Evolutionary studies of the Gnetales Chen Hou

Evolutionary studies of the Gnetales
Chen Hou
Academic dissertation for the degree of Doctor of Philosophy in Plant Systematics 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 Welwitschia with considerable divergence among them regarding morphological, ecological and molecular characters. A longstanding debate of the similarity 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 anemophilous 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. foeminea 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 proteins 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 provided. Thus I chose to continue with other projects.
In Gnetum, reconstruction of the genus phylogeny and assessments of divergence times of clades were performed using an extensive sampling of ingroup and outgroup accessions. The results show that the South American
lineage separated from the remaining genus in the Late Cretaceous. The continued 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 Cretaceous-Paleogene (K-Pg) boundary. In light of the genus phylogeny and estimated node ages, we suggest that the breakup of Gondwana influenced diversification 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 Chinese lianoid clade of Gnetum more in depth. Eleven chloroplast genomes
were generated, aligned and compared. Based on the information, four chloroplast markers were designed and applied to further resolve the species
relationships with an extensive sampling. The results show, with strong support, that G. parvifolium is sister to all the remaining species of the Chinese
linaoid clade. Another five lianoid species are confirmed using both morphological and molecular data, but several names are represented by type material that cannot be considered separate species. Modified keys for identification of male and female plants are presented, based on vegetative and reproductive structures. A subsequent dating analysis indicates that diversification
in the Chinese lianoid Gnetum clade took place mainly in the Neogene, during 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 Ephedra (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 81:127. 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 gymnosperm 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 gymnosperms 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 generated 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 responsible for the phylogenetic analyses and dating analyses in Paper IV and Paper V, genome assemblies individually in Paper IV and together with NW in
Paper III. CH has conducted the morphological studies in Paper V in cooperation 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 (Fig. 1b) and Gnetum (Fig. 1c-d), respectively. The monophyly of the Gnetales, first inferred based on morphology (Hooker 1863,
Crane 1985), has been repeatedly confirmed using molecular data, and
Ephedra is sister to the clade that comprises Gnetum and Welwitschia (Price
1996, Bowe et al. 2000, Rydin et al. 2002, Rydin et al. 2004, Rydin & Korall
2009). Ephedra comprises more than 50 species with the life forms shrubs,
climbers or small trees (Price 1996, Rydin & Korall 2009, Thureborn &
Rydin 2015). The geographic distribution of the genus covers arid and semiarid regions of northern Africa, western Europe to eastern Asia, and the
Americas (Fig. 1e, Kubitzki 1990a). Some Asian species of Ephedra, such as
E. equisetina, E. monosperma, E. regeliana and E. sinica are the natural
source of ephedrine, an alkaloid commonly used as stimulant and treatment
in traditional medicine (Huang 1998, Kitani et al. 2009, Kitani et al. 2011).
Welwitschia comprises a monotypic species, W. mirabilis, a shrub distributed in the Namib Desert (Fig. 1e, Hooker 1863, Pearson 1907, Pearson 1909,
Rodin 1953, Kubitzki 1990d). Gnetum comprises more than 30 species with
the life forms trees, shrubs and liana (Price 1996, Hou et al. 2015). The genus inhabits tropical and subtropical forests of South America, Africa and
South Asia (Fig. 1e, Kubitzki 1990c). Vegetative parts of Asian Gnetum
(Fig. 1d), such as bark of G. gnemon, G. latifolium and G. montanum can
provide high-quality fibres for fishing nets, threads and paper production
(Markgraf 1951, Fu et al. 1999). Mature seeds of Gnetum (Fig. 1c) can be
used for production of edible oil, soup and wine (Fu et al. 1999). Besides,
biochemistry extracted from the Asian species of Gnetum is reported to possess pharmacological effects, e.g., anti-oxidant (Yao & Lin 2005), antiinflammatory (Yao & Lin 2005, Yao et al. 2006) and anti-influenza (Liu et
al. 2010).
Gnetales are characterized by decussate and opposite phyllotaxis (sometimes
whorled), multiple axillary buds, ovules surrounded by one to two seed envelopes, a thin integument that protrudes and forms a micropylar tube, and a
granular infratectal layer in the pollen wall. Other distinctive characters of
the Gnetales are vessel elements with perforation plates evolved from a basic
plan of circular bordered pits, a character that used to be considered homologous with angiosperms but now believed to be independently evolved
(Thompson 1918, Carlquist 1996) because the perforation plates of angiosperm vessels are derived from pits of a scalariform shape. There are many
such examples; the direction of ovule development in Gnetales is acropetal,
which is different from the basipetal direction in angiosperms (Endress
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Hou, C. PhD thesis 2016
1996). Similarly, the seed envelopes that surround gnetalean ovules have
long been compared with the angiosperm carpel, but the Gnetales clearly
possess the gymnospermous mode of reproduction, with an exposed ovule.
The Gnetales have a double fertilization mechanism, but eventually generate
two zygotes rather than one embryo and one endosperm present in angiosperms (Friedman 1992, Carmichael & Friedman 1996). Therefore, the
Gnetales and angiosperm are believed independently derived and similarities
between the two groups are most probably superficial. Although most contemporary scientists consider the Gnetales most closely related to conifers,
the question of whether or not the Gnetales are most closely related to angiosperms remains unresolved despite many attempts to address the question
(Arber & Parkin 1908, Pearson 1929, Eames 1952, Carlquist 1996, Crane
1996, Endress 1996, Price 1996, Mathews 2009, Friis et al. 2011). Seed
plant phylogeny is still not resolved (Rydin et al. 2002, Rydin 2005,
Mathews 2009), despite many previous efforts. Thus, all these unresolved
questions make the evolutionary history of the Gnetales very intriguing.
Despite sharing a common ancestry and several uniquely derived features,
Ephedra, Gnetum and Welwitschia reveal dramatic differences in both vegetative and reproductive morphology (Thompson 1912, Pearson 1929,
Kubitzki 1990b, Endress 1996, Price 1996, Yang et al. 2015). For example,
leaves of Ephedra are highly reduced (Fig. 1a), often scale-like, and with 2-3
parallel veins that converge at the tip; leaves of Welwitschia are large and
strap-shaped (Fig. 1b), with parallel primary veins connected by Y-shaped
second-order veins; leaves of Gnetum are petiolate and similar to those of
many eudicots (Fig. 1e), with a venation that appears pinnate and reticulate.
The female cone of Ephedra has several decussately arranged bracts (Fig.
1a), but normally only the uppermost two bracts (rarely one or three) are
fertile and subtend female units; the female cone of Welwitschia have decussately arranged bracts piled in a cone axis (Fig. 1b), each of which subtends
a female unit; the female strobili of Gnetum are spike-shaped, bearing several whorls of irregularly placed female units on collars (Fig. 1h). In addition
to fertile male units, male cones of the three genera often comprise sterile
female structures (rarely present in Ephedra). Although the varied gross
morphology of the three genera provide valuable clues that have been used
to place some fossils phylogenetically, e.g., Cratonia cotyleton (Rydin et al.
2003), the substantial extinct diversity and unknown ancestral state of many
features make it difficult to place most fossils with certainty.
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Hou, C. PhD thesis 2016
Chapter 1 Pollination biology in the Gnetales
1.1 Anemophily versus entomophily
Pollination biology of the Gnetales interests plant scientists since the order
possesses morphologically bisexual reproductive organs (Pearson 1929,
Endress 1996), which are prevalent in angiosperms but rare in gymnosperms
(Friis et al. 2011). The bisexual reproductive organs are, however, functionally unisexual. Previous efforts have been performed to reveal the pollination
syndrome of the Gnetales. In Ephedra, anemophily (wind pollination) is
prevailing (Rydin & Bolinder 2015) and has been documented, for example,
for E. trifurca (Buchmann et al. 1989), E. distachya (Bolinder et al. 2016),
E. nevadensis (Bolinder et al. 2015a, Niklas 2015). Two species, E. foeminea
(= E. campylopoda) (Porsch 1910, Bolinder et al. 2016) and E. aphylla (Bino
et al. 1984, Meeuse et al. 1990) have been described as entomophilous (insect-pollinated). Welwitschia mirabilis, the monotypic species of Welwitschia, has also been described as entomophilous (Pearson 1907). Wetschnig and
Depisch (1999) argue that anemophily is present in the species, but unimportant for reproductive success(Wetschnig & Depisch 1999). In Gnetum,
entomophily is mostly likely prevailing and has been suggested for all studied species, i.e., G. gnemon (Kato & Inoue 1994, Kato et al. 1995), G. cuspidatum (Kato et al. 1995), G. luofuense (Corlett 2001). Authors of a recent
pollination study of G. parvifolium argue that the species possesses both
entomophily and anemophily (Gong et al. 2015). The statement is questionable, however, because the pollen grains of the species cannot be dispersed
far enough from the microsporangia by wind, indicating that anemophily is
unimportant in Gnetum, like in Welwitschia mirabilis.
1.2 Investigations of pollination drops in gymnosperms
Pollination drops play an essential role in pollination biology of gymnosperms. They are present in almost all gymnosperms (Strasburger 1872,
Pearson 1929, Ziegler 1959, Takaso 1990), and the main function is analogous to that of the angiosperm stigma, i.e., to act as pollen trappers and pollen carrier to the internal parts of ovules for subsequent fertilization
(Strasburger 1872, Owens et al. 1998, Nepi et al. 2009, Nepi et al. 2012).
Although this main function of pollination drops has long been known, studied closely e.g., in extant and extinct conifers (beginning with Doyle 1945),
details regarding pollen-pollination drop interactions are poorly understood
and the role of pollination drops has probably been underestimated (Coulter
et al. 2012, Little et al. 2014). A pioneer study inventories the biochemistry
of pollination drops in Taxus baccata and Ephedra distachya ssp. helvetica,
and demonstrates that sugars, amino acids, peptides and minerals are present
in pollination drops of the two species (Ziegler 1959). With the advents of
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Hou, C. PhD thesis 2016
promoted mass spectrometry techniques, proteomic studies have gained renewed focus on investigating unknown functions of pollination drops in
gymnosperms (Prior et al. 2013). O’Leary et al. (2004) found arabinogalactan proteins in pollination drops of Taxus × media. Poulis et al. (2005)
launched a proteomic study in the pollination drops of Douglas fir
(Pseudotsuga menziesii) and detected several types of proteins: invertases,
galactosidases, peroxidases and xylosidases. The proteins were considered to
participate in the process of pollen tube elongation and nutrition, but also
probably engage in filtering out external pollen from other species as a result
of heterospecific pollen selection. The initial attempts lead to extensive surveying of the protein profiles in pollination drops of various gymnosperm
species, conifers, Chamaecyparis lawsoniana, Juniperus communis, Juniperus oxycedrus, as well as members of the Gnetales, e.g. Welwitschia mirabilis (Wagner et al. 2007). Additional functions of pollination drops have
gradually been revealed, for example, anti-bacterial and anti-viral, and defense mechanisms (Wagner et al. 2007, Coulter et al. 2012).
