PHYLOGENETIC AFFINITIES OF AUSTRALASIAN SPECIMENS OF BATRACHOSPERMUM (BATRACHOSPERMALES, RHODOPHYTA) INFERRED FROM MOLECULAR AND MORPHOLOGICAL DATA A thesis presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Master of Science Sarah A. Stewart August 2006 This thesis entitled PHYLOGENETIC AFFINITIES OF AUSTRALASIAN SPECIMENS OF BATRACHOSPERMUM (BATRACHOSPERMALES, RHODOPHYTA) INFERRED FROM MOLECULAR AND MORPHOLOGICAL DATA by SARAH A. STEWART has been approved for the Department of Environmental and Plant Biology and the College of Arts and Sciences of Ohio University by Morgan L. Vis Associate Professor of Environmental and Plant Biology Benjamin M. Ogles Dean, College of Arts and Sciences Abstract STEWART, SARAH A., M.S., August 2006, Plant Biology PHYLOGENETICU AFFINITIES OF AUSTRALASIAN SPECIMENS OF BATRACHOSPERMUM (BATRACHOSPERMALES, RHODOPHYTA) INFERRED FROM MOLECULAR AND MORPHOLOGICAL DATA U (67 pp.) Director of Thesis: Morgan L. Vis The phylogenetic affinities of five Australasian species of the freshwater red algal genus Batrachospermum were investigated using molecular and morphometric data. Specimens attributed to B. pseudogelatinosum, B. campyloclonum, B. kraftii, B. theaquum and B. bourrellyi, were collected from eastern Australia, Tasmania, New Caledonia and New Zealand. DNA sequence data from the plastid rbcL gene was used to infer interspecies relationships for all taxa. The mitochondrial cox2-3 gene spacer region was utilized to infer the intraspecific relationships among specimens of B. pseudogelatinosum, B. campyloclonum and B. bourrellyi. Two clades were resolved for B. pseudogelatinosum specimens in the rbcL, with B. bourrellyi placed equivocally as sister or within the clade. B. theaquum formed a separate clade in all analyses. B. kraftii specimens were placed within both of the B. pseudogelatinosum clades. Morphometric data was incongruent with the molecular data, and implications for species concepts in these Australasian taxa will be discussed. Approved: Morgan L.Vis Associate Professor of Environmental and Plant Biology Acknowledgements Many thanks to my advisor, Dr. Morgan Vis, for her mentoring, patience, and, most importantly, for introducing me to the fascinating realms of phycology and molecular systematics. I thank my committee members, Dr. Philip Cantino, Dr. Matthew M. White and Dr. Harvey E. Ballard, Jr. for their guidance. I thank my collaborator, Dr. Timothy J. Entwisle, of the Royal Botanic Gardens and Domain Trust, Sydney, Australia for access to specimens and furthering my understanding of the species concept for B. campyloclonum. Hannah McPherson,Virginia Lehmkuhl and the Global Studies 2005 New Zealand students are acknowledged for their collection of specimens from Australia, Tasmania and New Zealand. Catherine Gallagher, collections manager at the National Herbarium of Victoria, Melbourne, Australia (MEL) and Kristina McColl, technical officer of plant science at the Royal Botanic Gardens and Domain Trust, Sydney, Australia, are acknowledged for assistance in obtaining herbarium accession numbers for specimens of Batrachospermum used in the study. Jhy-Min Chiang, Matthew Albrecht, Ryan McEwen, Jason Zalack and Gregory Wahlert are acknowledged for technical assistance with statistical and phylogenetic analyses. Funding through a National Science Foundation grant (DEB 0235676) to Dr. Morgan L. Vis and the Department of Environmental and Plant Biology of Ohio University is gratefully acknowledged. Finally, I thank my parents, Jim and Grace Stewart, for support and encouragement. 5 Table of Contents Page AbstractTU UT .............................................................................................................................3 AcknowledgementsTU UT ...........................................................................................................4 ListTU of TablesUT ....................................................................................................................6 ListTU of FiguresUT ...................................................................................................................7 IntroductionTU UT .......................................................................................................................8 MaterialsTU and MethodsUT ....................................................................................................17 CollectionTU of SpecimensUT .............................................................................................17 MolecularTU AnalysisUT .....................................................................................................18 PhylogeneticTU AnalysisUT .................................................................................................19 