Proteomics and Phylogenetics of the Gnetales

Home , Ephedra (plant), Gnetum, Welwitschia, Welwitschiaceae

Proteomics and phylogenetics of the Gnetales

Chen Hou Department of Ecology, Environment and Plant Sciences Stockholm University Supervisor: Catarina Rydin Licentiate thesis, 2014

Hou, C. Licentiate thesis, 2014

Abstract

A central point of Darwin’s theory of evolution is that accumulation of many small changes during the evolutionary process can result in significant change over time. In light of his theory, plant scientists seek for and compare different plant traits among species e.g., from morphology, DNA or proteins in order to discover the underlying evolutionary patterns and processes. The Gnetales, an intriguing family that comprises Ephedra, Gnetum and Welwitschia, have puzzled scientists for over a century. Their features are evolutionarily difficult to understand in comparison with other seed plants and this has hampered analyses of evolution and phylogeny regardless of whether morphological or molecular data has been utilized. In this thesis, I first attempt (Paper I) to seek for a new evolutionary indicator; a protein profile from pollination drops of Ephedra is compiled, and the results are compared with those from conifers and other seed plants. The aim of this proteomic study was also to investigate whether proteomic profiles vary among Ephedra species and are affected by different selection factors, e.g., pollination mode, ovule protection etc. The results indicate, however, that proteins are present only in very small amounts in pollination drops of Ephedra, and mainly as waste products from degrading cells. This is surprising since proteins are considered important for defense of the naked ovules of gymnosperms, e.g., against pathogens. Pollination drops of Ephedra have a very high sugar concentration and it is possible that carbohydrates are responsible for ovule defense in Ephedra. The second chapter of my thesis (Paper II) is devoted to Gnetum; a phylogenetic study based on genetic markers derived from both nuclear ribosomal regions and chloroplast regions is conducted. Previous studies have been hampered by difficulties with outgroup comparison and homology assessments of informative gene regions. A few attempts have been made to estimate the deepest splits in the genus, all with a limited ingroup sampling. We address the phylogeny of Gnetum and make a first assessment of the monophyly of species, using a denser sampling of taxa and a combination of faster and more slowly evolving molecular markers. The results are discussed in comparison with previous classification and morphology, and will provide a basis for further studies of taxonomy, ecology, and biogeography in Gnetum.

Hou, C. Licentiate thesis, 2014

List of Papers

The following papers, referred to in the text by their roman numerals, are included in this thesis.

I Chen Hou and Catarina Rydin, Proteome of pollination drops in Ephedra (Gnetales). Manuscript.

II Chen Hou and Catarina Rydin, Phylogeny and morphology of Gnetum (Gnetales). Manuscript.

Hou, C. Licentiate thesis, 2014

Contents

Abstract…………………………………………………………………………….…2

List of papers……………………………………………………………………….…3

Pollination biology and evolutionary history exploration of Gnetales…..…………...5

Introduction of Gnetales………………………………………………….…….…….5

Pollination biology of Gnetales……………………………………..…………….6

The evolution history of Gnetales…………………………………….…………..8

Ephedra……………………………………………………………………….8

Welwitschia………………………………………………….………..………10

Gnetum……………………………………………………………………….10

Future perspective……………………………………………………………………13

Acknowledgement………………………………………………………………...…14

References……………………………………………………………………….…...15

Hou, C. Licentiate thesis, 2014

Pollination biology and evolutionary history of the Gnetales

Introduction to the Gnetales

The Gnetales are a seed plant family that consists of three genera: Gnetum L. Ephedra L. and Welwitschia Hook.f. Ephedra comprises 45-55 species of shrubs, climbers or small trees, distributed in arid and semiarid regions of the world (Kubitzki 1990a). Gnetum comprises 30-40 species that are evergreen pantropical lianas or trees (Kubitzki 1990c). The monotypic species Welwitschia mirabilis is endemic to the Namib Dessert (Kubitzki 1990d) and southern Angola. Research on gnetalean megafossils and microfossils indicates that the family had a broader distribution in the past (Rydin et al. 2003, Rydin et al. 2004a, Dilcher et al. 2005, Rydin et al. 2006a, Rydin et al. 2006b, Rydin and Friis 2010), and the striking morphological and ecological variation, as well as the almost non-overlapping distributions of the three gnetalean genera, indicate a long evolutionary history with a greater diversity in the past than in the present.