1.3 A proteomic study of pollination drops of Ephedra
The achievements of previous proteomic studies in pollination drops encouraged us to survey and deepen the understanding of proteomics in pollination
drops of the Gnetales. The female ovules of the Gnetales, like those of other
gymnosperms, lack physical barriers for protection, e.g., against pathogens,
which can be accidently engulfed from external environment during the
course of pollination. In the Gnetales, only chitinase (a pathogen defense
protein) had been found in pollination drops of Welwitschia mirabilis
(Wagner et al. 2007), but little was known about protein profiles in drops of
Ephedra and Gnetum. In addition, unlike anemophilous conifer species, proteomics might be shifted in entomophilous species of the Gnetales as the
result of co-evolution with pollinators. A proteomic study of pollination
drops in Ephedra (Paper I) was conducted using four species, E. foeminea,
E. minuta, E. likiangensis and E. distachya. Pollination drops of E. foeminea
and E. distachya were collected in Asprovalta, Greece during 2011-2012,
whereas pollination drops from E. minuta and E. likiangensis were obtained
in the botanical greenhouse of Stockholm University, Sweden during 20112012. Daily collections from different specimens of Ephedra were manually
pooled with the total amount up to 50 μl and stored at -20℃. To roughly
assess amounts and types of proteins, partial samples of each species were
applied using gel electrophoresis. To reveal protein profiles of each species,
proteins were liquid-liquid extracted and analyzed using tandem mass spectrometry (see details of methods in Paper I and Paper II). The functions of
detected proteins were predicted by inquiring a set of proteomic databases.
The results of Paper 1 indicate that amounts and types of detected proteins
are considerably reduced in pollination drops of Ephedra compared with
those in conifers. The number of detected proteins vary among the four spe-
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Hou, C. PhD thesis 2016
cies, ranging from eight (E. foeminea) to two (E. minuta), but they all occur
in very low concentrations compared to what has been found in conifers.
Furthermore, degradome proteins account for the majority of the detected
proteins. Such proteins are waste products, and in this case are derived from
intracellular tissues as a result of nucellus degradation. It probably occurs
during the process of pollination and pollen chamber formation. Degradome
proteins have never before been found or carefully studied in pollination
drops of gymnosperms, and the reason is probably that studied conifer species do not produce a pollen chamber by degradation of the nucellus caps
during the course of ovule development. Unlike degradome proteins, secretome proteins (products of apoplastic secretion) are thought to be useful for
plants. For example, thaumatin-like proteins were found (Paper I) and predicted to destroy external pathogens in situ. These proteins have also been
found in pollination drops of conifers (O'Leary et al. 2007, Wagner et al.
2007). In addition, protein profiles of the entomophilous E. foeminea were
compared with those of the other, anemophilous, species (Paper I). The result shows that the pollination drops of E. foeminea are relatively “proteinrich”, probably an evolutionary consequence of plant-insect interactions.
However, the ecological functions of identified proteins are often poorly
understood and will be difficult to test in the future because all detected proteins are present in very low concentration in the pollination drops of Ephedra.
1.4 A new proteomic study
A subsequent proteomic study of pollination drops was conducted on seven
Ephedra species, i.e., E. compacta, E. distachya, E. foeminea, E
.likiangensis, E. minuta, E. monosperma and E. trifurca (Paper II). In the
study, extensive efforts were made to further reveal the differences of protein profiles among different species. Analytical methods were identically
applied in Papers I and II. The results of Paper II support those of Paper I
that the majority of detected proteins were degradome proteins, and that the
number of detected proteins differ dramatically among species, ranging from
twenty (E. foeminea) to six (E. monosperma). It is, however, very surprising
and worrying that the protein profiles shown in Paper I differ considerably
from those in Paper II (see Table 4 in Paper I), even though the same samples were applied in the two studies. It may indicate that the results of these
studies are not repeatable. Furthermore, the new proteomic study (Paper II)
assesses the protein profile of E. monosperma over time. The result shows
that the protein profile changes over time in the pollination samples of the
species. Taken together, these findings question the robustness of the proteomic results in pollination drops of Ephedra as well as in previous proteomic
studies in conifers. Pollination drops of conifers are very small and therefore
routinely pooled to ensure sufficient sample size for the proteomic analyses.
If temporal variation of protein components is present broadly in pollination
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Hou, C. PhD thesis 2016
drops, it is of highest importance not to pool the samples in the field. Even if
not pooled, it may be difficult to obtain results that are comparable across
species if it requires that the samples are taken from ovules of the exact same
developmental stage. New proteomic studies of pollination drops require
robust and more careful methods for collection and analyses of proteins in
gymnosperms.
1.5 Prospective studies
Pollination biology of the Gnetales has gained an increasing attention in the
recent five years, not only in the field of proteomic studies of pollination
drops, but also in fields of gross morphology of reproductive organs, pollen
morphology as well as field observation. Studies of carbohydrates are more
promising for investigations of ovule defense and plant-insect interactions
than are proteomic studies. Pollination drops of the Gnetales, like nectar in
angiosperms, have high sugar concentrations: as much as 25% was measured
in E. distachya (Ziegler 1959), 14-16% in G. cuspidatum and 3-13% in G.
gnemon (Kato et al. 1995), all of which are much higher than the 1.25%
described for Pinus nigra (McWilliam 1958). The sugary pollination drops
of the Gnetales can probably function both to attract and reward pollinators,
at least in Ephedra (Bolinder et al. 2016). Gnetum and Welwitschia might
not only use pollination drops as the attraction, in fact, scent emission has
also been documented in the two genera (Kato et al. 1995, Endress 1996). A
few studies (Bino et al. 1984, Kato et al. 1995) have indicated that extraovular nectar occurs in a few species of the Gnetales disregarding pollination
drops (in two species, Ephedra aphylla, Bino et al. 1984, and Gnetum cuspidatum, Kato et al. 1995). A recent study reveals, however, that pollination
drops secreted from female reproductive units provide the reward for pollinators in Gnetum cuspidatum, not nectar produced by extraovular nectaries
(Jörgensen & Rydin 2015). In addition, Rydin & Bolinder (2015) found a
correlation between pollination drop secretion and phases of the moon in the
entomophilous Ephedra foeminea. The system has most likely evolved in coevolution with the nocturnal pollinators, who utilize moonlight for efficient
navigation. The studied anemophilous species, however, do not possess such
a trait. It is highly unlikely that a similar system is present in Gnetum, which
inhabits tropical rain forests, but the entomophilous Welwitschia, which like
Ephedra exists in an open environment with low precipitation, may potentially have the same system although it has not been studied. Another field of
pollination study, investigation of pollen morphology has also proven helpful to predict the pollination syndromes of the Gnetales. Bolinder et al.
(2015a) found that the pollen wall ultrastructure of E. foeminea is denser
than that of other studied (anemophilous) species. It further lacks ability to
create the aerodynamic microenviroments created around the female cones
of anemophilous species of Ephedra, which use pollination drops to capture
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Hou, C. PhD thesis 2016
air-borne pollen grains. Similar comparisons are made in pollen ultrastructure of Gnetum. The granular layer of the pollen wall in G. africanum is spacious and possesses few but large granules (Tekleva & Krassilov 2009). This
stands in a sharp contrast with the pollen walls of other studied Gnetum species whose granular layers are filled with small granules (Yao et al. 2004,
Tekleva & Krassilov 2009). Together with the presence of unisexual male
cones, these findings indicate that G. africanum could be anemophilous rather than entomophilous (Jörgensen & Rydin 2015, Rydin & Hoorn 2016).
Entomophily is probably the ancestral state in the Gnetales and anemophily
has thus evolved at least once in the Gnetales (Bolinder et al. 2015a, Rydin
& Bolinder 2015, Bolinder et al. 2016), perhaps twice. Additional studies
are, however, needed to better understand the pollination biology of the
Gnetales.
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Hou, C. PhD thesis 2016
Chapter 2 Phylogenetic studies and dating analyses of
the Gnetales
2.1 Phylogenetic studies in Ephedra and taxonomic implications
Early phylogenetic studies of Ephedra, e.g., Rydin et al. (2004), Ickert-Bond
& Wojciechowski (2004), Huang et al. (2005), indicate that it is difficult to
reveal relationships in the genus. The results were poorly resolved and supported, and deep divergences differed among the studies. A subsequent study
(Rydin & Korall 2009) applied an increased sampling of taxa (104 ingroup
accessions and 100 outgroup accessions, which represent the main lineages
of vascular plants) and seven molecular markers (18S, 26S, nrITS, rbcL,
rpl16, rps4, trnS-trnfM). The reconstructed phylogeny of Ephedra places E.
foeminea as sister to all the remaining species. Among the remaining species
several Mediterranean groups are successive sisters to a monophyletic group
that comprises American and Asian species.
However, several problems remain in phylogenetic reconstructions of
Ephedra. Although the early divergences in the Ephedra phylogeny is resolved in Rydin & Korall (2009), it receives low statistic support, and species delimitations, especially the Mediterranean species complex, are uncertain. Besides of interspecific variation, intraspecific variation is investigated
and discussed in population genetic studies of the Old World species (Qin et
al. 2013, Wu et al. 2016) as well as the New World species (Loera et al.
2012, Loera et al. 2015). These studies corroborate that the interspecific
variation can be as large as intraspecific variation, indicating that the boundaries are very vague in closely related species of Ephedra. The statement is
also congruent with results in studies of morphological, anatomical and histological characters in female reproductive units (Rydin et al. 2010), micromorphology of seeds (Ickert-Bond & Rydin 2011) and pollen (Bolinder et al.
2015b). Nevertheless, the reconstruction of phylogenies in Ephedra allows
for tests of traditional classification schemes. Stapf (1889) divided Ephedra
into three sections based on cone morphology, i.e., E. section Pseudobaccatae, E. section Alatae and E. section Ascarca. The classification scheme of
Ephedra, however, did not gain support using molecular data (Ickert-Bond
& Wojciechowski 2004, Huang et al. 2005, Rydin & Korall 2009), indicating that previous classification of the genus is artificial. A new taxonomy of
the genus is urgently needed. In addition, it will be quite interesting to test
the phylogenetic position of several new species proposed in the last decade
using molecular data, for example E. sumlingensis (Sharma & Singh 2015).
2.2 Dating analyses and evolutionary history of the Ephedraceae
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Hou, C. PhD thesis 2016
Node ages in Ephedra, especially the ancestral nodes in the phylogeny, remain uncertain. The crown age of Ephedra has been previously assessed
about 8-32 Ma under a strict clock (Huang & Price 2003, Won & Renner
2003). The results, however, are not convincing because the taxon sampling
of the studies is quite small. A subsequent dating analysis of Ephedra
(Ickert-Bond et al. 2009) applies a broad sampling with 53 accessions as the
ingroup and 41 accessions as the outgroup. The study utilized ten molecular
markers generated in previous phylogenetic studies, and also applied a Welwitschia-like fossil, Cratonia cotyledon (Rydin et al. 2003), to constrain the
age of the Gnetum-Welwitschia clade. The results show that the crown age of
Ephedra is about 30 Ma. The estimated ages of extant species are thus much
younger than discovered Ephedra-like meso- and megafossils from the Early
Cretaceous (Crane 1996, Rydin et al. 2004, Rydin et al. 2006a, Rydin et al.
2006b, Wang & Zheng 2010, Yang et al. 2013, Liu & Wang 2015). These
fossils are not members of the crown group Ephedra but represent extinct
stem lineage(s) (Rydin et al. 2010).
