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Biological Journal of the Linnean Society, 2015, ••, ••–••. With 4 figures
First steps in studying the origins of secondary
woodiness in Begonia (Begoniaceae): combining
anatomy, phylogenetics, and stem transcriptomics
CATHERINE KIDNER1,2, ANDREW GROOVER3,4, DANIEL C. THOMAS5,6,7,
KATIE EMELIANOVA1,2, CLAUDIA SOLIZ-GAMBOA5 and FREDERIC LENS5*
1
Royal Botanic Gardens, Edinburgh, UK
Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh, UK
3
US Forest Service, Pacific Southwest Research Station, Davis, CA, USA
4
Department of Plant Biology, UC Davis, Davis, CA, USA
5
Naturalis Biodiversity Center, Leiden University, P.O. Box 9517, 2300RA Leiden, The Netherlands
6
School of Biological Sciences, University of Hong Kong, Hong Kong
7
Research and Conservation Branch, Singapore Botanic Gardens, Singapore
2
Received 14 November 2014; revised 19 December 2014; accepted for publication 19 December 2014
Since Darwin’s observation that secondary woodiness is common on islands, the evolution of woody plants from
herbaceous ancestors has been documented in numerous angiosperm groups. However, the evolutionary processes
that give rise to this phenomenon are poorly understood. To begin addressing this we have used a range of
approaches to study the anatomical and genetic changes associated with the evolution and development of
secondary woodiness in a tractable group. Begonia is a large, mainly herbaceous, pantropical genus that shows
multiple shifts towards secondarily woody species inhabiting mainly tropical montane areas throughout the world.
Molecular phylogenies, including only a sample of the woody species in Begonia, indicated at least eight instances
of a herbaceous–woody transition within the genus. Wood anatomical observations of the five woody species studied
revealed protracted juvenilism that further support the secondary derived origin of wood within Begonia. To
identify potential genes involved in shifts towards secondary woodiness, stem transcriptomes of wood development
in B. burbidgei were analysed and compared with available transcriptome datasets for the non-woody B. venustra,
B. conchifolia, and Arabidopsis, and with transcriptome datasets for wood development in Populus. Results
identified a number of potential regulatory genes as well as variation in expression of key biosynthetic enzymes.
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••.
ADDITIONAL KEYWORDS: molecular wood pathway – wood anatomy.
INTRODUCTION
Charles Darwin was one of the first scientists to
mention the occurrence of woodiness in some island
species belonging to otherwise herbaceous plant
groups (Darwin, 1859). He identified ‘insular woodiness’ as a derived or secondary state, which evolved
after ancestral herbaceous progenitor populations
reached the islands. For many of these woody island
*Corresponding author. E-mail: frederic.lens@naturalis.nl
species this shift in habit was later confirmed by
molecular phylogenetics, and anatomical studies
revealed that the wood in such species was characterized
by
protracted
juvenilism
(Carlquist,
1974,
2012;
Givnish,
1998;
Whittaker
&
Fernández-Palacios, 2007; Lens et al., 2009, 2013a).
There is growing evidence that evolutionary shifts
towards the woody habit occur convergently within
families, on single islands, but also in continental
areas with at least some consecutive dry months per
year (Carlquist, 1974; Lens et al., 2013a; F. Lens, in
prep).
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••
1
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C. KIDNER ET AL.
Distinguishing between herbaceousness and
woodiness is not always easy due to the continuous
variation between both growth forms. Plants from
most dicotyledonous angiosperm lineages – including
herbaceous groups – retain the ability to produce a
limited amount of wood in the basal parts of their
stems (Dulin & Kirchoff, 2010; Lens et al., 2012a;
Lens, Smets & Melzer, 2012b). The limited wood
formation in these herbaceous lineages may contribute biomechanical strength to the stem, and could
explain the retention of wood forming genes in herbaceous groups. In addition, pleiotropic activity of
cambium genes in shoot apical meristem function
may also contribute to their retention (Robischon
et al., 2012; Zhang et al., 2014). The potential
genetic simplicity of a habit switch from
herbaceousness back to the ancestral woody habit in
angiosperms is supported in Arabidopsis by the
2-gene model of Melzer et al. (2008). In this case,
loss of function in two MADS box transcription
factors enable the herbaceous wild-type to develop
into a woody shrub.
Secondary woodiness appears to be correlated with
extreme conditions in at least some groups. Drier
habitats and competition have been suggested as
drivers (Lens et al., 2013a, b) and different drivers
appear to operate even within the same clade (Lens
et al., 2009). It has also been suggested that woody
growth is a response to more favourable climatic
environments (especially lack of frost; Carlquist,
1974) or to promote outcrossing (Böhle, Hilger &
Martin, 1996), and that flexible developmental genetics allowing lineages to switch between herbaceous
and woody forms may have contributed to the evolutionary success of angiosperms (Bond, 1989). A better
understanding of the evolution of secondary woodiness will help us assess the advantages that the
woody habit confers.
Genomic and molecular tools are becoming available to study the environmental drivers and proximal genetic causes of this derived wood formation in
otherwise herbaceous lineages in a wide array of
angiosperms. In particular, transcriptome sequencing can reveal useful information about biosynthetic
pathways, regulatory pathways and targets of
selection even in non-model species (e.g. Wu et al.,
2014; Xu et al., 2014; Zhu et al., 2014). Comparative
transcriptomics allows us to identify potential candidate genes regulating or driving the production of
wood in secondarily woody species. This descriptive
approach can only provide a ‘snap shot’ of the
situation, but it is a valuable first step in identifying the developmental pathways and changes
involved.
The pantropical genus Begonia provides an ideal
group to investigate the evolution of secondary woodi-
ness. Begonia contains at least 1550 species comprising mostly herbaceous species, but also a number of
species that grow as woody shrubs (Doorenbos, Sosef
& de Wilde, 1998). The derived nature of Begonia
wood was described by Carlquist (1985) who studied
the wood anatomy of four woody South American
species and observed characters demonstrating protracted juvenilism, namely the presence of tall
multiseriate rays with mainly upright ray cells and
wide scalariform intervessel pitting. The estimated
number of woody begonias is difficult to assess
because of incomplete collection efforts in some
regions combined with the lack of thorough regional
flora treatments in these areas, but up to ca. 50
woody species might be possible. In published
phylogenies, only three woody African, one woody
Asian, and six woody neotropical Begonia species
are included, and these woody species represent at
least seven independent shifts towards secondary
woodiness, suggesting this is a fairly labile trait
within Begonia (Plana, 2003; Forrest, Hughes &
Hollingsworth, 2005; Goodall-Copestake et al., 2010;
Thomas et al., 2011; Moonlight et al., 2015).
Most of the woody Begonia species are native to
wet tropical mountain peaks of SE Asia (Beaman,
Anderson & Beaman, 2001; Hughes & Pullan, 2007),
Andean South American (Mark Tebbitt, pers. comm.),
moist East African montane forests (Reitsma, 1984;
Plana, Sands & Beentje, 2006) or moist tropical West
African islands (São Tomé and Principe). Consequently, for a majority of the woody begonias,
drought stress is definitely not involved in wood formation, although this has been suggested by recent
experimental results based on embolism resistance
measures in stems of herbaceous and woody
Arabidopsis thaliana individuals (Lens et al., 2013b).
