between the two remains unclear, It is possible that the fieshwater populations represent invasions by marine foms into inland habitats (Skuja,
1938). DNA sequence analyses of marine and fieshwater Hildenbrandia representatives fiom North Amenca demonstrated a clear distinction between the two kinds using some analyses (Chapter 2; Sherwood & Sheath, 1999). However, H angolensis, the freshwater
Hildenbrandia species in North America, was paraphyletic among the marine collections for the rbcL gene (this relationship was unresolved for the 18s rRNA gene), which does not indicate that a single invasion event established the freshwater populations on that continent.
En this study we employ DNA sequence and morphometric analyses to examine the p hy logenetic and biogeo graphic patterns of marine and fkeshwater Hilden brandia in
Europe, and to compare our findings to those of our previous study (Chapter 2; Sherwood
& Sheath, 1999). In addition, because the freshwater Hildenbrandia foms are largely distinct between Europe (H. rivztlaris)and North America angolensis), this study provides greater opportunity to clarïfjr the relationship between the rnarine and fieshwater 69 species and to examine the possible origins of fieshwater populations.
3.2. Materials and Methods
3.2.1. Sample collection and DNA extraction
Collection uiformation and identifications based on morphological characters for specimens included in the analyses are shown in Table 3.1 and collection locations are illustrated in Fig. 3.1. The following taxonomie references were used for identifications:
Rosenvinge (19 17): Denizot (1 968), Starmach (1 969) and Irvine & Pueschel (1 994).
Freshwater species identification was based on the criteria of thailus ce11 dimensions and presence of gemmae (Sheath et al. 1993; Chapter 2 this thesis; Sherwood & Sheath,
1999). Al1 fieshwater samples collected corresponded morphologically to a single species, H rivularis. Marine sarnples were examined for tetrasporangial appearance and division pattern, conceptacle size and shape and thallus and ce11 dimensions. Marine specimens with shallow conceptacles (up to 80 ,um deep) and tetrasporangia with divisions not al1 pardel were designated H rubra (Denizot, 1968; Abbott & Hollenberg,
1976). Specimens with slightly deeper conceptacles (up to 110 j.m deep) and tetrasporangia with parallel divisions only were designated H. crozranii (Denizot, 1968;
Irvine & Pueschel, 1994). No specimens corresponding to occidentalis var. lusitanica were collected during this study. Eleven fieshwater Hildenbrmdia collections were obtained fiom locations in Ireland, Wales, Gemany, France, Austria, Spain and Italy
(Table 3.1, Fig. 3.1). Ten marine sarnples were collected from Sweden, Nonvay, Wales,
Scotland, Northern Ireland, Germany, the Netherlands, and France. Samples were collected as described în Chapter 2. DNA extraction procedures were performed using CIm OQcc; zzA- eO39 .ss x-
Fig.3.1. Locations of European marine and fieshwater collection sites of Hildenbrandia specimens. Numbers refer to collection numbers in Table 3.1.
74
either the protocol described in Saunders (1993) with modincations given in Vis &
Sheath (1997), or with a Qiagen DNeasy Plant Mini Kit.
3.2.2. Morphometric Analysis
Morphometric anaiysis of the European Hildenbrandia samples was performed as
descnbed in Chapter 2. Reproductive character data codd ody be obtained for several
marine European samples. Therefore, only analyses based on vegetative characters,
marine versus fieshwater habitat and tetrasporangid division pattern were carried out.
3.2.3. Amplification and sequencing of the rbcL and 18s rRNA genes
Several primer combinations were used to ampliS. 1010 bp (base pairs) of the
rbcL gene of as many collections of Hddenbrandia as possible, corresponding to
positions 394-1404 of the complete rbcL sequence for Antithamnion sp. (GenBank). The
primer pairs HILF2 and rbcLR or HILFZ and COME'2 were used to amplify the rbcL gene
(Appendix 1). Partial sequences (ca. 1600 bp) of the 18s rRNA gene were arnplified for
as many samples as possible using the primer pairs described in Chapter 2, except that the
3' reverse primer was GIS. 1 (which terminated amplification of the fragment just
upstream of the intron reported for some marine collections in Chapter 2). The intemal
reverse primer G14 (Saunders & Kraft, 1994) was also employed in some reactions
(Appendk 1). Amplification procedures followed those in Chapter 2.
Sequence data generated of the rbcL and 18s rRNA genes were submitted to
GenBank, and the accession numbers are listed in Table 3.1. The coding of samples on
the phylogenetic trees and rnorphometric dendrogams indicates species designation by
morphology and collection location. The letters "SW" are used to denote marine collections.
3.2.4, rbcL and 18s rRNA gene sequence analyses
Analyses of the rbcL and 18s rRNA genes were carried out as described in
Chapter 2. Support for nodes on parsimony trees was determined using both bootstrap resampling and decay analysis (Bremer, 1988; Eriksson, 1997). Outgroups were selected and used in the phylogenetic analyses as described in Chapter 2- The following North
Amencan KiZdenbrandia representatives for the rbcL gene were added for phylogenetic context in both the parsimony and distance analyses (Chapter 2): AKSW 1 (AF 10781 l),
CASWl (AFlO78 l4), CASW3 (Ml078 l5), BCSW4 (to submit), CTSWl (AF107820),
CR20 (AF107816), CR24 (AF107817), MASWl (AF107821), NFSWl (AF107824),
NSS W 1 (Al? 107835) and ORSW2 (AF 107826)- The following North American
Hildenbrandia representatives for the 185 rRNA gene were also employed in the analyses
(Chapter 2): AKS W1 (AF108399), BCSWl (AF108400), BCSW4 (to submit), CASW3
(AF lO8402), CASW 1 (AFlO84O l), CTSW1 (AF1O84O8), CR20 (AILlO84O4), MAS W l
(AF lO8409), NFSW1 (A.10841l), NSSWl (AF108412), SL9 (AF108416), TX7
(AF108417) and TX9 (AF108418).
3.3. Results
3.3.1. Morphometric Analysis
Cluster and PCO analyses distinguished two groups of specimens among the
European Hildenbrandia collections (Figs. 3 -2 & 3 3). One-way analysis of variance indicated that these two groups are significantly different based on the characters of ce11 height and ce11 diameter @ < 0.00 1) (Table 3.2). No significant differences were Fig. 3.2. Cluster dendrogram of European marine and fieshwater HiZdenbrandia specimens. The numerical scale indicates similarity according to the Gower similarity coefficient. Group A contains al1 marine samples and Group B contains al1 freshwater samples. Marine specimens are denoted by tetrasporangial illustrations with the corresponding division pattern. SCOSWl SCOSW3 - 1 scosw4 GERSWI marine specimens B FRASWI NORSWI 8 and 1 SWESWl 8 NlSWl NEDSWl WALSW3 FRAI ITAI GER1 SPA1 SPA2 freshwater specimens AT1 5 AT14 AT1 O WAL2 WAL3 IR11
Gower General Similarity Coefficient Fig. 3.3. Principal CO-ordinatesbiplot of marine and fieshwater Hildenbmdia collections fiom Europe. Group A contains al1 marine samples and Group B contains aii fieshwater samples. Marine specimens are denoted by tetrasporangial illustrations with the correspondhg division pattern.
Table 3.2. Means and ranges (in parentheses) of characters used in morphometric analyses of marine and freshwater Hildenbrandia from Europe.
-- Morphoinetric ce11 diameter cell length filament height basal layer reproductive tetrasporangial Group (~m) @ln) (~m) lieight (pm) body division pattern
Group A 3.6 4,2 82.1 9.6 ' tetraspores both "rubra- (H. rubra & H. (2.9 - 4.3) (3.6 - 5.0) (45.1 - 120) (6.0 - 12.5) like" and crouanii) "crouanii-like" Group B 6.2 7.0 70.9 10.2 gemmae NIA (H.iivularh) (4.4 - 7.5) (5.8 - 8.4) (48.9 - 108) (5.2 - 15.6) 81 determined in the filament height (p < 0.30) or the basai layer height @ < 0-66) of the two groups (Table 3.2). Measurements of the groups of specimens indicated that the two marine species, H rubra and H. crouanii (Group A), had much smaller ce11 dimensions than the fieshwater species, H. rivularis, and no overlap in these dimensions was observed (Table 3.2). Gïoup B contained al1 of the fieshwater HiZdenbrandia specimens
(H. rivularis), and was separated fiom the other group by its freshwater habitat, Lack of tetrasporangia and larger ce11 dimensions (H. rivularis ce11 diameter = 4.4 - 7.5pm and ce11 length = 5.8 - 8.4pm; marine Hildenbmndia ceil diameter = 2.9 - 4.3pm and ce11 length = 3.6 - 5.Oprn). Within Group A, specimens with different tetrasporangial morphologies were not significantly different based on any characters (Figs. 3 -2 & 3.3).
3.3.2. Analysis of transitional saturation of the rbcL and 18s rRNA genes
The painvise p-distances (uncorrected sequence divergences) were plotted against the corresponding nurnber of transitions between pairs of sequences for both the rbcL and
18s rRNA genes of European collections of Hildenbrandia (Fig. 3.4 a, b). The graphs for both genes appear to fonn straight lines; thus, transitional saturation does not appear to be occurring in either gene for the European collections of HiZdenbrandia, and both genes were empioyed in phylogenetic reconstruction.
3.3.3. Parsimony, distance and quartet puzzling analyses of the rbcL gene
A £inal alignrnent of 10 10 bp was generated for the rbcL region of the
Hilaenbrandia collections. Of these 1 O 1O positions examined 623 were invariant, 305 were phylogenetically informative and 82 were autapomorphies. Parsimony analysis of the rbcL gene yielded one most-parsimonious tree with a length of 1526 steps and a Fig. 3.4. a) Graph of the number of transitions versus p-distances for al1 pairwise combinations of European KiZdenbrandia samples for the rbcL gene. No transitional saturation is evident in the data set.
b) Graph of the number of transitions versus p-distances for al1 pairwise combinations of European Hildenbrandia samples for the 18s rRNA gene. No transitional saturation is evident in the data set. 83 Hildenbrandia rbcL gene (Europe)
0.00 0.05 0.10 0.1 5 0.20 0.25 p-distances
Hildenbrandia 18s rRNA gene (Europe)
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 p-distances Fig. 3.5. Single most-parsimonious tree generated fiom European Hildenbrandia sarnples for the rbcL gene (CI = 0.44, Iength = 1526 steps). Freshwater specimens are denoted by a gemma illustration (cluster of cells) and marine specimens are denoted by a tetrasporangial illustration with the corresponding division pattern. H. rivulans (Austria 15, France 1, ltaly 1, Spain 1, Wales 3) H. rivularis (Spain 2) H. rivularis (Ireland 11) 2 93 H- rivularis (Gemany 1) 6 1oc H, nVularis (Austria 14, Wales 2) 14c H. angolensis (Costa Rica 20) H. angolensis (Costa Rica 24) 1 H occidentalis (California SW3 8 British Columbia SW4) H. mbra (Oregon SW2) H. mbra (California SW1) H. rubra (France SWI) H rubra (bilales SW3) aqrH. rubra (Norway SW1) H mbra (~or~ieklreland SWI, ~assachkettsSW1) H. mbra (Netherlands SW1) H mbra (Sweden SW1) -H. mbra (Connecticut SW1, ii-1 ii-1 1;; 5 Nova Scotia SWI) H. crouanii (Scotland SW4) H. rubra (Alaska SW1, Newfoundland SWI) 2 97 H. crouanii (Scotland SWI) 1 n I U - H. crouanii (Genany SW1) Bangia
86 Srnithora Erythrotnchia Fig. 3.6. Neighbor-joining tree generated by distance analysis of European marine and fieshwater Hildenbrandia samples for the rbcL gene. Freshwater specimens are denoted by a gemma illustration (cluster of cells) and marine specimens are denoted by a tetrasporangial illustration with the correspondhg division pattern. H. rivularis (Austria 15, France 1, ltaly 1, Spain 1, Wales 3)
H. fivulaRS (Spain 2) H. rivularis (Germany 1) H. rivularis (Ireland II) @Dl00 1 H rivularis (Austria 14, Wles 2) I H. angolensis (Costa Rica 20) H. angolensis (Costa Rica 24) H-occidentalis (Califomia SW3 & British Columbia SW4) 94 H. mbra (Oregon SW) 1O0 H. rubra (California SW1) 8 84 W. nibra (France SW1) I 1H. rubra (WaIes SM) nql- H. mbra (Norway SW1) H. mbra (Northern Ireland SW1, Massachusetts SWl)
I I H. rubra (Sweden SW1) 100 H. rubra (Connecticut SW1) 'L-QQ-~' H, crouaniiH. nibra (Scotland(Nova Scotia SW4) SW1)
810 H-rubra(AlaskaSW1) .: .: 98 llOOp H. rubra (Newfoundland SW1) H. crouanii (Swtland SWI ) -H crouanii (Gemany SW1) Bangia Porphyra 88 consistency index (CI) of 0.44 (Fig. 3.5)- The parsimony tree strongly resembled the neighbor-joinLng tree generated by distance analysis (Fig. 3 -6). The quartet puzzling maximum likelihood analysis tree yielded the same clades as the parsimony tree, and thus is not shown. The rbcL gene sequences of Austria 14 and Wales 2 (H. rivularis) were determined to be identical, as were Austria 15, France 1, Italy 1, Spain 1 and Wdes 3 (H rivularis), and Northem Ireland SW I and MAS W 1 (H. rubra). Thus, only one representative sequence for each of these groups was included in the analyses. In addition, the rbcL gene sequences of Spain 1 and Spain 2 (H. rivularis) had identical phylogenetically informative sites and were collapsed in the parsimony analysis, as were
AKSWl and NFS Wl (H rubra) and CTSWl and NSSWl (H. rubra). Sequences of the rbcL gene could not be deterrnined for two samples (Austria 10 [H. rivularis] and
Scotland SW3 CH. crouanii]),but several other representatives of their species were successfüIly sequenced. In both analyses the ingroup is separated fiom the outgroups
(Ban,0r,Ora,Eïythrotrichia, Porphyra, Porphyridiurn and Srnithora) wiih stmng bootstrap
(BP 100%) and decay support (decay 21 steps). The fieshwater samples (K. rivularis and
K. angolemis) in both the parsimony and distance analyses forrn a distinct clade with
100% bootstrap support and a decay value of 40 steps in the parsimony analysis (Fig. 3.5,
3.6). In addition, the two samples of H. angûlensis (CR20 & CE4) are positioned on a separate, well-supported branch (100% BP) in this fieshwater clade. Relationships arnong the marine samples, however, are less clear. The marine samples are contained in two clades, each with moderate to high support. However, there is Iittle to no support for a relationship between the two clades. The three samples of H crouanii included in the 89 analyses f Scotland SW1, Scotland SW4 and Germany SW1) do not form a monophyletic group. All three H. crozranii samples group with H. rubra samples with strong support
(97 - 100% BP; decay 10 - 25 steps). North Amencm and European samples of H. mbra group closeIy together in the rbcL analyses, and the samples are not separated into clades for the western and eastem Atlantic basin. For ex ample^ the H rubra samples fkom
Massachusetts and Northern Ireland form a clade with the samples fiom the Netherlands and Sweden. The two sarnples of H. occidentulis fiom California and British Columbia are unresolved in their position with respect to other samples in both the parsimony and distance analyses.
The ten sarnples of H- rivularis in the rbcL analyses were collected fiom a large geographical range, yet they form a well-supported monophyletic clade (93 - 100% BP; decay 6 steps) with Iittle or no sequence divergence arnong samples f?om al1 locations exarnined (O - 1.9%). This trend may indicate a recent dispersai into the varied fieshwater European habitats. Their positioning in the phylogenetic trees also indicates that they are derived with respect to some of the marine samples. Sequence divergence values for the rbcL gene among marine samples of HiZdenbrandia f?om Europe were extremely variable, ranging fiom 4.7 - 24.9%. Sequence divergence ranges among H rubra samples (4.7 - 24,9%) were larger than those of H. crouanii (8.7 - 15.9%).
Divergences between marine and fieshwater Hildenbrandiu ranged fiom 17.6 - 2 1.996, supporting the distinctiveness of the fieshwater species from the marine species.
3.3.4. Parsimony and distance analyses of the 18s rRNA gene
A final aiignment of 1387 bp was generated for the 18s rRNA gene regions of the 90
Xddenbrandia collections fiom Europe. Of these 1387 positions 120 1 were invariant, 73 were phylogenetically informative and 113 were autapomorphies- Parsimony analysis of the 18s rRNA gene yielded 108 most-parsimonious trees with a lenOOth of 841 steps and a consistency index of 0.71. A strict consensus of these 108 trees is show in Fig. 3.7. The neighbor-joining tree generated by distance analysis (Fig. 3.8) strongly resembles the parsimony tree, although bootstrap support for intemal nodes is much stronger in the former. The DNA sequences of the 18s rRNA gene used in the analyses for Spain 2,
Italy 1, Wales 2 and Wales 3 were identical, as were Germany 1, Austria 10 and Austria
14, Norway SW 1 and France SW 1, AKS W 1 and NFS W 1, and France 1 and Austria 1 S.
These pairs of samples were coliapsed into single representatives in the analyses. Fully alignable sequences of the 185 rRNA gene for the following samples could not be determineci: Scotland SW1 (H. crouanii), Scotland SW3 crouanii), ScotIand SW4 (H- crouanii), Germany SW1 (H crouanii) and Spain 1 (H rivularis). Unlike many of the marine Nildenbrandia collections analyzed fiom North Amenca (Chapter 2; Shenvood &
Sheath, 1999) and the European H. rubra collection analyzed by Saunders & Bailey
(1999),no Group IC1 introns were detected in any of the European collections, but this may have been due to the use of amplification pnmers that excluded the 3' end of the 18s rRNA gene.
In both analyses, the outgroups are separated fiom the ingroup with high support
(100% BP; decay 148 steps). In general, support is moderate in the parsimony anaiysis for the major clades. In both analyses the fieshwater samples (H rivuluris and H angolensis) form a distinct clade (72 - 85% BP; decay 2 steps). The North Amencan H. Fig. 3.7. Strict consensus of 108 most-parsimonious trees generated fiom European marine and fieshwater Hildenbrandia samples for the 18s rRNA gene (CI = 0.71; length = 841 steps). Freshwater specimens are denoted by a gernma illustration (cluster of cells) and marine specimens are denoted by a tetmsporangial illustration with the correspondkg division pattern. 1- 1- H fivuIaris (Austria 15, France 1) 79 H. n'vuIais (Austria 10, 2 Austria 14, Germany 1) 88 H. niwlans (Spain 2, lreland 11, 3 Wales 2, Wales 3, ltaly 1) @72 H. angolensis (Costa Rica 20) H, angolensis (St. Lucia 9) H. angolensis (Texas 7) H. angolensis (Texas 9) 7H, nrbra (British Columbia SW1) H. mbra (California SW1) H. mbra (Alaska SWI, Newfoundland SW1)
, 68 K mbra (~assachu&s SW1) 88 1 H. mbra (Nova Scotia SW1) 8 62 4 3 H mbra (Northern freland SW1) H. mbra (Sweden SW1) H. mbra (Norway SW1) H. mbra (Connecticut SWI) tH. mbra (Wales SW3) a H. mbra (Netherlandç SW1) H. occidenfalis (California British Columbia SW4) Bangia
100 , Srnithora 73 29 EryfhmtnChia 4 Porp hyridium Fig. 3.8. Neighbor-joining tree generated through distance analysis of marine and fieshwater Hildenbrandia samples &om Europe for the 185 rRNA gene. Freshwater specimens are denoted by a gemma illustration (cluster of cells) and marine specirnens are denoted by a tetrasporangial illustration with the corresponding division pattem. H,rivularis (Austria 15, France 1) H. fivularis (Austria 10, AustrÏa 14, Germany 1) H. rivulais (Spain 2, lreland 11, Wales 2, Wales 3, ltaly 1) H. angolensis (Costa Rica 20) H. angolensis (St. Lucia 9) H- angolensis vexas 7) 1O0 ( angolensis (Texas 9) H. mbra (British Columbia SW1)
Newfoundland SW1) rubra (Massachusetts SWI) rubm (Nova Scotia SWI ) -H. mbra (Northern lreland SWI) -H. rubra (Sweden SWI)
1O0 mbra (Connecticut SW?) & H. mbra (Wales SW3) 93 H. mbra (Netherlands SWI) H. occidentslis (California SW3 8 British Columbia SW4) 97 Bangia Porphyra Srnithora Erythmtnchia 95 angoknsis samples are positioned on two separate branches (CR20 versus TX7, TX9 and
SL9) in this analysis. The H rubra samples form a weakly supported clade (62 - 76%
BP; decay 3 steps). The two samples of H. occidentalis are resolved as basal to the other
HiZdenbrandia colIections with weak support (75 - 79% BP; decay 3 steps). Low support within the major clades means that few biogeographic trends cmbe ascertained from the data set.
Percentage sequence divergence values for the 18s RNA gene among European ffildenbrandia samples are quite low, and are not as varied as for the rbcL gene. Among freshwater samples the divergence ranges between O and 3.6%, while among marine sarnples it ranges Eom 1-0 - 5.8%. Between fieshwater and marine sarnples the divergence ranges from 2.9 - 5.8%.