Based on palynological data, the species diversity of Ephedra was shown to
be elevated in the Early Cretaceous but declined in the Late Cretaceous
(Crane & Lidgard 1989, Lidgard & Crane 1990). Cenozoic diversity has
never been assessed in such a summarizing way, but preliminary results indicate substantial fluctuations also during this era (Bolinder and Rydin in
prep.). The fluctuating pattern of biodiversity is probably influenced by both
abiotic and biotic factors. For example, orogenetic movements and gradual
acidification in Pliocene and Pleistocene may have facilitated the speciation
process in Old World clades (Qin et al. 2013) as well as New World clades
(Loera et al. 2012). Also biotic factors, such as varied pollination modes
(Bolinder et al. 2015a, Rydin & Bolinder 2015, Bolinder et al. 2016) and
seed-dispersal vectors (Hollander & Vander Wall 2009, Hollander et al.
2010, Loera et al. 2015) could play indispensable roles in the speciation and
extinction processes of Ephedra.
2.3 Phylogenetic studies in Gnetum and taxonomic implications
The species diversity of Gnetum has been poorly understood although efforts
have been made to delimit species based on gross morphology and geographic distribution. In the most recent monograph of Gnetum (Markgraf
1930), 30 species from South America, Africa and Asia were distinguished.
Markgraf 1930 divided the genus into two sections G. sect. Gnemonomorphi
and G. sect. Cylindrostachys, which roughly corresponds to the taxonomic
framework proposed by Griffith (1859). The arborescent species were considered to constitute an ancestral lineage of Gnetum (Markgraf 1930). The
phylogeny of the genus was first assessed in a modern framework in a series
of studies using molecular data (Won & Renner 2003, 2005b, a, 2006). The
studies revealed that Gnetum was a monophyletic group and indicated, alt-
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Hou, C. PhD thesis 2016
hough with poor support, that the South American lineage diversifies early
in the evolutionary history (Won & Renner 2006). Several questions remained, however, i.e., 1) the deepest splits in the genus 2) the monophyly of
sections and subsections 3) the phylogenetic relationships and monophyly of
closely related species.
In paper IV, phylogenetic analyses were launched using an extensive sampling of 58 accessions (representing 27 species of Gnetum) and 39 outgroup
accessions representing the remaining Gnetales, other seed plants, ferns, and
lycopods. Besides, a subsequent analysis was conducted using an increased
taxon sampling of outgroup species of the Gnetales (16 accessions of Ephedra and one accession of Welwitschia) plus a conifer accession (Calocedrus
sp), to root the trees. Six species that had never been sequenced before was
included, i.e., G. buchholzianum, G. camporum, G. indicum, G. leptostachyum, G. leyboldii and G. montanum. The results reveal that Gnetum section
Erecta Griff. 1859 (section Gnemonomorphi Markgr. 1930) is nonmonophyletic and comprises several clades. South American species (G.
subsect. Araeognemones in Markgr. 1930) form a clade that is sister to the
remaining Gnetum species with strong support. The African species comprise a monophyletic group (G. subsect. Micrognemones in Markgr. 1930),
which separates from a monophyletic Asian group (the “core” Gnetum). The
Asian clade comprises a clade of arborescent species (G. subsect. Eugnemones in Markgr. 1930) and a clade of lianoid species (G. sect. Scandentia
Griff. 1859, G. sect. Cylindrostachys in Markgr. 1930). The Asian lianoid
clade comprises a clade of G. gnemonoides and G. raya and the remaining
clade, which consists of a South East Asia clade and a Chinese clade. The
results reveal that G. subsections Stipitati and Sessiles (Markgr. 1930) are
polyphyletic.
Although paper IV aimed at resolving major relationships in Gnetum, the
results also provide resolution among closely related species in most clades.
There is one exception though; phylogenetic relationships and species delimitations in the Chinese lianoid Gnetum clade are not resolved and specimens
representing several species are nested. The reasons are probably several: 1)
genetic markers applied in Paper IV do not have sufficient sequence variation to resolve the phylogeny in this clade; 2) the sampling of the clade is
restricted and mainly based on cultivated plants; 3) misidentification of the
specimens might occur due to contradictory taxonomic conclusions in previous literatures; 4) other reasons, for example, hybridization, incomplete lineage sorting and horizontal gene transfer.
Hence, new efforts were made to investigate the phylogeny and species delimitations in the Chinese clade of Gnetum (Paper V). The study applies
freshly collected material from tropical and subtropical forests of southern
China (Guangdong, Guangxi, Hainan, Fujian, Yunnan and Hong Kong), as
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Hou, C. PhD thesis 2016
well as herbarium material. To begin with, phylogenetic analyses were conducted based on eleven chloroplast genomes representing G. gnemon, G.
luofuense, G. montanum, G. pendulum and G. parvifolium. Besides, we also
build phylogenies using a concatenation of protein coding sequences. Pairwise comparisons of the five chloroplast genomes were performed to reveal
sequence divergences, and four targeted molecular markers i.e., matK,
rpoC1, rps12 and trnF-trnV were designed and applied for Sanger sequencing. The subsequent phylogenetic reconstruction was conducted using five
molecular markers (nrITS, matK, rpoC1, rps12 and trnF-trnV) and a dense
taxon sampling, including 49 ingroup accessions and 16 outgroup accessions.
The trees were rooted on an African species (G. africanum) according to
results of paper IV. The results of Paper V indicate that Chinese lianoid Gnetum form a monophyletic group, which is congruent with previous phylogenetic studies. The accessions of Gnetum parvifolium form a monophyletic
group, which is sister to the remaining Chinese lianoid species. To further
define species delimitations and make taxonomic conclusions, a morphological study was performed based on 156 herbarium sheets including 40 type
sheets. The material used in Paper V represents all currently accepted Chinese lianoid species, and a few more with uncertain taxonomic status. Subsequent these investigations, new taxonomic conclusions were made based
on molecular as well as morphological data. We conclude that Chinese lianoid Gnetum comprise six species, i.e., G. catasphaericum H.Shao, G. formosum Markgr., G. luofuense C.Y.Cheng, G. montanum Markgr., G. pendulum C.Y.Cheng and G. parvifolium (Warb.) W.C.Cheng. Gnetum giganteum
H.Shao and G. gracilipes C.Y.Cheng are considered synonymous with G.
pendulum. Gnetum hainanense C.Y.Cheng ex L.K.Fu, Y.F.Yu &
M.G.Gilbert is synonymous with G. luofuense. The validity of G. cleistostachyum C.Y.Cheng and G. indicum (Lour.) Merr. is also discussed, both of
which are considered questionable about the name application. In addition,
modified taxonomic keys of Chinese lianoid Gnetum based on male and
female plants are provided in Paper V.
2.4 Dating analyses and evolutionary history of Gnetum
Compared with Ephedra, the evolutionary history of Gnetum is poorly understood. It is partially because the distribution of Gnetum is restricted to
(sub)tropical forests, from which both mega- and microfossils are usually
poorly preserved. So far two megafossils i.e., Khitania (Guo et al. 2009) and
Siphonospermum (Rydin & Friis 2010) are considered to be of possible Gnetum affinity, both discovered from the Early Cretaceous Yixian Formation,
China. Unfortunately, only parts of the plants are preserved and the preservation state is relatively poor, resulting in uncertain phylogenetic positions.
Consequently, the fossils cannot be used as calibration points in dating analyses. In addition, Gnetum-like microfossils, i.e., small, spherical and echinate pollen (Yao et al. 2004, Tekleva & Krassilov 2009, Tekleva 2015) are
20
Hou, C. PhD thesis 2016
largely unknown in the fossil record (Friis et al. 2011). This stands in sharp
contrast with the record of polyplicate microfossils of Ephedra and Welwitschia, which is common in Cretaceous strata (Crane & Lidgard 1989),
and in some Cenozoic localities as well (Han et al. 2016, Bolinder et al. in
progress). The reason may be that the ectexine of Gnetum is thinner than that
of Ephedra and Welwitschia and sensitive to taphonomical activities
(Tekleva 2015). Initial efforts of dating analyses were made in Won &
Renner (2006). The crown age of Gnetum was estimated to the Oligocene
(ca. 26 Ma) and the Asian grown group to the Miocene (ca. 22 Ma), indicating that the vicariance, break up of old continents, exerts no impact on clade
formation of Gnetum. The results of new dating analyses in Paper IV, however, indicate that these estimated ages of early divergences in Gnetum are
underestimated. It is partially because methods of dating analyses have advanced considerably in recent years and stratigraphic information has been
recently updated, but may also be because the sample size applied in previous dating analyses (Won and Renner 2006) is limited.
A new dating analysis was, therefore, conducted in Paper IV, based on the
dataset comprising 20 ingroup accessions as well as 39 outgroup accessions.
To calibrate the results to absolute time, several well-preserved fossils with
updated stratigraphic knowledge were applied. A strict clock was rejected
and a relaxed uncorrelated clock was thus applied, using two alternative tree
priors i.e., Yule process and birth-death process, of which the latter had significantly better fit to the data. The results of Paper IV date the crown age of
Gnetum to the Campanian (Late Cretaceous, ca. 81 Ma, 95% highest posterior density, HPD, 64-98 Ma). The South American clade was dated to the
Paleogene-Neogene boundary (22 Ma, 95% HPD: 8-39 Ma), and the Asian
clade to the Cretaceous-Paleogene (K-Pg) boundary (65 Ma, 95% HPD: 4882 Ma). Discrepancies of the assessed ages are considerable between the
results of Paper IV and the previous study in Won & Renner (2006), and it
intrigues us to reconsider the underlying geographical and environmental
factors that may have influenced evolution in Gnetum. Early divergence
events of Gnetum appear to correlate well with the final separation of South
America and Africa in the mid-Cretaceous, as assessed both from absolute
dates and phylogenetic patterns. Later dispersal, e.g., among continents and
islands of South East Asia, is also apparent from the results in Paper IV, and
is likely to have influenced speciation and geographic distribution of extant
species within a region or continent. The seed dispersal modes of Gnetum
are documented to be zoochory (by birds, civet-cates, fishes and rodents) as
well as hydrochory (ocean trends) (Ridley 1930, Markgraf 1951, Kubitzki
1985, Forget et al. 2002), but knowledge of seed dispersal in the genus is
patchy and incomplete.
Divergence times were also estimated among Chinese lianoid species (Paper
V). Normally distributed age priors (based on results in Paper IV) with en-
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Hou, C. PhD thesis 2016
forced monophyly were used to calibrate the phylogeny to absolute time.
The results reveal that the mean crown age of Chinese lianoid Gnetum is 21
Ma (HPD: 11-34 Ma) corresponding to the Oligocene to Miocene, and diversification events during the latter epoch gave rise to the major clades
within the Chinese lianoid group. The crown age of G. parvifolium is 6 Ma
(late Miocene to Pliocene, HPD: 2-11 Ma). The crown age of G. pendulum
and G. montanum is 2 Ma (HPD: 1-4 Ma), which is slightly younger than the
crown age of G. luofuense (3 Ma, HPD: 1-5 Ma) and the crown age of G.
catasphaericum (4 Ma, HPD: 2-7 Ma). The diversification in the Chinese
lianoid Gnetum during the Neogene coincides with a period of expansion of
tropical and subtropical areas in southern China (Yao et al. 2011).
