Coevolution between lichen mycobionts and photobionts in Clødoniø secfion Clødoníø (Ctadoniaceae) BY Sara Beiggi A Thesis submitted to the Faculty of Graduate Studies in partial Fulfillment of the Requirements For the Degree of Master of Science Department of Botany University of Manitoba Winnipeg, Manitoba April 2006 THE UNIVERSITY OF MANITOBA FACULTY OF GRADUATE STUDIES JJgg¿ COPYRIGHT PERMISSION Coevolution Between Lichen Mycobionts and Photobionts in Cktdonia section Clarlonía (Cladoniaceae) by Sara Beiggi A ThesislPracticum submitted to the Faculty of Graduate Studies of The Universify of Manitoba in partial fulfillment of the requirement of the degree of Master of Science Sara Beiggi O 2006 Permission has been granted to the Library of the Universify of Manitoba to lend or sell copies of this thesis/practicum, to the National Library of Canada to microfilm this thesis and to lend or sell copies of the film, and to University Microfilms Inc. to publish an abstract of this thesis/practicum. This reproduction or copy of this thesis has been made availabte by authority of the copyright owner solely for the purpose of private study and research, and may only be reproduced and copied as permitted by copyright laws or with express wriffen authorization from the copyright owner. Abstract In this study coevolution between the Ascomycetous lichen fungi in section Cladonia and its chlorophyte algal partner Trebouxia was evaluated by comparing phylogenetic trees of each symbiont. Phylogenetic trees were produced from the internal transcribed spacer (lTS) regions and the 5.8S nuclear ribosomal DNA (rDNA) sequences in algal and fungal partners of 17 speci es of Cbdonia section Cladonia and 2l others. These sequences were obtained using primers designed in conserved regions flanking the ITS regions that are specific to each of the fungal or algal rDNA. Results revealed that fungal species complexes were polyphyletic. Four Trebouxia species (7. glomerata, T. pyriþrntis, T. magna and T. erici) are associated with lichens of genus Cladonia in Manitoba. There was no evidence of cospeciation in section Cladonia. However an isolated event of cospeciation was observed in Cladonia section Cladonia (in C. macrophyllodes and C. pocillum) as well as an algal switch by C. pyxidata. Algal switching may be very common in lichens. In addition, existence of cryptic and ecological species within section Cladonìa may obscure cospeciation in this section. Results from this study indicate that further investigation is required to examine a larger number of individuals within each fungal species. This may reveal existence of cryptic species. In addition, the effect of microenvironmental conditions on algal selection requires more study. Acknowledgements I rvould like to sincerely thank my advisor, Dr. Michele Piercey-Norrnore, without whose dedication, patience, encouragement and advice this thesis would not have been published. An NSERC grant to Dr. Piercey-Normore provided the funding for this research. I would like to thank the members of my committee, Dr. Bruce Ford and Dr. George Hausner for their advice and guidance. I would also like to thank Dr. Teuvo Ahti for his comments and confirmation of my vouchers' identification. I would like to thank Mr. Keith Travis and Mr. Narinder Kalkat for their assistant. I would like to extend my thanks to all graduate students in the department for their support. I finally would like to thank my family and friends for their endless understanding and encouragement. lll Table of Contents Title. .......i Abstract. ......ii Acknowledgements..... ......iii Table of Contents.... ..........iv List of Tables .. .........vi List of Figures. ... .....viii 1. Introduction. ..... ....1 2.Literature Review.... ......3 2.1. The Mycobiont...... ............6 2.2. The Photobionr. .. .. .. .. ...15 2.3. Ribosomal DNA as a molecular marker. .......24 2.4. Coevolution and Theory of the Geographic Mosaic of Coevolution... ..........27 3. Materials and Methods. ..........35 4. Results ........49 4.1. Fungal Phylogeny.. .........49 4.2. Algal Phylogeny... ....61 4.3. Overall evolution... ...67 lv 5. Discussion. .........69 5.1. phylogenetic History of the Fungal parrner. ..........69 5.2. Phylogenetic Hisrory of the Algal partner. .....77 5.3. Coevolution. .....g4 6. Conclusion and Future Research ......... g9 7. Literature Cited. .....g1 Appendices 1-13.... ........r02 List of Tables Table 1. List of cradoniø species in Section cladoniaused in this study, their TLC numbers, colrection numbers and sampring location 36 Table 2. chemical and morphological characters and character states Table 3' The binary (0, r) dara matrix built based on chemicar (1-5) and morphological (6-23) characters of 17 Clodoniaspecimens used in