3.4. Discussion
With the addition of European samples, we have further clarified the relationship between marine and freshwater foms of Hildenbrandia and provided support for their continued taxonornic distinction. The H. rivularis and H angolemis samples form a monophyletic group in both the rbcL and 18s rRNA gene sequence analyses, although the resolution of the fieshwater samples is greater in the rbcL sequence analyses than in those of the 18s rRNA gene. The likelihood of a marine origin for some fieshwater red algal genera was proposed by Skuja (1938), and Hildenbrandia was cited as one possible example of this phenornenon. Two mechanisms that could explain an invasion of marine
Hildenbrandia into fieshwater habitats are long-term spread up estuaries and vector- assisted transport (Sheath, 1984; Chapter 2 this thesis). The single clade of fieshwater 96
HiZdenbrandia samples (contrary to some analyses in Chapter 2) suggests that the North
American and European collections have a common biogeographic origin, and the
positioning of the H. occidentalis samples in the 18s rEWA gene sequence analyses
supports a marine origin for these fkeshwater species. Although many studies have
attempted to elucidate the reproduction and Iife history of marine and freshwater
Hildenbra~dia(e-g. Budde, 1926; Starmach, 1969; Umezaki, 1969; DeCew & West,
1977; Seto, 1977), these biological processes in Hildenbrmdia are still poorly
understood, and thus the necessary modifications to reproductive structures during the
marine-freshwater transition (i. e. tetrasporangia to gemmae) are uncertain. Studies thus
far support the lack of a sexual stage in both the marine or the fieshwater species of
Hildenbrandia (Sheath et al., 1993; Irvine & Pueschel, 1994).
Phylogenetic analyses of the rbcL gene of rhe European and North American
samples indicate that neither marine species (H. rubra nor H crouanii) is monophyletic,
and these two species together do not form a monophyletic grouping. A complete 18s
rRNA gene sequence of H. crouanii could not be obtained for this study to examine the relationship between the two marine species. Neither the parsirnony nor the distance
analyses of the rbcL gene group the two H. crouanii collections f?om Scotland together.
In addition, the positioning of al1 three K. crounnii samples among the H rubra
collections and the separate H. nlbra clades indicate that H rubra is not a monopl~yletic taxon. The two samples of H. occidentalis in the analyses, which have a similar tetrasporangial division pattern to H. crouanii (Denizot, 1968), did not associate with the
H. crouanii samples in our analyses. As was determined in Chapter 2 (Shenvood & 97
Sheath, 1999), some morphological characters used to distinguish marine species of
Hildenbrandia, such as tetrasporangial division pattern, need to be fürther examùied to establish their utility for taxonomie purposes. Other cases of molecuiar evidence questioning traditional morphological characters have been reported in the Rhodophyta, such as the occurrence of monoecy versus dioecy in Baîrachospermum (Vis & Sheath,
1998)kd the validity of rhizoidal filaments in distinguishing Compsopogolz and
Compsopogonopsis Wtoul et al., 1999).
The range of sequence divergence values among samples of Europeui
Hildenbrandia was large for the rbcL gene (O - 24.9%), but was much smaller for the 18s rRNA gene (O - 5.8%). The North Amencan study also dernonstrated a large range in sequence divergence values for the rbcL gene (O - 25.8% within Hildenbrandia), and a much lower divergence range for the 18s rRNA gene (O - 9.7%) (Chapter 2). Given the higher Ievels of sequence divergence for the rbcL gene in sarnples fiom both continents, it appears that the rbcL gene is evolving at a faster rate than the 18s rRNA gene in
Hildenbrarzdia. The much lower rate of evolution of the 18s rRNA gene may be due to strong functional constraints on the gene, or the us2 of highly consei-ved regions in the phylogenetic analyses (Raué et al., 198 8). Sequence divergence values of up to 15% within Porphyra (Oliveira et al-, 1995) and up to 10.6% within i3angia (Müller et al.,
1998) for the rbcL gene have been reported, but the freshwater European Hildenbrandic collections examined in this study are less than these maximum values. For the 18s rRNA gene, values up to 8.4% for Bangia have been reported (Mü1Ier et al., 1998), but the greatest divergence was only 5.8% arnong European Hildenbrandia collections. 98
The value of traditional morphoIogical characters used to delimit species of
Hildenbrandia will be addressed through additional mo lecular phy logenetic studies of
Hildenbrandia samples of varying tetrasporangial morphology. Further clarification of the systematics of Hildenbrandia will also require examination of representatives of other species and samples fiom other continents, as weli as morphological analyses of the type specimens.
3.5. Literature Cited
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Agardh, J. 1852. Species, Genera et Ordines FZoridearzm. C.W.K. Gleerup, Lund, 1291 PP-
Ardré, F. 1959. Un intéressant Hildenbrandia du Portugal. Revue Algologique 4: 227- 23 7,
Bremer, K. 1988. The lïmits of amino acid sequence data in angiosperrn phylogenetic reconstruction. Evolution 42: 795-803.
Budde, H, 1926. Erster Beitrag zur Entwicklungsgeschichte von Hildenbrandia rivularis (Liebmann) Bréb. Deutsche Botanische Gesellschaff Berichte 44: 280-289.
DeCew, T.C. & West, J.A. 1977. Culture studies on the marine red algae Hildenbrandia occidentalis and H. prototypus (Cryptonemiales, Hildenbrandiaceae). Bulletin of the Japanese Society of Phycology 25: 3 1-4 1.
Denizot, M. 1968. Les Algues Floridees Encrozttantes (à l 'exclusion des Corallinacées). Museum National d'Histoire Naturelle, Paris, 3 10 pp.
Eriksson, T. 1997. AutoDecay ver. 2.9.7. (HyperCard stack distributed by the author). Botaniska Institutianen, Stockholm University, Stockholm.
Hoffmann, L. 1987. Répartition et écologie dYHildenbrandiarivularis (Liebrn.) J. Agardh (Rhodophyceae) en Belgique et au Grand-Duché de Luxembourg. Durnortiera 38: Irvine, L.M. & Pueschel, C.M. 1994. Hildenbrandiales. In: Seaweeds of the British Ides. Volume 1. Rhodophyta- Part 2B. Corallinales, Hildenbrandiales (Eds. L.M. Irvine & Y.M. Chamberlain). The Natural History Museum, London, pp. 235-241.
Israelson, G. 1942. The Freshwater Florideae of Sweden: Studies on their Taronomy. Ecologv, and Distribution. Symboiae Bo tanicae Upsalienses IV, 13 5 pp.
Lingelsheim, A. 1922. Eine bemerkenswerte Rotalge des Sül3wassers und ihre Erhaltung. Beitrag JU NaturdenkmaZpflege 9 : 348 -3 60.
Müller, K.M., Sheath, R.G., Vis, M.L., Crease, T.J. & Cole, K.M. 1998. Biogeography and systematics of Bangia (Bangiales, Rhodophyta) based on the Rubisco spacer, rbcL gene and 18s rRNA gene sequences and morphometric analyses. 1. North Amenca. Phycologia 37: 195-207.
Nardo, G.D. 1834. De novo algurum cui nomen est Hildenbrandia prototypus. Isis von Oken 6: 675.
Nichols, H. W. 1965, Culture and development of Hildenbrandia rivularis fiom Denrnark and North America. Arnerican Journal of Botany 52: 9- 1 5.
Oliveira, MC, Kurniawan, J., Bird, CL, Rice, E.L., Murphy, C.A., Singh, R.K., Gutell, R.R. & Ragan, M.A. 1995. A preliminary investigation of the order Bangiales (Bangiophycidae, Rhodophyta) based on sequences of nuclear small-subunit ribosomal RNA genes. Phycological Research 43: 7 1-79.
Palik, P. 1961. Neue Algen-Species, Subspecies, Varietaten und Formen. Annales Universiratis Scientinris Budapestiensis Rolando E~tvosSectio BioZogiu 4: 15 1- 154,
Raué, H.A., Klootwijk, J. & Musters, W. 1988. Evolutionary conservation of structure and function of high molecular weight ribosomal RNA. Progress in Biophysics and Molecztlur Biology 51 : 77- 129.
Raven, J.A., Johnston, A.M., Newman, J.R. & Scrimgeour, C.M. 1994. Inorganic carbon acquisition by aquatic photolithotrophs of the Dighty Burn, Angus, U.K.: uses and limitations of natural abundance measurements of carbon isotopes. New Phytologist 127: 271-286.
Rintod, TL., Sheath, R.G. & Vis, M.L. 1999. Systematics and biogeography of the Compsopogonales (Rhodophyta) with emphasis on the fieshwater families in North America. Phycologia 38: 5 17-527.
Ros, M.D., Jiménez, E.L., & Aboal, M. 1997. Primera cita de Hildenbrandia angolensis Welwitsch ex W. West & G.S. West (Hildenbrandiales, Rhodophyceae), para la flora algd epicontinentd Espaiïola. Anales del Jardin Botanico de Madrid 55: 458-460.
Rosenvinge? L.K. 1917. The Marine Algae of Denmark - Contributions tu their Natural Hislory. Vol. 1. Rhodophyceae. Andr. Fred. Host & Son, Copenhagen, 486 pp.
Saunders, G.W. 1993. Gel purification of red algd genomic DNA: an inexpensive and rapid method for the isolation of polymerase chab reaction-fiendly DNA. Journal of Phycology 29: 25 1-254.
Saunders, G. W. & Bailey, J-C. 1999. Molecular systematic analyses indicate that the enigmatic Apophlaea is a rnember of the Hildenbrandiales (Rhodophyta, Florideophycidae). Journal of Phycology 35: 171-1 75.
Saunders, G.W. & Kraft, G.T. 1994. Small-subunit rRNA gene sequences from representatives of selected families of the Gigartinales and Rhodyrneniales (Rhodophyta). 1. Evidence for the Plocamiales ord. nov. Canodian Journal of Botany 72: 1250-1263.
Seto, R. 1977. On the vegetative propagation of a fieshwater red aiga, Hildenbrandiu rivuluris (Liebm.) J. Ag. Bzrlletin of the Jupanese Sociep of Phycology 25: 129- 136.
Sheath? R.G. 1984. The biology of the fieshwater red algae. Progress in Phycological Reseurch 3: 89-157.
Sheath, R.G., Kaczmarczyk, D. & Cole, K.M. 19%. Distribution and systematics of fieshwater Hildenbrandia (Rhodophyta, Hildenbrandiales) in North America. European Journal of Phycology 28: 11 5- 12 1.
Sherwood, A.R. & Sheath, R.G. 1999. Biogeography and systematics of Hildenbrandia in North Amerka (Rhodophyta, Hildenbrandiales): inferences from morphometrics and rbcL and 18s rRNA gene sequence analyses. European Jotrrnal of Phycology 34: 523-533.
Silva, P.C., Basson, P.W. & Moe, R.L. 1996. Catalogue ofrhe Benthic Marine Algae of the Indian Ocean. University of California Press, Berkeley, 1291 pp.
Skuja, H. 1938. Comments on the fiesh-water Rhodophyceae. Botanical Review 4: 665- Stannach, K. 1969- Growth of thalli and reproduction of the red alga Hildenbrandia rivularis (Liebm.) J. Ag. Acta Societatis Botanicorum Poloniae 38: 523-533.
Starmach, K. 1984. Red algae in the Kryniczanka Stream - Krasnorosty potoku Kryniczanka. Fragmenta Floristicu et Geobo~unicu28: 257-294.
Umezaki, 1. 1969. The germination of tetraspores of Hildenbrandiaprororypus Nardo and its life history. Jozrrnal of Jupanese Botany 44: 17-32.
Vis, M.L. & Sheath, R.G. 1997. Biogeography of Batrachospermum gelatinosum (Batrachospermales, Rhodophyta) in North Arnerica based on molecular and morphological data Journal of PhycoZugy 33: 520-526.
Vis, M.L. & Sheath, R.G. 1998. A molecdar and morphological investigation of the relationship between Batrachospermum spermatoinvolucrum and B. gelatinosum (Batrachospermales, Rhodophyta). European Journal of PhycoZoa 33: 23 1-23 9.
West, G.S. & Fritsch, F.E. 1932. A Treatise on the British Freshwater Algae. Cambridge University Press, Cambridge, 534 pp. 102
CEFAPTER 4: The relationship between marine and freshwater Hildenbrandia along an historical salinity gradient
4.1. Introduction
The red algd genus Hildenbrandia contains both marine and fieshwater forms, both of which are widespread in Europe. Marine foms have been reported fkom diverse
European locations, including the Baltic Sea (Waem, 1952), the British Isles (Irvine &
Pueschel, 1994), the North Sea (Den Hartog, 1959), the Atlantic Coast of France and
Portugal (Agardh, 185 1; Ardré, 1959) and the Mediterranean (Nardo, I 834; Zanardini,
1840). Freshwater forms of Hildenbrandia are common in European stream and lake systems, and reports include locations such as Sweden (Israelson, 1942), Denmark
(Liebman, 183 9), Finland (Luther, 1954), the British Isles (Fritsch, l929), Poland (Palk,
1957), France (Bourrelly, 1955), Luxembourg (Hoffmann, 1987), Belgiurn (Compère,
1Wl), Germany fBudde, 1926), Spain (Sabater et al.,1998) and Italy (Zanardini, 1840).
The marine and freshwater forms strongly resemble one another morphologically, but they differ in their mode of reproduction. Sexual reproduction is unknown within the genus, but the marine foms produce tetrasporangia in conceptacles on the thallus surface, and the fieshwater forms produce gemrnae (Sheath et aL, 1993). Skuja (2938) proposed that the fieshwater forms of HiZdenbrandia may have arisen through invasions of marine
HiZdenbrandia into fieshwater habitats, based on the strong morphological resemblance between the two forms. The red algal genus Bangia, which is also believed to be a mernber of the "invader" group of fieshwater red algae, has been shown to acclimate to a range of salinities, demonstrating such a potential (Sheath & Cole, 1980). Previous 1O3 studies have examined the phylogenetic relationships between marine and freshwater foms of Hildenbrandia at the continental level for both North America and Europe
(Chapters 2 & 3; Sherwood & Sheath, 1999,2000). However, the possibility of a marine ongin for the fieshwater forms of Hildenbrandia ES not been examined in detail within a more limited geographical area, using molecular data. Molecular markers can help overcome problems of morphological comparisons stemming fiom a paucity of characters and questions of homology (e.g. Brodie et al., 1996). Both of these problems apply to
HiZdenbrandia, given the simple crustose thallus as well as differences in reproductive structures in marine and fieshwater foms (tetrasporangia versus gemmae).
The region of Sweden irnrnediately north of Stockholm, and the adjacent Baltic
Sea, is an ideal location for comparative studies of marine and fieshwater Huenbrandia.
Since the end of the last glaciation in the area, approximately 15,000-9,000 ya
(Winterhalter et al., 1981), glacial isostatic rebound has resdted in a gradual uplifting of the land at a rate of approximately 50 cm century-' (Risberg et al., 199 1). This rate ailows 81 the approximate age of freshwater bodies in the region to be inferred fiom their present day elevations. Freshwater Hildenbrandia (H. rivularis &iebrn.] J. Agardh) is very cornmon in the lakes and strearns of this region (haelson, 1942), and a marine species
(lX rubra [Somme&] Menegh.) is present in the adjacent brackish water Baitic (Waem,
1952). Although the study region has been deglaciated since approximately 9000 ya, the subsequent dynamics in the area, including the formation and demise of the Yoldia Sea,
Ancyclus Lake and Littorina Sea, indicate that the present day fieshwater bodies in the region are most likely younger than 5,000 y (Hutchinson, 1975). Thus, this system is 1O4 ideal for the testing of Skuja3s(1 93 8) hypothesis since it contains both marine and fieshwater fonns of HiZdenbrandia in close proximity to one another, and the fieshwater forms are known to have only coIonized the region relatively recently (Le. within the last
5,000 y), In addition, the diversity of elevations of lakes and streams indicates that water bodies were isolated fiom the Baltic Sea at different times, so that an histoncal salinity gradient is present in the region.
A number of molecular markers have been employed to address biogeographic questions, including DNA sequences of the internal transcribed spacer (ITS) regions of the rRNA genes (e.g. Lindstrom et al., l996), as well as techniques such as random amplified polymorphic DNA (RAPD7s- eg. Lindstrom et al-,1997)' restriction fiagrnent length polymorphisms (RFLP's - e.g. Stiller & Waaland, 1993) and intersimple sequence repeats (ISSR's - e.g. Vis, 1999). Sequence comparisons of more conserved genes, such as the RuBisCO large subunit (rbcL) and the nuclear nbosomal small subunit (18s rRNA) genes, are more comrnonly employed for investigations above the species level
(e.g. Freshwater et al., 1994; Ragan et al., 1994). In this study a combination of morphological analysis and ITS sequences and ISSR analyses was used to investigate the relationships among fieshwater Hilaenbrandia sarnples dong an historical salinity gradient. Ln addition, the biogeographic ongins of these sarnples were examined in relation to our previous collections of marine and fieshwater Hildenbrandiu fkom Europe, using comparisons of sequences of the rbcL and 18s rRNA genes (Chapter 3), which are appropriate for these data since representatives of several different species (both marine and fieshwater) were included. 4.2. Materials and methods
4.2.1. Collection of materials and morphological examination
Eleven fieshwater collections and three marine collections of Hildenbrandia fiom
Sweden were made in June 1999 (Table 4.1, Fig. 4.1). Two additional specimens of marine Hildenbrandia collected in December 1998 fkom the shoreline of the Stockholm
Archipelago were also employed in the analyses (Fig. 4.1, Table 4.1). Collections were made by scraping individual HMenbrandia thalli fkom a rock using a clean razor blade, and samples were cIeaned of epiphytes using cotton swabs and forceps before Mer processing. As a precaution, replicate samples for DNA extraction were dried in silica gel and fkozen at -20°C. For morphological examination, samples were futed in 2.5%
CaC0,-buffered glutaraldehyde (to prevent morphological distortion).
Fixed samples were examined using an Olympus BH-2 compound light microscope to determine whether each sample consisted of marine or fieshwater
Elildenbrandia. Sarnples were identified based on presence or absence of tetrasporangia and conceptacles (to indicate marine versus fieshwater collections), tetrasporangial division pattern and conceptacle dimensions, and cellular dimensions (Denizot, 1968;
Sheath et al., 1993). Gemrna production in rivularis (fieshwater species) is relatively rare (Chapter 3; Sherwood & Sheath, 2000), but the presence of conceptacles in H. rubra samples (marine species) is very common, since they are produced erosively (Pueschel,
1982) and are perennial structures. Thus, although gemrnae were not obsenred in the fieshwater samples, they were morphologically distinct fiom the marine samples owing to Table 4.1. Collection information, elevation, estimated age of sampled water bodies and GenBank accession numbers for Swedish samples of marine and freshwater Hildenbrandia. Collection numbers refer to codes on map.
- - -- Sampte number Collection information est. elevation rbcL 18s rRNA ITS 1 ITS 2 and identification a@ (masl)
swel2 Vramsan, near Kdpinge. Coll. A, Sherwood to submit to submit to submit to submit (H.rivirlaris) & R. Bengtsson. 20 June 1999,
swe 13 Skrabean at Nymolla. Coll. A. Sherwood & to submit (H. rivirlaris) R. Bengtsson. 20 June 1999, swel5 Frotuna Kyrksjo (lake). Coll. A, Shenvood, to subrnit to submit to submit to submit (H. rivirlaris) R. Bengtsson, D. Mollenhauer & T, Odelstrdm. 22 June 1999. O swe 16 Lama KyrksjU (lake). Coll, A. Shenvood, R, to submit to submit o\ (H rivularis) Bengtsson, D, Mollenhauer & T. Odelstrom. 22 June 1999. swe 17 Penningbyin (stream) at Penningby. Coll. A. to submit to submit (H.rivitlaris) Sherwood, R. Bengtsson, D,Mollenliauer & T, Odelstrom, 22 June 1999,
swe 18 Lake Vailoxen, Knivsta. Coll, A, Shenvood, to submit to submit to submit to submit (H. rivlrlaris) R, Bengtsson, D. Mollenhauer & T. Odelstrdm. 22 June 1999. swel9 Lake Erken at Erken Laboratory, Coll. A, to submit to submit (H. rivttlaris) Shenvood. 26 June 1999. swe20 Hargsan (stream) at Harg Village, Hwy 76. to submit to submit (H. rivzrlaris) Coll, A. Shenvood & T,Odelstrdm. 24 June 1999. nSWE12and SWE13 are located in the southern region of Sweden where the assumption of a constant rate of land uprise is invalid. Table 4.1, Continued.
Sample number Collection information est. elcvation rbcL 18s rRNA ITS 1 ITS 2 and identification age (masl) (Y) swe22 JarsUstrUmrnen (outlet stream of Lake to submit to submit (H. rivitlaris) Erken), Hwy 76. Coll, A. Sherwood. 26 June 1999. swe23 Jumkilsh Cstrearn) at Jumkil-Vallhov. Coll. 5000 25.0 to submit to submit (H.rivjilaris) A, Sherwood. 26 June 1999. swe24 FunboAn (stream) at Funbo, Coll, A, 1500 7.5 to subinit to submit to submit (H. rivularis) Sherwood. 26 June 1999,
swesw 1 Eastern Ask6, Stockholni Archipelago, to submit to submit to submit to submit O (H. rttbra) outside Trosa (rock 1). Coll. K. Eriksson, 4 December 1998. swesw2 Eastern AskU, Stockholm Archipelago, to submit to submit (H. nrbra) outside Trosa (rock 2), Coll, K. Eriksson. December 1998.
swesw3 Baltic Sea at Estonia Ferry Terminal. Coll. to submit to submit (H. rubra) A. Sherwood & T, Odclstr6m. 24 June 1999. swesw4 Baltic Sea at Nothamn, VaddU. Coll. A, to submit to submit (H, rubra) Sherwood & T,Odelstrom. 24 June 1999. swesw5 Baltic Sea, exposed Coast near Nothamn. to submit to submit (H, riibra) Coll. A. Sherwood & T. OdelstrUm. 24 June 1999, Fig. 4.1. Map of sampling locations for brackish and fkeshwater Hildenbrandia in Sweden. Numbers correspond to those in Table 4.1.
110
their lack of conceptacles and tetrasporangia, as well as ceU size merences.