2.5 Prospective studies
Despite various challenges, plant systematic studies of the Gnetales have
made considerable progresses in the current decade. Among other things,
continuous efforts have been made recently to resolve the early divergences
and estimate divergence ages in Ephedra (Thureborn & Rydin 2015). The
new phylogenetic study applies four nuclear ribosomal (18S, 26S, nrITS,
nrETS) and five chloroplast markers (rbcL, matK, rpl16, rps4 and trnStrnfM) using an increased sampling of Mediterranean specimens. The study
well resolves the relationships of E. foeminea, the other Mediterranean
clades, and the remaining species, and also discusses monophyly and delimitations of Mediterranean species. In addition, ongoing dating analyses apply
new understanding of the fossil pollen records (Bolinder et al. 2015b) to
calibrate the phylogeny within the crown group, resulting in considerably
older ages than previously estimated for the early divergence of Ephedra
(Thureborn & Rydin 2015). Previous hypotheses of long distance dispersal
in Ephedra (Ickert-Bond et al. (2009) may need to be revised. Prospective
studies of Ephedra can focus on the biogeography to inspect the possible
underlying mechanisms. Future phylogenetic studies of Gnetum should focus
on the species delimitation within other (sub)clades of the genus (other than
the Chinese clade), and test the taxonomic conclusions at the species and
subspecies levels. One ongoing project concerns species delimitations in the
paraphyletic assemblage Gnetum section Erecta Griff. 1859 (=section
Gnemonomorphi Markgr. 1930) using extensive sampling from several African and arborescent Asian species. Besides, to well explain the historical
diversification and biogeography of Gnetum, continuous studies are needed.
They could utilize ecological niche models to address the impacts of biotic
and abiotic factors on abundance and distribution of extant species.
Last but not least, speciation and extinction processes of the Gnetales seem
to correlate with global climate change. Between the late Eocene and Oligocene, the global climate became much drier and cooler (Zachos et al. 2001).
During the early phase of Miocene, the global climate became warmer again
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Hou, C. PhD thesis 2016
before it turned into the present cooler environment (Dutton & Barron 1997,
Zachos et al. 2001). The underlying mechanisms that drove the diversification of the Gnetales seem often associated with paleoclimatic fluctuations.
One example from my work is diversification in the Chinese lianoid clade of
Gnetum, which correlates in time with the expansion of tropical forests in
South East Asia (Paper V). However, other factors are conceivably also important. Preliminary results in an ongoing project of Bolinder and Rydin,
which aims to uncover the correlation between dynamic patterns of diversity
in Ephedra and altered paleoclimate, indicate that also biotic factors are likely to have had a strong impact on ephedran evolution.
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Chapter 3. Chloroplast genome investigations in the
Gnetales
3.1 Nuclear genomes of the Gnetales
Nuclear genome sizes among the three gnetalean genera are considerable
different (Leitch & Leitch 2013): Gnetum has the smallest nuclear genome
(1C values = 2.25-3.98 pg), including diploids (2n = 22) and tetraploids (2n
= 44). Welwitschia mirabilis has a larger nuclear genome than Gnetum (1C =
7.2 pg) with the chromosome number 2n = 42 (Leitch & Leitch 2013).
Ephedra possesses the largest and most variable nuclear genomes in the
Gnetales (1C = 8.8-18.22 pg), with the chromosome numbers 2n = 14 and 2n
= 28 (Leitch & Leitch 2013). The nuclear genome size of Ephedra has been
recently investigated using flow cytometry and chromosome count methods
(Ickert-Bond et al. 2015). In the study, the variation of genome size in the
genus was shown to be even greater than previously estimated (1C = 8.09 38.34 pg), which in addition indicates that Ephedra may have the largest
genome size among gymnosperms (i.e., 38.34 pg, 2n = 8x = 56 in Ephedra
antisyphilitica (Ickert-Bond et al. 2015). The study also highlights the predominance of polyploidization in Ephedra and demonstrates that whole genome duplication have facilitated speciation in the genus. The statement is
corroborated by a very recent study of population genetics and ecological
niche modelling among 12 Chinese Ephedra species (Wu et al. 2016). Unfortunately, completely sequenced nuclear genomes of the Gnetales are, so
far, not available.
Compared with nuclear genomes, chloroplast genomes are better understood
in the Gnetales. The size of the chloroplast genome varies among the three
genera according to Wu et al. (2009): Ephedra has the smallest chloroplast
genome in the order (E. equisetina, 109 518 bp) and Welwitschia the largest
(W. mirabilis, 118 919 bp). Gnetum is in between the two (G. parvifolium,
114 914 bp). The chloroplast genomes of the Gnetales are believed to be
more compact and reduced than those of other land plants (Wu et al. 2009),
except for some species of Pinaceae (e.g., Pinus koraiensis, 116 866 bp) (see
Table 1 in McCoy et al. 2008). Intraspecific variation of genomic sizes in the
Gnetales are poorly investigated but may occur as indicated by comparison
of the two chloroplast genomes of W. mirabilis, generated by McCoy et al.
(2008) and Wu et al. (2009) respectively. There is a 807 bp difference in
length between the two. Regarding the structure, the chloroplast genome of
Ephedra is characterized by fewer introns, short intergenic spaces and higher
gene densities compared with the chloroplast genomes of Welwitschia and
Gnetum (Wu et al. 2009). Moreover, the gene content shifts among the chloroplast genomes in the Gnetales. For example, the gene chlL is found in
Ephedra but is deficient in the other two genera (McCoy et al. 2008, Wu et
al. 2009).
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Hou, C. PhD thesis 2016
3.2 Structural differences among chloroplast genomes in the Gnetales
Compared with nuclear genomes, chloroplast genomes are better understood
in the Gnetales. The size of the chloroplast genome varies among the three
genera according to Wu et al. (2009): Ephedra has the smallest chloroplast
genome in the order (E. equisetina, 109 518 bp) and Welwitschia the largest
(W. mirabilis, 118 919 bp). Gnetum is in between the two (G. parvifolium,
114 914 bp). The chloroplast genomes of the Gnetales are believed to be
more compact and reduced than those of other land plants (Wu et al. 2009),
except for some species of Pinaceae (e.g., Pinus koraiensis, 116 866 bp) (see
Table 1 in McCoy et al. 2008). Intraspecific variation of genomic sizes in the
Gnetales are poorly investigated but may occur as indicated by comparison
of the two chloroplast genomes of W. mirabilis, generated by McCoy et al.
(2008) and Wu et al. (2009) respectively. There is a 807 bp difference in
length between the two. Regarding the structure, the chloroplast genome of
Ephedra is characterized by fewer introns, short intergenic spaces and higher
gene densities compared with chloroplast genomes of Welwitschia and Gnetum (Wu et al. 2009). Moreover, the gene content shifts among the chloroplast genomes in the Gnetales. For example, the gene chlL is found in
Ephedra but is deficient in the other two genera (McCoy et al. 2008, Wu et
al. 2009).
3.3 A newly generated chloroplast genome of Ephedra
Despite previous efforts mentioned above, additional chloroplast genomes
are required to reveal potential interspecific variation in terms of genomic
structure and gene content, as well as to assess interspecific sequence variation for prospective phylogenetic studies. In Ephedra, the knowledge of
chloroplast genomes was restricted to a single Chinese species, E. equisetina
(Wu et al. 2009), and little was known about other species of Ephedra. The
early diversifying lineages are especially interesting, not least E. foeminea,
which differs from all other species of Ephedra in several respects. It is sister to the remaining genus (Rydin & Korall 2009, Thureborn & Rydin 2015),
and differs morphologically from other species of Ephedra in that it has bisexual male cones. Besides, in contrast with most species of Ephedra including E. equisetina, which are anemophilous, E. foeminea has been confirmed
to be entomophilous with a distinct ecological status (for details of pollination biology in Ephedra, see chapter 1). Therefore, we investigated the chloroplast genome of this Mediterranean species (Paper III). Plant material of E.
foeminea was collected in Asprovalta, Macedonia, Greece in 2012. Extracted
DNA was sequenced using the Illumina Hiseq 2500 (Illumina Inc., San Diego, CA) and generated raw-reads were assembled into a chloroplast genome
using reference-guided and de novo methods. Gene assessment and annota-
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Hou, C. PhD thesis 2016
tion were largely transferred from the known chloroplast genomes of the
Gnetales but were also assessed.
Our results reveal that the chloroplast genome of E. foeminea possesses a
typical quadripartite structure, 109 584 bp in length (Fig. 2), comprising two
identical inverted repeats (IRs, each 20 739 bp), a large single copy region
(LSC, 60 027 bp) and a small single copy region (SSC, 8 079 bp). The assembled chloroplast genome of E. foeminea is 66 bp longer than that of E.
equisetina. There are 118 genes in the chloroplast genome of E. foeminea,
comprising 73 protein coding genes, 37 transfer RNA genes and 8 ribosomal
RNA genes. The gene density of E. foeminea is 1.076 (genes per 1000 bp),
which is slightly larger than the 1.068 of E. equisetina. The overall GC content is 36.7%, i.e., approximately the same as the 36.6% in E. equisetina.
The LSC, SSC and IRs have GC contents of 34.1%, 27.5%, and 42.1%, respectively. A pairwise comparison of chloroplast genomes of E. foeminea
and E. equisetina (Paper III) reveals 2352 variable sites, of which 1018 are
point mutations and 1334 insertions and deletions (indels). The detected
sequence variation is of great importance for designing highly variable
markers to well resolve species delimitation of Ephedra in prospective studies.
3.4 Chloroplast genome investigation in Chinese lianoid Gnetum
Compared with Ephedra, the chloroplast genomes of Gnetum are better understood; the genomes of several species are available on Genbank (G.
gnemon NC_026301, G. montanum NC_021438, G. parvifolium
NC_011942, G. ula AP014923). However, all these species belong in the
Asian clade. In light of the results from previous studies (Wu et al. 2009,
Hsu et al. 2015), chloroplast genomes of Gnetum have been known to possess a quadripartite structures i.e., LSC, SSC and IRs (see Fig. 3). In paper
V, five chloroplast genomes (G. gnemon, G. luofuense [=G. hainanense], G.
montanum, G. parvifolium and G. pendulum) were assembled using the same
methods as mentioned above. We found that the overall length of the chloroplast genomes vary among species, ranging from 114,405 (G. gnemon) to
115,011bp (G. parvifolium). Also intraspecific variation is present, for example, three specimens of G. parvifolium have been studied and the assembled chloroplast genomes reveal remarkable variation in length (being 114
850 bp, 114 950 bp and 115 011 bp, respectively). Our results show that
overall length of chloroplast genomes cannot act as a species-specific trait in
the Gnetales. The discrepancies of overall length of chloroplast genomes are
probably due to the presence and absence of repeats. Thus in paper V (unpublished results), sequences of the five studied Gnetum species were analyzed in REPuter (Kurtz et al. 2001). The setting of detection followed
Huang et al. (2014): hamming distance 3, minimal repeat size 30 bp and
three types of repeats, i.e., forward, reverse and palindromic repeats were
26
Hou, C. PhD thesis 2016
taken into account. The results reveal that the total numbers of repeats differ
slightly in the chloroplast genomes of the five species (Fig. 4a): 43 repeats
were found in G. parvifolium, 42 repeats in G. pendulum, and 40 each in G.
gnemon, G. luofuense (=G. hainanense) and G. montanum. The lengths of
the detected repeats vary from 35 bp to 153 bp across the five species, of
which direct repeats are prevailing and reverse repeats are fewest (Fig. 4a-d).