Nevertheless, for some other woody begonias drought
stress is an issue, such as some of the South East
Asian woody begonias native to dry coralline limestone hills with low water-holding capacity (Kiew,
1998, 2001), and some neotropical woody species
inhabiting dry habitats in the Andes. It even appears
that the woody Andean species in these drier areas
are woodier than the ones growing in more mesic
Andean habitats, such as the narrow Peruvian
endemic B. gorgonea exhibiting strikingly woody rhizomes in xeric environments (Mark Tebbitt, pers.
comm.).
The occurrence of secondarily woody species in both
wet and dry climates, makes this an excellent model
genus to investigate the diverse array of factors that
may drive shifts to secondary woodiness (Kiew, 1998,
2001; Beaman et al., 2001; Hughes & Pullan, 2007).
Begonia also has the advantage of having an array of
genomic resources including a draft genome sequence,
transcriptome datasets, and genetic maps (Brennan
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••
SECONDARY WOODINESS IN BEGONIA
et al., 2012; C. Kidner in prep), making questions
about the genetic underpinnings of the evolution and
development of secondary woodiness tractable.
Here we take an interdisciplinary approach using
three methods to investigate the evolution and development of secondary woodiness in Begonia: (1) we
describe detailed stem anatomical differences
between mainly herbaceous and woody species from
Borneo; (2) use an updated phylogenetic analysis
focusing on Bornean species to provisionally estimate
the number of shifts towards secondary woodiness in
Borneo and world-wide; and (3) characterize gene
expression in wood forming tissues of the secondarily
woody B. burbidgei Stapf native to Borneo. These
results provide us with preliminary data to inform
further discussion on the diversity of environmental
and abiotic factors that might be associated with
secondary woodiness in Begonia.
MATERIAL AND METHODS
OF WOODINESS VS. HERBACEOUSNESS
DEFINITION
We recognize the fuzzy boundary between woodiness
and herbaceousness creates difficulties (Dulin &
Kirchoff, 2010; Lens et al., 2012a, b, 2013a). We are
only interested in the evolutionary processes that
underlie the dramatic transition from ancestral ‘herbaceous’ species with no or limited wood formation to
derived woody shrubs with extensive wood development. A strict botanical definition of a woody species
is lacking, but in practice there are striking differences among groups including ‘herbaceous’ and
‘woody’ begonias (Figs 1, 2). We define secondarily
woody species as shrubs producing a distinct wood
cylinder extending towards the upper stem parts as
shown in Figure 4. This criterion only applies to the
following species in our sampling: B. burbidgei,
B. beryllae Ridl., B. fruticosa A. DC. and two species
new to science found in Crocker Range Park (Malaysia; Begonia sp. nov. spec. 2 and spec. 3). Based on
this definition, all the 45 Begonia species that were
investigated by Lee’s study of stem anatomy in
Begonia (Lee, 1974) should be called herbaceous,
although the author mentioned ‘considerable secondary growth’ in some species studied. We aim here to
investigate the switch to production of a robust wood
cylinder extending throughout the stems in some
woody species.
TAXONOMIC
SAMPLING, SEQUENCING PROTOCOL AND
PHYLOGENETIC ANALYSIS
During the September 2012 expedition to Mount
Kinabalu and Crocker Range Park, the corresponding
author collected material from 14 Begonia species
including four woody species and three new to
3
science. The frequency of the woody growth habit in
this locality suggested this would be a good starting
point for investigating the evolution of woody growth
within the genus. Voucher material is deposited in the
herbaria of Sabah Parks (Sabah, Malaysia) and the
Forest Research Institute Malaysia, voucher data are
presented in Table S1.
To understand the phylogenetic context of the
woody species, the species collected in Borneo were
incorporated into a plastid phylogeny of South East
Asian begonias (Thomas et al., 2011, 2012). Three
non-coding plastid DNA regions (ndhA intron, ndhF–
rpl32 spacer, rpl32–trnL spacer), which were shown
to be of considerable phylogenetic utility at the interand infrasectional level in Begonia were amplified
(Thomas et al., 2011, 2012; Moonlight et al., 2015). In
total, the ingroup comprised 105 accessions sampled
broadly from all major Asian Begonia sections. A focus
was put on accessions of the large section
Petermannia (> 250 species), which includes several
distinctly woody species. Accessions of the woody
B. beryllae, B. burbidgei Stapf, B. vaccinioides and
B. spec. 2, all of which were derived from our recently
collected material, were included. Accessions of seven
herbaceous species from Kinabalu, from which 19
species have been described in total (Hughes, 2008),
as well as accessions of an additional 42 species from
the entire geographic range of section Petermannia
were included in the analyses. Two African species,
Begonia dregei Otto and Dietr. and Begonia
sutherlandii Hook, were selected as outgroup based
relationships indicated in previous molecular
phylogenetic studies (Plana et al., 2004; GoodallCopestake et al., 2010). DNA sequences generated in
previous studies (Thomas et al., 2011, 2012) were
downloaded from the nucleotide database of the
National Centre for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/), and 55 sequences
were newly generated for this study (GenBank accession numbers are listed in Table S2).
Total genomic data was extracted from living material or silica gel dried material using the innuPrep
Plant DNA Kit (Analytika Jena, Jena, Germany)
according to the manufacturer’s protocols. Primers
and amplification protocols for the three chloroplast
markers were the same as in Thomas et al. (2011).
Sequencing polymerase chain reaction (PCR) products were purified and sequenced by MACROGEN
(Amsterdam) using an AB 3730 DNA Analyser
(Applied Biosystems).
Sequences were assembled and edited using
Geneious v6.1.7 (Drummond et al., 2010). The
sequences were pre-aligned using the multiple
sequence alignment software MUSCLE (Edgar, 2004)
implemented in Geneious using default settings, and
subsequently manually checked and optimized in
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••
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C. KIDNER ET AL.
Figure 1. Overview of variation in herbaceousness (A–C) and distinct woodiness (C, D) among Asian begonias, and
growth habits with reference to specialised organs: tuberous (A), rhizomatous (B), non-tuberous/rhizomatous (C–E).
A, Begonia tenuifolia Dryand, Begonia obovoidea Craib (Lens and Tisun 62). C, Begonia gambutensis Ardi and DC
Thomas D, Begonia vaccinioides Sands (photography credit Rogier van Vugt). E, Begonia sp. nov. (Lens and Tisun 78).
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••
SECONDARY WOODINESS IN BEGONIA
5
Figure 2. Light microscope sections of Begonia stems showing marked anatomical diversity between herbaceous species
(A, B) and woody species (C–E). A, Begonia chlorocarpa, cross-section showing intact primary vascular bundles,
interfascicular cambium is developing (arrows). B, Begonia aff. cauliflora, cross-section through basal stem part showing
narrow wood cylinder (arrow). C, Begonia sp. nov. (Lens and Tisun 78), cross-section at the stem base showing marked wood
cylinder with tall rays. D, Begonia sp. nov. (Lens and Tisun 82), tangential section illustrating tall rays with mainly upright
ray cells (arrows). E, Begonia fruticosa, tangential section showing wide gaping scalariform intervessel pitting (arrows).
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••
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C. KIDNER ET AL.