4.2.2. DNA extraction, rbcL and 18s rRNA gene amplifcation, purifkation,
sequencing and gene sequence analyses
Samples were ground in liquid nitrogen and DNA was extracted using the Qiagen
DNeasy Plant Mini Kitm. The rbcL and 18s rRNA genes were amplified fiom a
geographically widespread subset of the collections (four fieshwater and one marine -
SWE12, SE15, SWE18, SWE24 & SWES Wl) to veriQ that they were conserved in their gene sequences (see Appendix 1 for prirners). Double-stranded PCR amplifications were perfonned in either a Perkin Elmer 2400 Gene Arnp PCR System or a Perkin Elmer
DNA Thermal Cycler 480. ApproximateIy 1100 base pairs (bp) of the rbcL gene were
amplfied using the primer pairs described in Chapters 2 & 3, as follows: initial
denaturation at 95 OC for 2 min, followed by 35 cycles of denaturation for 1 min at 93 OC, primer annealing at 47 OC for 1 min, extension for 4 min at 72°C and a final extension for
6 min at 72OC. Three separate but overIapping pieces of the 18s rRNA gene were
amplified for a total of 1443 bp, using the primer pairs listed in Appendix 1. The
amplification procedure for the 185 rRNA gene pieces was as follows: initial denaturation at 95 OC for 2 min, followed by 37 cycles of 1 min at 93 OC,primer annealing
at 55°C for 1 min, extension for 2 min at 73°C and a final extension for 3 min at 7S°C.
Reaction volumes for both amplification procedures were as described in Chapter 2,
except that bovine serum albumin (BSA) was added to reactions (5% ha1volume) to
enhance amplification, and in some cases 25pL reactions (at the same concentrations)
were employed. PCR products were visualized on 2% agarose gels stained with ethidium 111
bromide, and successfüi amplification products were cleaned using the QIAquick PCR
Purification Kit. Sequencing was performed as described in Chapter 2-
Sequences were assembled with DNA sequencing software (SeqEd, ABI).
Sequences of both genes were easily aligned since they were highly conserved among collections. The number of base pair changes between sequences for the two genes was calculated using the program MEGA (Molecular Evolutionary Genetics Analysis; Kumar et al., 1993)- Unrooted neighbor-joining trees were produced Tom a Kimura-2-parameter distance matrix through cornparisons of these sequences to other European Hildenbrandia sequences for these two genes (Chapter 3; Shenvood & Sheath, 2000), using PHYLIP
(Felsenstein, 1993). Trees were subjected to bootstrap resampling (1000 replicates).
Parsimony analysis was performed using PAUP *4.0 (Swofford, 2000) with the branch- and-bound algorithm and bootstrap resampling (1 000 replicates).
4.2.3. ITS 1 and ITS 2 amplification and analysis
The ITS regions were amplified separately for each sample using the primer pairs listed in Appendix 2, Amplification procedures were the same as for the rbcL gene except an annealing temperature of 49 OC was empIoyed. PCR products were visudized, purified and sequenced, and the resulting sequences were assembled as described in the previous section. Sequences were aligned (where possible) using the program
CLUSTAL-X (Thompson et al., 1997) and were manually adjusted by eye. The number of bp changes between sequences was calculated using MEGA (Kurnar et al., 1993).
Unrooted neighbor-joining and UPGMA trees were constmcted f?om a Kimura-2- parameter distance rnatrix using PHYLIP, and were subjected to bootstrap resampling 112
(1,000 replicates). Parsimony trees were dso calculated as described above fiom the sequences using PAUP, where possible, to examine phylogenetically informative changes arnong sequences. Combined analyses were run, where possible, for ITS I and ITS2 as described above (both parsimony and distance analyses).
4.2.4. ISSR amplification and analyses
Fifteen primers were initiaily screened using the same subset of samples as in the rbcL and 18s rRNA gene sequence analyses (Appendix 1). Three primers yielded amplification products for al1 collections (ISSR8, IS SR1O and ISSRl 1), and these were used to produce band profiles for al1 collections in a Perkin Elmer DNA Thermal Cycler
480 by the following single-primer amplification reaction: initial denaturation of 94°C for
2 min, followed by 3 5 cycles of denaturation at 94 OC for 3 0 s, primer annealhg at 44°C for 45 s, extension at 72OC for 1 min 30 s, and a fmal extension of 10 min at 72OC. The total reaction volume of XpL consisted of OSpL of genomic DNA, 20mM each of dATP, dTTP, dGTP and dCTP, 0.4rnM primer, 2mM MgCl2, and 10X reaction buf5er
(Perkin Elmer) with 1.O unit Taq polymerase. Al1 ISSR-PCR reactions were duplicated to ensure reproducibility. PCR products were electrophoresed on a 1.5% agarose gel containing 1X TBE baer and stained with ethidium bromide for 4 h at 100 kV. Gels were viewed under W light and recorded as digital images, which were exarnined using
Adobe Photoshop v.5.0 (Adobe Systems, Inc.). Individual bands were scored as present
[il or absent [O] for each sample.
Simila~3ybetween Hildenbrandia sarnples was estimated using both the Dice coefficient [2NJN, + N,,)] and the Jaccard coefficient [N,/(N, + N, + N,)] where NN is 113 the number of bands shared by sarnples x and y and N, and N, are the number of bands shown by samples x and y. These coefficients are appropriate for cornparisons based on rnolecular band patterns since they do not consider shared absence of bands to be a similarity between samples (Esselman et al-, 1999; Vis, 1999). UPGMA and neighbor- joining trees were calculated from the corresponding dissimilarity matrices using the
NEIGHBOR program in PHYLIP.
4.2.5. Determination of time since isolation from the Baltic Sea
Ages of freshwater bodies sarnpled for the study were calculated using the elevation of the water body above sea level and the rate of land nse in the region.
Elevations were obtained fiom topograpfiical maps (Lantmateriverket topographicai map series, 1986 - 199l), and were rnultiplied by a land-rise factor of 50 cm - 100 y-' (Ignatius
et al. ? 1981) (Table 4.1). The two samples of H rivularis collected fiom the southem region of Sweden (S WE 12 and SWE13) were not included in the age analysis since the assumption that uplifting has been constant is not valid for that region (Agrell, 1979).
4.3. Results
4.3.1. Morphological examination of collections
Al1 freshwater collections (SWE12 - SWE24) were determined to be H. rivularis based on the complete lack of conceptacles and tetrasporangia, and the relatively large ce11 size (H rivularis z cell dimensions 5.7 x 5.2pm versus ET. rzrbra x ce11 dimensions
3.6 x 2.9pm) (Sherwood & Sheath, 2000). Marine collections (SWESW1 - SWESW5) contained nurnerous conceptacIes with tetrasporangia possessing division patterns where al1 planes of division were not parallel, and hence they were attributable to H rubra. 114
4.3.2. Analyses of the ITS 1 and ITS 2 regions
Al1 fiesinvater and marine samples were successfiilly arnplified for ITS 1, but PCR amplification of SWEI 3 and SWE24 was not successful for ITS2 (no products generated). The final ITS 1 alignment for the fieshwater sequences was 450 bp in length, with the sequence Iength ranging fiom 439 - 441 bp. The ZTS 1 sequences for ail five marine samples were identical (656 bp), but were unalignable with the fieshwater samples except for a 15 bp region at the beginning and a similar region at the end of the spacer, highlighting a major distinction between the two groups of collections. The ITS2 final alignment for the fieshwater sequences was 3 18 bp in length, with the sequence lengths ranging fiom 307 - 3 17 bp. The ITS2 sequences for the marine samples were again identical, and were 288 bp in length. The cornbined ITS 1 and ITS2 alignment for the fieshwater sequences was 735 bp in lenad, and the individual sequences ranged from
71 7 - 727 bp. The ITS 1 and ITS2 regions of the freshwater samples were analyzed both separately and combined by distance and parsimony analyses.
The unrooted neighbor-joining and parsimony trees of the ITS 1 sequences are presented in Fig. 4.2 (a &b). UPGMA trees were dso constmcted for al1 sets of samples, but are not shown since they yield the same groups as the neighbor-joining trees.
Estimated time (years) suice isolation fiom the Baltic Sea of each sampling location is listed beneath or beside the sarnple name for each tree. Many samples were very similar or identical in sequence (cg.SWE 16, SWE20, SWE22, S WEZ) and others were very distant (e.g. SWEl2 & SWE24), which, to some extent, corresponds to geographical distance between sites (Fig. 4.1). Exarnining only phylogenetically informative changes Fig. 4.2. a) Unrooted neighbor-joining tree of ITS 1 sequences for fkeshwater Hildenbrandia samples fkom Sweden (bootstrap proportions out of 1,000 repiicates) Time (years) since isolation fiom the Baltic Sea is noted by . each sample.
b) One of three most parsimonious trees (unrooted) cdcdated for ITS 1 sequences for fieshwater HiZdenbrandia samples (bootstrap proportions out of 1,000 replicates). Tree length = 68 steps. Time (years) since isolation fiom the Baltic Sea is noted by each sampie. hhhh )*)r)i)r Fig. 4.3. Unrooted neighbor-joining tree for ITS2 sequences of fkeshwater Hildenbrandia fiom Sweden (bootstrap proportions out of 1,000 replicates). Tirne (years) since isolation fiom the Baltic Sea is noted by each sample.
Fig. 4.4. a) Unrooted neighbor-joining tree for combined ITS 1 and ITS2 sequence data for fkeshwater Hildenbrandia samples fkom Sweden (bootstrap proportions out of 1,000 replicates). Time (years) since isolation fiom the Baltic Sea is noted by each sarnple-
b) Single most-parsimonious tree (unrooted) for combined ITS 1 and ITS2 data for fkeshwater Hildenbrandia fiom Sweden (bootstrap proportions out of 1,000 replicates). Tree length = 41 steps. Time (years) since isolation fkom the Baltic Sea is noted by each sample. Ac- % 121
(Fig. 4.2 b) yielded similar groupings but did not disringuish SWE 15, SWE 17 and
S WE 18 fiom one another. A gradient of water body ages corresponding to genetic distance is not evident fiom the analyses. An unrooted neighbor-joining tree for the ITS2 sequences is presented in Fig. 4.3. Too few phylogenetically informative sites were available in this data set to construct parsimony trees. Only four distinct sequences were obtained for the nine samples sequenced, and although some groups resembled those fiom the ITSi data set (e-g. SWEIS, SWE17 & SWE19), others did not (e.g. SM12&
S WE18). Again, water body ages do not appear to correlate with the genetic distances displayed on the tree. The cornbined ITS 1 and ITS2 neighbor-joining and parsimony trees (Fig. 4.4 a & b) generally demonstrated groupings intermediate between the ITS 1 and ITS2 analyses.
4.3.3, ISSR-PCR results
Three primers produced band patterns for al1 samples: ISSR8 yielded 21, ISSRlO yielded 16 and ISSRl1 yielded 29 distinct bands, for a total of 66 scored bands. In general, similarities were lower using the Jaccard than Dice coefficient (Table 4.2).
Average similarities were hïgher between freshwater samples (x Jaccard = 42.2%, R Dice
= 58.1%) and between marine samples (n Jaccard = 4l.l%, z Dice = 60.3%) than between marine and fieshwater collections (R Jaccard = 17.7%, R Dice = 29.8%). Trees produced using both the neighbor-joining and the UPGMA algorithm were very similar
(Fig. 4.5 a & b); thus, only the trees produced through the neighbor-joining method are presented in Fig. 4.5. In these trees the length of the branches is directly proportional to the genetic distance between pairs of samples. The trees produced by both similarity Table 4.2. Percent similarity among marine and freshwater Ifildenbrundia samples from ISSR analyses based on the Dice coefficient (above the diagonal) and the Jaccard coefficient (below the diagonal).
Sample swe swe swe swe swe swe swe swe swe swe swe swe swe swe swe swe 12 13 15 16 17 18 19 20 22 23 24 swl sw2 sw3 sw4 sw5
swel2
swel3
swel5
swel6
swe 17
swel8 swe I9
swe20
swe22
swe23
swe24
swesw 1
swesw2
swesw3
swesw4
swesw5 Fig. 4.5. Unrooted neighbor-joining trees of fieshwater and brackish Hildenbrandia samples Eom Sweden based on ISSR analyses with the Dice (a) and Jaccard (b) coefficients. Time suice isolation fiom the Baltic Sea is indicated beneath each of the fieshwater collections. No tirnes are given for SWE 12 and SWE13 since the assumption of constant rate of land nse is not valid for this geographicai area. Group A includes ail of the brackish water H. rubru samples, and Group B includes ail of the fieshwater H rivularis samples.
125 coefficients demonstrate a clear distinction between the marine and the freshwater samples of Hildenbrandia, as seen fiom the average similarities, with the marine samples
(Group A) clustering together on a separate branch fiom the freshwater samples (Group
B) in both cases. The Eeshwater samples are fiom water bodies whose ages ranged from approximately 800 - 5,000 y, with an average age of 2,300 y since isolation fkom the
Baltic Sea. No obvious correlation cmbe seen between the age of the water body sampled and the clusters formed in the ISSR analyses, as was found for the ITS analyses.
For example, a cluster consisting of SWEl6, SWESO, SWE22, SWE23 and Sm24is present in both ISSR networks; however, the ages of the water bodies corresponding to these samples range fkom 1,500 - 5,000 y. Another exarnple is the group of samples
SWElS- SWE17, Sm18 and SWEI9, whose water body ages range fiom 900 - 3,100 y.
4.3.4. rbcL and 18s rRNA gene sequence analyses
The rbcL and 18s rRNA genes were successfÜlly amplified and sequenced for the five representative collections among the Hildenbrandia samples from Sweden. The final alignment of the rbcL gene consisted of 1,000 bp, and no insertions or deletions were required. The final alignrnent for the 18s rRNA gene consisted of 1,443 bp, and three deletions were observed in the marine HMenbrandia sequence (SWESWI), with respect to the fieshwater samples. An analysis of the number of bp changes among sequences revealed that both regions were moderately to well conserved among fieshwater sarnples, but were more divergent between marine and fi-eshwater collections (Table 4.3). For the rbcL gene, arnong keshwater samples, the number of bp changes ranged fiom O - 19. For the 18s rRNA gene the number of bp changes ranged fiom O - 2. Between marine and Table 4.3. The number of base pair changes in gene sequences among five representative samples of Hildenbrandia from Sweden. Above the diagonal is the number of base pair changes for the rbcL gene and below the diagonal is the number of base pair changes for the 18s rRNA geiie (swe = freshwater collections, swesw = marine collections). Sarnple codes refer to collections on map.
Sample swe12 swe15 swel8 swe24 swesw 1 , fi 0.*-' swel2 .l 19 19 19 182
sweswl 55 56 55 55 k 'ig. 4.6. a) Unrooted neighbor-joining tree of European marine and fieshwater Hildenbrandia samples and representative Swedish samples based on the rbcL gene. Bootstrap proportions based on 1,000 replicates. Al1 fieshwater samples form a gxoup with little to no sequence divergence among them (circle). The following fieshwater samples are included: SWE 12, SWEI S, S WEI 8, SWEX, France 1, Wales 2, WaIes 3, Ireland 11, Germany 1, Austria 14, Auda 15, Spain 1, Spain 2 and Italy 1. The following marine samples are included: Wales SW3, France SW 1, Scotland S W 1, ScotIand SW4, Germany SW 1, Nonvay SW 1, Northern Ireland SW 1, Netherlands S W Z and Sweden SW 1.
b) One of two most-parsirnonious trees based on the same data set as (a). Tree Iength = 607 steps.
Fig. 4.7. a) Unrooted neighbor-joining tree of European marine and fieshwater Hildenbrandia samples and representative Swedish samples based on the 18s rRNA gene. Bootstrap proportions based on 1,000 replicates. Al1 fieshwater samples form a group with Little to no sequence divergence among them (circle). The following fieshwater samples are included: SWE12, SWEIS, SWE18, SWE24, Wales 2, Wales 3, Ireland 11, Germany 1, France 1, Austria 10, Austria 14, Austria 15, Spain 2 and Italy 1. The following marine samples are included: Northern Ireland SW 1, Sweden SWl, Nonvay SWZ, Netherlands SW1 and France SW1.
b) One of 24 most-parsimonious trees based on the same data set as (a). Al1 trees showed the fieshwater samples distinct fiom the marine samples. Tree length = 56 steps.
131 fieshwater samples the number of bp changes was 182 for the rbcL gene and ranged fiom
55 - 56 for the 18s rRNA gene (Table 4.3).
The rbcL and 18s rRNA gene sequences for the Swedish H ridaris and H- rubra samples were compared to other sequences of these two genes for representative
European specimens using unrooted neighbor-joining and parsimony trees (rbcL - Fig.
4.6 a & b; 18s rRNA - Fig. 4.7 a & b). For both genes, the fieshwater HiZdenbrandia fkom Sweden and other European locations (see figure legends for Est of specimens included) formed a very distinct group with little or no corrected sequence divergence among samples (O - 2.0% for rbcL, O - 0.5% for 18s rRNA). This contrasts to the marine samples, which were more divergent fiom one another (4.9 - 23.4% for rbcL, 0.6 - 3.4% for 18s rRNA).
4.4. Discussion
A number of molecular markers were employed in this study to examine the evolutionary ongins of fieshwater HiZdenbraPidia in Sweden and, although al1 methods highlighted the strong differences between marine and fieshwater samples, many of the data sets were incongruent with respect to relationships among fieshwater samples. For example, trees generated Eom ITS 1 data showed the fieshwater samples S WE16,
SWEZO, SWE22 and SWEZ to be identical, while SWE24 was on a long branch fiom these samples. In contrast, the ISSR trees showed SWE24 to be quite similar to SWE22.
These discrepancies in the relationships among the freshwater samples may be due to differences in the techniques. ISSR's are based on banding patterns generated fiom amplified regions between microsatellites, which are dispersed throughout the genome 132
(Li, 1997), while the ITS regions are part of a tandemly repeated array in the nuclear
genome (Singer & Berg, 1991), and ~~LLSthe two techniques may reveal differences in the
level of conservation and sources of variation-
It is clear fiom this study that the Swedish and other European fkeshwater
Hildenbrandiu collections analyzed represent a very distinct lineage fiom those of marine habitats. In addition, multiple marine iineages are indicated by the analyses (Figs. 4.6 &
4.7). The rbcL gene, 18s rRNA gene and ITS sequences al1 highlight the conservation of sequence and low divergence among fieshwater European collections. The results of the
ISSR analyses also support a greater molecular similariQ within a habitat type (Le. marine or fieshwater) than between habitats; however, the ISSR results were not as clear as the other techniques- Thus, our attempts to uncover a genetic gradient of
Hildenbrandia dong the historical salinity gradient fkom fieshwater lakes and strearns to the present day Baltic were not successful because the fieshwater samples are not immediately derived fi-om a Baltic source. The previous studies of HiZdenbrandia in
North Arnerica and Europe (Chapters 2 & 3) indicated that the evolutionary histories are distinct for the fieshwater species on the two continents. In North Arnenca, the fieshwater species, H angolensis Welw. ex W.West et G.S. West is not a distinct genetic entity, and its variability may be due to establishment in keshwaters by multiple invasion events (Chapter 2). In contrast, the European fieshwater forms appear to be genetically homogeneous (Chapter 3), as confirrned in this study.
The Baltic Sea has a varied and well-studied Quatemary history. Since the end of the last glaciation, approximately 15,000-9,000 ya, the Baltic has alternated between 133 freshwater and various degrees of salt water (Russell, 1985). The most recent phase during which the Baltic seems to have been entirely fkesh was the Ancyclus Lake stage, beginning approximately 7,500-6,500 ya. This was followed by the formation of the
Littorina Sea (6,000-5,000 ya), a marine stage, and then the brackish water Baltic Sea
(-3,000 ya) as the waters became cooler and less saline (Hutchinson, 1975; Agrell, 1979).
The Baltic currently possesses a largely marine flora- It is possible, however, that a subtidal marine algal flora (which could have included Hildenbrandia) could have been present in the Baltic as early as the Yoldia Sea stage, since the subsequent Ancyclus Lake may have only affected the surface water layers, and the bottom water may have remained saiine or brackish (Agrell, 1979). Marine Hildenbrandia has been reported fiom depths of 3 1 m in the North Sea (Rosenvinge, 1917), demonstrating that the alga can live at moderate depths.
The present day Baltic Sea is a reduced salinity environment, with salinities ranging fiorn approxirnately 1-10%0 (cornpared to the average value for seawater of
3 5%"); salinities cm be even lower in the imer basins (Waern, 1952). Raven (1 999) has commented on challenges faced by marine algae living in waters of reduced salinity, specifically addressing Baltic macroalgae. Osmoregulatory stress (on al1 cells but especially on naked gametes) and lower inorganic carbon availability in brackish waters than saline waters are likely to impose strong constraïnts on the ability of Baltic rnacroalgae to live in this habitat (Raven, 1999). In addition, an increased tendency to polyspermy (fertilization by more than one sperm) has been reported for some Baltic macroalgae (Serriio et al., 1999), which cmbe a lethal condition since the electrical 134 polyspermy block usually employed by marine organisms becornes less effective under decreased Na" concentrations. However, the distribution of Hildenbrundia in the Baitic basin may not be affected by dl of these stresses. Gametes have not been convincingly reported for HiIdenbrandia (Irvine & Pueschel, 1994), and thus the marine fonn present in the Baltic Sea, W. rubra, does not have wall-less gametes which would require strong compensatory action (i-e. active transport of solutes across the plasmalemma) to maintain osmotic equilibrium; Thus, the absence of sexual reproduction may be an asset in this environment.
Although marine forms of Hildenbrandia cm be abundant in estuarine environments (cg Waern, 1952; Bird & McLachlan, 1992), the fkeshwater forms of the genus (possessing gemmae as reproductive structures) are not reported fiom habitats with increased salinities. Even in brackish water of much reduced salinity, tetrasporangia are the only reproductive structures found, Intermediate structures between tetrasporangia and gernmae have not been found. Thus there is no evidence for homology of tetrasporangia and gemmae, and this, in combination with our rnolecular results in this study, is consistent with the European fieshwater species of Ni[denbrandia not having undergone multiple, recent invasions into fkeshwaters from the marine environment.