Among the detected 205 repeats, the majority (121 repeats) are located in
intergeneric regions, whereas the remaining 84 repeats are situated in protein
coding regions (Table 1). These detected repeats are considered to play a
crucial role of rearranging architectures of the chloroplast genomes (Palmer
1991). Based on previously known chloroplast genomes and those generated
in paper V, gene content and order are considered consistent in Asian Gnetum. The chloroplast genomes consist of 115 genes, of which 66 are protein
coding genes, eight ribosomal RNA (rRNA) genes, and 40 genes transfer
RNA (tRNA) genes. The gene psbA located in the IRb region is a pseudogene with a reduced size compared with the counterpart located in the conjunction of IRa and LSC regions. The 5’end exon of gene rps12 is located in
LSC and two 3’d end exon ends in IRa and IRb, respectively. In addition,
chloroplast genomes of the five studied Chinese Gnetum species were
aligned and variable sites were detected using a SNP/variation finder program (Paper V). The results show that there are 9345 variable sites, comprising 5600 base substitutions (60%) and 3745 insertions and deletions (indels,
40%). The majority of sequence variation was detected in non-protein regions (i.e., rRNA and tRNA genes, introns and intergenic spacers) whereas
the protein regions are relatively conserved. The sequence variation provides
useful information regarding the possibility to design variable molecular
markers for further resolving relationships among closely related species of
Asian Gnetum.
3.5 Prospective studies
The investigation of chloroplast genomes provides indispensable opportunities to deepen the understanding of the evolutionary history of the Gnetales
in terms of genome architecture and sequence variation. Alignments and
comparisons of chloroplast genomes may reveal interspecific variation as
well as intraspecific variation in the Gnetales. Besides, genomic data can
also be applied to investigate population genetics and biogeographic patterns
(Powell et al. 1995, Weising & Gardner 1999, Provan et al. 2001, Petit et al.
2005, Mariac et al. 2014). For example, we assessed single sequence repeats
(SSRs) from the five chloroplast genomes of the Chinese lianoid Gnetum
clade generated in paper V (unpublished results). The detection and identification of SSRs was conducted in Phobos with the selection criteria described
in Huang et al. (2014). In total, 283 single sequence repeats (SSRs) were
detected (Table 2), of which 61 SSRs were detected in G. montanum, 58 in
G. parvifolium, 58 in G. pendulum, 56 in G. luofuense and 50 in G. gnemon.
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Hou, C. PhD thesis 2016
The detected SSRs are promising to be further applied for population genetic
studies in Gnetum. In addition, it would be very interesting to generate the
complete nuclear genomes of the Gnetales in future studies. The nuclear
genomes of Gnetum and Welwitschia are much smaller than the single one
known in conifers, the Norway spruce (Nystedt et al. 2013), and may therefore be easier to assemble de novo. Besides, although the mitochondrial genome of Welwitschia mirabilis has been published very recently (Guo et al.
2016), knowledge is still lacking for Ephedra and Gnetum, and more efforts
can be made in this field.
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Chapter 4 Taxonomy and species delimitation in the
Gnetales
Taxonomy and species delimitation is challenging in Ephedra and Gnetum.
It is partially because gross morphology does not reveal considerable interspecies variation, particularly in Ephedra as well in as some Gnetum lineages, e.g., Chinese lianoid Gnetum. In addition, intraspecific variation is also
present in some species that possess a broad geographic distribution, e.g., E.
distachya (Kakiuchi et al. 2011) and G. parvifolium (Huang et al. 2010),
which further complicates taxonomic choices and assessments of species
delimitation. In addition, vegetative parts of the Gnetales may exhibit plasticity as a response to the environment. This is for example the case for Chinese lianiod Gnetum, as plant height and leave size vary dramatically among
and within species. In addition, recent hybridization clearly occurs between
some wild populations; apparently resulting for example in blurred species
delimitation of anemophilous Ephedra specie (Ickert-Bond & Rydin 2011),
but this has not been well studied so far.
Nevertheless, several issues can be considered to better resolve taxonomy
and species delimitation in the Gnetales. First, alpha-taxonomic work requires an extensive sampling of specimens, preferably reproductive material.
Besides, an extensive number of morphological characters should be investigated in order to avoid misidentification. For example, to resolve the taxonomy and species delimitations of 11 putative species of the Chinese lianoid
Gnetum clade (Paper V), 40 type specimens and more than 126 sheets of
supporting material were reviewed, and 21 morphological characters were
measured and compared (see also paragraph 2.3 above). In addition to gross
morphology, there is an ongoing project on anatomical structure and histology patterns of female reproductive units of Chinese lianoid Gnetum (Hou
and Rydin, unpublished). The study aims to generate anatomical and histological characters, and to find relevant characters useful for wider comparative studies of the entire genus and potentially also extinct relatives. Furthermore, it should be noted that qualitative and quantitative morphological
characters often require new and rigorous studies in the Gnetales. For example, the number of sterile ovules in male spikes of certain Asian Gnetum
species was recently reassessed and showed considerably different numbers
compared with the original descriptions (Jörgensen & Rydin 2015). Last but
not least, to combine morphological and molecular data (as is done in Paper
V) is a powerful strategy to provide better corroborated results on taxonomy
and species delimitation, in particular in groups like the Gnetales where
morphological investigation and species recognition are difficult.
29
Hou, C. PhD thesis 2016
Concluding remark
Biodiversity of the Gnetales has been the focus of my PhD studies, but more
efforts are needed in the future. At first, a robust and complete phylogeny of
the Gnetales is important, but still not fully achieved. To make further progress, informative genetic markers can be designed based on initial investigations of nuclear and chloroplast genomes. An efficient but much more
costly and time-consuming approach would be to analyze entire genomes for
all investigated specimens. However, morphological data is also extremely
important and useful in this old group with ample extinct diversity. With
comprehensive morphological datasets at hand, simultaneous analyses of
living and extinct species can be made. The aim of such future studies can
for example be to understand relationships of fossils to living species, to
assess the evolution of features over time, and to resolve the relationships of
the Gnetales to other seed plants. But it can also be to provide comprehensive assessments of alpha-taxonomy, species diagnoses and identification of
new living and fossil species. Futures studies should also explore poorly
understood topics such as pollination biology, ecology, biogeography and
population genetics. I hope more efforts can be made to continuously explore “the beauty and complexity of the evolutionary process” in the
Gnetales.
30
Hou, C. PhD thesis 2016
Legend
Fig. 1. Gross morphology and biogeographic distribution of the Gnetales (a) mature seeds of Ephedra likiangensis (b) opposite leaves and strobili of
Welwitschia mirabilis (c) Three female spikes of Gnetum gnemon with several developing seeds; (d) stems and leaves of G. gnemon; (e) geographic
distribution of Ephedra (in red), Gnetum (in blue) and Welwitschia (in
green). Photographs by CH.
Fig. 2. A map of the chloroplast genome of Ephedra foeminea (see also Paper III). Genes, which are transcribed at clockwise and counter-clockwise
directions are arranged inside and outside of the genome map, respectively.
Genes that possess various functions are labelled with different colors.
Genes that contain introns are marked with bold black lines. The large single
copy region (LSC), the small single copy region (SSC) and the inverted repeats (IR) are marked inside the genome map. Percentage of GC content is
shown with a threshold line of 50%. The length of the chloroplast genome of
Ephedra foeminea is shown in the middle.
Fig. 3 A map of the chloroplast genomes of G. gnemon, G. luofuense (=G.
hainanense), G. montanum, G. parvifolium and G. pendulum (see also Paper
V). Genes, which are transcribed at clockwise and counter-clockwise directions are arranged inside and outside of the genome map, respectively. Genes
that possess various functions are labelled with different colors. Genes that
contain introns are marked with bold black lines. The large single copy region (LSC), the small single copy region (SSC) and the inverted repeats (IR)
are marked inside the genome map. Percentage of GC content is shown with
a threshold line of 50%. The lengths of the cp genomes of the five Gnetum
species are shown in the middle.
Fig. 4 Repeated sequences detection in the five cp genomes of Gnetum studied in Paper V. (a) number of the three repeat types; (b) frequency of the
direct (forward) repeats by length; (c) frequency of the reverse repeats by
length; (d) frequency of the palindromic repeats by length.
31
Hou, C. PhD thesis 2016
Svensk sammanfattning (Swedish Summary)
Gnetales omfattar tre olika släkten, Ephedra, Gnetum och Welwitschia, som
skiljer sig mycket åt gällande utseende, ekologi och molekylära egenskaper.
Långvariga vetenskapliga debatter kring likheter mellan Gnetales och
blomväxter, samt rent allmänt oklara släktskapsrelationer inom fröväxter,
har inspirerat forskare att fortsätta studera Gnetales evolutionära historia,
decennium efter decennium. De presenterade projekten handlar om flera
tvärvetenskapliga frågor, från proteinfunktion och kloroplastens genom, till
släktskapsförhållanden, morfologi och taxonomi. Här sammanfattas nya rön,
kvarstående problem, och insikter och framtidsutsikter, baserat på resultaten
av mina slutförda och pågående projekt.
Inom släktet Ephedra har medelhavsarten E. foeminea visat sig vara
insektspollinerad, till skillnad från släktet i övrigt, och arten har en viktig
fylogenetisk status som syster till alla övriga Ephedra arter. Därför är det en
intressant nyckelart som studerats mycket på senare år. Här presenteras dess
kloroplast-genom, som jag sekvenserat och jämfört med det enda kloroplastgenom inom släktet som är känt sedan tidigare, det av den vindpollinerade,
asiatiska arten E. equisetina. Genomet är indelat i fyra huvudområden och
omfattar 118 gener och totalt 109 584 kvävebaspar. En parvis jämförelse
mellan E. foeminea och E. equistetina visar på över 2000 variabla
baspositioner, data som kan användas för framtida släktskapsstudier. Jag
studerade också vilka proteiner som finns i pollinationsdropparna hos E.
foeminea och tre vindpollinerade Ephedra-arter, främst i syfte att hitta
försvarsprotein som skyddar reproduktionen. Resultaten visar dock att
protein förekommer i mycket små mängder hos Ephedra jämfört med andra
studerade gymnospermer, och i huvudsak som nedbrytningsprodukter, det
vill säga rester av döende celler. Några tänkbart funktionella proteiner
hittades också, men även dessa förekommer i mycket små mängder. På
grund av problem med analysmetoderna, vilket gör att resultaten eventuellt
kan ifrågasättas, samt den låga förekomsten av proteiner i Ephedras
pollinationsdroppar, valde jag att gå vidare med andra projekt.
Inom Gnetum har jag arbetat med rekonstruktion av släktskapsrelationer och
analyser om när arter och grupper av arter divergerade från varandra.