Geneious. Inversions were identified in the ndhFrpl32 spacer region of all Philippine samples of
Begonia section Diploclinium (309–355 bp), the rpl32trnL spacer of Begonia pendula Ridl. (37 bp), as well
as in the rpl32-trnL spacer of six distantly related
species (11 bp, see Thomas et al., 2011). These inversions were reverse-complemented, thereby retaining
substitution information in the fragments.
Bayesian phylogenetic reconstructions were performed using the XSEDE application of MrBayes
v3.2.2 (Ronquist & Huelsenbeck, 2003) provided by
the CIPRES Science Gateway (Miller, Pfeiffer &
Schwartz, 2010). Three partitions based on spacer
and intron identity (ndhA intron, ndhF-rpl32 spacer,
rpl32-trnL spacer) were defined a priori. Models of
sequence evolution of each nucleotide sequence partition were determined using MrModelTest (Nylander,
2004) under the Akaike Information Criterion (AIC).
Parameters for character state frequencies, substitution rates of nucleotide substitution models, and rate
variation among sites were unlinked across partitions. The mean branch length prior was set from the
default mean (0.1) to 0.01 to reduce the likelihood of
stochastic entrapment in local tree length optima
(Brown et al., 2010; Marshall, 2010). Four independent Metropolis-coupled MCMC analyses were run.
Each search of 10 million generations used four
chains, a temperature parameter setting of 0.6 and
was sampled every 1000 generations. Convergence
was assessed by using the standard deviation of split
frequencies with values < 0.005 interpreted as indicating good convergence. Tracer v1.5 (Rambaut &
Drummond, 2009) was used to check for stationary
and adequate effective sample sizes for each parameter (ESS > 200). Convergence of posterior probabilities of splits from different runs were checked using
the Compare and Cumulative functions of AWTY
(Nylander et al., 2008). The initial 25% of samples of
each run were discarded as burnin and the remaining
trees were summarized as 50% majority-rule consensus tree with nodal support summarized as posterior
probabilities.
STEM
ANATOMY
Stem samples of the four woody species collected on
Mount Kinabalu (B. beryllae, B. burbidgei, B. sp2 and
B. sp3) were compared to stem samples from herbaceous species in the same area and sampled more
widely across SE Asia. A stem sample from a fifth
woody species, the large liana B. fruticosa (Sao Paolo,
Brazil), was included to address the range of wood
anatomy in Begonia. In total, eight wood samples
from the five species investigated were sectioned
using a sliding microtome. Wood sections were coloured with safranin-alcian blue mix and mounted
with euparal (standardized protocol explained in Lens
et al., 2007). Nine samples representing the basal
stem part of herbaceous species were embedded in LR
White resin (hard grade, London Resin, UK), sectioned with a rotary microscope, and stained with
toluidine blue according to the protocol described in
Hamann, Smets & Lens (2011). Transverse sections
and longitudinal sections were made for the woody
species, while the herbaceous species were represented by transverse sections only.
STEM
TRANSCRIPTOME ANALYSIS
Samples from a woody B. burbidgei individual were
collected on the Mount Kinabalu summit trail at
2870 m asl in ultramaphic soils (Lens and Tisun 51,
Fig. 3, see Table S1 for detailed voucher data). Green
and more basal woody stem samples were collected
(Fig. 4A), sliced longitudinally into smaller fragments, and stored immediately in RNA later
(Ambion). Samples from the same regions of the same
stems were also collected for histological analysis.
Figure 4B shows the green tissue sample with
cambial growth just initiating and Figure 4C shows
much more wood formation in the woody stem
sample. Material used for transcriptome sequencing
was from the same stem section as for the histological
analysis.
Due to the difficulty in determining the cambial
layer in the preserved tissue sections, material from
the entire stem cylinder (as shown in Fig. 4a, c) was
used for the RNA preps. RNA was extracted using
Invitrogen’s Plant RNA extraction solution with modifications to the protocol to include an acid phenol
extraction after the chloroform extraction and a final
LiCl precipitation. Illumina-compatible sequencing
libraries were made using the Illumina Truseq RNA
Sample Preparation Kit according to the manufacturer’s protocol. Samples were multiplexed 6 per lane of
HighSeq 2000. 161 874 378 raw reads were generated
from the two samples. Reads were quality trimmed
using FASTQ groomer in Galaxy (cut-off value 20,
minimum percentage 90; Blankenberg et al., 2010)
and the cambial stage (28 221 304 reads) and woody
stage (49 756 127 reads) reads were jointly assembled
into 98 258 contigs using Trinity (Haas et al., 2013).
24 707 contigs were over 1 kb, 1525 over 3 kb. 280
chloroplast and mitochondrial sequences were
removed by comparison to the Begonia chloroplast
genomes and previously identified mitochrondrial
sequences (Brennan et al., 2012; Harrison, 2012).
Reads were mapped back to the assembly using
bowtie 2.0 (Langmead & Salzberg, 2012). We used
blast matches to the draft Begonia conchifolia A.
Dietr. genome (C. Kidner, unpublished) to the
TAIR set of Arabidopsis thaliana proteins
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••
SECONDARY WOODINESS IN BEGONIA
B. dipetala
.97
1
.64
B. samahensis
B. socotrana
1
.55
1
1
.64
1
B. malabarica
B. floccifera
B. tenuifolia
B. elisabethae
B. smithiae
B. grandis
1
1
B. hymenophylla
B. alicida
B. brandisiana
1
1
B. aceroides
1
1
.96
1
1
.90
.65
0.01
7
B. flagellaris
B. sikkimensis
1
B.
palmata
1
1
B. sizemoreae
B. versicolor
1 .96
B. venusta
1
B. decora
.81
B. pavonina
.5
B. hatacoa
.92
B. robusta
B. multangula
1
B. areolata
.99
B. obovoidea
1
B. roxburghii
1
B. acetosella
B. silletensis
1
B.
longifolia
1
B. aptera
B.
masoniana
1
B. morsei
B. spec. 4 PH115
B. amphioxus
B. pendula
B. aff. cauliflora FL065
1
B. aff. cauliflora FL066
1
B. burbidgei
1
B. burbidgei FL040
B.
vaccinioides
SNP25535_1
1
B. vaccinioides SNP25535_2
1
B. spec. 1 FL062
B. beryllae FL071
B. cf. beryllae FL074
.97
B. imbricata FL075
B. kingiana
B. goegoensis
1
B.
sudjanae
1
B. muricata
B. nigritarum
B. cleopatrae
1
1
B. gueritziana
.88
B. fenicis
1
B. hernandioides
1
B. chloroneura
B. lepida
1
B. resecta
.67
B. verecunda
B. aff. congesta
1
B. corrugata
.88
B. harauensis
1
B. laruei
1
B. multijugata
B. spec. 2 FL078
1
.73
1
B. spec. 2 FL072
.98 .81
B.
aff. erythrogyna FL002
.99
B. chlorosticta
1
B. mamutensis FL029
B. oblongifolia FL014
.96
B. inostegia FL021
1
B. inostegia FL013
B. oblongifolia FL028
1
B. weigallii
B. symsanguinea
.98
B. strigosa
1
.99
B. argenteomarginata
B.
negrosensis
1
B. poliloensis
.8
B. serratipetala
1
B. brevirimosa
B. hekensis
1
B. varipeltata
1
B. stevei
1
B. macintyreana
.99
1
B. chiasmogyna
1
B. mendumiae
1
B. masarangensis
1
B. capituliformis
B. hispidissima
B. siccacaudata
1
B. bonthainensis
B. ozotothrix
1
B. nobmanniae
1
B. prionota
.52
B. didyma
.56
B.
watuwilensis
1
B. flacca
.87
B. koordersii
B. lasioura
.92
B. comestibilis
B. guttapila
1
B. rantemarioensis
B. sanguineopilosa
.9
B. vermeulenii
1
B. torajana
B. demissa
B. wrayi
Herbaceous
Woody
Non-tuberous/rhizomatous
Tuberous
Rhizomatous
Swollen stem base
Figure 3. Bayesian 50% majority-rule consensus tree (cpDNA data: ndhA intron, ndhF-rpl32, rpl32-trnL; three data
partitions; 110 accessions). Posterior clade probabilities are indicated next to the nodes. Squares next to the terminals
indicate growth habit types: black, swollen stem base; blue, tuberous; brown, woody; green, herbaceous; purple,
rhizomatous; white, non-tuberous/–rhizomatous.