Instead, fieshwater Hiklenbrandiu in Europe (K. rivularis) appears to be a completely separate entity fkom the marine forms as illustrated by the genetic uniqueness of the fieshwater forms. This trend suggests that few invasions or a single invasion event occurred into fieshwaters in Europe, followed by dissemination among fieshwater habitats. In addition, the strong genetic distinction between marine and fkeshwater forrns suggests that these invasions were not recent (Le. not since the end of the last glaciation
in this region). Skuja's (1938) theory of invasion by some marine red algae into freshwater habitats is not supported for Hildenbratidia on the scale of the last 10,000 y in southern Sweden.
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CHAPTER 5: Microscopie analysis and seasonality of gemma production in the freshwater red alga aildenbrandia angolensis
5.1. Introduction
The crustose red alga Hildenbrandia Nardo is widespread on intertidal and mbtidal coastlùies worldwide (e-g. Taylor, 1937; Abbott & Hollenberg, 1976;
Womersley, 1994; Silva et al., 1996). The fieshwater representatives, although morphologically very similar to the marine hs,are more locaiized in their distribution
(Sheath et al., 1993)- The thallus of both marine and fieshwater forms of Hildenbrandia has a heterotrichous construction with a prostrate basal layer of cells fkom which extend upright, branched filaments of cells (hine & Pueschel, 1994). The fieshwater species H rivularis (Liebrn.) J. Agardh is commonly reported fiom Europe and Asia (e.g- Jao, 1941;
Bourrelly, 1985), while H. nngolensis Welw. ex W. West et G.S. West is the common species reported fiom North America, South Arnerica and northem Afnca (West & West,
1897; Necchi, 1987; Sheath et al., 1993) and H ramanaginaii M. Khan is described fiom
India (Khan, 1974). The first two species are easily distinguished by ce11 size; the Srpe specimen of angolemis \vas observed to have significantly smaller ce11 dimensions than that of H. rivularis (ce11 diameter R = 5Spm versus 8.4pm, ce11 length x = 4.6pm versus 8.6prn; Chapter 6). The third species, H. raman~~naii(Khan, 1974), is of dubious validity since the taxonomie description does not resemble the genus
Hildenbrandia (descnbed as green in color and lacks gemmae) and the type material is not accessible for examination (Chapter 6).
The marine and fieshwater species of Hildenbrandia are distinguished both by 141 their habitat and their mode of reproduction. The marine species reproduce through the production of tetrasporangia in conceptacles on the thallus surface (Irvïne & Pueschel, l991), while the fieshwater species reproduce through the production of gemmae, which are flattened, cylindrical clusters of cells that develop on the thallus surface (Sheath et al.,
1993). The gemmae of fieshwater HiIdenbrandia appear to be a unique type of propagule among the red dgae. Reproduction is believed to be entirely asexual and vegetative in
Hildenbrandia, as no sexual stage has been documented convincingly (Fritsch, 1945;
DeCew & West, 1977).
Gemmae were first accurately described and recognized as reproductive structures by Starmach (1952), who noted their presence on collections of fieshwater Hildenbrandia fiom Poland, but earlier authors (Fritsch, 1929; Geitler, 1932) may have seen these structures as well. Since that heseveral authors have examined the morphology of gernmae using light microscopy and culture experiments (e.g. Nichols, 1965; Seto, 1977;
Necchi, 1987), but no studies have reported the developmentd sequence of gemma production in fieshwater Hildenbrandia using a combination of light microscopy, scanning and transmission electron microscopy and histochemistry for cornparison of gemma and thallus cells. In this study 1 examined the processes of gemrna development and release fiom the thallus in HiIdenbrandia angolemis. In addition, coIlections of H- angolensis fiom two spring-fed streams in Texas were made over a 12-month penod to examine the seasonality of gemma production.
5.2. Materials and Methods
5.2.1. Collection of Hildenbrandia angolensis 142
Multiple collections were made fiom two sprîng-fed streams in Texas fkom
October 1996 to September 1997. Four collections were made dong the San Marcos
River, San Marcos, Texas, U.S.A- (29 "54N, 97O54'W) (December 1996, April 1997,
May 1997 and September 1997) and six collections were made near the spring source of the Coma1 River, New Braunfels, Texas, U.S.A. (29 "42N, 98 "6'W) (October 1996,
December 1996, February 1997, April 1997, May 1997 and September 1997). Samples were either scraped fiom the rock substratum with a razor blade and fked irnrnediately for microscopy or transportecl [ive to the laboratory for culture in dilute (1 :20 strength)
Alga-Gro Freshwater Medium (Carolina Biological Supply). Pieces of thallus were fixed for light microscopy, histochemistry and seasonal rneasurements in 2.5% CaC0,-baered glutaraldehyde to prevent rnorphological distortion (e.g. Sheath et al., 1993). Collections for transmission and scanning electron microscopy were fixed in KamovsSf's fixative
(KarnovsS., 1965). The following strearn conditions were also measured at each sarnpling tirne: water temperature, pH, specific conductance, current velocity and maximum depth as described in Shenvood & Sheath (1999a). Daylength data were obtained fiom the Astronornical Applications Department of the US. Naval Observatory.
5.2.2. Microscopical and histochemical techniques
Light microscopy: Samples from both the field-collected material and the cultured materid were used to examine the processes of gemma development and release.
Collections fixed in 2.5% CaC0,-buffered glutaraldehyde were dehydrated in a graded ethanol series and embedded in LR White Resin. Thick sections were eut with glas knives and stained with Toluidine blue O in sodium tetraborate for morphological 143 observations (MiUonig, 1976). Sections were viewed on an Olympus BK-2 compound microscope and photographed with an Olympus PM- 1OAK photographie systern.
Scanning electron microscopy: Fixed samples were dehydrated in a graded ethanol series and critical point dried. Dried samples were mounted and sputter coated with a
AOdailoy and viewed on a JEOL 35-C SEM at 20-25 kV-
Transmission electron microscopy: Fixed samples were processed according to Sheath et al. (1977) and viewed with a JEOL 100-CX TEM at 60 kV.
Histochemistry and X-ray microanalysis: Thick sections of material embedded in LR
White Resin were cut with glass knives. Sections were stained with Toluidine blue O in benzoate buffer at pH 4.4 (Feder & O'Brien, 1968), the Penodic acid - Schiff (PAS) reaction (O'Brien & McCully, 1981)' 1% Amido black in 7% acetic acid (Fisher, 1968),
Alcian blue (Sheath & Cole, 1!BO), 1% Acid füchsin in water (Feder & O'Brien, 1968),
0.3% Sudan black B in 70% ethanol (BeneS, 1964) and 0.02% Coomassie bdliant blue
(in Clarke's solution) (Massaro & Markert, 1968; Gahan, 1984). Controls consisted of applying solutions to sections without the staining component (e-g. omission of penodic acid application in the PAS reaction), or removal of the targeted structures (e-g. methanol
/ chlorofonn lipid extraction for lipid stains). The reaction for each stain was evaluated for the thallus cells, the gemma cells and the material contained in the space between the gemmae and the region of the thallus fiom which the gernrnae were released. For each stain the reaction was characterized for the three areas as absent, weak, moderate or strong. Energyaispersive X-ray microanalysis was employ ed to determine the composition of dense, osmophilic globules present in both thallus and gemma cells, since 144 similar bodies have previously been found to be polyphosphate bodies in red algae
(Chopin et al, 1997). Fixed material was embedded in LR White resin (without OsO, post-fixation), thui sectioned and mounted on copper grïds. Sections were not stained.
Energy dispersive X-ray analyses were performed using a Link Analytical LZ-5 X-ray detector on a Philips 400T TEM operating at 100kV.
5.2.3. Seasonality of gemma production
To examine seasonal trends in gemma production and thallus morphology for H. angolemis in two Texas spring-fed streams the following characters were rneasured or detennined for each sample: ce11 diameter (n = 30), ceil Iength (n = 30), filament height
(n = 20), basal layer height (n = 20), gemma diameter (n = 10) and gemma height (n =
10). To quanti@ gemma production, the nurnber of gemmae in 1.0 mm' was counted for each sarnple (n = 10). Ln addition, the number of recently released gemmae was counted
(seen as "holes" on the thallus surface) in the same manner. Means for each measurement and sample were plotted over tirne (error bars calculated as standard error of the mean). Correlations of sample measurements and stream conditions were performed using the software program SigmaStat (Jandel Scientific) with the Spearman
Rank Order Correlation @ < 0.05).
5.3. Results
5.3.1. Gemma morphology and anatomy
The gemmae of H angolensis develop on the upper suiface of the crustose thallus, and appear as outwardly domed, round bodies that are not contiguous with the sukouoding thailus (Fig. 5.1). The cylindricd gemmae appear circular when viewed 145 fiom above (Fig. 5.2), and range in diameter fiom 48.6 - 71.0pm (x = 59.6,~~)and in height fiom 25.8 - 37.9pm (x = 3 1-8pm). Mer settling and adhering to a substratum some gemmae collected in Comal Springs were observed to have rhizoids (Fig. 5.3).
5.3.2. Histochemistry and X-ray microanalysis
The results of the staining reactions for each of the histochemical stains are sumrnarized in Table 5.1. Two stains did not yield a positive reaction for any of the three areas examinecl: Toluidine blue O in benzoate buffer pH 4.4 (phenolics) and Alcian blue
(mucopolysaccharides) (Table 5.1). The absence of a positive stainulg reaction with
Alcian blue for H. angolensis gemmae was previously demonstrated by Sheath & Cole
(1 990). A strong staining reaction was observed using Coomassie brilliant blue (proteins) in both the thallus and gemma cells, with a weak reaction in the region between them.
Sirnilar results were obtained for Amido black (protein), with a more moderate staining reaction observed in the cells of the gemmae. Thus, these stains indicate that both the thallus and the gemma cells contain moderate to large amounts of protein and that the region f?om which the gemmae are released contains a small arnount of protein (Table
5.1). The PAS reaction stained both gemmae and thallus cells positively for starch, but yielded a more intense reaction in the gemrna cells (Fig. 5.4). Sudan black B stained positively for lipids in both thallus and gemrna cells (Table 5.1). Energy dispersive X-ray microanalysis of dense, osmophilic globules indicated that they did not contain large amounts of phosphorus (spectnim not shown), and thus were not polyphosphate bodies
(Chopin et al., 1997). Figs. 5.1 - 5.4. General morphology of gemmae of H angolemis.
Fig. 5.1. Scanning electron micrograph of the cmst surface showing gemmae as raised bumps (small arrow) and regions where gemmae have previously been released fiom the thdus (large arrow).
Fig. 5.2. Light micrograph of a released gemma.
Fig. 5.3. Gemma that has settled and adhered to substratum, as indicated by the growth of rhizo ids (arrows) .
Fig. 5.4. Periodic acid-Schiff reaction-stained section of LR White-embedded material. The gemma cells (small arrow) stain more darkly than thallus cells (large arrow), indicating larger amounts of starch in the gernmae.
5.3.3. Gemma development
The developmental sequence of gemmae in H angolemis was examined by light
rnicroscopy using toluidine blue O-stained sections of resin embedded material. Fully
formed gemmae were visible in transverse sections of the thallus as wedge-shaped
clusters of cells (Fig. 5.5). Gemmae detached laterally fiom the thallus first by a peripheral ceil layer (Fig. 5.6) or simultaneously by peripheral and basal ce11 layers (Figs.
5-7, 5.8). Afier gernmae were completely detached from the H. angolensis thallus, they are released by an unknown mechanism (Figs. 5.9,5.10). The areas from which gemmae have been released cm sometimes be seen to produce additional gemmae (Fig. 5. IO), which may indicate the presence of localized meristematic regions for the production of gemmae.
Released gemmae are cornposed of unbranched or branched, upright filaments four to seven cells ta11 (Figs. 5.1 1, 5.12). Cells at the ends of the filaments are more dome-shaped than the imer cells. Gemmae appear to be released through a process of ce11 separation, since regions where partial connections of the gemma to the thallus can still be observed (Fig. 5.12). The cells of the gemmae have thick walh (Fig. 5.13) and contain numerous floridean starch granules that fil1 >50% of the ce11 volume (Figs. 5.13,
5.14). In many of the gernrna cells the floridean starch granules form a ring surroundhg the nucleus of the ce11 (Fig. 5.14). Copious amounts of material are present in the region between the released gemma and the thallus (Figs. 5.15,5.16), including bacterial cells, lipid globules and starch granules (Fig. 5.16). Figs. 5.5 - 5.10. Light microgaphs of gernrna formation and detachment in HiIden brandia angolemis.
Fig. 5.5. Gernmae first become recognizable within the thallus by their slightly larger cell size.
Fig. 5.6. Detachment of the gernma fkom the thdus can occur by apparent ce11 separation fiom the sides of the gemma (arrows).
Fig. 5.7. Detachment cm also occur by ceIl separation of both the bottom (small arrow) and the sides (large arrow) of the gemma.
Fig. 5.8. Detachment can also occur by ce11 separation of the bottom cells (arrow) of the gemma.
Fig. 5.9. After most cellular connections with the thallus have been broken the gemma is released ikom the thallus.
Fig. 5.10. Thallus cells in the region beneath the released gemma appear larger than the surroundhg thallus cells (arrow). An additional gemma is being formed directly below the released gemma.
Figs. 5.1 1 - 5.16. Transmission electron rnicrographs of gemma structure and development of HiIdenbrandia angolensis,
Fig. 5.11. An almost complete gemma demonstrating the organization into vertical £iles of cells. The cells at the ends of the filaments (small arrows) are more rounded in shape than the cells in the inner parts of the filaments (large arrows).
Fig. 5.12. A partial connection (arrow) between the gemma (g) and the thallus (t) is evident,
Fig. 5.13. A group of gemma cells with thick ce11 walls (arrows).
Fig. 5.14. A filament of a gemma. Cells of the fiIament have large numbers of floridean starch granules (s), which fiequently form a ring around the nucleus (n).
Fig. 5.15. Copious arnounts of material (arrow) present in the region between the gemma (g) being released and the thallus (t).
Fig. 5.16. Higher magnification view of the material in the region between the gemma (g) and the thallus (t). Both lipid globdes (1) and starch granules (s) are present, as well as bacterial cells (b).
154
5.3.4. Seasonalïty of gemma production
The mean numbers of gemmae and released gemmae counted per 1.O mmz were plotted over time to determine seasonal trends in gemma production by H. angolensis in the San Marcos River and Comal Springs (Fig. 5.17). The trends of gemma abundance are quite different for the two strearns, indicating that either there is not a distinct period of gemma production, or the sampling regime was not adequate to reved seasonai patterns. At least sorne gemma production was observed in ail collections, and in the samples in Comal Springs, the number of holes left on the thallus by released gemmae was highest in the May 1997 and September 1997 collections. Hildenbrandiu angolensis was ody found in the San Marcos River on four collection dates, so few counts could be made. However, both the number of gemmae and the number of released gemmae were higher in the May and September collections than the December and Apnl collections.
Correlation analyses of H. ungolensis thallus morphometry and physical and chemical conditions in the strearns for each sampling date (data from Shenvood & Sheath
1999a) revealed few significant correlations. For the samples collected fkom Comal
Springs, genima height was significantly and positively correlated with daylength (r =
1.O, p < 0.003). Thus, the gernmae of the Comal Springs population were largest in the summer months and smallest in the winter months. In addition, thallus ce11 Length was significantly and negatively correlated with the number of released gemmae counted (r =
-0.37, p < 0.02). No significant correlations were determined for the samples collected fiom the San Marcos River. Fig. 5.17. The number of gemmae and the number of "holes" counted per 1.0 mm' produced by Hildenbrandia angolemis over the period f?om October 1996 to September 1997. Collections are fiom Comal Springs, New Brauafels, Texas (circles) and the San Marcos River, San Marcos, Texas (squares). Although distinct periods of gemma production could not be determined, gemma production was observed to continue throughout the year. Error bars represent standard errors of the mean. Number of gernmae per unit area during sarnpling period 22 20 - - - 18 -
Oct Dec Feb Apr Jun Aug 1996 1996 1997 1997 1997 1997 sampling date
-O- Comal Springs 4- San Marcos River
Number of released gemmae per unit area during sampling period
Oct Dec Feb Apr Jun Aug 1996 1996 1997 1997 1997 1997 sampling date . 5.4. Discussion
The gemmae of H- angolensis are more appropriately referred to as propagules
than spores, since they are composed of clusters of undifferentiated vegetative cells rather
than a single ceU (Guiry & IMne, 1989). The uniqueness of these propagules should be
emphasized, given that most other algae, where it occurs, have asexual reproduction via
spores. Aithough several marine red algae are known to produce asexual propagules (e.g.
Deucalion and Centroceros wsman& Kraft, l982]), the 00other fieshater rhodophyte with propagules is Bahachospermum breuîelii Rabenh., and these propagules
are formed as products of fertilization (Sheath & Whittick, 1995). Other groups of algae
produce propagules, (such as the brown alga Sphacelaria [e-g. Kitayama et al, 199 1J),
and several mosses, livenvorts and fems also produce propagules known as gernmae,
some of which superficially resemble those described here (e.g. Farrar & Johnson-Groh,
1990; Ligrone ef al., 19%).
Both the marine and the freshwater fonns of Hildenbrandia are believed to lack a
semal stage (Chapter 2; Shenvood & Sheath, 1999b), presumably having lost it
secondarily. Whether the tetraspores of marine Hildenbrandia and the gemmae of the eeshwater species are produced meiotically or mitotically has not been demonstrated, and thus it is unknown whether some representatives of the genus are homothallic ploidy
variants or if al1 cells of Hildenbrandia have the same chromosome number. Starmach
(1952) regarded the gemmae of H. rivularis as homologous to the tetrasporangia of
marine species, and suggested they were sufficiently similar for the gemmae to have been
denved from the tetrasporangia. However, without evidence of meiosis in the formation 158 of tetrasporangia in marine species of the genus, this is speculation.
The present rnicroscopical observations cohthat the marine and the fieshwater forms of Hildenbrandia are anatomically similar. Previous reports of
Hildenbrandia ultrastructure have demonstrated that cells and filaments of the thallus are connected by numerous prixnary and secondary pit connections (Pueschel, 1988; 1989), which most likely enhance the toughness of the thallus. Pueschel (1989) reported the freshwater species, H. rivularis, to have morphologically similar pit plugs to the marine species of Hildenbrannia, and additional phylogenetic evidence has demonstrated a close relationship between marine and fieshwater forms of the genus (Chapters 2 & 3).
The rhizoids observed growing from released gemmae were described previously
(e.g. Starmach, 1969; Seto et AL, 1974; Seto, 1977). Rhizoids were present only in gemmae that had been released fiom the thallus and that were attached to a substratum, suggesting that the rhizoids serve an anchoring purpose for the newly developing cmsts of the alga. The gonimoblas t propagules of Batrachospermum breutelii produce similar rhizoids (Sheath & Whittick, 1995).
The ring of floridean starch grandes observed surrounding the nucleus has been reported previously for other florideophyte taxa (Sheath, 1977). The large amount of floridean starch observed in the gemma cells is in contrast to the thallus cells, as noted previously by Starmach (1 969), and may be used as a carbohydrate reserve during the germination process. Flondean starch is digested in the germination of gonimoblast propagules of Batrachospermum bueutelii (Sheath & Whittick, 1995). The histochemical results for tests other than starch did not, for the most part, reveal strong differences between the gemma and the thallus cells-
This study provides the first micrographs of sectioned matenal demonstrating reproduction in fieshwater Hilaenbrundia. Both Seto (1 977) and Necchi (1987) illustrated the development of gemmae within the cmst of fieshwater Hildenbrandia, but the findings of this study differ substantially. The gemmae of X angolensis were observed to develop within the thallus, forming only superficial bumps on the surface of the crust. In contrast, Seto (1977) illustrated gemma development in rivularis as forming protuberances on the crust surface in which almost the entire height of the gemma extends above the cmst. In addition, locations on the cmst where gemmae had been previously released appear as small craters in our micrographs instead of raised structures (Seto, 1977).
The early literature on fi-eshwater HiIdenbrandia includes a nurnber of unsubstantiated fïndings on reproduction other than by gernmae. A number of studies reported sexual reproduction in fieshwater specimens (Boni, 1880; Petit, 1880; Palik,
1957). However, in al1 cases, later interpretations showed that the illustrations depicted either rhizoidal growths i?om the gemmae or the thallus, or other species of algae living in close association (Starmach, 1969). The earliest report of fireshwater Hildenbrandia in
North Arnerica (Wolle, 1887) indicated reproduction by tetrasporangia; later Flint (1 955) provided drawings of tetrasporangial production within conceptacles of fi-eshwater
Hildenbrandia. However, given that many of our observations in this study are based on matenai hmthe same stream (Coma1 Springs, New Braunfels, Texas) as that studied by
Flint (1955), and given that no additional reports have been made of tetrasporangial 160 production in fkeshwater Hildenbrandia, we question the validity of these earïier observations.
This work represents the fîrst study of the development and seasonality of fieshwater Hildenbrmdia in North America, and is the fïrst to incorporate findings i?om diverse techniques such as light microscopy, scanning and transmission electron
Wcroscopy, histochemistry and seasonai observations. Further studies into the details of reproduction in both the marine and fieshwater forms of Hildenbrandia are necessary to confirm the Life history of this genus and investigate the ongins of the fieshwater representatives.
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Sherwood, A-R. & Sheath, R.G. 1999b. Biogeography and systematics of Hildenbrandia (Rhodophyta, Hildenbrandiales) in North America: inferences fiom morphometrics and rbcL and 18s rRNA gene sequence analyses. European Journal of PhycoZogy 34: 523-532.
Shenvood, A.R. & Sheath, R.G. 2000. Biogeography and systematics of Hildenbrandia (Rhodophyta, Hildenbrandiales) in Europe: inferences fiom morphometrics and rbcL and 18s rRNA gene sequence analyses. European Journal of PhycoIogy 35: 143-152.