Despite apparent differences, several key morphological characters are prevalent in all the gnetalean genera. For example, phyllotaxis is strictly decussate or sometimes whorled; vessels may be present and have perforation plates with circular bordered pits; all species have compound strobili; ovules are surrounded by at least one outer covering (future as seed envelope); and a micropylar tube extends beyond the seed envelope and exposes pollination drops (Kubitzki 1990b, Friis et al. 2011). These morphological characters, in combination with molecular evidence, show that the Gnetales are monophyletic and that Ephedra is sister to a clade comprising the genera Gnetum and Welwitschia (Crane 1985b, Price 1996, Ickert-Bond and Wojciechowski 2004, Rydin et al. 2004a, Won and Renner 2006, Rydin and Korall 2009).

The Gnetales share morphological similarities with angiosperms, and whether or not these similarities reflect a common ancestry or parallel evolution has been debated for at least a hundred years (e.g. Arber and Parkin 1908, Eames 1952, Mathews 2009). For example, Gnetum and Welwitschia have (angiosperm-like) leaves with reticulate venation, tetrasporic female gametophytes and reduced male gametophytes (Friis et al. 2011). Double fertilization has been documented in the Gnetales (Friedman 1992, Carmichael and Friedman 1996), but the final output of the process yields two zygotes instead of one embryo and endosperm as in angiosperms (Carmichael 2012). Vessel elements responsible for efficient water conduction were originally thought to be homologous with those of angiosperms, but are now considered independently evolved in angiosperms and the Gnetales (Carlquist 1996). Further, pollen tubes are reported to germinate very quickly in the female ovules of Ephedra and Gnetum, as they do in angiosperms but not in other gymnosperms (Williams 2008). From a molecular perspective, it is also interesting to note that Gnetum shares a mitochondrial

Hou, C. Licentiate thesis, 2014

sequence (nad1 and two adjacent exons) with angiosperms (Won and Renner 2003), although this is clearly a result of horizontal transfer, not most recent common descent (Won and Renner 2003).

Thus, many of these similarities (but not all of them) to some extent substantiate a possible phylogenetic relationship between Gnetales and angiosperms, i.e. the “anthophyte hypothesis” (Crane 1985a, Loconte and Stevenson 1990, Donoghue and Doyle 2000). This hypothesis has, however, been questioned by a series of subsequent studies based mainly on molecular data, in which other hypotheses on seed plant relationships have been put forward, e.g. “gnetifer hypothesis” (Gnetales sister to conifers) (Chaw et al. 1997, Rydin and Korall 2009), “gnepine hypothesis” (Gnetales sister to Pinaceae) (Bowe and Coat 2000, Chaw et al. 2000, Braukmann et al. 2009), the “gnecup hypothesis” (Gnetales sister to cupressophyte conifers) (Raubeson et al. 2006, Braukmann et al. 2009), “gnetophyte-sister hypothesis” (Gnetales sister to all other seed plants) (Magallón and Sanderson 2002, Rydin et al. 2002), and the Gnetales sister to all other gymnosperms (Schmidt and Schneider-Poetsch 2002). Most of these hypotheses are well supported in a statistical sense and in spite of ever-increasing efforts, it has not been possible to discriminate between them; seed plant phylogeny remains unresolved.