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••
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C. KIDNER ET AL.
Figure 4. Woody Begonia burbidgei individual that was used for stem transcriptome analysis (A) and its stem
developmental stages (B, C) sampled in RNA later in the field (mount Kinabalu summit trail, Sabah, Borneo). A, Habit
(Lens and Tisun 51). B, Cross-section through inflorescence stem that has initiated a vascular cambium (arrows, yellow
cross in A) – cambial stage transcriptome. C, Cross-section through more basal stem part showing extensive wood
formation (yellow circle in A) – wood stage transcriptome.
(TAIR10_pep_20101214_updated
at
http://www
.arabidopsis.org) and the Populus trichocarpus Torr.
and A. Gray protein dataset from phytozome (http://
www.phytozome.com,
Ptrichocarpa_210_protein.fa
.gz) to annotate the sequences. 54 030 sequences had
a blastn hit at 1e-40 or better in the Begonia
conchifolia draft genome to 17 743 unique ORFs. As
B. conchifolia is a distant relative of B. burbidgei
(Thomas et al., 2012), we expect this set to include
conserved Begonia genes. 25 042 sequences had a
blastx hit at 1e-10 or better to 10 423 unique TAIR
proteins. The TAIR hits were used to annotate the
B. burbidgei sequences. RSEM and edge-R were used
to detect genes differentially expressed between the
cambium stage and wood stage samples (Robinson,
McCarthy & Smyth, 2010; Li & Dewey, 2011; Haas
et al., 2013).
We compared coding sequences for orthologous
genes in B. burbidgei and the herbaceous B. venustra,
for which a transcriptome dataset from vegetative
buds has already been published (Brennan et al.,
2012) using custom scripts produced by K.
Emelianova (available on request). To run Reciprocal
Best BLAST Hits to identify orthologs in the two
species, longest open reading frames were extracted
from ortholog pairs using getORF (Rice, Longden &
Bleasby, 2000) and translationally aligned using
MAFFT (Katoh & Standley, 2013), Virtual Ribosome
(Wernersson, 2006) and RevTrans (Wernersson &
Pedersen, 2003). Translational alignments were used
as input to the Codeml package of PAML (Yang,
2007). The site test was used in Codeml to test for
positive selection, using the one-ratio model, producing values of kN, kS and kN/kS for each orthologous
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••
SECONDARY WOODINESS IN BEGONIA
pair. Orthologs which were more than 75% divergent
and thus unsuitable for the NG86 method were not
included in the final results. The ortholog pairs with
indications of positive selection were annotated by
blastx to Arabidopsis proteins; only those with a
match of at least 1e-10 or better were included in the
rest of the analysis.
We used reciprocal tblastx to identify orthologs
between the Begonia burbidgei and Populus
trichocarpus sequences from a published cambium
transcriptome (Liu et al., 2014), and compared relative expression levels by reads per million reads per
kb of the Populus coding sequence (as Begonia
burbidgei sequences were often not the full coding
sequence) (Table S6).
RESULTS
VARIATION
IN HABIT
Secondarily woody Begonia species are characterized
by a shrubby growth habit and can usually be easily
distinguished from herbaceous relatives (Figs 1, 3). In
general, herbaceous species have semi-succulent
stems, and may have specialized storage organs such
as tubers (Fig. 1A) or rhizomes (Fig. 1B) (Figs 1C, 3).
In contrast, secondarily woody species are shrubs
(Fig. 1D, E) characterized by wood formation extending towards the upper parts of the stem; rhizomes or
tubers are generally absent in the Bornean clade we
examined, though they seem to occur in some
neotropical woody Begonia (Carlquist, 1985; Fig. 3;
see also Materials and Methods for further discussion
about the definition between woodiness vs.
herbaceousness).
DESCRIPTION
OF WOOD ANATOMY IN
Begonia
In order to develop a clade of Bornean Begonia as a
model for understanding the secondary evolution of
woodiness we investigated mature wood anatomy in
detail for four of these woody species from samples
collected in the field (B. burbidgei Stapf (Lens and
Tisun 44), B. beryllae Ridl. (Lens and Tisun 71), B. sp.
nov. 2 and 3 (Lens and Tisun 78 and 82). The only
pre-existing description of wood anatomy in Begonia
is for three neotropical species and a hybrid
(Carlquist, 1985). We included the large neotropical
liana B. fruticosa A. DC. to allow direct comparisons
of our South East Asian species with an unrelated
woody species from a parallel radiation (Fig. 2,
Table S1). The wood description for all these samples
is similar and can be summarized as follows:
Growth ring boundaries absent. Wood diffuse porous. Vessels
concentrated in the intrafascicular regions, (7)-17-59-(82)/
mm2, usually solitary, sometimes in short radial multiples of
9
2–3 and occasionally in short tangential multiples of 2-(3),
vessel outline angular. Vessel perforation plates simple.
Lateral wall pitting typically wide gaping scalariform
(Fig. 2E), pits with minute borders, pit cavities 18–50 μm in
horizontal size, non-vestured. Tangential vessel diameter (20)30-100-(130). Vessel elements (150)-220-380-(470) μm long.
Length-on-age curve of vessel elements flat or slightly
decreasing. Tyloses present in B. fruticosa and occasionally in
B. sp. nov. 3. Tracheids absent. Fibres sometimes septate,
occasionally septate in B. fruticosa, thin-walled, (250)-430520-(680) μm long, with mostly simple to occasionally
minutely bordered pits distributed in radial and tangential
walls. Axial parenchyma scanty paratracheal, 2-4-(5) cells per
strand. Rays exclusively multiseriate and confined to the
interfascicular regions, (16)-20-30-(36) cells wide, and very tall
(Fig. 2C, D), at least 1500 μm but often much higher than the
length of the sections, 0–2 rays/mm2. Exclusively upright ray
cells present in B. burbidgei, B. beryllae and B. sp. nov. 2 and
3 (Fig. 2D), but mainly procumbent to sometimes also square
to upright in B. fruticosa. Sometimes thick-walled sclereids in
the tall rays. Sheath cells and mineral inclusions not
observed. A tendency towards layering in fibres, vessels elements and axial parenchyma strands in B. fruticosa.