Silva, P.C., Basson, P.W. & Moe, R-L.1996. Catalogue of the Benthic Marine Algae of the Indian Ocean. Universiq of California Press, Berkeley, 1291 pp.
Starmach, K. 1952. The reproduction of the fiesh water Rhodophyceae Hildenbrandia rivularis (Liebm.) J. Ag. Acta Societatis Botanicorum Poloniae 21: 447-474.
Starmach, K. 1969. Growth of thalli and reproduction of the red alga Hildenbrandtia rivularis (Liebm.) J. Ag. Acta Societatis Botanicorum Poloniae 38: 523-533.
Taylor, W.R.T. 1937. Marine Algae of the Northeastern Coast of North America. The University of Michigan Press, Ann Arbor, 509 pp.
West, W. & West, G.S. 1897. Welwitsch's Afiican fieshwater algae. Journal of Botany Wolle, F. 1887. Fresh-water Algae of the United S'rates (exclusive of the Diatomnceae). The Comenius Press, Pennsylvania, 364 pp.
Womersley, H.B .S. 1994. The Murine Benthic Flora of Southern AustraIia- Rhodophyr~- Part IIL4. Australian Biological Resources Sîudy, Canberra, 508 pp. 165
CEIAPTER 6: Global biogeography and systematics of the mdenbrandiales: inferences from rnorphometrics of type speeimens and global collections, and sequence analysis of the rbcL and 18s rRNA genes
6.1. Introduction
The red algal order Hildenbrandiales contains two genera: Apophlaea, which is limited in distribution to the marine coastlines of New Zealand (Hawkes, 1983), and
Hildenbrandia, which is globally distributed in both marine and fieshwater habitats (e-g.
Rosenvinge, 1917; Bourrelly, 1955; Silva et al., 1996). Despite the prevalence of the order, little work has been conducted until now to cl- its systematics by comparing results fiom modem molecular analyses with those fiom traditionai morphometric analyses of type specimens, although past attempts have been made to summarize the systematics of Hildenbrnndia morphologically (Denizot, 1968). Current systematic treatments in phycology usually employ a combination of morphological and molecular analyses to obtain support for taxonomie decisions kom severai sources (e.g. Saunders et al., 1995 [Acrochaetiales-Palmarialescornplex]; Vis & Sheath, 1998
[Ba~achospermum];Bailey, 1999 [Corallinaceae]; Pueschel et al., 2000 [Audouinella]), and this will be employed in the current study.
The biogeography and systematics of Nildenbrandia in both North America and
Europe were examined in previous chapters (Chapters 2 & 3; Sherwood & Sheath, 1999,
2000). In this chapter 1 expand upon these analyses through the inclusion of collections of Hildenbrandiales representing taxa not previously available for analysis, including H. lecannellieri, H. patula, H. dawsonii, Apophlaea sindairii and Apophlaea Zyallii, as well 166 as additional collections fiom scattered global regions, including the Philippines,
Australia, the Cmary Islands, Chile, Uruguay and South Afnca. Phylogenetic reconstruction of members of the order using DNA seqriences of the rbcL and 18s rRNA genes is presented here, as well as morphometric analyses of global collections and the type specirnens.
6.2. Materials and Methods
6.2.1. Type specimens, historicaily significant specimens and global collections analysed
Type and historicdly significant specirnens of the order Eldenbrandiales
(HiZdenbrandia and Apophlaea) were analyzed as foIIows (herbarium abbreviations according to Holmgren et al. [1990]):
1. Apophlaea lyallii Hook. f. et Hm. (1855: 244). Locality: On rocks, Preservation
Harbour, Middle Island, New Zealand, i. 185 1, D. Lyall, sy-ntype, No. E0004447 1, E.
2. A. lyallii var. gigarhoides Hook. f. et Harv. (1855: 244). Locality: Otago, New
Zealand, iii. 1850, D. Lyall, syntype, No. 000530643, BM.
3. A. sinclairii Harv. ex Hook. f. et Harv. (1845: 550). Locality: New Zealand, no date given, Sinclair, syntype, No. 000530644, BM.
4. Hildenbrandia ["Hildenbrandtia"] angolensis Welw. ex W.West et G.S. West (1 897:
3). Locality: Golungo Alto, Ad silices in rivulis sylv. primit. de Quibanga pr. Sange,
Angola, vi. 18 57, Welwitsch, syntype, Welwitsch Collecticm No. 150, BM 3435 slide collection, BM.
5. H. canariensis Bmgesen (1929: 15). Locaiity: Gran Canaria: South of Las Palmas near 167
Christobdlo, 29.iii.1921, F. Bmgesen, syntype, No. 3986, C.
6. H. rHiZdenbrandtiayy]crouanii ["crouanr"] (J. Agardh) J. Agardh (1876: 379).
Basionyrn: Haematophlaea crouani (1852: 495). Locality: Sur les roches "Dit" ansi du
Cortem, environs de Brest, no date available, ''fières" Crouan, holotype, No. 27613, LD.
7. H rHiZdenbrandtia"] expansa Dickie (1874: 357). Locality: St. Paul's Rocks
(Challenger Expedition), 29 O 15' W, one degree N of equator, 27.viii. 1873, H.N. Moseley, syntype, No. 000530646, BM-
8. "H f7uviatiZi.s Bréb.". Specimen on a rock, glued to a card, historically significant specimen, Falaise, PC.
9. "H. fluviatilis Bréb.". Specimen on a rock, glued to a card, histoncally signincant specimen, Falaise, S.
10. H [;CHildenbrandtia'~]galapagemis Setch. et N.L. Gardner (1937: 91). Locality:
Charles Island, Galapagos, 26.i~.1932, J.T. Howell, holotype, No. 2365 19, UC.
1 1. H kerguelensis (Askenasy) Y.M. Chamb. (1 962: 372). Basionym: K- rcfiildenbrandtiayy]prototypus var. kerguelensis Askenasy (1 888 : 3 0). Locality:
Kerguelen (Gazelle Expedition), slides of the holotype (made by F. Schmitz), ix. 1888, L.
Askenasy, No. 00530647, BM.
12. H. ["'Hildbrandtia"] Zecannellieri rcLe Cannellieri", "Le Cannelier?'] Har. (1 887:
74). Locality: Ad rupes maritimas Baie Orange (Fuegia), viii. 1883, Hariot, slide of the holotype, PC.
13. H. ["'Hildenbrandfia"] occidentalis Setch. ex N.L. Gardner (1 9 17: 393). Localiîy :
Land's End, San Francisco, California, U.S.A., 4.i. 19 16, N.L. Gardner, holotype, No. 188974, UC,
14. H r'HiZdenbrandtiay '1 occidentaZis var. Zusitanica Ardré (1 959: 23 3). Locality: Supra rupes in oceano atlantico ad oras Lusitaniae, Parede, 26.vi.1957, F. Ardré, holotype, PC.
15. H. ["H'denbrandtiu"] occidentalis var. yessoensis (Yendo) Ardré (1959: 233).
Basionym: H. ["'HiZdenbrandkia"] yessoensis Yendo (1920: 11). Locality: In mpibus marïtimas ad oras Yesso, Oshoro, Hokkaido, Japan, 30.iii.1915, K. Yendo, sections of holotype, SAP,
16. H. putula ["'expansa", homonym (1 996: 3 57)] Womersley (1 994: 145). Localiîy :
Apollo Bay, Victoria, Australia, 6.ii. 1990, H.B.S. Womersley, isotype, No. ADA60088,
AD.
17. H. rCHiZdenbrandria"]rivularis (Liebm.) J. Agardh (185 1: 495). Basionym:
Erythroclathrus rivularis Liebm. (1 839: 174). Locality: Stream at Kiugs Milis, Seaiand,
Denmark, vi. 1826, S. Hornemann, C [measurements fiom Sheath et al. (1 993)l.
18. rivuluris var. drescheri ["Drescheriyy]Lingelsh. (1922: 355). Locality: Mühlgraben bei Ellguth K. Othnackan, 1920, E. Drescher, lectotype here designated, BRSL.
19. H- ["'HiZdenbruntiaY~]rubra (Sommerf.) Menegh. (1 84 1: 426). B asionym: Verrucaria rubra Sommerf. (1826: no pagination). Locality: Ydtdalen, vii. 1822, Sommerfelt, holotype, Herb. Univers. Christianensis, Nonvay, O.
20. H. sanjuunensis Hollenb. (1 969: 164). Locality : High intertidal one-half mile east of
Fnday Harbour Laboratones, Sm Juan Is., Washington, U.S.A., 19.vi. 1968, D. Russell, microslide of holotype, No. 00061194, US. .
21. H sanjuanensis Hollenb. Locality: about 100 yards south of Small Pox Bay, San Juan 169
Island, San Juan County, Washington, U.S.A., l4.vi. 1968, G.J. Hollenberg, microslide,
No- 066265 OJS slide #-1419), US.
The following type specimens were requested but were mavailable for
examination:
1. Hildenbrandia ["Hildenbrandtia"] arracana ["Amna"] G. Zeller (1873 : 192); MB
2. H. dawsonii (Ardré) Hollenb. (1971 : 286) (basionym H canariensis var- dawsonii
Ardré [1959: 2301); PC
3. H nardiana Zanardini (2840 :134); Venezia
4. H paroliniana Zanardini (1 840: 135); Venezia
5. H. cHiZdbrandfia"]prototypus Nardo (18 3 4: 676); Venezia
6. H ramanaginaii M. Khan (1974: 238); BHAV, BSD, BSIS, BURD, CAL, DD
7. H. ["Hildenbrandtia"] rivularis spp. chalikophila Palik (1961: 152); BP
8. H ["Hildenbrundtia"] rosea Kütz. (1843 : 384); L
9. H ['CHiZdenbrandtia'~]sangzrinea Kütz. (1843 : 384); KRAM, L
The following type specirnens of taxa previously synonomized with H. rubra were
examined for verification of synonomy :PalmeZla rubru Hornem., Erythro clathrus peZZitus Liebm. and Rhododermis drummondii Harv. No reproductive structures
(gemmae or tetrasporangia) were visible on the specimen of Palmella rubra, and thus the
synonomy of this species with rubra is uncertain. The type specimens of both
Erythroclathrtcs pellitus and Rhododermis drummondii corresponded to H rubra.
Additional global collections of Hildenbrandia and Apophlaea included in the
analyses are listed in Table 6.1 and locations are illustrated in Fig. 6.1.
Table 6.1. cont.
Sample code Collection or source information Taxon GenBank Accession rbcL 18s rRNA
SL9 Chapter 2 (Sherwood & Sheath, 1999) H,angolensis " AFlO8416 PR19 Chapter 2 (Sherwood & Sheath, 1999) If angolensis - AF108415 FL63 Chapter 2 H. angolensis to submit to submit SWESWI Chapter 3 (Shenvood & Sheath, 2000) H. rubra AF2O8 8 12 AF208828 NORSW 1 Chapter 3 (Shenvood & Slieath, 2000) H rubra AF208807 AF208826 SCOSWl Chapter 3 (Shenvood & Sheatli, 2000) H. crouanii AF208808 to submit SCOSW3 Chapter 3 (Sherwood & Sheath, 2000) H. crouanii - - SCOSW4 Chapter 3 (Shenvood & Sheath, 2000) H, crouanii AF208809 - WALSW3 Chapter 3 (Shenvood & Sheath, 2000) H, rubra AF2O88 15 AF20883 1 WAL2 Chapter 3 (Sherwood & Sheath, 2000) H. rivularis AFZO88 13 AF208829 WAL3 Chapter 3 (Shenvood & Sheath, 2000) H. rivularis AF2O88 14 AF208830 NISWl Chapter 3 (Shenvood & Sheath, 2000) W. rubra AF208799 AF2088 19 IR1 1 Chapter 3 (Sherwood & Sheath, 2000) H. rivularis AF208805 AF208824 GERSW 1 Chapter 3 (Shenvood & Sheath, 2000) H. crouanii AF208803 to submit GERI Chapter 3 (Shenvood & Sheath, 2000) H, rivularis var. AF208804 AF208823 drescheri
Fig. 6.1. Collection locations of marine and fieshwater Hildenbrandia, as well as two Apophlaea specirnens used in global analyses. Circles indicate marine HildenbraPldta colIections, diarnonds indicate fieshwater Hikienbrandia collections and squares indicate Apophlaea collections. For collection and sample details see Table 6.1.
6.2.2. Morphomehic analyses
Type and histoncally significant specimens of Apophlaea were requested and examined, but only the upright portions of the thallus were present. Thus, morphometric cornparisons to Hildenbrandia specimens could not be made due to the absence of the crustose portion of the thallus. However, measurements of length and diarneter of tetrasporangia were made, and tetrasporangial division pattern nnted for cornparison with
Hildenbrandia specimens.
Samples were fixed in 2.5% CaC03-buffered glutaraldehyde to prevent morphological distortion, or re-hydrated after preservation in silica gel and they were analyzed to determine associations based on sirnilarïty. The following characters were measured or determined for al1 collections as well as type and historically significant specimens: ce11 diarneter (n = 3 O), ce11 length (n = 30), filament length (n = 30), basal layer height (n = 30) and habitat (marine or fi-eshwater). Where possible, marine collections were measured for the following reproductive characters: maximum conceptacle depth and diameter (n = IO), and maximum tetrasporangial length and diameter (n = 10). Presence or absence of protuberances on the thallus surface and the tetrasporangial division pattern for each sample were also noted. Tetrasporangial division pattern was coded as follows: divisions parallel or some divisions not parallel, and other aspects of tetrasporangial rnorphology were also noted (such as whether tetraspores are in contact with one another or separated by a space, and whether planes of division were transverse or oblique). The presence or absence of gemmae was noted for fieshwater ccllections. The data rnatrix contained Ê. cornbuiation of qualitative (binary-coded) and 178 quantitative data, The data were ranged so that characters with large numencal values were not given more weight tlian characters with srnaUer numericd values (Dunu &
Everitt, 1982). For each of the following analyses both cluster analysis (UPGMA algorithm) and ordination (principal components PCA] or principal CO-ordinatesPCO] analysis) were performed on the data:
Analysis 1 - Al1 type specimens and historicaiiy significant specimens.
Analysis 2 - Marine type specimens and histoncdy significant specimens inciuding reproductive character measurements,
Analysis 3 - AU specirnens (using only vegetative characters). Binary coding was employed for marine [Ilj versus fieshwater [O] specimens, and the tetrasporangial division pattern; divisions not al1 paralfel [l], divisions parallel [O].
Analysis 4 - Ail marine specimens for which reproductive character data were available.
Analysis 5 - Ail fieshwater specimens.
For analyses 1,2,3 and 5 the Gower similarity coefficient was used for both cluster and PCO analyses since it alIows the incorporation of both qualitative and quantitative data. For analysis 4, cluster and PCA based on Euclidean distances were used since only quantitative characters were involved. Significance of groups resulting fiom the analyses was tested using one-way analysis of variance (ANOVA) at a significance Ievel ofp < 0.05. Ciuster and ordination analyses were carried out using
MVSP 3 -0 (Multi-Variate Statistical Package; Kovach Computing Services, 1986- 1W8), and ANOVA's were carried out using Minitab (Ryan et al., 1985). 179
6.2.3. rbcL and 18s rRNA gene sequence analyses
For as many collections as possible the rbcL and 18s rRNA genes were amplified and sequenced (excluding types and historically significant specimens). The following methods were carried out as descrïbed previously (Chapters 2 & 3): PCR amplincation of the rbcL and 18s rRNA genes, purincation, sequencing and alignment, outgroup selection, maximum parsimony (MF) analysis, neighbor-joining (NJ) analysis and quartet puzzling maximum likelihood (QP) reconstruction.
6.3. Results
6.3.1. Morphometric analyses
Means of each quantitative character and binary designations for qualitative characters for each specimen are provided in Appendk 2. Tetrasporangial dimensions of
Apophlaea type and historically significant specimens (mean length 23.9 - 26.1 pm; mean diameter 6.0 - 7.5pm) were well within the range of dimensions of marine Hildenbrandia specimens (mean length 15.0 - 45.0prn; mean diameter 4.3 - l3.Sprn) (Table 6.2). The tetrasporangiai divisions of al1 ApophZaea specimens were parallel.
Analysis 1 - Axis 1 of the PCO biplot (x-axis) accounted for 57.7% of the total variation in the data set and axis 2 (y-mis) accounted for 13.5%, for a total of 7 1.2%. Type and historically significant specimens were separated into two groups based on cluster (Fig.
6.2) and PCO (Fig. 6.3) analyses, which corresponded to marine (A) and fieshwater (B) specimens. H. kcannellieri was distinct from the remainder of the marine specimens due to its thick thallus (Z = 929 pm for H. Zecannellieri, x = 274 pm for rest of marine specimens) and presence of protuberances on the thallus surface. Marine specimens were Table 6.2. Mean tetrasporangid dimensions for Apophlaea and Hildenbrandia type and histoncaliy significant specimens.
Specirnen mean tetrasporangial mean tetrasporangial leWh(Pm) diameter (pm) Apophlaea lyallii 23 -9 6.0 A. lyallii var. gigartinoides A. sinclairii Hilden brundia canariensis 28.2 9.8 H crouanii 33.4 H. galapagemis 30.3 H. kerguelensis 25.1 H. lecannellieri 34.0 H occidentalis 3 6.9 H occidentalis var. Iusitanica 45.0 H. occidentalis var. yessoensis 19.2 H- patula 28.7 H. rubra 34.0 H. sanjuanensis 15.0 Fig. 6.2. Cluster dendrogram of type and historicdy signincant specirnens of Hilden brandia based on vegetative datq marine versus fieshwater coding and tetrasporangial division pattern coding. Two groups are evident which correspond to marine (A) and fieshwater (B) specirnens. Tetrasporangia division pattern is indicated for the marine specirnens. The numericd scale indicates the level of similarity at which clusters are formed, according to the Gower similarity coeEcient. H. lecannellieri Eryfhroclath rus pellitus H. occidentalis H. patula H. occidentalis var. yessoensis H. sanjuanensis H. occidentalis var. lusitanica H. kerguelensis H. sanjuanensis #2 H. crouanii
H. canariensis H, rivularis H. fluviatilis (S) H. f/uviati/is (Pc) freshwater H,rivularis var. drescheri H. angolensis
Gower General Similarity Coefficient Fig. 6.3. Principal CO-ordinatesbiplot of type and historically signincant specimens of Hildenbrandiu. Two main groups are evident corresponding to the marine (A) and freshwater (B) specimens. Tetrasporangial division pattern is noted for the marine specimens.
185
largely, but not completely, separable based on tetrasporangial division pattern in the
cluster dendrogram (Fig. 6.2), and were more cleady separable in the PCO biplot (Fig.
6.3). Groups A (marine) and B (fieshwater) were also signifïcantly separated based on
cell diameter @ < 0.00 l), ceii length (p < 0.004), filament height (p < 0.0 11) and basal
layer height (p < 0.001), with the freshwater specimens on average having larger cell
dimensions but smaller filament and basal layer heights.
Analysis 2 - Analyses of marine types and histoncally significant specimens with
reproductive character data demonstrated two groupings (A & B), corresponding to the
two tetrasporangial division patterns (Figs. 6.4 & 6.5). Axis 1 of the PCO biplot
accounted for 28.5% of the total variation and axis 2 accounted for 17.4%, for a total of
45.9%. The two groups are not significantly separated on characters other than tetrasporangial division pattern. Again, lecannellieri is distinct fiom the remainder of the specimens within Group B, according to the cluster dendrogram. H. occidentalis is
morphometrically more similar to different species (e-g. H. pahrla and H. crouanii) than
to its own varieties (H. occidentalis var. Zusitanica and H. occidentalis var. yessoensis),
based on the criteria of vegetative and reproductive measurements.
Analysis 3 - Cluster (Fig. 6.6) and PCO (Fig. 6.7) analyses of al1 specimens based on
vegetative characters and tetrasporangial division pattern revealed two main groups,
corresponding to fieshwater (A) and marine (B) specimens. Axis 1 of the PCO biplot
accounted for 53.8% of the total variation and axis 2 accounted for 12.9%, for a total of
66.7%. The marine and fieshwater specimens have significantly different ce11 dimensions
@ < 0.001), filament lengths @ < 0.001) and basal layer heights @ < 0.004). Within the Fig. 6.4. Cluster dendrogram of ody type and histoncally significant marine specimens ernploying vegetative and reproductive character data. Two groups are evident corresponding to specimens with tetrasporangid divisions that are not al1 parallel to one another (A) and those with transversely divided tetrasporangia (B). The numencal scale indicates the level of similarity at which clusters are formed, according to the Gower similarity coefficient.
Fig. 6.5. Principal CO-ordinatesbiplot of type a~dhistoncally significant marine specimens employing both vegetative and reproductive character data. The same groups are evident as for Fig. 6.4.
Fig. 6.6. Cluster dendrogram of all Hildenbrandia global collections as well as type and historically signïfïcant specimens, based only on vegetative data, marine versus fieshwater coding and tetrasporangial division pattern. Only type and historically significant specimens are indicated on the dendrogram. The numerical scale indicates the level of similarity at which clusters are formed, according to the Gower simî1arity coefficient.
Fig. 6.7. Principal CO-ordinatesbiplot of dl Hilaenbrandia global collections as weII as type arZ tiistorically signïficant specimens, based only on vegetative data, marine versus freshwater coding and tetrasporangial division pattern. Only type and historïcaiiy significant specimens are indicated on the dendrogram.
194 fieshwater group (A), two Mergroups are delineated which correspond to the Europe and Canary Islands specimens versus the North American and Philippines specimens.