Resultaten visar att den sydamerikanska linjen separerades från övriga arter i
släktet under yngre krita. Fortsatt diversifiering gav upphov till en afrikansk
grupp och en asiatisk grupp. Den asiatiska gruppen, som omfattar två
trädformerande arter, syster till de återstående liaonida arterna, är från kritapaleogen (K-Pg) gränsen. Mot bakgrund av släktskapen och gruppernas
åldrar kan man anta att uppdelningen av superkontinenten Gondwana har
påverkat basala diversifieringsmönster inom Gnetum. Senare spridning har
32
Hou, C. PhD thesis 2016
också bidragit till den nuvarande utbredningen av Gnetum. Utifrån mina
resultat var det dock tydligt att taxonomi och artavgränsningar är dåligt
underbyggda, och måste studeras vidare för alla undergrupper inom Gnetum.
Jag har påbörjat denna uppgift genom att studera den kinesiska lianoida
Gnetum-gruppen mer på djupet. Elva kloroplast-genom genererades och
jämfördes. Baserat på dessa utformades fyra kloroplast-markörer som
sekvenserades för ytterligare ett stort antal individer. Informationen
användes för att undersöka släktskapsrelationer. Resultaten visar att G.
parvifolium är syster till alla övriga arter i den kinesiska lianoida gruppen.
Ytterligare fem arter bekräftas baserat på både morfologiska och molekylära
data, men flera namn representeras av växtmaterial som inte kan anses vara
egna arter. Modifiering av tidigare nycklar för identifiering av han- och
honplantor presenteras. En dateringsanalys visar att diversifiering i den
kinesiska lianoid Gnetum-kladen skedde främst under neogen, då
miljöförändringar ledde till utbredning av tropiska skogar i det som nu är
södra Kina. Detta bör ha gynnat diversifiering inom Gnetum.
33
Hou, C. PhD thesis 2016
Acknowledgement
I would like to give my deepest gratitude to my supervisor Catarina Rydin
for providing a precious opportunity to be enrolled as a PhD student. Many
thanks for sharing her wisdom with me and your kind encouragement and
patience. Your always-sweet smiles are really impressive, as well as being so
warm-hearted to care of my daily life in Sweden. In addition, I would like to
thank Ove Eriksson and Tanja Slotte for providing comments and suggestions on my PhD thesis.
I will give many thanks to Niklas Wikström, for being the opponent of my
Licentiate dissertation. In addition, it was very interesting to discuss with
you about phylogenetic knowledge and methods during the book exam, and
learning bioinformatics from you using next generation data in my research.
Besides, thanks for helping me editing and providing many suggestive comments on the Chinese lianoid Gnetum paper.
I will give many thanks to Anbar Khodabandeh for helping me in the molecular lab and answering my questions. I have really enjoyed working with you
in the morphological lab, too.
I will also give many thanks to other members of our Gnetales group, at first,
to my roommate Kristina Bolinder, I am so fortunate to share the office with
you, it is very exciting to discuss with you about the research, and our chats
during and after work brought me a lot of pleasure. Thanks to Aelys Humphreys and Olle Thureborn for comments and sequences during our collaboration with the TAXON paper, as well as to Eva Larsen. It has been unforgettable to work with you all in the undergraduate course and having pleasure together during journeys to conferences abroad.
I would like to thank other persons in the former plant systematic division.
Thanks to Per-Ola Karis, I really enjoy the discussion during the book exam
with you and your personal sense of humour is very impressive. Thanks to
Sylvain Razafimandimbison, I really benefit from the book exam of plant
taxonomy and enjoyed your docent presentation of angiosperms evolution.
Thanks to Barbro Axelius for learning undergraduate pedagogy from you,
and other previous members, Birgitta Bremer, Kent Kainulainen, Åsa
Krüger, Frida Stångberg, Annika Bengtson for the “PhD-sitting” in the first
year, and for making my work so pleasurable and convenient.
I would like to thank the persons who gave me a warm-hearted hosting and
assisted me for the field collection and material transportation in southern
China. Many thanks to Jenny Lau, Tang Chin Cheung, Laura Won from the
University of Hong; thanks to Richard Corlett, Bo Pan, Jian-tao Yin, Ma-
34
Hou, C. PhD thesis 2016
reike Roeder, Daniele Cicuzza from Xishuangbanna Tropical Botanical Garden; thanks to Si-jin Zeng, Hai-jun Yang from South China Agricultural
University; thanks to Nan Deng, Sheng-qing Shi, Ze-ping Jiang from Chinese Academy of Forestry; thanks to Zhu-qiu Song, Shi-xiao Luo from
South China Botanical Garden; thanks to En-de Liu from Kunming Institute
of Botany, thanks to Joeri Strijk, Kun-fang Cao from Guangxi University.
I would like to give my sincere gratitude to my parents that assisted me for
the field collection in Hainan and their continuous encouragement and guidance in my daily life. At the end, thanks to treasured Chinese friends in
Stockholm, Xiong-zhuo Tang, Xiao Wang, Kun Wang, Si-mei Yu, Li-min
Ma, Ning Sun, Liqun Yao, Yun-po Zhao and Yan Wang for bringing a lot of
happiness in my daily life.
35
Hou, C. PhD thesis 2016
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44
(a)
(b)
(c)
(d)
Ephedra
Gnetum
Welwitschia
(e)
G. gnemon L. 114,405 bp
G. parvifolium (Warb.) C.Y.Cheng
114, 950 bp
G. luofuense C.Y.Cheng 114,947 bp
G. montanum Markgr. 114,922 bp
G. pendulum C.Y.Cheng 114,917 bp
(a)
(b)
Number of direct repeats
Total number of detected repeats
50
20
18
16
14
12
10
8
6
4
2
0
45
40
35
30
25
20
15
10
5
0
G. gnemon 102
G. parvifolium 108
G. luofuense 110
G. montanum106
30-49bp
7
6
10
8
G. pendulum 103
10
G. gnemon 102
G. parvifolium 108
G. luofuense 110
G. montanum106
G. pendulum 103
50-69
3
18
12
13
15
Palindric
16
2
1
6
6
Reverse
11
0
0
2
4
70-99
1
12
15
8
4
Forward
13
41
39
32
32
100-160
2
5
2
3
3
(c)
(d)
Number of palindromic repeats
Number of reverse repeats
12
10
9
10
8
7
8
6
6
5
4
4
3
2
2
1
0
G. gnemon 102
G. parvifolium 108
G. luofuense 110
G. montanum106
G. pendulum 103
G. gnemon 102
G. parvifolium 108
G. luofuense 110
G. montanum106
G. pendulum 103
30-49
10
2
0
4
5
30-49
9
0
0
2
4
50-69
3
0
0
2
1
50-69
2
0
0
0
0
70-99
3
0
1
0
0
0
Table 1. Repeat type and locations detected in the five chloroplast genomes of Gnetum produced in
Paper V. Total number of repeat detected: 205.
Chloroplast genomes
Gnetum gnemon 102
G. luofuense 107 (=G.
hainanense)
Repeat
Type
Forward
Forward
Forward
Palindromic
Palindromic
Palindromic
Forward
Forward
Palindromic
Reverse
Reverse
Palindromic
Forward
Palindromic
Forward
Reverse
Reverse
Palindromic
Palindromic
Forward
Palindromic
Reverse
Reverse
Palindromic
Forward
Palindromic
Reverse
Reverse
Palindromic
Forward
Palindromic
Palindromic
Reverse
Reverse
Forward
Palindromic
Palindromic
Forward
Reverse
Length
(bp)
138
120
93
87
86
82
63
55
54
51
51
50
50
50
49
49
49
49
48
48
48
47
47
46
46
46
45
45
44
44
44
44
43
43
43
43
43
43
39
Forward
39
62671
Forward
128
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Palindromic
107
92
89
86
86
83
81
81
79
77
73
70
69
Position A
Locus
Position B
Locus
33561
70431
5208
28754
9491
28457
28476
51966
4474
4475
4476
4475
4475
4477
17493
4475
4478
70407
4475
4475
4479
4475
4480
4475
4475
4481
4475
4482
4475
4475
4483
48434
4475
4484
13310
13310
13451
52447
4490
ycf1
psbB/rps12
trnT/trnK
trnN/ycf1
trnH/trnI
trnN/ycf1
trnN/ycf1
ycf2
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbB/rps12
psbD/trnT
psbD/trnT
trnL/rps7
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
trnL/rps7
psbD/trnT
psbD/trnT
ycf2
ycf2
ycf2
ycf2
psbD/trnT
rpl36/rps1
1
33702
70574
5298
37407
56358
37431
28754
52020
4474
4475
4476
4475
4477
4477
48418
4475
4478
70425
4475
4479
4479
4475
4480
4475