MOLECULAR
PHYLOGENETICS
Three non-coding plastid DNA regions (ndhA intron,
ndhF–rpl32 spacer, rpl32–trnL spacer) from 105
Asian species and two African species were used for a
Bayesian phylogenetic reconstruction of the sampled
Bornean Begonia species (Table S2; Fig. 3). Secondarily woody species native to Borneo, all of which are
assigned to Begonia section Petermannia, are
retrieved in two distantly related clades of Bornean
begonias: clades A and B. The strongly supported
clade A (posterior probability, PP: 1) includes accessions of the woody species B. burbidgei and
B. vaccinioides Sands, the woody species B. beryllae
Ridl. and accessions of the herbaceous species
B. imbricata Sands and B. spec. 1. Clade B (PP: 0.98)
includes the woody species B. spec. 2, as well as
several herbaceous species (B. chlorosticta Sands, B.
aff. erythrogyna Sands, B. mamutensis Sands,
B. oblongifolia Stapf, B. inostegia Stapf).
STEM
TRANSCRIPTOMICS OF
Begonia burbidgei
Tissue from young and older stages of a stem of one
B. burbidgei individual was collected from a mature
Mount Kinabalu individual (Fig 4A, Lens and Tisun
5, see Table S2 for more details on individual and
locality), and total RNA was extracted. Histology of
the samples showed that the younger sample had just
initiated cambial growth (shown in Fig. 4B) and the
older sample had extensive woody growth (Fig. 4C).
Transcriptomes were produced from each sample (all
tissues in a cross-section of the stem) and annotated
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••
10
C. KIDNER ET AL.
by comparison to Arabidopsis thaliana Heynh proteins and Populus trichocarpa Torr and A. Grey.
We used three search strategies to mine this data
for information on the molecular wood development in
this species. The first approach was to use RSEM and
edge-R (Robinson et al., 2010; Li & Dewey, 2011; Haas
et al., 2013) to detect genes differentially expressed
between the initiating and fully woody stems of
B. burbidgei. These genes are expected to include
those promoting vascular cambium activities versus
wood differentiation in Begonia. The second approach
was to compare coding sequences for orthologous
genes in B. burbidgei and the herbaceous B. venustra,
for which a transcriptome dataset from vegetative
buds has already been published (Brennan et al.,
2012). Genes showing a high Ks/Kn ratio in this
comparison are expected to include the targets of
selection during the evolution of woody growth. Our
third approach was to compare the genes expressed in
the primarily woody Populus trichocarpa cambium
with those found in secondarily woody B. burbidgei
stems. Genes found in P. trichocarpa but missing from
the B. burbidgei sample are expected to include those
which are involved in cambium activity in the primarily woody P. trichocarpa but not in the secondarily
woody B. burbidgei. We also examined the expression
levels in B. burbidgei of genes which had been implicated in secondary growth and lignin biosynthesis in
a range of model species.
COMPARATIVE
TRANSCRIPTOMICS OF WOOD AND
CAMBIUM STAGE
B. burbidgei
STEMS
Edge-R analysis produced a list of 577 contigs
showing differential expression between wood and
cambium stage stems by more than two-fold and a
P-value of less than 1e-3. These contigs were annotated by blastx matches to the Arabidopsis thaliana
and the P. trichocarpa protein databases (Table S1).
287 of these 577 contigs had no good blastx match (at
1e-10 or better) in Arabidopsis or poplar. These
sequences will require further analysis to determine
function.
The genes with best support for differential expression between the wood and cambium stage of Begonia
burbidgei stems and with good annotation include
a number involved in meristem determinacy
(REBELOTE AT3G55510.1), growth (the expansin
AT1G26770.1), cell wall biosynthesis (xyloglucan
endotransglycosylase AT4G25820.1), many lipid
metabolism associated genes including HOTHEAD
(AT4G25820.1) and FIDDLEHEAD (AT2G26250.1) as
well as some genes thought to be involved in secondary cell wall synthesis such as the FASCICLIN-like
arabinogalactan protein 8 (FLA8) (AT2G45470.1)
(Table S3).
The Gene Ontology (GO) terms of the differentially
expressed annotated contigs were counted and compared to the annotations for all B. burbidgei
sequences using agriGO (Du et al., 2010). 26 GO
terms were enriched in the differentially expressed
set (Table 1). These include carbohydrate transporters, cell wall associated genes and serine-type
exopeptidases.
COMPARATIVE TRANSCRIPTOMICS OF THE WOODY B.
burbidgei AND THE HERBACEOUS B. venustra
B. venustra King is an herbaceous species from
Malaysia, not closely related to the woody
B. burbidgei (Thomas et al., 2012). A transcriptome
for vegetative buds of B. venustra had been previously
analysed (Brennan et al., 2012). The vegetative bud
sample includes some young stem tissue but the
range of cell types present differ too much from the B.
burbidgei stem samples for differential expression
analysis to be useful. However, comparison of the
sequences suggests some interesting variation
between these two species.
A python pipeline was used to analyse sequence
variation between orthologous genes from the woody
B. burbidgei and the herbaceous B. venustra. The 256
orthologs showing kN/kS ratios exceeding 1 – suggestive of positive selection – are listed in Table S4. This
set includes interesting candidate regulators of woody
growth such as orthologs of two myb transcription
factors (AT5G41020 and AT1G26780 (LOF1)). It also
includes orthologs of genes that may be linked to
growth on ultramafic soil such as AT4G34050, an
enzyme involved in response to cadmium, and
orthologs of secondary metabolism enzymes such
as caffeoyl coenzyme A O-methyltransferase 1
(AT4G34050) (Table S4). The GO terms for those with
a Kn/Ks over 1 were compared to the annotations for
all pairs using agriGO (Du et al., 2010). No significantly over-represented term was identified. We find
no evidence supporting the hypothesis that the
evolution of woody growth in B. burbidgei involved
concerted protein sequence change for genes in a
particular pathway.
COMPARATIVE
B. burbidgei
Populus trichocarpa
TRANSCRIPTOMICS OF
AND
To compare wood development in B. burbidgei to that
in model wood forming species, we used the genetic
resources of P. trichocarpa (Tuskan et al., 2006).
3974 B. burbidgei contigs had a blastx hit to a
P. trichocarpa protein but did not have matches (at
1e-40) in the B. conchifolia genome. This set of potential wood-associated genes comprised matches to 2522
P. trichocarpa genes including meristem regulatory
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••
SECONDARY WOODINESS IN BEGONIA
11
Table 1. Gene Ontology (GO) terms enriched in the set of genes differentially expressed between cambium stage and
wood samples of B. burbidgea stems
GO term
Description
GO:0015295
GO:0005402
GO:0005351
GO:0051119
Solute:hydrogen symporter activity
Cation:sugar symporter activity
Sugar:hydrogen symporter activity
Sugar transmembrane transporter
activity
Carbohydrate transmembrane
transporter activity
Symporter activity
Solute:cation symporter activity
Secondary active transmembrane
transporter activity
Serine-type peptidase activity
Serine hydrolase activity
Substrate-specific transporter activity
Substrate-specific transmembrane
transporter activity
Ion transmembrane transporter activity
Cation transmembrane transporter
activity
Serine-type exopeptidase activity
Exopeptidase activity
Transmembrane transporter activity
Serine-type carboxypeptidase activity
Carboxypeptidase activity
Active transmembrane transporter
activity
Transporter activity
Peptidase activity, acting on L-amino
acid peptides
Extracellular region
Apoplast
External encapsulating structure
Cell wall
GO:0015144
GO:0015293
GO:0015294
GO:0015291
GO:0008236
GO:0017171
GO:0022892
GO:0022891
GO:0015075
GO:0008324
GO:0070008
GO:0008238
GO:0022857
GO:0004185
GO:0004180
GO:0022804
GO:0005215
GO:0070011
GO:0005576
GO:0048046
GO:0030312
GO:0005618
genes such as a ZPR2 ortholog (Potri.002G149600)
and arabinogalactans (Table S5).