The type and historically signincant specimens of H. rivularis, H rivularis var. drescheri and H jhviatilis, the oldest of which is rivularis, associated with the first group, and the type specimen of H angolensis associated with the second group. These two groups are siggificantly dif5erent based on ce11 dimensions @ < 0.001), filament Iength @ <
0.032) and basal layer height @ < 0.00 l), with the H. rivularis group having larger characters than the H angolensis group. Within the marine group (B), two distinct groupings are evident fiom the cluster dendrogram, corresponding to the type specimen of
H. Zecannellieri and one collection fiom South Afnca versus al1 other marine specimens.
These two groups are significantly different based on ceII length @ < 0 .O 18) and filament length @ <0.00 1). Marine specirnens do not associate by tetrasporangial division pattern in these andyses.
Analysis 4 - Cluster (Fig. 6.8) and PCO (Fig. 6.9) analyses of al1 marine specimens for which reproductive character data were obtainable demonstrated two groups (A & B) which corresponded to tetrasporangial division pattern. Axis 1 of the PCO biplot accounted for 38.3% of the total variation and axis 2 accounted for 1 1.8%, for a total of
50.1%. Groups A and B were significantly different based on ceIl length @ < 0.034), filament height @ < 0.001), conceptacle diarneter @ < 0.023), conceptacle depth @ -=
0.002) and tetrasporangial diameter @ < 0.010). Wi'ithin Group A (Fig. 6.8), the type specimens of H occidentalis and H. lecannellieri are distinct fiom the remainder of the specimens in that group based on their large filament length @ < 0.001) and large cell Fig. 6.8. Cluster dendrogram of ail marine specimens (including type and histoncally signiticant specimens) for which reproductive character data were available, based on dl characters. Two groups are evident, corresponding to specimens with parallel tetrasporangial division (A) and those with not only parallel tetrasporangial division (B). The numerical scale indicates the level of similarity at which clusters are fomed, according to the Gower similarity coefficient.
Fig. 63. Principal co-ordinates biplot of al1 marine specimens (including type and historically significant specimens) for which reproductive character data were available, based on ali characters. Two groups are evident, corresponding to specimens with completely parallel tetrasporangial divisions (A) and those with some non-parallet tetrasporangial divisions (B) . I H. /ecannellieriA AH I H sanjuanensis #2A ABCSW~ -0,lO CRSWl A AMEXSWJ A H. sanluanensis ~SCOSWî SCOSW4& -0.20 AÜS SW1
-0.29 RlSWl wGERSWl Fig. 6.10. Cluster dendrogram of all freshwater specimens (mcluding types and historically significant specirnens). Two groups are evident correspondïng to H rivularis sarnples (A) and H angolensis samples (El). The numerical scale indicates the level of dissimilarity based on Euclidean distance. r H, rivularis H, fluviatilis (S) FRAI lTAl I GER1 Cl1 H, rivularis var, drescheri SPA1 CanaryEurope Islands and 1 AT1 4 I AT1 O
I WAL2 North America and I 1 WAL3 Philippines IR11 1 H. angolensis
Dissimilarity based on Euclidean distance Fig. 6.11, Principal components anaiysis biplot of all fkeshwater specimens (including types and historically significant specimens). Two groups are evident conesponding to W. rivularis samples (A) and H. angobnsis samples (B). B North America and 0.13- Philippines
AAH. tïuviatilis (SI AH. rivularis length (p < 0.003).
Analysis 5 - Cluster (Fig. 6.10) and PCA (Fig. 6.1 1) analyses of all fieshwater specimens demonstrated two groups (A & B) that correspond to the groups in Analysis 3. In the
PCA biplot axis 1 accounted for 71.4% of the total variation and axis 2 accounted for
23 .O%, for a total of 94.4%- Groups A and B are significantly different based on ce11 dimensions @ < 0.00 l), filament length (p < 0.032) and basal layer height (p < 0.00 1), with the H. rivularis group having larger characters than the H angolemis group.
6.3.2. Analysis of transitional saturation of the rbcL and 18s rRNA genes
The painvise p-distances (uncorrected sequence divergences) were plotted against the corresponding number of transitions between pairs of sequences for both the rbcL
(Fig. 6.12 a) and 18s rRNA (Fig. 6.12 b) genes fiom the global collections of the
Hildenbrandiales. The rbcL gene sequence data approached transitional saturation at approximately L 5% divergence, with most of the data points occuring beyond this level of divergence. Thus, the rbcL does not appear to be appropriate for comparisons within the order at a global level. In contrast to this trend, the 18s rRNA gene sequence data do not appear to be transitionally saturated for the Hildenbrandiales at a global level of cornparison, since the plot is essentially linear. As a result, global molecular analyses of the Hildenbrandiales were restricted to the 18s rRNA gene sequence data.
6.3.3. 18s rRNA gene sequence analyses - parsimony analysis
Parsimony analysis of 175 phylogenetically informative characters for the 18s rRNA gene (alignment included 11 O 1 invariable sites and 164 autapornorphic sites) yielded four most-parsimonious trees (CI = 0.67; length = 1269 steps). One of the four Fig. 6.12. a) Graph of the number of transitions versus thep-distances for al1 pairs of sarnples in the global Hildenbrandiales alignment for the rbcL gene. Transitional saturation occurs at approximately 15% for the rbcL gene.
b) Graph of the number of transitions versus the p-distances for al1 pairs of samples in the global Hildenbrandiales alignment for the 18s rRNA gene. Transitional saturation does not appear to be occuning in the data set. 205 Global rbcL (Hildenbrandiales)
0.00 0.05 0.10 0.15 0.20 0.25 p-distances Global 18s rRNA (Hildenbrandiales)
0.05 0.10 p-distances Fig. 6.13. One of four most-parsimonious trees generated using parsimony analysis of the 18s rRNA gene for the Hildenbrandiales (CI = 0.67, length = 1269 steps). Groups A, B, C and D include specimens of H. rubrn. Group E includes most fieshwater specimens (both H. rivularis and H. angolensis) and Group F includes specimens of H. crouanii, H. dawsonii and H patula. Freshwater specimens are denoted by a gemma illustration (cluster of cells) and marine specimens are denoted by tetrasporangial illustrations with the corresponding division pattern. SWESW NlSWl NSSW , NORSW1, MASW CTSWl ORSW AUSSW
CHISWl
VWLSw3 A, sinclaini A. lyallii
lTAl IR11, SPA; PHI1 FL63 SL9 & TX7 URUSWl SCOSWl AUSSWl MEXSW BCSWI MU Bangia Pophyra Erythroftichia 27 Srnithora Porp hyndium 208 trees is shown in Fig. 6.1 3; the others differed in the position of the WALSW3 sample within the rubru group and the position of several other North American and European H rubra samples with respect to one another. Little or no support was indicated for these relationships (e.g grouping of SWES W 1 and NISW 1). The ingroup (Hildenbrandiales) is well supported as monophyletic (100% BP, 154 decay steps). H. rubru is depicted as a paraphyletic taxon (Groups A, B, C and D), and there is Little support for the relationships among these groups. However, some relationships are evident within the groups. Several H. rubra collections fiom both Europe and North America were identical in their phylogenetically informative sites (NSS W 1, NORS W 1 and FRAS W 1), indicating a closer relationship among these samples from different continental coastlines than among some fiom the same continent. The position of WALSW3 is unresolved. Group D contains four Pacific North Amerka H. rubra samples @CS W 1, MEXS W 1, CASW 1 and AKSW 1), as well as one collection ti-om eastem Canada (NFSW1). The two col~ections morphologically corresponding to N. lecannellieri (SAS W 1 and CHISW 1) are strongly supported as monophyletic (98% BP, 8 decay steps), as are the two morphological desigxations of H occidentalis (BCSW4 & CASW3; identical in 18s rRNA gene sequence). Several marine species with only parallel tetrasporangial divisions form a clade (F), including H. crouanii, N. patula and H dawsonii. However, one additional sample of H. crouanii (GERSW1) is in an unsupported position near the base of the tree. This is most Iikely due to the large number of undetennined bases (N's) in this sequence. The two collections of the second genus within the Hildenbrandiales, Apophlaea, group tightly together (97% BP; 6 decay steps); however, the phylogenetic position of 209 Apophlaea with respect to the genus Hildenbrandia is unresolved as there is little or no support for the positioning of the major clades with respect to one another. The freshwater species of Hildenbrandia form a monophy letic group (E) with the exception of two North Amencan samples (W and PR19), which are poorly supported at the base of the tree. Support for Group E is poor (60% BP; decay I step), but is stronger with the omission of SL9 and TX7 (80% BP; decay 1 step). As previously reported (Chapter 3), many of the European H rfuzdaris samples are identical in sequence for the 18s rRNA gene, and the relationships among these samples are unresolved due to this lack of phylogenetic signal. The North American fieshwater collections are supported as basal to the European collections, and the one fieshwater collection fiom the Philippines groups with a North Amerïcan collection (FL63) with weak support (58% BP; 1 decay step). 6.3.4. 18s rRNA gene sequence analyses - distance analysis The neighbor-joining tree based on distance analysis of the 18s rRNA gene sequences is presented in Fig. 6.14. This tree shows many of the same trends as the parsimony analysis, but there are several differences. The H rubra samples AUSSW2 and ORSW2 are basal to the order, and the Apophlaea samples are supported as being derived fiom these (89% BP). Although unsupported, the H. occidentalis samples (CASW3 and BCS W4) are positioned much closer to the other sarnples of Hildenbrandia with only parallel divisions than in the parsimony tree. The freshwater samples in this analysis form a monophyletic group with the exception of TX9. Otherwise, the neighbor- joining tree strongly resembles the parsimony tree. Fig. 6.14. Neighbor-joining tree based on distance analysis of the 18s rRNA gene for the global alignment of the Hildenbrandiales- Fig. 6.15. Quartet puzzling maximum Likelihood tree based on the 18s rRNA gene for the global Hildenbrandiales alignment. - IR11 , SPA2, WAL2 & WAL3 ITA1 AT1 5 8 FRA1 AT1 0, AT14 & GER1 82 97 Cm0 CR24 84 PHI1 i( c FL63 52 73 -Lx7 HsL9 A n PR19 51 URUSW? 54 SCOSWl 77 AUSSWl MEXSW3 53 CASW1 63 NFSWl 88 GERSWl CTSWl 95 ORSW2 1AUSS, 55 r BCSW4 & CASW3 214 6.3.5.18s rRNA gene sequence analyses - quartet puzzling analysis The tree resulting fÏom the quartet puzzling variant of maximum likelihood analysis (1000 puzzling steps) is shown in Fig. 6.15. Again, many of the same groups indicated by parsimony and distance analysis are depicted in this tree. One major exception, however, is the lack of monophyly for the Hildenbrandiales ingroup. Here the two H. occidentalis samples (BCSW4 and CASW3) are shown grouping with the Bangides (Bangia and Porphyra) (supported in 55% of puzzling steps). Relationships among major clades are again not supported in this analysis. However, essentially the same sample groupings are evident, including support for the two collections of Apophlaea @O%), the two collections of H. lecannelieri (87%) and the remaining "crouanii-like" samples (77%), again with the exception of GERSW1. Again, several rubra groups are evident (A, B, C, D and E), and the fieshwater samples are monophyletic with the exception of TX9. 6.3.6. Sequence divergence values of the rbcL and 18s rRNA gene Corrected sequence divergence values for the rbcL gene among global collections of the Hildenbrandiales ranged fiom O - 18.8% among fieshwater samples of Hildenbrandia, O - 26-6% among marine collections of Hildenbrandia and 8 -9% among the two Apophlaea species. Values between marine and fieshwater Nildenbrandia ranged fiom 14.2 - 25.9%, and between Apophlaea and Hildenbrandia ranged fiom 17.3 - 25.1%. Values for the 18s rRNA gene were approximately one quarter of those for the rbcL gene. Among fieshwater Hildenbrandia collections the corrected sequence divergences ranged fiom O - 6.79% for the 18s rRNA gene. Arnong marine samples of 215 Hildenbrarzdia divergences ranged fiom O - 9.5%, and between the two Apophlaea species the divergence was 1.2%- Values between marine and fieshwater Hildenbrandia ranged fkom 1.2 - 9.7% and between Apophlaea and Nildenbrandia ranged fiom 3.1 - 7,4%. 6.4. Discussion The Hildenbrandiales has traditionally been plagued with taxonomic problems due to a proliferation of taxonomic names where few morphological characters are available for their separation. Severd characters comrnonly employed within the genus Hildenbrandia for taxonomic purposes, such as thallus thickness and conceptacle dimensions (in marine species), are known to Vary with the age of the plant (e.g. Pueschel, 1982). In addition, cellular dimensions are variabIe in different parts of the thallus due to the branching filaments composing the crust (Starmach, 1969), and the measurement of this character mut be combined with representative sarnpling of cells dong the Iengths of the filaments. Although differences in these characters are evident for several species (e-g. the reportedly thicker thalli of H lecannefieri and N. occidentalis), they must be interpreted with caution, given the variation present in these characters over the life of the plant. Other characters of doubthl validity (such as the presence of paraphyses to separate H. dawsonii fi-om H canariensis and H crouanii), have been used previously to distinguish species (Hollenberg, 1971). Reports of paraphyses are quite common in the Hildenbrandia literature (e.g. Hariot in Askenasy, 1888; Womersley, 1994), and their interpretation has been much debated. For example, the presence or absence of paraphyses was used, in part, to delimit sections within the 216 genus Hildenbrandia by J. Agardh (Agardh, 1852), wMe more recent uivestigôtors have suggested that reports of paraphyses are actualiy empty sporangiai walls in the conceptacle or fimgal filaments (Ardré, 1959; Denizot, 1968). Pueschel (1 982) provided ultrastructural evidence to support the latter view. In contrast to these variabIe morphological characters, tetrasporangial morphology is more uniform within and among collections and thus is a more appropriate character for morphological distinction. Two widespread marine species, H. crouanii and H rubra, reportedly mermainly in their tetrasporangial morphologies (eeg lrvine & Pueschel, 1994). The rnolecular data show the two species to be distinct (Figs 6.1 3 - 6-13, although not rnonophyletic. The paucity of useful morphological characters within Hildenbrandia means that tetrasporangial morpho f ogy must be one of the primary characters used to distinguish species of HiZdenbrandia given its relative stability as a character. SeveraI unique morphological characters are also present for individual species of Hildenbrandin, which, although not usefid in determining cladistic relationships among species (since this relies on shared, derived characters piley et al., 199 Il), allow the distinction of species. For example, H Zecannellieri has protuberances on the thallus surface which result fiom filaments of the crust growing to different lengths (Womersley, 1994), and K-canariensis is unique in having oblique tetrasporangial divisions (personal observations of the type specimen). Given the small number of morphological characters that are taxonornically distinctive, molecular cornparisons have greatly aided in the assessrnent of this order. 217 The groups yielded by cluster and PCO anaLyses when vegetative characters and tetrasporangid cleavage ody were used differed substantially from those produced with vegetative and reproductive characters (e.g. Fig. 6.2 versus Fig. 6.4). Where only vegetative characters and tetrasporangial cleavage are used the specimens do not yield groups corresponding to tetrasporangial division pattern (Fig. 6.2), whereas they do when conceptacle and tetrasporangial dimensions are included in the analyses (Fig. 6.4). In addition, the similarity between pairs of samples is lower when reproductive characters are included, presumably because the extra characters increase the overall levels of variation in the data for each sarnple. The separation of samples based on tetrasporangial cleavage, as in Fig. 6.4, may be influenced by the qualitative nature of the character. Tetrasporangial division pattern was binary coded as either [O] (some divisions not pardel) or cl] (divisions al1 parallei), and the Gower similarity coefficient was employed to use these data with quantitative measurements of other thallus characters. The Gower similarity coefficient (Gower, 197 1) has been a usehi development since it allows alternative multivariate statistical techniques to be used where standard techniques (eeg principal components analysis and multidimensional scaling) will not tolerate mixed data types (Podani, 1999); however, binary character matches for pairs of OTU's are given a full score of 1 while calculated sirnilarities for quantitative characters range between O and 2. Thus, binary characters are, by default, scored more heavily when positive matches occur, and this may explain why tetrasporangial division pattern appears to be such a critical character in these morphometric analyses. Despite this, use of the Gower similarity coefficient seems justified both because tetrasporangial division pattern is one 218 of the few discrete morphological characters available for the Hildenbrandiales, and because our molecular evidence supports the importance of this character. Previous molecular analyses (Saunders & Bailey, 1999), as well as those presented here support continued placement of the genus Apophlaea within the Hildenbrandiales. Aithough the molecuiar analyses indicate that HiZdenbrandia is not monophyletic, the two sequences of Apophlaea do form a monophyletic group within the Hildenbrandia samples (Fig. 6.13 - 6.15). The morphometnc analyses of the type specimens could not indude the types of Apophlaea because only the upright portions of the Apophlaea thaili were preserved, and the basal, crustose portion is the ody part of the plant directly comparable to Hildenbrandia. This shortcoming of the type method \vas compensated for, at least in part, by including Apophlaea in the molecular analyses. Sequence divergence values between the sarnples of the two Apophlaea species are moderate (8.9% for the rbcL gene and 1.2% for the 185 rRNA gene). Given these values, in combination with the distinctive morphologies of the two species, continued recognition of both A. sinclairii and A. ZyaZZii is recommended. In general, the pa.sepercent sequence divergence values obtained for the rbcL and 18s rRNA gene for samples of Hildenbrandia and Apophlaea are within or higher than the range commojy reported for red algae. For the rbcL gene, values of 0.1 - 0.4% within species (Vis et al., 1998), interspecific values of 1.2 - 7.2% (Freshwater & Rueness, 1994; Hornmersand et aL, 1994) and interpeneric values of 2.8 - 14.5% (Hornmersand et al., 1994; Vis et al., 1998) have been reported. Our pairwise percent sequence divergences within a species range fiom O - 26.6%, the upper end of which is 219 very high compared to other red algae. For the 18s rRNA gene, values of up to 8.4% divergence within the genus Bangia (Müller et al., 1998) and within the genus Porphyra (Oliveira et al., 1995) have been reported, with values more typically in the range of several percent for cornparisons of Florideophyte genera (eg. Bailey, 1999). Within Hildenbrandia, our maximum pairwise percent sequence divergence was 9.7%, with divergences within a species ranging fkom O - 9.5%. Although several taxa included in the molecular analyses are monophyletic and demonstrate relatively small levels of sequence variation (e-g. H. occidentalis, K- rivularis and Apophlaea), others are genetically heterogeneous and do not form monophyletic groups (e-g. H: rubra and H. angolensis) (Fig. 6.13 - 6.1 5). Taxonomically, this presents a problem since in the cladistic sense, these c%mnatural" groupings should not be interpreted as species (Wiley et al., 1991). The present study includes both cladistic (phy logenetic trees) and phenetic (morphometric) analyses; a combination of these approaches will be used for taxonomic evaluation of the order. Several reasons exist for not followïng a strict cladistic approach with the Hildenbrandia data sets. Firstly, it has been argued that speciation in plants may be a paraphyletic process in many cases since most models of sympatric speciation generate monophyletic derivative species fi-om paraphyletic ancestors (Rieseberg & Brouillet, 1994). Thus, paraphyly may be a natural state for some species. In line with this argument, Ragan (1998) also illustrated how traditional concepts of algal taxa are incompatible with cladistic concepts, and that a phylogenetic systematic scheme would necessitate taxonomic changes fiom the level of species up to Kingdom, the results of which wodd 220 rernove the usefûhess and intuitiveness of the current scheme. Secondly, so few morphological characters are available for the Hildenbrandiales that the groups uidicated in the phylogenetic trees (which are only partially congruent among phylogenetic analyses) could not be reliably distinguished using a morphologically based taxonomic scherne. The reasons for incongmency in groupings fiorn the different analyses must be further investigated. Possibilities include inappropriateness of molecular markers and convergent morphology of several independent lineages. Although bo th the 18 S rRNA and the rbcL genes have been used extensively for phylogenetic reconstruction of red algal groups (see introduction for examples), the limits of phylogenetic signal (ie. where so many base changes have occurred between samples that phylogenetic history is obscured Fomoplasy]) are approached for some algal and hïgher plant lineages (e-g. Soltis et al., 1999). This phenornenon may be a distinct possibility for Hildenbrandia if the genus contains a broader genetic diversity than most other red algd genera. The simple morphology of Hildenbrandia may have arisen multiple times (e-g. by reduction of upright thallus portions to the crustose base, or by loss of an upright garnetophytic generation), resulting in non-monophyly of the genus andor some lineages within it. Future work to resolve these issues may include phylogenetic reconstruction with more conserved molecdar markers or more conserved regions of variably conserved genes (such as the rRNA genes). At the present tirne, and until the phylogenetic histones of these taxa can be better resolved with different markers or the reasons for the non- monophyly of the groups elucidated, taxonomic changes will be kept to a minimum. 