Proteomics and phylogenetics of the Gnetales

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.
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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.
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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
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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
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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
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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
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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
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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
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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, Welwitschiostrobus 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
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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
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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.
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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.
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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！)
14
Hou, C. Licentiate thesis, 2014
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Ephedra
Gnetales
Gnetum
Gnetum