We used expression data from P. trichocarpa
cambium (Liu et al., 2014) to find genes expressed
during wood development in P. trichocarpa that are
differentially expressed or missing in the wood of
B. burbidgei (Table S6). A number of interesting
genes were differentially expressed in this comparison. Potri.014G015600, an ortholog of the myb transcription factor NtLIM, required for full activation of
the phenylpropanoid pathway (Kawaoka & Ebinuma,
2001), is expressed at over 1000-fold higher in
P. trichocarpa cambium than in B. burbidgei stems.
Agusti et al. (2011) identified MOL1 and RUL1 as
opposing regulators of secondary growth. These two
Count in
significantly
differentially
expressed list
Count in
B. burbidgei
transcriptome
P-value
FDR
13
13
13
13
70
70
70
80
6.90E–11
6.90E–11
6.90E–11
4.00E–10
6.90E–09
6.90E–09
6.90E–09
2.90E–08
13
85
8.60E–10
5.10E–08
13
13
14
91
90
188
2.00E–09
1.80E–09
1.90E–06
8.70E–08
8.70E–08
7.20E–05
9
9
25
22
88
88
624
534
9.80E–06
9.80E–06
3.00E–05
5.50E–05
0.00029
0.00029
0.0008
0.0014
18
15
392
293
6.20E–05
7.20E–05
0.0014
0.0015
5
6
25
5
5
17
30
48
672
30
32
399
8.90E–05
9.50E–05
9.30E–05
8.90E–05
0.00012
0.00023
0.0016
0.0016
0.0016
0.0016
0.0019
0.0033
29
11
880
266
0.00022
0.0032
0.0033
0.044
11
8
12
12
97
63
213
209
3.80E–07
6.00E–06
0.00015
0.00012
7.80E–05
0.00062
0.0077
0.0077
genes are expressed at similar levels in the cambium
stage stem and the woody stem of B. burbidgei and
the P. trichocarpa cambium sample, but orthologs
of three of the targets identified in Agusti et al.
(2011) – At1g46480 (WOX1), At1g52340 (ABA2) and
At5g57130 – are upregulated in the Begonia samples,
suggesting this pathway may also be modified in
B. burbidgei wood development.
This analysis also highlighted a set of genes
expressed in the P. trichocarpa cambium sample but
not recovered from the B. burbidgei transcriptomes.
Most of these genes are not annotated in
P. trichocarpa but some suggest interesting differences between Populus and Begonia wood. Some
arabinogalactans, in particular FASCICLIN-like
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••
12
C. KIDNER ET AL.
Table 2. The reads per kilobase per million reads (RPKM) for genes encoding enzymes in the lignin biosynthetic pathway
for Begonia burbidgei (wood and cambium stage samples) compared to a sample of Populus trichocarpus cambium
Enzyme
Direction
Enzyme
code
Wood
stage
Cambium
stage
Poplar
cambium
Phenylalanine ammonia-lyase
Trans-cinnamate 4-monooxygenase
4-Coumarate-CoA ligase
Caffeoyl shikimate esterase
Shikimate O-hydroxycinnamoyltransferase
Caffeoyl-CoA O-methyltransferase
Cinnamyl-alcohol dehydrogenase
Peroxidase
4-Coumarate-CoA ligase
Beta-glucosidase
Coniferyl-aldehyde dehydrogenase
To lignin
To lignin
To lignin
To lignin
To lignin
To lignin
To lignin
To lignin
Away from lignin
Away from lignin
Away from lignin
4.3.1.24
1.14.13.11
6.2.1.12
3.1.1.5
2.3.1.133
2.1.1.104
1.1.1.195
1.11.1.7
6.2.1.12
3.2.1.21
1.2.1.68
333.6
522.97
175.85
59.69
95.01
11.63
3.78
645.27
175.85
114.89
43.88
281.76
388.13
118.93
63.27
73.58
9.35
5.61
484.26
118.93
147.88
45.26
474.6
1466.45
2.44
60.1
438.17
1857.5
83.9
1130.42
2.44
8.34
16.63
arabinogalactan proteins were not recovered from the
Begonia samples though they were expressed at a
high level in Populus cambium, as were a number of
calmodulin-like proteins (Table S7).
To provide an overview of the biosynthetic pathways that differed between the B. burbidgei and
P. trichocarpa samples, we summed the reads per
kilobase per million reads (RPKMs) for each enzyme
class (EC); these data are presented in Table S8. A
key enzyme in the lignin biosynthetic pathway,
Caffeoyl-CoA O-methyltransferase, is one of the
enzymes much more highly expressed in Populus
cambium than in the wood or cambium Begonia stem
samples, whereas 4-coumarate-CoA ligase, one of the
earlier enzymes in the pathway is expressed at much
higher levels in the B. burbidgei samples.
To focus on the lignin pathway, we compared
expression levels of all genes from this biosynthetic
pathway we could recover from B. burbidgei, including the newly identified lignin biosynthetic gene
Caffeoyl shikimate esterase (Vanholme et al., 2013).
The RPKM for genes encoding enzymes in the
lignin biosynthetic pathway are listed in Table 2.
B. burbidgei wood expresses many of the lignin
biosynthetic enzymes, but at a lower level than in the
P. trichocarpa cambium sample, particularly at the
distal end of the pathway, whereas the expression
level of enzymes leading away from lignin is higher.
Some genes have been proposed as regulators of
wood synthesis (reviewed in Andersson-Gunnerås
et al., 2006). Of the 22 genes listed in
Andersson-Gunnerås et al. (2006), including many
myb transcription factors and some cellulose synthase
genes, B. burbidgei orthologs were identified for 17
but none of these showed any significant difference in
expression levels between our cambium and wood
samples (data not shown).
DISCUSSION
HIGH
NUMBER OF CONVERGENT SHIFTS TOWARDS
SECONDARY WOODINESS WITHIN
Begonia
Our phylogenetic analysis is in agreement with other
available molecular phylogenies showing that all
woody Begonia species have been derived from herbaceous relatives (Plana, 2003; Forrest et al., 2005;
Goodall-Copestake et al., 2010; Thomas et al., 2011,
2012). The derived origins of woodiness in Begonia
species is also supported by the wood anatomy of the
five species studied in this paper and the four
neotropical Begonia shrubs investigated by Carlquist
(1985). All these wood samples show protracted
juvenilism, as demonstrated by the tall rays with
mainly upright ray cells, wide gaping scalariform
intervessel pitting, and the flat length-on-age curve
for vessel elements (Fig. 2; Carlquist, 1985, 2009,
2012). However, protracted juvenilism in wood is not
always associated with secondary woodiness, as some
studies have indicated a strong link between protracted juvenilism in wood and specific growth form
types in primarily as well as secondarily woody angiosperms (Carlquist, 2009; Dulin & Kirchoff, 2010; Lens
et al., 2013a).