6.5. Taxonornic proposals and revised descriptions ORDER: Eiidenbrandiales Pueschel et K.M. Cole (1982: 718) emend A.R. Sherwood et Sheath REVISED DESCRIPTION: Thalli entirely cmstose or with erect axes arïsiug fkom crustose base, closely adherent to substratum, composed of cylindrïcal cells arranged in verticai rows, rhizoids sornetimes present on gemmae of fkeshwater forms; pit plugs with a cap membrane but Iacking outer cap layer; gametangial reproduction unknown, tetrasporangia produced by marine forms, tetrasporangia zonate (divisions transverse or oblique) with divisions either all parallel to one another or with some not pardel, produced in conceptacles that enlarge by continuous conversion of vegetative cells lining chamber into tetrasporocytes; spore germination unipolar, cytoplasrnic contents of genninating spore evacuated into germ tube. FAMILY: Hildenbrandiaceae Rabenh. (1868: 408) [as HiIdenbrandtiaceae] ernetzd A.R. Sherwood et Sheath Description as for the order. GENERA: Apoplriuea Harv. ex Hook. f. et Harv. (1845: 550) TYPE SPECIES: A. sinclairii Harv. ex Hook. f. et Harv. (1845: 550) SPECIES AND SUBSPECIFIC TAXA: 1. A. IyaCIii Hook f. et Ham. (1855: 244) SYNONYMS:none 222 TAXONOMIC NOTES: Ody the upright thallus portions of the type specimens were preserved, and thus the specimens could not be compared morphometrically to HiZdenbrandia (aside fiom tetrasporangial dimensions and morphology [see 6.3 -11) since the two genera share only the cmstose basal thallus portion. Due to the superficiai analysis of the type specimen and lack of sufncient comparative material, the description will not be revised. 2. A. ZyaIiii var. gigarfinoides Hook. f. et Harv. (1855: 244) SYNONYMS: none TAXONOMIC NOTES: As for A. Zyallii, only the upright thallus portions of the type specimens were preserved. Due to the superficial analysis of the type specimen and lack of sufficient comparative material, the descnption will not be revised. However, this is considered a dubious taxon since it differs from the nominate variety only by overall dimensions, which codd be due solely to the age of the plant. 3. A. sinclairii Harv. ex Hook f. et Haw. (1845: 550) SYNONYMS : none TAXONOMIC NOTES: As for A. ballii, only the upnght thallus portions of the type specirnens were preserved. Due to the superficial analysis of the type specimen and lack of sufncient comparative material, the descnption will not be revised. Hildenbrannica Nardo (1834: 675) TYPE SPECIES: H rubra (Sommefi) Menegh. SPECIES AND SUBSPECIFIC TAXA: 1. W. ungoienslr Welw. er W.West et G.S. West (1897: 3) emend A.R Sherwood et Sheath SYNONYMS: none REVISED DESCRIPTION: Freshwater only, thalli crustose, composed of tightly adherent, branched nlaments which arise fiom a unistratose basal layer; cells 3.1 - 5.2pm in length, 3.2 - 5Spm in diameter, thalius 26.0 - 6l.7pm thick and basal layer of cells 3.6 - 8Spm thick; sexual reproduction unknown, asexual gemmae produced within the thallus and released fiom the thallus surface. TAXONOMIC NOTES: Until Sheath et al. (1993) published a morphometric study of North American fieshwater Hildenbrandia, H angolensis was not a generaliy recognized taxon, and most fieshwater specïmens worldwide were referred to as N. rivularis, H. angolensis was collected and examined by Welwitsch during his phycological explorations of Angola, but was only later validly published by the Wests (West & West, 1897). The morphometric and molecular analyses presented here, the morphometric analyses of Sheath et al. (1 993) and the separate distribution of angolensis and H. rivularis support the distinction of the two species, recognizable based on cellular dimensions. 2. H. crouanii (J. Agardh) J. Agardh (1876: 379) emend. A.R. Sherwood et Sheath B ASIONYM: Haernatophlaea crouanii J. Agardh (1852: 495) SYNONYMS: H. canariensis Bmgeson (1929: fi), canariensis var. dirwsonii Ardré (1959: 230), H. dawsonii (Ardré) Hoiienb. (1971: 286), H. prototypus var. kerguelensis sensu Dawson (1 953 : 96), H occidentalis var. lusitanica Ardré (1 959: 23 3), H patula Womersley (1994: 145), H. expansa Womersley (homonym) (1996: 357), rosea 224 Crouan (1 867: 148) (non Kütz.), Haematophlaea crouanii J. Agardh (1 852: 499, REVISED DESCRIPTION: Marine, thalli cnistose, composed of tightly adherent, branched filaments which arise fkom a unistratose basal layer; cells 3.7 - 5.7pm in length, 3.2 - 5. lpm in diameter, thdus 5 1-8 - 329pm thick and basal layer of cells 5.7 - 15.0pm thick; sexual reproduction unknown, transversely divided tetrasporangia produced in uniporate conceptacles; tetrasporangia 2 1.9 - 40.9pm in length and 7.1 - 1 1.6~min diameter, conceptacles 64.6 - 126prn deep and 63.3 - 114pm in diameter. TAXONOMIC NOTES : Examination of the type specimen of H. crouanii in this investigation revealed that al1 tetrasporangia have transverse, pardlel divisions (Fig. 6.16 a). This is in direct contrast to observations by Rosenvinge (1 9 2 7), who examined the same specimen and reported tetrasporangid divisions to be oblique. In addition, tetraspores within the tetrasporangia were observed in this study to be separated by small spaces, rather than being tightly connected to one another. This morphological feature was also noted in this study for the type specimens of H: occidentalis var. Zusitanica and H. patula, as well as the marine collection MEXS W3 (corresponding to H dawsonii), and is in contrast to the type specimens of H occidentalis, H kerguelensis, H sanjuanensis and H. occidentalis var. yessoensis. In addition, the type specimen of H canariensis was observed to have oblique tetrasporangial divisions, which contrasts with the protologue of that species @orgesen, 1929). The possibility that oblique versus transverse cleavage couid results fiom preservation artifact was not established since no live materid was exarnined containing oblique cleavages, and this, in combination with the differences observed in the type specimens fiom their descriptions, resulted in oblique versus Fig. 6.16. a) Transverse, parallel tetrasporangial morphology (most marine taxa of Hildenbrandia)- b) Tetrasporangial morphology of H mbra. 227 transverse cleavage not being used as a character in these analyses. The phylogenetic analyses of the 18s rRNA gene (Figs. 6.13 - 6.15) indicated a close relationship among the samples of these taxa (SCOSWI [K-crouanii], SCOSW4 [H. crouanii], MEXSW3 [H. dawsonii] and AUSSWl [K.patula]). Although no specimens of H occidentalis var. lusilrmica were available for the molecular comparisons, the morphometric analyses demonstrate a close association between this specirnen and crouanii (Figs. 6.2 - 6.7). The type specimen of H. dmsonii was mavailable for examination, but the one collection morphologically comesponding to this species (MEXS W3) is supported as having a close phylogenetic relationship with H crouanii and H parula specimens (Figs. 6.13 - 6.15). Thus, based on these molecular and rnorphological data, it is proposed tbat the foliowing taxa be synonymized under the oldest epithet, H crouanii: H occidentalis var. lusitanica, H. canariensis, H. dawsonii and H. patula. H. canariensis was included as a synonym and H. duwsonii was noted as a possible synonym of H. crouanii by Irvine & Pueschel (1994). 3. H. kergmiensis (Askenasy) Y.M. Chamb. (1962: 372) BASIONYM: H prorotypus var. kerguelensis Askenasy (1 888: 30) SYNONYMS : none TAXONOMIC NOTES: This taxon was descnbed as H pi-ototypus var. kerguelensis by Askenasy (1888), and later elevated to specific status by Chamberlain (1962). InswfTïcient data is available at present (e.g collections other than the type specimen) to recommend taxonomie changes. 4. H. occidentalis Setch. ex N.L. Gardner (1917: 393) 228 SYNONYMS: none TAXONOMIC NOTES: Two collections of this taxon were included in the molecular analyses. Although they were fiom distant geographical locations (one collection fiom northern California and one collection fiom British Columbia), they were identical in sequence for the 18s rRNA gene; demonstrating much lower divergence than most collections of H. rubra, and were not closely associated with other marine collections of Hildenbrandia. Based on these data, continued recognition of H occidentalis is recommended. 5. H. occidentalis var. yessoensis (Yendo) Ardré (1959: 233) SYNONYMS: none TAXONOMIC NOTES: Insufficient data is available at present (e-g. collections other than the type specimen) to recommend taxonomic changes. 6. H. sanjuanenris Eolleob. (1969: 164) SYNONYMS : none TAXONOMIC NOTES: InsuEcient data is available at present (e-g. collections other than the type specimen and one historically signïficant specimen) to recommend taxonomic changes. 7. H. lecannelfieri Har. (1887: 74)emend A.R. Sherwood et Sheath SYNONYMS: H. pachythallos Dickinson (nomen nudum) REVISED DESCRIPTION: Marine, thallus crustose and thick with distinct protuberances on surface, composed of tightly adherent, branched filaments which arise fiom a unistratose basal layer; cells 5.2 - 7.0pm in length, 4.1 - 4.8pm in diameter, thallus 229 704 - 929pm thick (based ody on the type specimen) and basai layer of cells 8.3 - 1 1.4prn thick; sema1 reproduction unknown, transversely divided tetrasporangia produced in uniporate conceptacles; tetrasporangia approximately 3 1pm in length and 7 - 8pm in diameter, conceptacles approxirnately 70pm deep and 75pm in diameter. TAXONOMIC NOTES: This distinctive species of Hildenbrandia was decribed by Hariot (1887) fiom the coast of Cape Hom and is commonly reported in the southern hemisphere (e-g. Chamberlain, 1 962; Womersley, 1 994)- H. pachythallos Dickinson (nomen nudurn) was reported fiom the South African coast, but was recognized as H. ZecanneZZieri by S tegenga et al. (1 997). Tetrasporangial morphology as in Fig. 6.16 a. 8. W. rivularis (Lieban.) J. Agardh (1852: 495) ernend A.R Sherwood et Sheath BASIONYM: Erythroclathrus rivularis Liebm. (1 83 9: 174) SYNONYMS: H. rivularis var. drescheri Lingelsh. (1922: 353, W.fluviatilis Bréb. (nornen nudum), H. rmea var. fluviatilis Kutz- (nornen nudum), H. puroliniana Zanardini (1841: 135) REVISED DESCRIPTION: Freshwater only, thalli crustose, composed of tightly adherent, branched fïiaments which arise fi-orn a unistratose basai layer; cells 5.8 - 8.6pm in length, 4.4 - 8.4prn in diameter, thallus 35-5 - 108pm thick and basal layer of cells 5.2 - 15.6pm thick; sexuai reproduction unknown, asexual gemmae produced within the thallus and released fiom the thallus surface. TAXONOMIC NOTES: This fieshwater taxon was originally described as Erythrocluthrus rivuraris by Liebman (183 9), five years after the circurnscription of Hildenbrandia by Nardo (which at that time encompassed only marine species). Based 230 on similar vegetative thallus morphology it was transferred to Hlldenbrandia by J. Agardh (1 852). Eleven years previous Zanardini had described a similar fieshwater Hildenbrandia as H. paroliniana, wbich was synonomized with H. rivularis by J. Agardh (18 52) at the same time that the transfer of Erythroclafhrus rivuluris to Hildenbrandia was made. Lingelsheim (1922) described a variety of H rivularis (var. drescheri) which he distinguished fiom the nominate variety as being dark blood red in color, rather than the lighter red to pink color often reported for rivularis (e.g. Budde, 1926; Geitler, 1932). Both our morphometric and rnolecular analyses indicated that H. rivularis var. drescheri is indistinguishable fkom H. rivularis, and their synonyrny is recornmended. The designations H. fluviatilis Bréb. and H. rosea var.fluviafilis Kütz. are nomenclaturally problematic. H: fltmiatilis Bréb. is presumably a narne written on a specimen sent to Kutzing by Brébisson, and Kützing interpreted it as a variety of H. rosea, but he gave no description. Thus, H. rosea var.fluviatilis Kütz. and HfluviatiZis Bréb. refer to the same specimen, and both names are nomina nuda (P.C. Silva, personal communication). H fruviafilis Bréb. was subsequently synonymized with rivularis by J. Agardh (1 852). We have exarnined several specimens Iabelled H. Jtuviatilis Bréb. (PC, S), and rnorphometrically they are similar to H. rivularis (Figs. 6.10 & 6.1 1)- 9. H. rubra (Sommerf.) Menegh. (1841: 426) emend. A.R. Sherwood et Sheath BASIONYM: Venucaria rubru Sommerf. (1 826: no pagination) SYNONYMS: H gaZapagensis Setch. ef N.L. Gardner (1937: 91), H prototypus Nardo (1 834: 676), Eryrhroclathrus pellitus Liebm. (1 839: 175), H. nardi Zanardini (1 841: 13) (nom. illeg. ), H. nardiana Zanardini (1 840: 134), H. rosea Kütz. (1 843 : 384), H. 23 1 sanguinea Kutz (1 843 : 384), H. rosea Kütz- (1 843 :384), Rhododermïs drummondii Harvey (1844: 27), Zonaria deusta Lyngbye (1 8 19: 19) REVISED DESCRIPTION: Marine, thalli cmstose, composed of tightly adherent, branched filaments which arise fiom a unistratose basal layer; cells 2.1 - 5.8pm in length, 2.2 - 4.8pm in diameter, thallus 45.1 - 1 88pm thick and basal layer of cells 3 -4 - 15.9pm thick; sexual reproduction unknown, tetrasporangia with divisions not al1 parallel (Fig. 6.16 b), produced in uniporate conceptacles; teûmporangia 17.6 - 32.4prn in Iength and 5.7 - l2Sprn in diameter, conceptacles 47.4 - 135pm deep and 55.9 - 1 17pm in diameter- TAXONOMIC NOTES: Hildenbrandia rubra (Sornmerf.) Menegh. was originally described in the lichen genus Verrucaria by Sommerfelt (1 826) as l? rubra. The genus Hildenbrandia was not erected until 1834 by Nardo, who decnbed H prorowpus Nardo as the first species of Hildenbrandia. Meneghini (1 841) recognized the conspecificity of K rubra and H protofypus, and applied Sornmerfelt's older (1826) specific epithet, rubra, to Nardo's generic epithet, Hilaenbrandia. Nurnerous other designations were published and subsequently synonymized with H. prototypus, including sanguinea, H. rosea and H nardiana (Mobius, 1893; Denizot, 1968). H nardi was synonymized with H rubra by Meneghini (184 1). Dawson (1 963) recornmended the synonymy of H. galapagensis with H. rubra, and Denizo t (1 968) noted that H. galapagensis and rztbra (as H. prototypus) are distinguishable only by H galnpagensis having a thinner thallus and smaller ce11 sizes. The morphometric analyses indicated that the type specimens were very similar (Figs 6.2 - 6.8) and synonymy of H galapagensis under the older epithet, H rubra, is recommended. 232 10. Doubffil species: the following type specimens were either unavailable for examination, or the specimens were not reproductive and therefore couid not be completely evaluated and used in cornparisons to other specimens: H arracana Zelier (1 873 : 192), expansa Dickie (1874: 3 57), H. ramanagindi M. Khan (1974:238) and H rivularis ssp. chalikophila PaLik (1 96 1: 15 1). ~etrasporangialmorphology was not described in the protologue of either H arracuna or H expansa (Zeller, 1873; Dickie, 1874), and thus these must be lefi as dubious species. The conspecificity of H expansa and rubra (as EL pratotypus) was suggested by Denizot (1 968). 6.6. Key to the species of Hiidenbrandia 1.a) Occurs exclusively in fieshwater habitats ...... 2 -l 1.b) Occurs in brackish or marine habitats ...... A 2.a) Cells smd and similar in size to marine species, 3.1 - 5.2 Pm in length and 3.2 - 5.5 pm in diameter ...... H angolensis 2.b) Cells mostly larger than marine species, 5.8 - 8.6 Pm in length and 4.4 - 8.4 pm in diameter ...... drivularis 3 .a) ThalIus with distinct protuberances on the surface ...... H lecannellieri 3.b) Thallus without distinct protuberances on the surface ...... ,...... 4 4.a) Tetrasporangia with divisions not al1 pardel to one another ...... H. rubra 4.b) Tetrasporangia parallel and transversely or obliquely dividrd ...... 5 5 .a) Tetrasporangial length <14,um ...... -H. sanjuanensis 5.b) Tetrasporangial length >14pm ...... -6 6.a) Conceptacles <100pm deep ...... crouanzz* * 233 6.b) Conceptacles >1 00pm deep ...... -...... -. - .-.- ..-. - *- .--.-.. -. - .. -. .. ------.- - .--.7 7-a) Tetrasporangial length >3 0pm ...... -.-.. --. .. . . --. .- - .-.-.-.-. .-. . . . . -. . -. -. .- .-. -HH occidentalis 7.b) Tetrasporangial length <3 0pm ...... , ...... -. - - - -.-...... -. .. . -. .-- - -..-. - -. -.- -. - - - -..-. 8 8.a) Conceptacles -400pm diameter .-..-...... H. occidentalis var. yessoensis 8.b) Conceptacles >100pm diameter ...... H kerguelensis 6.7. Literature Cited Abbott, I.A. & Hollenberg, G.J. 1976. Marine Algae of Calfornia. Stanford University Press, Stdord, 827 pp. Agar&¶ J. 1852. Species genera et ordines algarum, Volumen secundum: algas florideas complecter?s. Lund, 1291 pp. Agardh, J. 1876. Species genera et ordines algarum, Volurnen tertiurn: de Florideis czrrae posteriores. Lipsiae, 724 pp. Ardré, F. 1959, Un intéressant Hildenbrandtia du Portugal. Revue AlgoIogrgrque4: 227- Askenasy, E, 18 8 8. Algen (mit Unterstutzung der Herren E. Bornet, A. Grunow, P. Hariot, M. Mobius, O. Nordstedt), in IV Theil, Botanik (A. Engler), in Forschmgsreise S. MX "Gazelle ". Berlin, 68 pp. Bailey, J-C. 1999. Phylogenetic positions of Lithophyllum incrustans and Titanodema pustulatum (Corallinaceae: Rhodophyta) based on 18s rRNA gene sequence analyses, with a revised classification of the Lithophylloideae. Phycologia 38 : 208-216. Bsrgesen, F. 1929. Marine algae fi-om the Canary Islands, especially fiom Teneriffe and Gran Canaria. ILI. Rhodophyceae, part II, Cryptonemiales, Gigartinales and Rhodymeniales. Kongelige Danske Videnskabernes Selskab, Biologiske Meddelelser 8: 1-97. Bourrelly, P. 1955. Quelques stations fiançaises dYHiZenbrandiarivularis (Liebm.) Bréb. Revue Algologique 1: 168-169. Budde, K. 1926. Zweiter Beitrag zur Entwicklungsgeschichte von Hildenbrandia rivularis (Liebmann). Dezrtsche Botanische Gesellschafr Berichte 44: 3 67-3 72. Chamberlain, Y .M. 1962. Notes of two species of Hildenbrandia. Nova Hedwigia 4: 3 7 1- 373. Crouan, P.L. & Crouan, H.M. 1867. Florule du Finistère. Paris and Brest, 262 pp. Dawson, EX1953. Marine red algae of Pacific Mexico. Part 1. Bangiales to Corallinaceae subf. Corallinoideae. AlZan Hancock Pacz9c Expeditions 17: 1-239. Dawson, E.Y. 1963. New records of marine algae fiorn the Gaiapagos Islands. Paczj2 Naturalist 4: 1-23. DeCew, T.C. & West, J.A. 1977. Culture studies on the marine red algae Hildenbrandia occidentalis and H. prototypus. Bulletin of the Japanese Society of Phycology 25: 31-41. Denizot, M. 1968. Les algues Fïor idées encruutantes (à 1 'exclusion des Corallinacées). Museum National d'Histoire NatureHe, Paris, 3 10 pp. Dickie, G. 1874. Enurneration of the algae collected at St. Paul's Rocks by H.N. Moseley, M.A., Naturalist to the H.M.S. ccChallenger'7.Journal of the Linnaean Society [London] Botany 14: 355-359. Dunn, G. & Everitt, B.S. 1982. An Introduction fo Mathematical Tmonomy. Cambridge University Press, Cambridge, 152 pp. Freshwater, D. W. & Rueness, 1. 1994. Phylogenetic relationships of some European Gelidium (Gelidides, Rhodophyta) species, based on rbcL nucleotide sequence analysis. Phycologia 33: 187-1 94. Gardner, N.L. 1917. New Pacific Coast marine algae 1. University of CaZifornia Publications in Botany 6: 377-4 16. Geitler, L. 193 2. No tizen über Hilden brandia rivularis und Heribaudiella fluviatilis. 