this study .....3g Table 4. List of 32 cradonia, cradia and Trebouxia species and corresponding ITS sequence accession numbers in GenBank, downloaded for this study 45 vt Table 5. Range of pairwise similarities (,,p', values) within members of species complexes and members of species within the section cladonia based on fungar ITSi,5.gs and ITS2 nucleotide sequences 50 Table 6. Results of incongruence tests (ILD and KH tests) calculated for datasets in this study. ....57 Table 7. A comparison of the variation in algal and fungal aligned ITS I and ITS2 nucleotide sequences used in this study... .......60 Table 8. comparison of pairwise distances, using the uncorrected,,p,, distance method, and number of sequence substitutions between natural photobionts and two known Trebouxza species in clade A ofFig.g..... ....63 vn List of Figures Fig' 1: Variation of microtubular root configuration in green algae. Basal bodies are shown as rectangles and microtubullar roots as bundles of two or four lines. a, I o'clock-7 o'clock configuration in chlorophyceae; b, 5 o' clock- I I o'clock configuration in ulvophyceae, cladophorophyceae, Bryopsidophyceae, Dasycladophy ceae, Trentepohliaceae and Trebouxiaceae; and c, 6 o'clock-12 o'clock confìguration in zygnematophyceae, Klebsormidiophyceae and charophyceae as well as to the various classes of land plants (modified from van den Hoek 1995) Fig. 2: Geographic mosaic of coevorution between a pair of species. circles represent communities. Each affow within circles indicates selection on one species. Arrows between communities indicate gene flow. coevolutionary hotspots are indicated by bold circles in a matrix of evolutionary cold spots, indicated by fine circles. (Modified from Thompson 1999). ............2g Fig 3: cup morphology in the genus cladonia. Left, atrumpet-shaped cup, covered with coarse granular soredia; Right, a goblet-shaped cup, covered with farinose soredia. .......40 vlll Fig 4' illustration of a nuclear rDNA repeat unit showing annealing sites for amplification primers (1780F-5',l7g0A-5'and ITS4-3') used in this study. These taxon-specific primers amplify algal and fungal ITS rDNA. The ambiguous sites for the taxon specific primers are located on the small subunit of ribosomal DNA flanking the ITSI region. ........42 Fig. 5. one of 15 most parsimonious trees for the fungal morphology, ITS l, 5.gs and ITS2 nucleotide sequence data. Dataset includes 17 mycobionts of Cladonia from this study and 26 from GenBank. Indels in the sequences are scored and incorporated into the data.. ..........51 Fig. 6. one of 12 most parsimonious midpoint rooted trees for the fi.rngal morphological data. Dataset includes l7 mycobio nts of Cladoria from this study. 53 Fig. 7. 50%ó majority rule of 38 most parsimonious trees for the fungal ITS I nucleotide sequence data. Dataset includes 17 mycobio nts of Cladonia from this study and 26 from GenBank... ...... ..... .5 g Fig. 8' 50%o majority rule tree of 29 most parsimonious trees for the fungal ITS2 nucleotide sequence data. Dataset includes l7 mycobionts of Cladonia from this study and 26 from GenBank.. .. .59 lx Fig. 9: One of two most parsimonious midpoint rooted trees for the algal ITSI, 5.8S and ITS2 combined nucleotide sequence data. Dataset includes 1l photobionts of Cladonia from this study and six known Trebouxia species from GenBank... ............62 Fig. 10: The single most parsimonious midpoint rooted trees for the algal ITS I nucleotide sequence data. Dataset includes 11 photobionts of Cladonia from this study and six known Trebouxia species from GenBank... .....64 Fig. I I : 50%;o majority rule consensus of 22 mosfparsimonious midpoint rooted trees for the algal ITS2 nucleotide sequence data. Dataset includes 1l photobionts of Cladonia from this study and six Trebouxia species from GenBank. ......66 Fig 12: Comparison of the photobiont and the mycobiont phylogenies of natural lichen associations based on ITSI, 5.8 and ITS2 combined sequence data. Datasets include eight Cladoniø samples. Both photobionts and mycobionts are isolated from from the same specimen. a: The single most parsimonious midpoint rooted tree for the algal ITS sequence data; and, b: The single most parsimonious midpoint rooted tree for the fungal ITS sequence data.. ........6g Append. 1. 50% majority rule consensus of 15 most parsimonious midpoint rooted trees for the fungal morphology, ITSI, 5.8s and ITS2 nucleotide sequence data. Dataset includes I7 mycobionts of Cladonia from this study and 26 from GenBank. Indels in the sequences are scored and incorporated into the data. ....104 Append. 2. One of th¡ee most parsimonious midpoint rooted trees for the fungal morphology, ITS1,5.8S and ITS2 sequence data. Dataset includeslT mycobiontsof Cladoniafromthis