The number of shifts towards secondary woodiness
in Begonia is still unknown. The densest phylogenetic
sampling of South East Asian begonias (in total ca.
650 species) published to date includes only one
woody species, B. burbidgei from Borneo (Thomas
et al., 2012). Our analysis adds the woody
B. vaccinioides, B. beryllae, an unknown woody
species collected in the Crocker Range (Begonia sp. 2,
Lens and Tisun 78), and additional accessions of
B. burbidgei (Fig. 3), together with original sequences
from nine herbaceous Bornean species. Based on our
increased sampling, B. burbidgei falls together with
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••
SECONDARY WOODINESS IN BEGONIA
B. vaccinioides and B. beryllae in the same Bornean
clade, while Begonia sp. 2 is placed in an unrelated
Bornean clade. In the first clade, we hypothesize that
these three woody species represent one shift towards
secondary woodiness, although B. beryllae seems to
be more closely related to the herbaceous B. imbricata
and an unknown herbaceous species in the Crocker
Range (Lens and Tisun 62) than to B. vaccinioides
and B. burbidgei. Further increasing the Bornean
sampling – more than 100 Bornean Begonia species
are currently accepted (Hughes, 2008; Sang, Kiew &
Geri, 2013) – will identify whether the two herbaceous species are secondarily herbaceous or whether
this first Bornean clade includes more than one shift
towards secondary woodiness. In the second Bornean
clade, the woody Begonia sp. nov. 2 clusters together
with herbaceous species, with B. erythrogyna Sands
and B. chlorosticta as closest relatives (Fig. 3). It is
likely that more shifts in SE Asia have occurred,
because there are several other potentially woody
Asian species such as B. keithii Kiew (Borneo) and
B. merrittii Merr. (Philippines), for which DNA
sequence data are not available to date. B. merrittii,
native to moist montane forests in the Philippines
(Hughes & Pullan, 2007), is definitely woody, but for
B. keithii and several other species anatomical observations are required to assess whether these species
represent truly woody shrubs, or whether wood formation is only limited to the base of the stem as is the
case for many herbaceous species (see definition of
woodiness vs herbaceousness in Methods section).
Besides the two shifts found in the SE Asian clade,
there are also two clear shifts towards secondary
woodiness in the African clade (Plana, 2003), the
continent on which Begonia initially diversified (ca.
160 sp.). One shift leads to the tall shrubs B. baccata
Hook and B. crateris Exell native to the tropical West
African islands São Tomé and Principe (Reitsma,
1984), and the second shift is represented by the
woody climber B. meyeri-johannis Engl. inhabiting
moist montane East African forests (Plana et al.,
2006).
In South America, the situation is more complex
due to the high number of species (ca. 690 sp.), the
abundance of woody species (possibly up to ca. 30 sp.;
Mark Tebbitt, pers. comm.), the lack of taxonomic
revisions or detailed stem anatomical observations,
and the variation in habit ranging from herbaceous
species, towards different types of woody growth
forms, such as acaulescent suffrutescent growth forms
with thick woody rhizomes (e.g. B. gorgonea Tebbitt),
woody shrub-like species (e.g. B. parviflora Schott),
and even tall woody lianas reaching the canopy
(B. fruticosa). In the recent phylogenetic analysis of
neotropical begonias (Moonlight et al., 2015), six
woody species are included, leading to at least four
13
additional shifts towards secondary woodiness. Again,
this number is likely an underestimation and a much
denser sampling together with a detailed anatomical
survey is desired to obtain a more realistic view on
the plasticity of growth forms within Begonia.
TOWARDS
UNRAVELLING THE REGULATION OF WOOD
FORMATION IN
Begonia
After the Helianthus (Asteraceae) transcriptome
dataset of seedlings (Moyers & Rieseberg, 2013), our
Begonia burbidgei transcriptome dataset is the
second study that performs an RNA-seq experiment
in stems of a secondarily woody species. While our
sampling is limited, our work serves as an initial
investigation into the genetic changes which accompany the development of wood in this species.
The induction of genes associated with secondary
growth, such as expansins and xyloglucans, is to be
expected during the establishment of wood formation
in B. burbidgei and we do see such changes
(Table S3). The differential expression of lipid
metabolism genes is more surprising, though some
trees produce wood with substantial amounts of lipids
(Hoch, Richter & Körner, 2003) and lipid vesicles are
involved in transport of lignin precursors to the cell
wall. Alternatively, the differences in lipid-related
gene expression may be due to changes in tissues
other than the cambium. As the stem matures,
changes would also be expected in the periderm
(Fig. 4C) and this suberized tissue is a likely location
for products of the lipid metabolic pathway.
One aim of our work was to identify potential
candidate genes for the control and development of
secondary growth in Begonia burbidgei. Due to sampling constraints we cannot establish strong correlations between particular regulators and wood
development, but by using a variety of approaches we
have a short list of six sets of genes worthy of further
investigation. Firstly, an ortholog of REBELOTE is
upregulated in wood stages of the B. burbidgei stem
(Table S3). This gene promotes meristem determinacy
in Arabidopsis redundantly with ULTRAPETALA
and SQUINT (Prunet et al., 2008). In Begonia
conchifolia six loci encode REBELOTE-like genes,
only one of which has any transcripts in the vegetative bud transcriptome, and that at a very low level.
Based on alignments of the coding regions, this
paralog expressed in the vegetative bud is likely the
ortholog of the REBELOTE gene expressed at high
levels in the wood sample of B. burbidgei. This
ortholog is unlikely to have the same role in terminating meristems as its Arabidopsis ortholog as the
vegetative buds it is expressed in are active. A change
in role could be related to the lack of ULTRAPETALA
and SQUINT co-expression in Begonia. None of the
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••
14
C. KIDNER ET AL.
37 Arabidopsis genes co-expressed with REBELOTE
according to GeneMANIA (genemania.org) have
B. burbidgei orthologs with significantly differential
expression between the two samples (P < 0.05), suggesting the B. burbidgei ortholog of REBELOTE is
active in a different pathway to the Arabidopsis
original.
A second gene that could be involved in the maintenance of an active cambium is the ortholog of ZPR2
found in B. burbidgei and P. trichocarpa but not in
the genome of the herbaceous B. conchifolia
(Table S5). ZPR2 is part of a gene family, which
interacts with HD-ZIPIII proteins to regulate
meristem function in Arabidopsis (Wenkel et al.,
2007). Association of expression variation in these
genes and woody growth would establish whether
these regulators are key points in the evolution of
woody growth in Begonia.
Another of the differentially expressed genes in the
woody B. burbidgei without orthologs in the genome
of the herbaceous B. conchifolia is an ortholog of
AT5G23960, TERPENE SYNTHASE 21 (TPS21).
This gene encodes a sesquiterpene synthase involved
in generating all of the group A sesquiterpenes
found in the Arabidopsis floral volatile blend. Its
B. burbidgei ortholog (comp104430_c1_seq1) is
expressed at very high levels in the vascular cambium
stem stage, but goes down in the woody stage. The
Begonia ortholog could be involved in generating secondary products associated with woody tissue.
Fourthly, orthologs of two myb transcription factors
(AT5G41020 and AT1G26780 (LOF1)) show signs
of sequence divergence between the herbaceous
B. venustra and woody B. burbidgei (Table S4). Such
divergence could change their targets or their
co-regulators and so completely change their effects.