Pollination biology in the Gnetales

Anemophily or wind pollination has been broadly documented in non-flowering plants, for example, in Ginkgo (Del Tredici 2007), all conifers and Ephedra (e.g. E. trifurca, Buchmann et al. 1989 and E. distachya, Bolinder et al. submitted). It is probably less well known that entomophily or insects pollination plays an important role in pollination biology in non-flowering seed plants, as suggested by a series of studies on cycads (Norstog 1987, Tang 1987, Norstog and Nicholls 1997), Gnetum (Kato and Inoue 1994, Kato et al. 1995), Welwitschia (Wetschnig and Depisch 1999) and Ephedra foeminea (Bolinder et al. submitted). Entomophilous plants display distinctive strategies to attract potential pollinators for transmission of pollen. For example, cycads (Pellmyr et al. 1991) and Gnetum (Kato et al. 1995) are reported to produce specific volatilized chemicals to attract insects. Cycads can make use of self-heated system to “push and pull” insects for the purpose of pollen transportation (Terry et al. 2007); both male and female cones of the Gnetales are able to secret sugary pollination drops that attract pollinators (Ziegler 1959, Bino et al. 1984, Meeuse et al. 1990, Kato et al. 1995, Endress 1996, Wetschnig and Depisch 1999). At least some species in all gnetalean genera have morphologically bisexual reproductive organs, which however are functionally unisexual (Endress 1996). Bisexual structures are rare in gymnosperms but common in angiosperms (Friis et al. 2011).

Pollination drops are commonly produced by gymnospermous ovules but absent in

Hou, C. Licentiate thesis, 2014

the enclosed ovules of angiosperms (Nepi et al. 2009). In contrast with liquid secretion in flowering plants, for example nectar which is specifically produced as pollinator reward, pollination drops of gymnosperms have another primary function: that of trapping pollen grains and transporting them to the internal of ovules (Tomlinson et al. 1997, Owens et al. 1998, Gelbart and von Aderkas 2002). During the process, pollination drops probably play other roles as well, roles that have bearing on seed plant reproduction and plant-environment interaction. Thus, to uncover potential underlying functions in the context of pollination biology, investigations of biochemistry of pollination drops are relevant. The major biochemical components of pollination drops are carbohydrates, amino acids, minerals and proteins (Nepi et al. 2009, Nepi et al. 2012). Among these components, proteins are interesting because they are assumed to have several important ecological roles, e.g. to defend ovules from external pathogens (O'Leary et al. 2007, Wagner et al. 2007). Considering the fact that there is no physical protection of ovules in gymnosperms, like the carpel of angiosperms, chemical defense against external threats is probably very important in gymnosperms. Proteomics of pollination drops of the Gnetales would be interesting to study further, partly because the Gnetales are an isolated clade that often differs substantially from other seed plants, but also because there is variation in pollination biology within the order (Bolinder et al. submitted). It would therefore be interesting to compare protein profiles of pollination drops between anemophilous and entomophilous taxa of the Gnetales in order to address whether they differ with pollination mode. The results may also have implications for future studies of relationships and evolution of seed plants.

So far, protein profiles of pollination drops have only been documented for a restricted number of anemophilous gymnosperms, and for Welwitschia of the Gnetales (Poulis et al. 2005, O'Leary et al. 2007, Wagner et al. 2007). To make a first assessment of the protein profile of pollination drops in the remaining Gnetales, I conducted a study of pollination drops of Ephedra (Paper I). Drops from four species, i.e. E. foeminea, E. minuta, E. likiangensis and E. distachya, were collected both in the field and from cultivated specimens. Proteins were extracted from the pollination drops, and then detected using gel electrophoresis and mass spectrometry. Our result indicates that the average amount and number of proteins are very low in Ephedra compared with profiles reported for pollination drops of other gymnosperms (Paper I). All the investigated species of Ephedra mainly have degradome proteins, i.e. proteins that are considered to be remains of cell degradation. A degradome has not been discovered in pollination drops of other gymnosperms studied so far, and this variation among seed plants is probably correlated with formation of a pollen chamber, which occurs in some seed plants (e.g. the Gnetales), but not in others (Paper I). Another main type of proteins is secretome proteins, which are actively secreted and supposedly functional, for example, thaumatin-like proteins that act as defense proteins (O'Leary et al. 2007, Wagner et al. 2007). Results from the proteomic profiles

Hou, C. Licentiate thesis, 2014 in pollination drops from the four investigated species of Ephedra showed that E. foeminea has a higher number of detected proteins than the other three species. It is thus possible that pollination mode could account for the difference of protein profiles between Ephedra species. Ephedra foeminea is entomophilous whereas other species are anemophilous, and more proteins are thus present in pollination drops in E. foeminea, in which plant-insect interactions are known to occur. However, the amount of proteins detected in our study (Paper I) was so low that proteins are unlikely to be of any functional importance in Ephedra. Moreover, even though most species of Ephedra are anemophilous, other plant-insect interactions may occur. Ovules of E. distachya are for example often infested by parasitic wasps (Bolinder et al. unpublished work).