4481
4481
4475
4482
4475
4483
4483
48434
4475
4484
13451
52447
52588
52588
4490
ycf1
psbB/rps12
trnT/trnK
psaC/trnN
trnH/trnI
psaC/trnN
trnN/ycf1
ycf2
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbB/rps12
psbD/trnT
psbD/trnT
trnL/rps7
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
trnL/rps7
psbD/trnT
psbD/trnT
ycf2
ycf2
ycf2
ycf2
psbD/trnT
rpl36/rps1
1
70978
psbB/rps12
70999
psbB/rps12
70978
70447
34136
70978
34112
28535
70458
33812
70447
33822
34164
34136
9382
psbB/rps12
psbB/rps12
ycf1
psbB/rps12
ycf1
trnN/ycf1
psbB/rps12
ycf1
psbB/rps12
ycf1
ycf1
ycf1
trnH/trnI
71020
70460
34250
71041
34226
28600
70471
33914
70473
33924
34236
34178
57150
psbB/rps12
psbB/rps12
ycf1
psbB/rps12
ycf1
trnN/ycf1
psbB/rps12
ycf1
psbB/rps12
ycf1
ycf1
ycf1
trnH/trnI
62709
G. montanum 105
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Palindromic
Forward
Forward
Palindromic
Forward
Forward
Forward
Palindromic
Palindromic
Forward
Palindromic
Palindromic
69
66
65
64
64
64
62
62
58
58
58
57
57
55
55
55
53
53
53
51
50
49
47
44
40
40
153
128
107
86
83
81
77
76
69
68
67
66
65
62
58
58
57
57
55
53
53
53
50
50
50
48
47
47
46
46
46
44
44
44
33366
33924
70978
70462
70475
70449
34061
34142
13953
52587
33932
32993
33842
70458
34242
34256
70447
33822
34184
70488
33830
33941
34157
70978
13953
52587
34070
70935
70935
70935
28525
33790
33800
34147
33344
34033
33902
70430
70935
34039
13943
52562
33820
32971
70441
33800
34120
70430
4516
4516
33919
4516
4521
33820
34033
4513
4516
70935
49008
17529
ycf1
ycf1
psbB/rps12
psbB/rps12
psbB/rps12
psbB/rps12
ycf1
ycf1
ycf1
ycf2
ycf2
ycf1
ycf1
psbB/rps12
ycf1
ycf1
psbB/rps12
ycf1
ycf1
psbB/rps12
ycf1
ycf1
ycf1
psbB/rps12
ycf2
ycf2
ycf1
psbB/rps12
psbB/rps12
psbB/rps12
trnN/ycf1
ycf1
ycf1
ycf1
ycf1
ycf1
ycf1
psbB/rps12
psbB/rps12
ycf1
ycf2
ycf2
ycf1
ycf1
psbB/rps12
ycf1
ycf1
psbB/rps12
psbD/trnT
psbD/trnT
ycf1
psbD/trnT
psbD/trnT
ycf1
ycf1
psbD/trnT
psbD/trnT
psbB/rps12
trnL/rps7
ycf2
33432
34344
71062
70488
70488
70488
34091
34298
13971
52605
34352
33050
33944
70497
34284
34298
70499
34344
34298
70501
34352
34361
34313
71083
13989
52623
34184
70956
70977
70998
28590
33892
33902
34261
33410
34177
34322
70443
71019
34069
13961
52580
33922
33028
70454
34322
34276
70456
4516
4518
34339
4516
4525
34342
34291
4525
4516
71040
49008
17529
ycf1
ycf1
psbB/rps12
psbB/rps12
psbB/rps12
psbB/rps12
ycf1
ycf1
ycf1
ycf2
ycf2
ycf1
ycf1
psbB/rps12
ycf1
ycf1
psbB/rps12
ycf1
ycf1
psbB/rps12
ycf1
ycf1
ycf1
psbB/rps12
ycf2
ycf2
ycf1
psbB/rps12
psbB/rps12
psbB/rps12
trnN/ycf1
ycf1
ycf1
ycf1
ycf1
ycf1
ycf1
psbB/rps12
psbB/rps12
ycf1
ycf2
ycf2
ycf1
ycf1
psbB/rps12
ycf1
ycf1
psbB/rps12
psbD/trnT
psbD/trnT
ycf1
psbD/trnT
psbD/trnT
ycf1
ycf1
psbD/trnT
psbD/trnT
psbB/rps12
trnL/rps7
ycf2
G. parvifolium 108
G. pendulum 103
Forward
Forward
Forward
Forward
Reverse
Reverse
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Palindromic
Forward
Forward
Palindromic
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
44
40
40
39
37
37
127
120
111
107
102
97
94
94
88
84
81
81
80
80
77
72
71
69
69
68
68
66
65
64
63
63
62
59
58
58
57
56
56
55
55
53
52
51
50
50
50
48
46
153
128
107
86
83
81
77
69
68
67
66
17529
13943
52562
70432
4521
4522
4886
70382
28934
70382
5213
70379
70395
70408
70949
70379
70395
70408
70949
34227
70934
70965
70379
28656
33335
70395
70408
34154
34036
70965
13930
52546
70949
34053
70379
34004
32962
33903
34006
70395
70408
34167
34064
33960
4523
34012
34227
4523
4527
34072
70937
70937
70937
28527
33792
33802
33346
34035
33904
70432
ycf2
ycf2
ycf2
psbB/rps12
psbD/trnT
psbD/trnT
trnT/trnK
psbB/rps12
ycf1
psbB/rps12
trnT/trnK
psbB/rps12
psbB/rps12
psbB/rps12
psbB/rps12
psbB/rps12
psbB/rps12
psbB/rps12
psbB/rps12
ycf1
psbB/rps12
psbB/rps12
psbB/rps12
trnN/ycf1
ycf1
psbB/rps12
psbB/rps12
ycf1
ycf1
psbB/rps12
ycf2
ycf2
psbB/rps12
ycf1
psbB/rps12
ycf1
ycf1
ycf1
ycf1
psbB/rps12
psbB/rps12
ycf1
ycf1
ycf1
psbD/trnT
ycf1
ycf1
psbD/trnT
psbD/trnT
ycf1
psbB/rps12
psbB/rps12
psbB/rps12
trnN/ycf1
ycf1
ycf1
ycf1
ycf1
ycf1
psbB/rps12
49008
13979
52598
70471
4521
4522
5113
70395
29045
70408
5334
70418
70421
70421
70985
70431
70434
70434
70967
34257
70952
71001
70444
28833
33401
70447
70447
34184
34126
70983
13957
52573
71003
34143
70457
34034
33019
33960
34126
70460
70460
34197
34094
34317
4523
34042
34287
4523
4529
34186
70958
70979
71000
28592
33894
33904
33412
34179
34324
70445
ycf2
ycf2
ycf2
psbB/rps12
psbD/trnT
psbD/trnT
trnT/trnK
psbB/rps12
ycf1
psbB/rps12
trnT/trnK
psbB/rps12
psbB/rps12
psbB/rps12
psbB/rps12
psbB/rps12
psbB/rps12
psbB/rps12
psbB/rps12
ycf1
psbB/rps12
psbB/rps12
psbB/rps12
trnN/ycf1
ycf1
psbB/rps12
psbB/rps12
ycf1
ycf1
psbB/rps12
ycf2
ycf2
psbB/rps12
ycf1
psbB/rps12
ycf1
ycf2
ycf1
ycf1
psbB/rps12
psbB/rps12
ycf1
ycf1
ycf1
psbD/trnT
ycf1
ycf1
psbD/trnT
psbD/trnT
ycf1
psbB/rps12
psbB/rps12
psbB/rps12
trnN/ycf1
ycf1
ycf1
ycf1
ycf1
ycf1
psbB/rps12
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Forward
Palindromic
Forward
Forward
Palindromic
Forward
Forward
Palindromic
Palindromic
Forward
Forward
Palindromic
Forward
Palindromic
Forward
Forward
Forward
Forward
Forward
Reverse
Reverse
Reverse
Reverse
65
62
58
58
57
57
55
53
53
50
50
50
48
47
47
46
46
46
44
44
44
44
42
40
40
39
38
37
37
35
35
70937
34041
13945
52564
32973
33822
70443
70432
33802
4518
4518
33921
4518
4523
33822
4515
4518
34035
70937
17531
17531
49010
70443
13945
52564
70434
70460
4523
4524
4523
4526
psbB/rps12
ycf1
ycf2
ycf2
ycf1
ycf1
psbB/rps12
psbB/rps12
ycf1
psbD/trnT
psbD/trnT
ycf1
psbD/trnT
psbD/trnT
ycf1
psbD/trnT
psbD/trnT
ycf1
psbB/rps12
trnL/rps7
trnL/rps7
trnL/rps7
psbB/rps12
ycf2
ycf2
psbB/rps12
psbB/rps12
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
71021
34071
13963
52582
33030
33924
70456
70458
34324
4518
4520
34341
4518
4527
34344
4527
4518
34293
71042
17531
49010
49010
70469
13981
52600
70473
70473
4523
4524
4523
4526
psbB/rps12
ycf1
ycf2
ycf2
ycf1
ycf1
psbB/rps12
psbB/rps12
ycf1
psbD/trnT
psbD/trnT
ycf1
psbD/trnT
psbD/trnT
ycf1
psbD/trnT
psbD/trnT
ycf1
psbB/rps12
trnL/rps7
trnL/rps7
trnL/rps7
psbB/rps12
ycf2
ycf2
psbB/rps12
psbB/rps12
psbD/trnT
psbD/trnT
psbD/trnT
psbD/trnT
Table 2. A list of sequence repeats (SSRs) detected in the five chloroplast genomes of Gnetum
produced in Paper V. In total, 283 repeats.
Gnetum cp genomes
Repeat unit
G. gnemon var. brunonianum 102
A
Number
of repeat
units
11
A
12
7
A
A
13
14
1
5
A
AT
AAAG
15
6
8
9
10
17
4
1
1
1
1
1
1
2
AAATC
AAAAAG
AAAAAG
3
3
3
1
1
2
AATTCG
3
3
AAAAAG
AAAAAC
3
3
1
1
A
10
21
G. luofuense 110 (=G. hainanense)
Number
of SSRs
Position
A
Position
B
Region
Location
Locus
20
1,362
4,537
4,870
5,112
5,260
5,350
7,901
10,543
33,126
33,309
34,048
35,720
55,392
58,539
62,082
67,910
70,502
70,645
92,023
97,417
10,128
34,469
55,806
62,204
62,452
94,237
107,521
87,288
5,129
70,555
75,735
107,885
109,099
5,634
85,519
4,476
5,045
72,999
4,493
16,822
49,102
4,273
70,564
16,115
49,807
13,874
52,000
52,054
70,564
29,498
1,372
4,547
4,880
5,122
5,270
5,360
7,911
10,553
33,136
33,319
34,058
35,730
55,402
58,549
62,092
67,920
70,512
70,655
92,033
97,427
10,139
34,480
55,817
62,215
62,463
94,248
107,532
87,300
5,142
70,568
75,748
107,898
109,112
5,648
85,530
4,491
5,062
73,018
4,527
16,837
49,117
4,291
70,584
16,132
49,824
13,891
52,017
52,071
70,584
29,520
LSC
LSC
LSC
LSC
LSC
LSC
LSC
IRb
SSR
SSR
SSR
SSR
IRa
LSC
LSC
LSC
LSC
LSC
LSC
LSC
IRb
SSR
IRa
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
IRb
IRa
LSC
LSC
IRb
IRa
IRb
IRa
IRa
LSC
SSR
IGS
IGS
IGS
IGS
IGS
IGS
IGS
CDS
CDS
CDS
CDS
IGS
CDS
CDS
CDS
IGS
IGS
IGS
intron
CDS
CDS
CDS
CDS
IGS
IGS
IGS
IGC
IGS
IGS
IGS
IGS
intron
intron
intron
IGS
IGS
IGS
IGS
IGS
IGS
IGS
IGS
IGS
CDS
CDS
CDS
CDS
CDS
IGS
CDS
psbZ/trnS
psbD/trnT
trnK/trnT
trnK/trnT
trnK/trnT
trnK/trnT
psbA/trnK
ycf2
ycf1
ycf1
ycf1
trnP/ycf1
ycf2
rps19
rps8
psbB/psbT