Myb transcription factors that regulate wood development have already been characterized in a number
of species (Goicoechea et al., 2005; Zhang et al., 2014).
Further analysis of this pair through transgenic
experiments could show if they have the capacity to
affect wood development.
Another potential regulator of B. burbidgei wood is
an ortholog of NtLIM, which is expressed at a much
lower level in B. burbidgei than in P. trichocarpa
(Table S5). In Nicotiana, this gene regulates the
phenylpropanoid pathway (Kawaoka & Ebinuma,
2001). The first steps of this pathway are shared with
the lignin biosynthetic pathway, but expression levels
for biosynthetic enzymes from this level of the
pathway are not downregulated in the B. burbidgei
sample in comparison to P. trichocarpa.
A final set of regulators worth examining are
downstream targets of MOL1 and RUL1 (Agusti
et al., 2011). Though not significantly differentially
expressed themselves, three of their targets are
upregulated in B. burbidgei samples relative to
P. trichocarpa suggesting this pathway too may be
involved in the production of secondarily woody stems.
COMPARING
WOOD REGULATION IN
WITH
Begonia
Populus
Our B. burbidgei transcriptomes show lower expression of biosynthetic genes leading to lignin compared
to the situation in Populus. This is to be expected,
because apart from the fact that Populus has primary
woodiness and Begonia secondary woodiness, the
wood anatomy of Begonia and Populus is very different. For instance, fibre walls in Populus wood are
more heavily lignified than in Begonia wood, and
Begonia has much more unlignified parenchyma in its
wood (especially tall rays) compared with Populus
(Figs 2, 4, S1). Some of the differences between
Populus and Begonia lignification could be due to
sequence variation in lignin biosynthetic genes such
as that identified in an ortholog of caffeoyl coenzyme
A O-methyltransferase 1 by PAML analysis
(Table S4). The differential expression analysis also
suggests potential differences in lipid and terpenoid
biochemistry in Begonia wood (Table S3).
Fasciclin-like arabinogalactans have been implicated in shoot growth (Johnson et al., 2011), fibre
extension (Huang et al., 2013; Liu et al., 2013) and
secondary wall synthesis and wood formation
(reviewed in MacMillan et al., 2010). They are
thought to act through modification to the cell wall’s
elasticity. This could also contribute to the differences
seen between Begonia and Populus wood. Orthologs of
some FASCICLIN-like arabinogalactans are differentially expressed in wood and cambium stages of
B. burbidgei stems (Table S3). Other arabinogalactans are found in B. burbidgei but not in the
genome of herbaceous B. conchifolia (Table S5), and
some are not recovered from Begonia samples though
they are expressed at high levels in Populus cambium
(Table S7). Further analysis of this group of proteins
may reveal interesting differences between the formation of wood between Begonia and Populus.
POTENTIAL
ABIOTIC VARIABLES TRIGGERING
SECONDARY WOODINESS IN
Begonia
A comprehensive explanation why secondary woodiness has evolved across Begonia is not possible from
this initial analysis, but it is clear that a complex mix
of different factors is involved. Our Bornean collections point to a strong relationship between increased
woodiness, altitude and soil type. All our woody
Begonia collections were found in montane areas
between 1800–2900 m above sea level, and several of
these grow on ultramafic rocks, which is also
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••
SECONDARY WOODINESS IN BEGONIA
confirmed for the two known populations of
B. vaccinioides, the woodiest species of Borneo (Rimi
Repin, pers. comm.), and for B burbidgei collected
along the mountain trail of Mount Kinabalu at
2870 m asl (Lens and Tisun 51, Fig. 4A). Ultramafic
rocks and the derived serpentine soils are edaphically
stressful for plant growth due to their nutrient deficiencies (especially Ca), low water-holding capacity,
and high levels of heavy metals and Mg (Brady,
Kruckeberg & Bradshaw, 2005). The combination of
poisonous soils and low water-holding capacity must
cause stress to the plant, which could lead to wood
formation in analogy to the drought stress hypothesis.
However, the flora native to serpentine soils (in e.g.
California) is mainly composed of herbaceous or primarily woody genera, indicating that only these specific soil conditions are not sufficient to trigger wood
formation (Anacker et al., 2010; Anacker & Harrison,
2012).
In conclusion, woody Begonia species are derived
from herbaceous relatives and this trait has evolved
independently numerous times across Begonia. The
selective pressures behind these events remain
unclear. Precipitation, altitude and soil conditions
may play a role, but it seems more likely that a mix
of these factors combined with other abiotic and environmental cues can promote the expression of the
wood pathway in Begonia. Novel gene expression
features of Begonia wood in comparison with Populus
wood (changes in the lignin biosynthetic pathway,
changes in FASCICLIN-like arabinogalactans) may
be related to the differences in wood anatomy
observed. This variation suggests lines of research to
better understand the different properties of the wood
of primarily and secondarily woody species. The
transcriptomes we have generated also provide a
number of candidate genes for regulation of wood
formation in Begonia burbidgei through modification
of meristematic activity and cellular anatomy which
are worth investigating in other secondarily woody
species both in Begonia and other genera. The genus
Begonia offers an excellent opportunity to test the
importance of these candidate genes through examination of expression and sequence variation in
phylogenetically matched sets of woody and herbaceous species pairs.
ACKNOWLEDGEMENTS
The authors thank Naturalis Biodiversity Center and
Sabah Parks for organising and financially supporting the Mount Kinabalu & Crocker Range expedition.
We also thank Rimi Repin, Mark Tebbitt and Marc
Sosef for their valuable discussions, and Ruth Kiew
for identifying the collections from Kinabalu and
Crocker Range. Marcelo Pace is acknowledged for
15
collecting the wood sample of Begonia fruticosa, and
Sukaibin Sumail for sending leaf material of
B. vaccinioides. Frederic Lens received support from
the Alberta Mennega Foundation. We would like to
acknowledge the very useful discussions and presentations at the April 2014 Linnean Society meeting
‘Collection-based research in the genomic era’, which
greatly aided our analysis of this data.
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
Figure S1. Transverse (A) and tangential (B) wood section of Populus trichocarpa showing more lignified
tissue, smaller alternate intervessel pitting (long arrows), and narrower and shorter rays (short arrows)
compared to Begonia burbidgei.
Table S1. List of specimens used in this paper, with reference to their voucher data, place of origin and habitat.
Voucher material is deposited in Sabah Parks (Sabah, Malaysia) and Forest Research Institute Malaysia.
Species with an asterisk are woody.
Table S2. GenBank accession numbers. GenBank accession numbers in bold font indicate sequences newly
generated for this study. All other sequences were downloaded from the nucleotide database of the National
Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
Table S3. Sequences differentially expressed between cambium stage and wood stage Begonia burbidgei
samples.
Table S4. Sequences with high divergence between woody Begonia burbidgei and herbaceous Begonia venustra.
Table S5. Begonia burbidgei sequences with matches in Populus trichocarpa but no matches in the genome of
herbaceous Begonia conchifolia.
Table S6. Sequences with differential expression between Begonia burbidgei stems and Populus trichocarpa
cambium.
Table S7. Sequences present in a transcriptome from Populus trichocarpa cambium but not in the Begonia
burbidgei transcriptome.
Table S8. Relative expression levels for each enzyme class between Begonia burbidgei and Populus
trichocarpa.
© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••