In addition, the methodology currently employed in proteomic studies of pollination drops is perhaps unreliable. A recent study of proteomics of pollination drops of E. monosperma (von Aderkas, Hou et al. in progress) result in different proteomic profiles in drops collected at different time periods. This may indicate that the protein profile varies substantially over the reproductive period. So far proteomic studies have always been based on pooled samples of pollination drops, often collected from many specimens and during a longer time period. This has been considered necessary since the amount of pollination drops produced may be very low, in particular in conifers. The approach means, however, that variations over the reproductive cycle are not detected, and furthermore, results from different taxa may not be comparable if sampling unintentionally has shifted towards different time periods in the reproductive cycle of the species. Another cause of concern is that results indicate that the repeatability and validity of results produced with currently used methods could be questioned.

In conclusion, many questions remain on proteomic profiles of pollination drops in gymnosperms. Methodology as well as sampling strategies conceivably need considerable improvements. For Ephedra, it seem however clear that very low amounts of proteins are present, and mostly in the form of a degradome. The same appears to hold for Welwitschia (Wagner et al. 2007); Gnetum has not been studied for proteomics of pollination drops. Future studies of ovule defense, and biochemical profile and function of pollination drops of the Gnetales should probably focus on carbohydrates instead of proteins.

The evolutionary history of the Gnetales

Ephedra

Species relationships in Ephedra have been studied based on morphological data (Ickert-Bond et al. 2003, Liu et al. 2008, Rydin et al. 2010, Ickert-Bond and Rydin

Hou, C. Licentiate thesis, 2014

2011) and molecular data (Rydin et al. 2004b, Huang et al. 2005, Rydin and Korall 2009). The studies indicate that morphological and molecular divergence is generally low in Ephedra, and evolution of similar morphological traits have occurred in parallel in unrelated subclades in the genus (Ickert-Bond and Wojciechowski 2004, Huang et al. 2005, Rydin et al. 2004, Rydin et al. 2010). It has been widely accepted that Ephedra foeminea is sister to the remaining species in crown group Ephedra (Rydin and Korall 2009), although the result was weakly supported and partly poorly resolved. The hypothesis was recently tested and supported, using additional data (Thureborn and Rydin, unpublished work). Yet denser sampling is needed to resolve species boundaries and biogeography in some subclades (Rydin and Korall 2009, Kakiuchi et al. 2011, Qin et al. 2013).

Quantitative studies of fossil ephedroid pollen (Crane and Lidgard 1989, Lidgard and Crane 1990) indicate an increase in gnetalean diversity during the Early Cretaceous (Crane and Lidgard 1989, Lidgard and Crane 1990). The result is consistent with the discovery of Ephedra-like megafossils from the same time period (Rydin et al. 2004a, Rydin et al. 2006a, Rydin et al. 2006b, Wang and Zheng 2010, Yang et al. 2013). The ephedran diversity declined however dramatically during the latter part of the Cretaceous (Crane and Lidgard 1989), and the initiation age of the crown group Ephedra has been estimated to the early phase of Oligocene (ca. 30 Ma Ickert-bond et al. 2009). These findings are in agreement with the low genetic and morphological diversity observed among extant species of Ephedra (Ickert-Bond and Wojciechowski 2004, Huang et al. 2005, Rydin et al. 2010).