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Botanica Marina 37: 193-203. Hooker, J.D. 1855. The botany of the Antarctic voyage of H.M. Discovery ships "Erebus" and ccTerror"in the years 1839-1843 under the command of Sir James Clark Ross. II. Flora Novae-Zelandiae. II. Flowerless Plants, London, pp- 244-245. Hooker, J.D. & Harvey, W.H. 1845. Algae Nova Zelaodiae: being a catalogue of al1 the species of algae yet recorded as inhabiting the shores of New Zealand, with characters and bnef descriptions of the new species discovered during the voyage of H.M. Discovery ships "Erebus" and "Terrer", and of others communïcated to Sir H. Hooker by Dr. Sinclair, the Rev. W. Colenso, and M. Raoul. London Journal of Botany 4: 549-550. lrvine, L.M. & Pueschel, C.M. 1994. Hildenbrandiales. In: Seaweeds of the British Isles, Vol. I, Rhodophyta, Part 2B, Corallinales, Hildenbrandiales. (Eds. L. M. hine & Y.M. Chamberlain). The Natural History Museum, London, pp- 23 5-24 1. Khan, M. 1974. On a fresh water Hildenbrandia Nardo fiorn hdia. Hydrobiologia 44: 23 7-240. Kovach Cornputing Services. 1986- 1998. Multi-Variate Statistical Package, v.3.0. Pentraeth, Wales. Kutzing, F.T. 1843. Phycologia Generalis. Leipzig, 458 pp. Liebman, F. 1839. Bemzrkinger og tillæg til den danske algeflora. Naturhistorisk Tidsskrz# 2: 464-494. Lingelsheim, A. 1922. Eine bemerkenswerte Rotalge des Süawassers und ihre Erhaltung. Beitrag zu Naturdenkmalpfrege 9: 348-3 60. Lyngbye, H.C. 18 19. Tentamen Hydrophytologiae Danicae, Hafiiae [Copenhaged, 248 PP- Meneghùii, G. 1841. Memoria sui Rapporti di Organizzazionefia le Alghe Propriamente dette O Ficee e le Alghe terrestri O Licheni A tti della III Riunione degli scienz. Ifal. In Firenze-Torino, pp. 417-593. Mobius, M. 1893. Beitrag zur K~MI~~ssder Algenflora Javas. Berichte der Deutschen Botanischen Gesellschqft 11 : 11 8- 13 9. Müller, KM., Sheath, R.G., Vis, M.L., Crease, T.J. and Cole, K.M. 1998. Biogeography and systematics of Bangiu (Bangiales, Rhodophyta) based on the Rubisco spacer, rbcL gene and 185 rRNA gene sequences and morphometric analyses. 1. North America. Plzycologia 37: 195-207. Nardo, G.D. 183 4- De novo genere alganun cui nomen est Nildbrandtiu prototypus. Isis von Oken 6: 675. Oliveira, MC., Kurniawan, J., Bird, C.J., Rice, E.L., Murphy, C.A., Singh, R-K., Gutell, R.R. and Ragan, M.A. 1995. A preliminary investigation of the order Bangiales (Bangiophycidae, Rhodophyta) based on sequences of nuclear srnall-subunit ribosomal RNA genes. Phycological Research 43: 7 1-79. Palik, P. 1961. Neue Algen-Species, Subspecies, Varietaten und Formen. Annales Universitatis Scientiatis Budapestiensis Rolando Eotvos Sectio Bio Iogrgra4: 15 1- 154, Podani, J. 1999. Extending Gower's general sirnilarity coefficient to ordinal characters. Taxon 48: 33 1-340. Pueschel, C.M. 1982. Ultrastructural observations of tetrasporangia and conceptacles in Hildenbrandia (Rhodophyta: Hildenbrandiales). British Phycologial Journal 17: 333-341. Pueschel, CM. & Cole, K.M. 1982. Rhodophycean pit plugs: an ultrastructural survey with taxonomie implications. American Journal of ~0th~69: 703-720. Pueschel, C.M., Sciunders, G,W. & West, J.A. 2000. Affrnities of the freshwater red alga AudouineZla rnacrospora (Florideophyceae, Rhodophyta) and related forms based on SSU rRNA gene sequence analysis and pit plug ultrastructure. Journal of P/-yc~logy36: 433-439. Rabenhorst, L. 1868. FZora Europa Algarum. Aquae Dulcis et Sthnarinae. Lipsiae, 46 1 PP . Ragan, M.A. 1998. On the delineation and higher-level classification of algae. European Journal of Phycology 33: 1-15. Rieseberg, L.H. & Brouillet, L. 1994. Are many plant species paraphyletic? Taon 43: 21- 3 2. Rosenvinge, LX. 1917. The Marine Algae of Denmark: Contributions to their Natural History. Vol. 1. Rhodophyceae. Ande. Fred. Host & Son, Copenhagen, 486 pp. Ryan, B.F., Joiner, B.L. & Ryan, T.A. Jr. 1985. Minitab Hamibook, 2ndEdition. Duxb~uy Press, Boston, 374 pp. Saunders, G.W. & Bailey, J-C. 1999. Molecular systematic analyses indicate that the enigmatic Apûphlaea is a member of the Hildenbrandiales (Rhodophyta, Florideophycidae). Journal of PhycoZûgy 35: 17 1-1 75. Saunders, G.W., Bird, C.J., Ragan, M.A & Rice, E.L. 1995. Phylogenetic retationships of species of uncertain taxonornic position within the Acrochaetiales-Palmariales complex (Rhodophyta): inferences from phenotypic and 185 rDNA sequence data. Journal of Phycology 3 1: 60 1-61 1. Scagel, R.F., Garbary, D.J., Golden, L. & Hawkes, M. W. 1986. A Synopsis of the Benrhic Marine AZgae of British Columbia, Narthern Washington and Southeast Alaska. Phycological Contribution No. 1, University of British Columbia, Department of Botany, 444 pp. Setchell, W.A. & Gardner, N.L. 1937. The Templeton Crocker Expedition of the California Academy of Sciences, 1932, no.3 1. A preliminary report. on the algae. Proceedings of the Calijiurnia Academy of Science 22: 65-98. Sheath, R.G., Kaczmarczyk, D. & Cole, K.M. 1993. Distribution and systematics of fieshwater Hildenbrandia (Rhodophyta, Hildenbrandiales) in North Amenca. European Journal of Phycology 28: 1 15-121. Sherwood, A.R. & Sheath, R.G. 1999. Biogeography and systematics of Hildenbrandia (Rhodophyta, Hildenbrandiales) in North America: inferences fiom morphometrics and rbcL and 18s rRNA gene sequence analyses. European Journal of Phycology 34: 523-532. Shenvood, A.R. & Sheath, R.G. 2000. Biogeography and systematics of Hildenbrandia (Rhodophyta, Hildenbrandiales) in Europe: iderences fiom morphometrics and rbcL and 18s rRNA gene sequence analyses. European Journal of Phycology 35: 143-152. Silva, P.C., Basson, P.W. & Moe, R.L. 1996. Catalogue of the Benthic Marine Algae of the Indian Ocean. University of Cul$ornia Publicarions in Botany 79, 1259 pp. Soltis, P.S., Soltis, D.E., Wolf, P.G., Nickrent, D.L., Chaw, S.-M. & Chapman, R.L. 1999. The phylogeny ofland plants inferred fkom 18s rDNA sequences: pushing the limits of rDNA signal? Molecular Biology and Evolution 16: 1774- 1784. Somrnerfelt, S.C. 1826. Supplernentum Florae Zapponicae quam edidit Dr. Georgius Wuhlenberg. Christianise [Oslo], 33 1 pp. Starmach, K. 1969. Growth of thalli and reproduction of the red alga Hildenbrandtia rivularis (Liebm.) J. Ag. Acta Societatis Botanicorum Poloniae 38: 523-533. Stegenga, H., Bolton, J.J. & Anderson, R.J. 1997. Seaweeak of the South Afiican West Coast. Contributions fiom the Bolus Herbarium No. 18,655 pp. Vis, M.L., Saunders, G.W., Sheath, R.G., Duse, K. & Entwisle, T.I. 1998. Phylogeny of the Batrachospermales (Rhodophyta) iderred from rbcL and 18s ribosomal DNA gene sequences. Journal of Phycology 34: 341-350. Vis, M.L. & Sheath, R.G. 1998. A molecular and morphological investigation of the relationship between Batrachosperrnum spermatoinvolucmrn and B. gelatinosum (Batrachospermales, Rhodophyta). European Journal of Phycology 33 : 23 1-239. West, W. & West, G.S. 1897. Welwitsch's African fkeshwater algae. Journal of Botany 35: 1-7. Wiley, E.O., Siegel-Causey, D., Brooks, D.R. & Funk, V.A. 199 1. The Compleat Cladist: A primer ofphylogenetic procedures. The University of Kansas Museum of Natural History, Special Publication No. 19, 158 pp. Womersley, H.B.S. 1994. me Marine Benthic Flora of Aus~?aZia,Part IIU. Flora of Australia SuppIementary Series Number 1,508 pp. Womersley, H.B.S. 1996. me Marine Benthic Flora ofAustmZia, Part IIB. Flora of Australia Supplementary Series Number 5,392 pp. Yendo, K. 1920. Novae Aigae Japoniae. The Botanical Magazine 34: 1- 12. Zanardini, G. 1841. Alla dïrezione della Bibliotheca Italiana. [Lettera intomo ad alcune nouve specie di alghe et species alganim novae vel minos cognitae]. BibZioteca Italiana [Milano] 96: 13 1- 13 7. Zeller, G. 1873. Algae collected by Mr. S. Kurz in Arracan and British Burma, determined and systernatically arranged by Dr. G. Zeller. Journal of the Asiatic Society of BengaZ 42: 175-193. CHAPTER 7: General Sumrnary It is evident from the data presented in Chapters 2 - 6 that Hildenbrandia has had a complex evolutionary history. Most analyses support the order Hildenbrandiales as monophyletic, but in ail cases (including previously published studies) the inclusion of representatives of Apophlaea indicates that although this genus is monophyletic, Wildenbrarzdia is not. It will be necessary to CO- the non-monophyly of Wilderrbrandia through studies invohg different molecula. markers before all taxonomie implications can be realized. Several possible explmations exist for non-monophyfy of kiildenbrandia, which need to be investigated in the future, including convergence of morphology of several distinct lineages and genetic diversification without corresponding morphological diversification. Both possibilities deserve further attention; the first because the simple morphology of Hildenbrandia resembles the crustose phase of many marine red algae, and therefore could have arisen multiple times, the second because the assurned lack of sexual reproduction in Hildenbrandia may have allowed for somatic mutations to accumulate due to lack of recombination. At present, and until additional information regarding the biology of these organisms becomes r available, continued recognition of the monophyletic genus Apophlaea and the non-monophyletic genus Nildenbrandia is recommended. The following discussion summaries the results fiom this thesis, as they relate to the research objectives outlined in Chapter 1. 1. To analyze in detail the biogeographic and systematic patterns evident from Hildenbrandia collections from the North American and European 242 continents, and on a srnalier scale from other regions of the world, using a combination of morphological, morphometric and molecular analyses. The analyses of marine and fieshwater Hildenbrandia fkom North America (Chapter 2) and Europe (Chapter 3) indicated several key trends: fieshwater Hildenbrandia from the two continents constitute two distinct lineages which are distinguishable based on morphology as well as in the molecular trees; the srnalier celled North American form (H. angolensis) is paraphyletic and may have arisen fkom multiple invasion events by marine forms, while the larger celled European form (H rivularis) is monophyletic and geneticaliy homogeneous. The common marine species, H rubra, is not monophyletic and is very genetically diverse; however, some biogeographic patterns correspondkg to oceanic basin are disceniable. Several other marine species included in the analyses are morphometrically distinct based at least on some characters (H. crouanii, H dawsanii and H. occidentah), and according to some molecular analyses- The inclusion of additional global collections (Chapter 6) gave Mersupport to the conclusion that some morphological characters used to delimit species of Hildenbrandia do not correIate weil with clades on molecular trees. This must lead to one of severai conclusions: either these morphological characters are not taxonomically usefil within Hildenbrandia, the current concept of the genus Hildenbrandia encompasses multiple phylogenetic lineages, or the molecular markers used in the studies are not appropriate for those kind of comparisons. Further research is necessary to clac these possibilities. 2. To examine the possibility of a marine origin for the freshwater forms of Hildenbrandia through comparisons of marine and freshwater collections 243 from several continents based on the rbcL gene, 18s rRNA gene and ITS regions of the rRNA genes. A close phylogenetic relationship between the marine and fkeshwater forms of Hildenbrandia was indicated by most molecular analyses (Chapters 2 ,3,4 and 6), supporthg the concept of keshwater forms being derived fiom marine forms. A common origin for most representatives of the two fieshwater species exarnined (H. angolensis and rivularis) is suggested by the European and global analyses, but the subsequent diversification into fieshwater habitats may have occurred independently in the two species. Analyses of the rbcL and 18s rRNA genes in this thesis indicated that the North American fieshwater species, H. angolensis, is composed of a series of paraphyletic lineages; these may have arÏsen by multiple invasions of marine Hildenbrundia into fieshwater habitats. In contrast, the European species, H rivularis, exhibited little to no sequence divergence for the two genes, suggesting that a single invasion event, possibly of the North Arnerican freshwater stock, established the fieshwater populations on this continent. A smaller scale examination of H. rivularis (Chapter 4) from southern Sweden and its relationship to the geographicaliy close marine species, H. rubra, indicated that the two species are genetically distant fiom one another. Sequences of the ITS regions were very similar within marine collections and within freshwater collections, but were unaiignable between marine and fieshwater collections, and large differences in their lengths were notable. Analyses of the rbcL and 18s rRNA genes with representative European collections again indicated low genetic variation among fieshwater forms compared to marine forms, and large differences between sequences representing the two 244 habitats, indicating that invasion events fkom marine to fieshwater do not occur on a shoa time scale. 3. To study the processes of gemma development and release in the freshwater species of Hildenbrandia, H. angolensis, using several forms of microscopy and histochemistry. By employing a combination of light microscopy (whole mounts, sectioned matenal and histochemical stains), scanning electron microscopy and transmission electron microscopy the processes of gemma development and release were elucidated and compared to previous reports. It was observed that gemmae develop within the thallus fkom a possible meristematic region near the base of the thallus, and release through a ce11 separation process. The potential of repeated gemma production in a specific region of the thallus would allow for large numbers of these unique propagules to be formed and released. Gemma cells are generally larger than thallus cells, and contain appreciably larger amounts of floridean starch than thallus cells, as seen by light and transmission electron microscopy, and confinned by histochemistry. Observations of gemmae in H angolensis allowed for the clarification of previous misconceptions in this species, and corroborated results reported for the European species, H. rivularis. 4. To re-evaluate the systematics of the genus Hiidenbrandin through analyses of the type specimens, worldwide collections of the genus, and comparisons through molecular analyses. Tweive marine and five specific and subspecific taxa of Hildenbrandia were generally recognized pnor to these analyses. Based on analyses of the rbcL and 18s rRNA genes, 245 as well as morphometrics of the type specimens and other global colIections, it is suggested in this thesis that the number of marine species be reduced to seven and fkeshwater to four (Chapter 6). SeveraI type specimens could not be obtained and, in most cases, their taxonomy was not modified. Although representatives of dl taxa were not available for inclusion in the rnolecular trees, the morphometrïc analyses indicated that no morphological characters separated some groups of species (e-g.H. kerguelensis and H occidentalis), and that their separate taxonomic descriptions probably arose fiom the assumption that their different geographical ranges waxranted separation. Further studies incorporating representatives of taxa not included here may refine these taxonomic recornmendations. 5. To re-examine whether Witdenbrandia and Apophlaea have a close phylogenetic relationship based on rnolecular analyses. Previous work demonstrated a close phylogenetic relationship between the two genera of the Hildenbrandiales, based on two 185 rRNA gene sequences of marine Hildenbrandia and one of Apophlaea ZyaZZii. Inclusion of the second species of Apophlaea, A. sinclairii, in the analyses of this thesis (Chapter 6),as well as additional representatives of rnany marine and fieshwater Wenbrandia species, strongl y supported Apophlaea, but no t Hildenbrandia, as a monophyletic genus. Possible explanations for the non-monophyly of Hildenbrandia have been outlined in the introduction to this chapter; until these are further investigated the continued recognition of both Apophlaea and Nildenbrandia is recommended since the morphologies of the two genera are very distinct. Appendix 1. Sequences of prirners used in this thesis. -- Primer name Amplification Primer sequence (5' - 3') reaction HILFI ' rbcL forward TAG ATC CAA TTG AAG CTG CTG C EF2' rbcL forward TAA CAG CTT GTG ATC TAT ATA GAG C F 160' rbcL forward CCT CAA CCA GGA GTA GAT CC rbcLR2 rbcL reverse ACA TTT GCT GTT GGA GTC TC C0MPl3 rbcL forward GAA TCT TCT ACA GCA ACT TGG AC COMP~~ rbcL reverse GCA TCT CTT ATT ATT TGA GGA CC HF18s.2' 18s rRNA forward GAG CTA ATA CGT GCC AAA ACG CG GO4. l4 18 S rRNA internal GTC AGA GGT GAA ATT CTT GG GO8. l4 18s rRNA internal GAA CGG CCA TGC ACC ACC AAC G075 18 S rRNA reverse TCC TTC TGC AGG TTC ACC TAC G14' 18s rRNA internal CCT TGG CAG ACG CTT TCG CAG ITSH. 1' ITS 1 forward GGA AGG AGA AGT CGT AAC AAG G ITS 1O6 ITS 1 reverse GCT GCG TTC TTC ATC GAT ITs~~ ITS 2 forward ACA TCG ATG AAG AAC GTA GC AB28' ITS 2 reverse CCC CGG GAT CCA TAT GCT TAA GTT CAG CGG GT ISSM8 ISSR-PCR ISSR2' ISSR-PCR ISSlU8 ISSR-PCR ISSR4' ISSR-PCR ISSR5' ISSR-PCR ISSR6' IS SR-PCR ISSR7' ISSR-PCR ISSR8' ISSR-PCR Appendix 1. cont. Primer name Amplification Primer sequence (5' - 3') reaction ISSRg8 ISSR-PCR (GT),GG ISSRI08 ISSR-PCR ISSRl 18 ISSR-PCR (GT)&C ISSR12' ISSR-PCR (CAC),GC ISSR138 IS SR-PCR (GAG)@ ISSR34' ISSR-PCR (CTC),GC ISSR15' ISSR-PCR (GTG),GC designed for this thesis * fkom Vis et al., 1998 fiom Rintoul et al., 1999 fiom Müller et al., 1998 fiom Saunders & Kraft, 1994 hmWhite et al., 1990 'hm Vis & Sheath, 1997 fiorn Vis, 1999 Literature Cited Müller, KM, Sheath, R.G., Vis, M.L., Crease, T.J. & Cole, K.M. 1998. Biogeography and systematics of Ban@ (Elangiales, Rhodophyta) based on the Rubisco spacer, rbcL gene and 18s rRNA gene sequences and morphometric analyses. 1. North America. Phycologia 37: 195-207. Rintoul, TL., Sheath, R.G. & Vis, M.L. 1999. Systematics and biogeography of the Compsopogonaies (Rhodophyta) with emphasis on the fi-eshwater families in North America. Phycolo@-a38: 5 17-527. Saunders, G.W. & Kraft, G-T. 1994. SrnaIl-subunit rRNA gene sequences f?om representatives of selected families of the Gigartinaies and Rhodymeniales (Rhodophyta). 1. Evidence for the Plocamiales ord. nov. Canadian Journal of Botany 72: 1250-1263. Vis, M.L. 1999. Intersimple sequence repeats (IS SR) molecular markers to distinguish gametophytes of Batrachospermum boryanum (Batrachospermales, Rhodophyta). Phycologia 38: 70-73. Vis, M.L., Saunders, G.W., Sheath, R.G., Dunse, K. & Entwisle, T.J. 1998. Phylogeny of the Batrachospermales (Rbodophyta) inferred fiom rbcL and 18s ribosomal DNA gene sequences-JDurnuZ of Phycology 34: 341 -350. Vis, M.L. & Sheath, R.G. 1997. Biogeography of Baîruchospermum gelatinosum (Batrachospermales, Rhodophyta) in North America based on molecular and morphological data. Journal of Phycology 33: 520-526. White, T.J., Bruns, T., Lee, S- & Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Merhods and Applications (Eds. M.A. huis, D.H. Gelfand, J.J. Sninslq & T.J. White). Academic Press, San Diego, pp. 3 1 5-322. Appcndix 2. Means of niensurcd charactcrs and binruy coding for collections, type specimens and historically signiliant spccimens used in rnorpliometric analyses, Specimen name ccll cell filament basal layer conceplacle conceptacle tetrasporangiuin tetraspormgium marine or tetrasporangial protuberances O; coding diametcr length height hcight diamew depih length diameter freshwater code BCSWl 3.5 4.7 180.0 7,l 66,9 64,7 24.3 7.1 O O NSSWl 4.1 4.6 133.3 8.2 57.9 64,3 23,O 8.3 O O CASWl 3,3 3.8 88.4 9.2 59,l 56,3 26.2 8,5 O O CTSWl 3,6 4.3 83.9 9.2 559 53,7 2 1,8 8,3 O O MASW 1 2,8 2.6 88.0 5.5 - O O CRSW l 2.2 2.1 104.5 8.6 66,8 58.7 24,4 12,l O O CRSW2 3.3 3.1 78.6 3.4 - O O CR20 3.9 4.1 47.8 4.1 - I CR24 4.3 3.8 49,8 4.1 - I TX7 3.2 4,4 61.7 3,6 - 1 TX9 3.3 4.6 59.5 4,O - 1 SL9 3.7 3,l 50.7 3.9 - 1 SL2 3.4 3.4 26.0 4,2 - 1 PR19 3.7 4.3 33.4 4.4 - 1 BCSW2 2.8 3,5 100,6 11.6 74,s 59,5 32.4 8,s O O WASW l 3.0 3.3 1059 12,8 84S 93,6 298 9.5 O , O BCSW3 3.7 5.2 98,6 15,O 88.0 8 1,7 23.6 8.4 O O MEXSW l 4.0 3.1 187.8 14,3 102.0 1 1 1.7 22,1 10.6 O O MEXSW2 4.2 5.8 91,4 9.0 81.1 76,9 28.9 9,9 O O CASW3 2.3 4.6 334.8 7.9 113.3 138.2 20.7 4,9 O 1 CASW2 4.8 5.5 50,6 7.9 51.9 47,4 2 1,3 10.0 O O ORSWl 3.8 4.3 163.9 15.9 82,l 79.9 23.6 7,9 O O ORSW2 4.2 5.8 125.4 7.0 74.4 86,3 23,O 7.7 O O AKSW 1 2.5 3.0 70,9 10.9 69.8 67.8 18.0 5.9 O O NFSW 1 3.4 4.4 83.4 12,4 79.6 87,I 26,7 7.8 O O RIS W l 3,8 4.8 68,7 7.7 62.8 65.9 17.6 5.7 O O IR1 l 6.9 6.0 75.3 15.6 - 1 WAL2 6.6 5.8 99.4 12.5 - I WAL3 5.7 6.4 58,8 15.6 - 1 WALSW3 2.9 3.6 59.4 11,7 - O O AT10 4.4 6.8 80.1 12.7 - 1 AT14 5.4 6.9 48.9 11.4 - 1 AT15 6.7 8.4 56.7 9.5 - 1 NEDS W 1 3.0 4.3 88.4 12,2 - O O FMSWI 3.5 4.0 45,l 6,7 - O O Appendix 2 cont. Means of measured chaïacters and binary coding for collections, type specirnens and historically signifiant specimens used in morphometrics, Specimen name cell cell filament basal layer conceptacle conceptacle tetrasporangium tetrasporangium marine or tetrasporangial. protuberances. or coding diameter length height height diameter deptli length diameter fieshwater code BLZSW I 3,2 3.8 93.3 7.2 - - O O O CI 1 7.4 7.0 46.4 9.5 - - 1 O URUSWl 3.4 4.0 54.6 5.7 - O 1 O FL63 3.6 5.2 46.8 6.t - - - 1 O PHI 1 3,8 5.0 32.8 6.2 - 1 O SASW l 4.1 5.2 703.7 8.3 - - O 1 1