MorphometricTU AnalysisUT ..............................................................................................22 ResultsTU UT .............................................................................................................................23 DiscussionTU UT .......................................................................................................................31 ReferencesTU UT .......................................................................................................................39 6 List of Tables Table Page 1. Collection information................................................................................................46 2. Primers.……………………………………………………………………………...48 3. Pairwise distances for rbcL…………………………………………………………49 4. Pairwise distances for cox2-3….…………………………………………………....50 5.Morphometric data: means and ranges……………………………………………....51 6.CART classifications………………………………………………………………...53 7. Discriminant analyses variable-variate correlations ...................................................54 7 List of Figures Figure Page 1.: Batrachospermum gametophyte thallus and distinguishing features ........................55 2.: Batrachospermum life history………………………………………………………56 3.: Australasian species of Batrachospermum investigated……………………………57 4.: Morphometric characters…………………………………………………………...58 5.: Maximum Likelihood tree inferred from rbcL data………………………………..59 6.: Minimum Evolution tree inferred from rbcL data…………………………………60 7.: Maximum Parsimony tree inferred from rbcL data………………………………..61 8.: Bayesian tree inferred from rbcL data……………………………………………..62 9.: Maximum Parsimony tree inferred from cox2-3 data……………………………..63 10.: CART tree generated from clades obtained in cox2-3 analysis………………….64 11.: CART tree generated from taxonomic groupings………………………………..65 12.: Discriminant analysis of taxonomic groupings using all characters……………..66 13.: Discriminant analysis of taxonomic groupings using vegetative characters…….67 8 Introduction Division Rhodophyta, the red algae, includes ca. 5000 species, most of which are marine. Approximately 3-5% of rhodophyte taxa occur in freshwater or estuarine environments. Among the freshwater taxa, genus Batrachospermum is the most species- rich with ca.130 species. Originally described by Roth in 1797, the genus Batrachospermum was established to include uniaxial, whorled branch forms. The genus is characterized by having determinate carposporophytes with radially-branched gonimoblast filaments that arise directly from the fertilized carpogonium, carpogonial branches arising from the periaxial and fascicle cells, and symmetrical or somewhat asymmetrical carpogonia with elongate or club-shaped trichogynes (Kumano 2002). Batrachospermum occurs worldwide, from the arctic to the tropics. It inhabits well-oxygenated streams, where it is found attached to rocks or submerged woody debris and macrophytes. Like all members of Division Rhodophyta, Batrachospermum lacks flagellated cells in any stage of its life history, has intercellular connections with pit-plugs, unstacked thylakoids and chloroplasts lacking an external endoplasmic reticulum, and utilizes phycobilin pigments, phycoerythrin and phycocyanin in photosynthesis with floridean starch as a storage product (Sheath 1984, 2003). The macroscopic gametophyte is dominant in the life history, and morphological features of the gametophyte are often used in taxonomic discrimination at the species level (Fig. 1). Batrachospermum exhibits oogamous reproduction in which a non-motile spermatium (male reproductive cell) fertilizes the carpogonium (female reproductive cell). Unlike other florideophycean red algae, 9 Batrachospermum and other related genera lack a tetrasporic life history stage, but have two sporophyte stages and one gametophyte stage (Fig. 2) (Sheath 1984, 2003; Kumano 2002). The Order Batrachospermales Pueschel et Cole (1982) is characterized by a heterotrichous, or attached, growth habit in the gametophyte life history stage, absence of tetraspores in the life history, and a double-layered pit plug with a domed outer layer (Pueschel & Cole 1982; Sheath 1984, 2003). This order contains three families: Batrachospermaceae, Lemaneaceae and Psilosiphonaceae (Vis et al. 1998). The order also includes a genus, Balliopsis, that was transferred to Batrachospermales on the basis of nuclear rDNA sequence data, but its placement within a family is uncertain (Saunders & Necchi 2002). In addition, the family Thoreaceae is no longer included in the Batrachospermales based on sequence data, and has been raised to ordinal status (Vis et al. 1998; Müller et al. 2002). The family Batrachospermaceae,