Processes behind diversity fluctuations in Ephedra have recently been inferred from several ecological and biogeographic studies. Studies on pollination biology suggest that a conversion from entomophily to anemophily could have been essential for the radiation of Ephedra in the Paleogene (Bolinder et al. submitted). A niche conservatism study indicates the recent diversification of New World Ephedra species could be partially influenced by expansion of arid areas coupled with climatic changes and orogenetic process in the past (Loera et al. 2012). Speciation in Old World Ephedra clades is likely to be coupled with similar processes, for example, the uplifts of Qinghai-Tibetan Plateau and gradual Asian aridification processes are considered to cause the divergence of Chinese Ephedra during the middle or late Micocene (Qin et al. 2013). Biogeographic analyses of Ephedra are rare, but Ickert-Bond et al. (2009) suggest that Old World Ephedra dispersed from Central Asia to the Mediterranean area through long distance dispersal mechanism; whereas the New World Ephedra dispersed back and forth between the North and South American continents. Explanations for the disjunct distribution of New World and Old World Ephedra are however not well studied. Seed dispersal mechanisms have been tested for New World Ephedra, and both zoochory and anemochory have been found (Ickert-Bond and Wojciechowski 2004, Hollander and Vander Wall 2009, Hollander et

Hou, C. Licentiate thesis, 2014

al. 2009). No similar studies of Old World species have been conducted, and it is in addition uncertain from current studies on New World species how far the seeds can disperse. However, based on available knowledge on the history of Ephedra and the observed biogeographical patterns of the clade, dispersal over long distances, even oceans, appears possible.

Welwitschia

Welwitschia has received ample attention from botanists for several decades due to the abnormal shape of the plant, considered a “monster” in the Namib Dessert (Rodin 1953, von Willert et al. 1982, Crane and Hult 1988, Cooper-Driver 1994, Di Salvatore et al. 2013). Although evolutionary isolated and monotypic today, it is now well-documented that the Welwitschia lineage was much more diverse and widespread in the past. Welwitschia-like fossils, e.g. Cratonia cotyledon (Rydin et al. 2003) Priscowelwitschia austroamericana, Welwitschiophyllum brasiliense, Welwitschio- strobus murili (Dilcher et al. 2005) from Brazil, Drewria potomacensis from Eastern North America (Crane and Upchurch Jr. 1987), Chaoyangia liangii from China (Duan 1998), and Heerala antiqua from Siberia (Krassilov 2009) indicate that the genus and its close relatives had a broader, perhaps world-wide, distribution and a greater morphological diversity than today. Till now there are no convincing explanations why the Welwitschia lineage has found a last refuge in the Namib Dessert and in Angola. Welwitschia is in fact not well adapted to desert conditions (Gaff 1972). It has repeatedly been suggested that it is confined to areas very near drainage canals, and survives mainly by means of a long taproot (Pearson 1906, Kers 1967, Gaff 1972). Welwitschia is also capable of recovery from injury (Henschel and Seely 2000) and is known to build up a strong mutual relationship with underground mycorrhiza (Jacobson et al. 1993, Strullu-Derrien and Strullu 2007). Welwitschia is well adapted to insect pollination (Wetschnig and Depisch 1999), and to seed dispersal via wind (Kubitzki 1990d), both of which could result in less inbreeding among closely related individuals (Jacobson and Lester 2003).

Gnetum

Diversity and interspecific relationships of Gnetum were extensively studied by Markgraf in a series of papers and floras (Markgraf 1930, 1950, 1965). Based on comparative studies of morphology and geographic distribution, Markgraf divided Gnetum into 30 species (Markgraf 1930). Now, little over 40 species are recognized (WCSP 2014). Markgraf (1930) considered the arborescent species of Gnetum most ancestral. The statement however did not yield support from subsequent studies based on molecular data (Won and Renner 2003, 2005, 2006). These studies confirmed monophyly of Gnetum and tentatively indicated that South American taxa are sisters to the remaining genus, but attempts to resolve deep divergences were hampered by

