Evolutionary studies of the Gnetales Chen Hou Academic dissertation for the degree of Doctor of Philosophy in Plant Sys- tematics presented at Stockholm University 2016 Evolutionary studies of the Gnetales Chen Hou ©Chen Hou Stockholm University 2016 ISBN 978-91-7649-371-7 Printed in Sweden by US AB, Stockholm 2016 Distributor: Department of Ecology, Environment and Plant Sciences, Stockholm University, Sweden ”Although relatively few people have chosen to study the Gnetales, those who have had the opportunity to work with these organisms experience a profound sense of extraordinary beauty and complexity of the evolutionary process.” - Willian E. Friedman, 1996 Abstract The Gnetales consist of three distinct genera, Ephedra, Gnetum and Wel- witschia with considerable divergence among them regarding morphologi- cal, ecological and molecular characters. A longstanding debate of the simi- larity between the Gnetales and angiosperms and the unresolved seed plant phylogeny intrigues plant scientists to further investigate the evolutionary history of the Gnetales. The presented projects deal with interdisciplinary questions on proteomics, chloroplast genomes, phylogenetic relationships, gross morphology and taxonomy. The thesis aims to summarize general problems encountered in previous studies, and to provide new insights and future perspectives based on the results of completed and ongoing projects. In Ephedra, the Mediterranean species E. foeminea has been shown to be entomophilous and it possesses an important phylogenetic status as the sister of the remaining genus. Therefore, the chloroplast genome of E. foeminea was assembled and compared to that previously presented (of the anemophi- lous Asian species E. equisetina, nested in the core clade of Ephedra). The genome has a quadripartite structure and comprises 118 genes and 109,584 base pairs. A pairwise genome comparison was conducted between E. foe- minea and E. equisetina, resulting in the detection of 2,352 variable sites, the obtained data can be used for prospective phylogenetic studies. A proteomic study was also conducted on E. foeminea along with three anemophilous Ephedra species, in order to investigate the biochemistry of the pollination drops. The results show that detected proteins in the pollination drops of Ephedra vary dramatically among species but always occur in very low amounts. The majority of the detected proteins are degradome proteins, i.e., waste products from degrading cells of the nucellus. Some secretome pro- teins were also found, which are putatively functional, but also these proteins occur in very low amounts. The repeatability of the proteomic studies can, however, be questioned. The sampling methods and proteomic analyses are probably problematic although some suggestions for improvement are pro- vided. Thus I chose to continue with other projects. In Gnetum, reconstruction of the genus phylogeny and assessments of diver- gence times of clades were performed using an extensive sampling of in- group and outgroup accessions. The results show that the South American lineage separated from the remaining genus in the Late Cretaceous. The con- tinued diversification event gave rise to an African lineage and an Asian lineage. The crown age of the Asian clade, which comprises two arborescent species sister to the remaining liaonid species, was estimated to the Creta- ceous-Paleogene (K-Pg) boundary. In light of the genus phylogeny and esti- mated node ages, we suggest that the breakup of Gondwana influenced di- versification patterns in Gnetum. Later dispersal events also contributed to the current distribution of Gnetum, and to the phylogenetic patterns within each of the major clades. From my results, it is however clear that taxonomy and species delimitations are poorly defined, and needs to be further studied for all subclades of Gnetum. I have initiated this task by studying the Chi- nese lianoid clade of Gnetum more in depth. Eleven chloroplast genomes were generated, aligned and compared. Based on the information, four chlo- roplast markers were designed and applied to further resolve the species relationships with an extensive sampling. The results show, with strong sup- port, that G. parvifolium is sister to all the remaining species of the Chinese linaoid clade. Another five lianoid species are confirmed using both morpho- logical and molecular data, but several names are represented by type mate- rial that cannot be considered separate species. Modified keys for identifica- tion of male and female plants are presented, based on vegetative and repro- ductive structures. A subsequent dating analysis indicates that diversification in the Chinese lianoid Gnetum clade took place mainly in the Neogene, dur- ing which environmental