psbB/rps12
psbB/rps12
atpF
rpoC2
ycf2
ycf1
ycf2
infA/rps8
infA/rpl36
atpl/rps2
trnS/ycf3
psbL/trnQ
trnK/trnT
psbB/rps12
petL/psbE
ycf3
ycf3
trnK
trnF/trnL
psbD/trnT
trnK/trnT
rpl20/rps12
psbD/trnT
trnL/ycf2
trnL/ycf2
psbD/trnT
psbB/rps12
ycf2
ycf2
ycf2
ycf2
ycf2
psbB/rps12
ycf1
1,574
4,444
4,574
6,796
6,920
29,058
30,597
31,572
32,737
33,749
35,320
60,624
61,861
70,436
70,861
87,582
97,824
104,426
105,421
1,583
4,453
4,583
6,805
6,929
29,067
30,606
31,581
32,746
33,758
35,329
60,633
61,870
70,445
70,870
87,591
97,833
104,435
105,430
LSC
LSC
LSC
LSC
LSC
SSR
SSR
SSR
SSR
SSR
SSR
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
IGS
IGS
IGS
CDS
CDS
IGS
CDS
CDS
CDS
CDS
CDS
intron
IGS
IGS
IGS
IGS
CDS
CDS
CDS
psbC/trnS
psbD/trnT
psbD/trnT
matK
matK
trnN/ycf1
ycf1
ycf1
ycf1
ycf1
ycf1
rpl16
rpl14/rpl16
psbB/rps12
psbB/rps12
trnE/trnQ
rpoC2
rpoB
rpoB
G. parvifolium 108
A
11
10
A
12
5
A
13
3
A
14
2
A
15
2
C
10
2
C
C
11
13
1
2
AT
AAG
6
9
16
5
1
1
1
2
AAAAAG
3
3
A
10
27
A
11
9
A
12
4
108,418
108,435
5,449
31,158
60,746
60,841
62,304
62,680
66,937
67,396
73,588
80,502
10,197
35,092
56,396
76,348
91,977
36,281
71,224
108,085
72,855
105,657
5,688
109,649
84,708
86,470
84,685
66,924
86,174
86,145
4,538
5,070
16,149
50,453
4,287
16,200
50,400
108,427
108,444
5,459
31,168
60,756
60,851
62,314
62,690
66,947
67,406
73,598
80,512
10,208
35,103
56,407
76,359
91,988
36,293
71,236
108,097
72,868
105,670
5,702
109,663
84,717
86,479
84,695
66,936
86,186
86,156
4,555
5,101
16,164
50,468
4,308
16,217
50,417
LSC
LSC
LSC
SSR
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
IRb
SSR
IRa
LSC
LSC
SSR
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
IRb
IRa
LSC
IRb
IRa
intron
intron
IGS
CDS
intron
intron
IGS
CDS
intron
intron
IGS
IGS
CDS
CDS
CDS
IGS
IGS
IGS
IGS
IGS
IGS
CDS
intron
intron
IGS
intron
IGS
intron
IGS
IGS
IGS
IGS
CDS
CDS
IGS
CDS
CDS
ycf3
ycf3
trnK/trnT
ycf1
rpl16
rpl16
rps8/rps14
rps8
petB
petB
rpl20/rps12
psaI/trnR
ycf2
ycf1
ycf2
petE/petL
atpA/atpF
trnP/ycf1
psbB/rps12
trnS/ycf3
psbB/rps12
rpoB
trnK
ycf3
atpE/trnfM
trnL
atpE/trnfM
petB
trnF/trnL
trnF/trnL
psbD/trnT
trnK/trnT
ycf2
ycf2
psbD/trnT
ycf2
ycf2
4,451
6,789
6,913
17,508
30,545
31,544
32,709
33,718
34,628
35,277
36,257
49,050
60,580
62,254
62,750
62,994
65,727
70,824
76,017
84,668
86,450
92,615
97,806
104,408
105,406
107,234
108,051
4,248
4,586
62,630
66,875
71,171
74,704
80,483
87,554
109,622
10,190
4,460
6,798
6,922
17,517
30,554
31,553
32,718
33,727
34,637
35,286
36,266
49,059
60,589
62,263
62,759
63,003
65,736
70,833
76,026
84,677
86,459
92,624
97,815
104,417
105,415
107,243
108,060
4,258
4,596
62,640
66,885
71,181
74,714
80,493
87,564
109,632
10,201
LSC
LSC
LSC
IRb
SSC
SSC
SSC
SSC
SSC
SSC
SSC
IRa
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
IRb
IGS
CDS
CDS
CDS
CDS
CDS
CDS
CDS
CDS
CDS
IGS
IGS
intron
IGS
IGS
IGS
intron
IGS
IGS
IGS
intron
intron
CDS
CDS
CDS
IGS
IGS
IGS
IGS
CDS
intron
IGS
IGS
IGS
IGS
intron
CDS
psbD/trnT
matK
matK
rps7/trnL
ycf1
ycf1
ycf1
ycf1
ycf1
ycf1
trnP/ycf1
rps7/trnL
rpl16
rpl14/rps8
infA/rps8
infA/rpl36
petD
psbB/rps12
petG/petL
atpE/trnfM
trnL
atpF
rpoC2
rpoB
rpoB
psbM/rps4
trnS/ycf3
psbD/trnT
psbD/trnT
rps8
petB
psbB/rps12
psaJ/rpl33
psal/trnR
trnE/trnQ
ycf3
ycf2
G. montanum 105
A
13
2
A
A
A
A
A
C
C
AT
AAG
AGA
ATA
ATT
AAAAAG
14
16
18
19
26
11
12
13
20
5
5
5
5
3
1
1
1
1
1
1
1
1
1
1
1
1
1
2
AAAAGA
3
1
A
10
22
A
11
10
A
12
8
A
13
6
A
14
2
A
C
C
15
10
11
1
1
2
C
13
1
35,049
56,353
76,330
72,822
91,957
105,642
1,363
5,678
71,145
72,663
66,875
86,143
5,074
4,528
50,422
16,130
14,017
52,534
16,181
50,369
4,286
35,060
56,364
76,341
72,834
91,969
105,655
1,378
5,695
71,163
72,688
66,885
86,154
5,100
4,567
50,437
16,145
14,033
52,550
16,198
50,386
4,307
SSC
IRa
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
IRa
IRb
IRb
IRa
IRb
IRa
LSC
CDS
CDS
IGS
IGS
IGS
CDS
IGS
intron
IGS
IGS
intron
IGS
IGS
IGS
CDS
CDS
CDS
CDS
CDS
CDS
IGS
ycf1
ycf2
petL/psbE
psbB/rps12
atpA/atpF
rpoB
psbZ/trnS
trnK
psbB/rps12
psbB/rps12
petB
trnF/trnL
trnK/trnT
psbD/trnT
ycf2
ycf2
ycf2
ycf2
ycf2
ycf2
psbD/trnT
1,574
4,444
4,559
5,136
6,790
6,914
30,575
31,550
32,715
33,727
35,298
36,259
60,818
66,919
67,313
67,378
82,488
87,548
97,790
104,392
105,387
107,235
4,580
29,035
60,599
61,837
62,655
73,545
73,968
76,309
84,662
108,385
1,370
10,191
31,135
35,070
56,371
60,722
91,943
108,403
70,416
71,181
80,462
86,139
108,052
109,619
72,812
105,623
5,682
84,673
84,648
86,435
66,906
1,583
4,453
4,568
5,145
6,799
6,923
30,584
31,559
32,724
33,736
35,307
36,268
60,827
66,928
67,322
67,387
82,497
87,557
97,799
104,401
105,396
107,244
4,590
29,045
60,609
61,847
62,665
73,555
73,978
76,319
84,672
108,395
1,381
10,202
31,146
35,081
56,382
60,733
91,954
108,414
70,428
71,193
80,474
86,151
108,064
109,631
72,825
105,636
5,696
84,682
84,658
86,445
66,918
LSC
LSC
LSC
LSC
LSC
LSC
SSR
SSR
SSR
SSR
SSR
SSR
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
SSR
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
IRb
SSR
SSR
IRa
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
IGS
IGS
IGS
IGS
CDS
CDS
CDS
CDS
CDS
CDS
CDS
IGS
intron
CDS
CDS
CDS
IGS
IGS
CDS
CDS
CDS
IGS
IGS
IGS
intron
IGS
CDS
IGS
IGS
IGS
IGS
intron
IGS
CDS
CDS
CDS
CDS
intron
IGS
intron
IGS
IGS
IGS
IGS
IGS
intron
IGS
CDS
intron
IGS
IGS
intron
CDS
psbC/trnS
psbD/trnT
psbD/trnT
trnK/trnT
matK
matK
ycf1
ycf1
ycf1
ycf1
ycf1
trnP/ycf1
rpl16
petB
petB
petB
atpB/rbcL
trnE/trnQ
rpoC2
rpoB
rpoB
psbM/rps4
psbD/trnT
trnN/ycf1
rpl16
rpl14/rpl16
rps8
rpl20/rps12
rpl20/rps18
petE/petL
atpE/trnfM
ycf3
psbZ/trnS
ycf2
ycf1
ycf1
ycf2
rpl16
atpA/atpF
ycf3
psbB/rps12
psbB/rps12
psal/trnR
trnF/trnL
trnS/ycf3
ycf3
psbB/rps12
rpoB
trnK
atpE/trnfM
atpE/trnfM
trnL
petB
AT
G. pendulum 103
AAG
6
10
19
5
1
1
1
2
AAAAAG
3
3
A
10
22
A
11
9
A
12
9
A
13
4
A
14
2
A
C
C
15
11
13
1
1
2
AT
AAG
6
10
19
5
1
1
1
2
AAAAAG
3
3
86,110
5,077
4,522
16,139
50,428
4,287
16,190
50,375
86,121
5,096
4,559
16,154
50,443
4,308
16,207
50,392
LSC
LSC
LSC
IRb
IRa
LSC
IRb
IRa
IGS
IGS
IGS
CDS
CDS
IGS
CDS
CDS
trnF/trnL
trnK/trnT
psbD/trnT
ycf2
ycf2
psbD/trnT
ycf2
ycf2
1,575
4,561
5,138
6,792
30,577
31,552
32,717
33,729
35,300
36,261
60,820
62,283
66,922
67,316
67,381
82,490
84,664
87,546
97,788
104,390
105,385
108,381
4,445
4,582
29,037
60,601
61,839
62,658
73,547
73,970
76,311
10,193
31,137
35,072
56,373
60,724
70,419
91,941
108,049
108,398
1,370
71,183
80,464
109,614
72,814
105,621
5,684
84,650
66,909
86,139
86,110
5,079
4,524
16,141
50,430
4,288
16,192
50,377
1,584
4,570
5,147
6,801
30,586
31,561
32,726
33,738
35,309
36,270
60,829
62,292
66,931
67,325
67,390
82,499
84,673
87,555
97,797
104,399
105,394
108,390
4,455
4,592
29,047
60,611
61,849
62,668
73,557
73,980
76,321
10,204
31,148
35,083
56,384
60,735
70,430
91,952
108,060
108,409
1,382
71,195
80,476
109,626
72,827
105,634
5,698
84,660
66,921
86,151
86,121
5,098
4,561
16,156
50,445
4,309
16,209
50,394
LSC
LSC
LSC
LSC
SSC
SSC
SSC
SSC
SSC
SSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
SSC
LSC
LSC
LSC
LSC
LSC
LSC
IRb
SSC
SSC
IRa
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
LSC
IRb
IRa
LSC
IRb
IRa
IGS
IGS
IGS
CDS
CDS
CDS
CDS
CDS
CDS
IGS
intron
IGS
intron
intron
intron
IGS
IGS
IGS
CDS
CDS
CDS
intron
IGS
IGS
IGS
intron
IGS
CDS
IGS
IGS
IGS
CDS
CDS
CDS
CDS
intron
IGS
IGS
IGS
intron
IGS
IGS
IGS
intron
IGS
CDS
intron
IGS
intron
IGS
IGS
IGS
IGS
CDS
CDS
IGS
CDS
CDS
psbC/trnS
psbD/trnT
trnK/trnT
matK
ycf1
ycf1
ycf1
ycf1
ycf1
trnP/ycf1
rpl16
rpl14/rps8
petB
petB
petB
atpB/rbcL
atpE/trnfM
trnE/trnQ
rpoC2
rpoB
rpoB
ycf3
psbD/trnT
psbD/trnT
trnN/ycf1
rpl16
rpl14/rpl16
rps8
rpl20/rps12
rpl20/rps18
petE/petL
ycf2
ycf1
ycf1
ycf2
rpl16
psbB/rps12
atpA/atpF
trnS/ycf3
ycf3
psbZ/trnS
psbB/rps12
psaL/trnR
ycf3
psbB/rps12
rpoB
trnK
atpE/trnfM
petB
trnF/trnL
trnF/trnL
trnK/trnT
psbD/trnT
ycf2
ycf2
psbD/trnT
ycf2
ycf2