Hou, C. Licentiate thesis, 2014 difficulties with outgroup comparison. A new phylogenetic study (Paper II) was performed, in which a much larger set of ingroup taxa were analyzed together with outgroup information. The resulting topology resolves South American taxa (Gnetum subsection, Araeognemones Markgraf 1930) as sister to the remaining genus. Although the statistical support for this relationship was low, analyses consistently result in this sister relationship and it conceivably reflects the true evolutionary history of the genus. The African lineage (Gnetum subsection Micrognemones Markgraf 1930) is the next diverging clade and sister to Asian Gnetum which is the most species-rich clade within Gnetum. The arborescent species of Gnetum (Gnetum subsection Eugnemones Markgraf 1930) is sister to the remaining Asian taxa. Our study assigned the position of sex species (G. leyboldii, G. camporum, G. buchholzianum, G. montanum, G. indicum and G. leptostachyum) that have not been included in previous phylogenetic studies, and species boundaries are discussed in light of a denser sampling than in previous studies (Markgraf 1930, 1950, 1965, Won and Renner 2003, 2005, 2006). Further, our results show that Gnetum section Erecta (Griffith 1859) is not monophyletic but comprises a paraphyletic assemblage of clades outside of the “core” Gnetum clade. Gnetum section Scandentia (Griffith 1859) is monophyletic but the Gnetum subsections Stipitati and Sessiles within the section are non-monophyletic. The division was based on the length of pedicils but this feature is apparently variable among and within clades.

Some interesting morphological characters, previously discussed as informative, or with potential relevance for evolution of form and function in Gnetum, were discussed in Paper II. It is worthwhile to note that morphological differences among Gnetum species are apparent but poorly investigated and understood. At first, we attempted to map both vegetative and reproductive structures on the new phylogeny to investigate character evolution in Gnetum. The resulting pattern was, however, very difficult to interpret. This is partially because morphological characters described by Markgraf (1930, 1950, 1965, 1967) may be non-overlapping among species and groups are partly defined by ancestral traits; the purpose of Markgraf’s work was that of species recognition and classification, not necessarily to trace evolution. For example, leaf features in G. raya are documented as “shiny” (Markgraf 1930, 1967), which is incomparable with the leaf feature “fleshy” in G. microcarpum (Markgraf 1930). Regarding female spikes of Gnetum, internodes between collars are clearly visible in some species, e.g. G. gnemon but not apparent in other species, but the criteria that defines the two character states (visible and invisible) seems arbitrary. Other characters are poorly studied. Sterile ovules present on male spike is the prevailing state in Gnetum but is reported to be absent in African species (Pearson 1912, Markgraf 1930). Kato et al. (1995) question the presence of sterile ovules in male spikes of an Asian species, G. cuspidatum, based on field observation, but the statement contradicts information in Markgraf (1930). We have not yet conducted the detailed morphological studies needed to confirm the absence or presence of tiny

Hou, C. Licentiate thesis, 2014

sterile ovules in the male spikes of G. cuspidatum. Additional studies of morphological variation are required to assess character evolution in Gnetum.

Being tropical evergreen plants, Gnetum may have low preservation potential, and both megafossils and microfossils of Gnetum are lacking. The characteristic polyplicate pollen of Welwitschia and Ephedra (Traverse 2008) often preserved in fossil strata, are not present in Gnetum. Pollen of Gnetum is echinate (Rydin et al. 2004b, Yao et al. 2004) with a thin exine that may be less resistant to taxonomical change. So far, only two fossils with possible affinity to Gnetum have been described: Khitania columnispicata (Guo et al. 2009) and Siphonospermum simplex (Rydin and Friis 2010), both from the Yixian Formation, China. The phylogenetic positions of the two fossils are uncertain, which means that these fossils are difficult to apply as calibration points in analyses of divergence times of clades. A previous dating analysis has been conducted on Gnetum, based on a dataset of 10 Gnetum species plus 27 outgroup taxa from other seed plant clades (Won and Renner 2006). The result indicates that extant Gnetum stems from a relatively recent radiation during the late Oligocene (ca. 26 Ma). Asian Gnetum and African Gnetum share a common ancestor from the Miocene (ca. 22 Ma) (Won and Renner 2006). If these results are correct, they exclude the possibility of vicariance having influenced the major phylogenetic patterns in Gnetum.