changes probably facilitated diversification in the lineage. List of Papers This thesis is based on the following five papers, referred to in the text by their respective roman numerals. Paper I. Hou, C. & Rydin, C. 2013. Proteome of pollination drops in Ephed- ra (Gnetales). Manuscript. Paper II. von Aderkas, P.; Prior, N.; Gagnon, S.; Little, S.; Cross, T.; Hardie, D.; Borchers, C.; Thornburg, R.; Hou, C. & Lunny. A. 2015. Degradome and secretome of pollination drops of Ephedra. The Botanical Review 81:1- 27. DOI:10.1007/s12229-014-9147-x Paper III. Hou, C.; Wikström N. & Rydin, C. 2015. The chloroplast genome of Ephedra foeminea (Ephedraceae, Gnetales), an entomophilous gymno- sperm endemic to the Mediterranean area. Mitochondrial DNA. DOI:10.3109/19401736.2015.1122768 Paper IV. Hou, C.; Humphreys, A. M.; Thureborn, O. & Rydin. C. 2015. New insights into the evolutionary history of Gnetum (Gnetales). Taxon 64:239-253. DOI:10.12705/642.12 Paper V. Hou, C.; Wikström N.; Strijk J. S. & Rydin, C. 2016. Resolving phylogenetic relations and species delimitations in closely related gymno- sperms using high-throughput NGS, Sanger sequencing and morphology. submitted Reprints were made with permission from the publishers. Contributions of the thesis Papers I, III, IV, V were written by CH with comments and suggestions from the co-authors, and the designs of the studies were performed by CH and CR. CH did analyses of gel electrophoresis in Paper I and Paper II. CH has gen- erated the majority of sequences in Paper IV together with OT, all sequences in paper V, and the sequences in Paper III together with NW. CH is respon- sible for the phylogenetic analyses and dating analyses in Paper IV and Pa- per V, genome assemblies individually in Paper IV and together with NW in Paper III. CH has conducted the morphological studies in Paper V in cooper- ation with the co-authors. Content Introduction …………………………………………………………………...…………………………..10 Chapter 1. Pollination biology in the Gnetales 1.1 Anemophily versus entomophily ………………………………………………..……………………12 1.2 Investigations of pollination drops in gymnosperms……………………………………...…………..12 1.3 A proteomic study of pollination drops of Ephedra…………………………………….………….…13 1.4 A new proteomic study……………………………………………………………...…………...........14 1.5 Prospective studies………………………………………………………………………….…………15 Chapter 2. Phylogenetic studies and dating analyses of the Gnetales 2.1 Phylogenetic studies in Ephedra and taxonomic implications……………………………..…………17 2.2 Dating analyses and evolutionary history of the Ephedraceae………………………………..………17 2.3 Phylogenetic studies in Gnetum and taxonomic implications………………………………...………18 2.4 Dating analyses and evolutionary history of Gnetum…………………………………………………20 2.5 Prospective studies…………………………….………………………………………………………22 Chapter 3. Chloroplast genome investigations in the Gnetales 3.1 Nuclear genomes of the Gnetales………………………………………………………………..……24 3.2 Structural differences among chloroplast genome in the Gnetales……………………………………25 3.3 A newly generated chloroplast genome of Ephedra………………………………………..…………25 3.4 Chloroplast genome investigation in Chinese lianoid Gnetum………………………………..………26 3.5 Prospective studies…………………………………………………………………………………….27 Chapter 4. Taxonomy and species delimitations in the Gnetales…………………………………...…….29 Concluding remarks……………………………………………………………………………………….30 Legend………..……………………………………………………………………………………………31 Svensk sammanfattning (Swedish summary)……………………………………………………..………32 Acknowledgement……………….…………………………………………………………………..……34 Literature cited…………………………………………………………………………………….………36 Abbreviations 1D SDS PAGE One dimensional SDS polyacrylamide gel electrophoresis AICc Corrected Akaike information criterion AICM A posterior simulation-based analogue of Akaike information criterion CIPRES Cyber-infrastructure for Phylogenetic Research CTAB Cetyl trimethylammonium bromide DDT Dichlorodiphenyltrichloroethane DNA Deoxyribonucleic acid GTR + Γ The general time reversible model with gamma distributed rates HPD Highest posterior density IRs Inverted repeats K-Pg Cretaceous-Paleogene LC-MS Liquid chromatography-mass spectrometry LR Likelihood ratio LSC Large single copy MCMC Markov Monte Carlo chains NGS Next generation sequencing nrITS Nuclear internal transcribed spacers (including ITS1, 5.8S and ITS2) PP Posterior probability RJ-MCMC Reversible-jump Markov chain Monte Carlo RNA Ribonucleic acid rRNA Ribosomal RNA SSC Small single copy TBR Tree-bisection reconnection tRNA Transfer RNA UCLN Uncorrelated lognormal relaxed clock WCSP World Checklist of Selected Plant Families Hou, C. PhD thesis 2016 Introduction The order Gnetales consists of three families, Ephedraceae, Gnetaceae and Welwitschiaceae. Each family is monogeneric and comprises Ephedra (Fig. 1a), Welwitschia