If so, seed dispersal ability is a key factor for understanding the disjunct distribution of Gnetum at present, as is the case in Ephedra. The probability of long distance dispersal in Gnetum was preliminarily discussed based on, on one hand, a series of observation of seed dispersal in the field, e.g. by birds (Ridley 1930), civet-cats (Markgraf 1950), rodents (Forget et al. 2002) and fishes (Kubitzki 1985), and compared with physical transportation ways, e.g. by ocean drifts (Ridley 1930, Markgraf 1950). So far, however, there is no comprehensive study to validate the candidate dispersal vectors that Gnetum could make use of for seeds dispersal in the past and presence. Furthermore, seed dispersal to new geographic regions is not enough; the plants also have to be able to survive and reproduce in a new continent/region/habitat, even if they no longer have access to mutualistic partners.

Hou, C. Licentiate thesis, 2014

Future perspective

Preliminary studies (Hou et al. in progress) indicate that divergences of clades in Gnetum are generally older than previously estimated. It is also possible that biogeographical hypotheses need to be revised in light of results based on additional sampling of data and specimens compared with previous studies. The Gnetum lineage seems to have a long history considering a (phylogenetically) long stem-lineage and ample fossil documentation with affinity to Welwitschia (and perhaps also Gnetum; mainly from the Aptian and Albian, see Rydin et al. 2003, Dilcher et al. 2005, Guo et al. 2009 and Rydin et al. 2010). Thus a new study including both estimation of divergence times of clades, and a biogeographic analysis is carried out in order to further investigate the evolutionary history of Gnetum. The study focuses on 1) the geographic origin of extant Gnetum, 2) studies of the potential of Gnetum taxa to migrate and colonize new regions, and 3) the possible correlation between diversification events and plate tectonics and/or paleoclimatic changes. Further, during the summer of 2014 I will do field work in China, and these studies will form the basis for additional systematic and ecological work on the “core” Gnetum clade in continental Asia, the “hot-spot” region of the genus.

Fig. 1 The phylogeny of Gnetales, the red lines represent phylogeny of Ephedra, the green lines represent phylogeny and Gnetum and the black line represents Welwitschia mirabilis. Taxa have been labeled with their corresponding distribution. The topology is based on results in Rydin and Korall 2009 and Paper II.

Hou, C. Licentiate thesis, 2014

Acknowledgment

I am very grateful to my supervisor Catarina Rydin for providing the opportunity to be involved in the PhD studies. Many thanks for her tremendous support with knowledge, inspiration, biotechniques and care of my daily life in Sweden. I am so happy to yield always-sweet smiles, encouragement and great patience from her especially the most difficult research period that we met in 2012.

I will give many thanks to Anbar Khodabandeh who gave me a lot of help in the lab and answered my never-ending questions about the DNA sequencing technique.

I will also give many thanks to other members of our Gnetales group: thank you for lively discussions and helps from my roommate Kristina Bolinder, and also postdoc Aelys Humphreys who provides me very nice ideas and constructive suggestions in my research. And thanks also to the master students, Olle Thureborn, Lena Ivarsson, Eva Larsen and Lina Jerrå for the nice talks.

I would like to thank to Åsa Krüger for the “PhD-sitting” in the first year and many thanks to other people in the former plant systematic division, Birgitta Bremer, Per-Ola Karis, Sylvain Razafimandimbison, Niklas Wikström, Kent Kainulainen, Frida Stångberg and Annika Bengtson for different helps of both researches and my daily life, especially Barbro Axelius for her kind endurance of the noise from our heated debates in the past. I will also thank all other lovely persons in our new department to make my work so pleasurable and continent. I also thank Birgitta Bremer for valuable comments on the text.

Finally, I would like to give my sincere gratitude to my parents that have raised me up and continuously provided me with many encouragement and guidance in my daily life outside of China. (谢谢我的爸妈，衷心地感谢你们的鼓励和支持，我会努力的！)

Hou, C. Licentiate thesis, 2014

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Ephedra Welwitschia

Gnetales

Gnetum Gnetum