Follow-up Proposal - Claudia Voelckel
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Extension of Feodor Lynen Research Fellowship
Claudia Voelckel
Signatures of Speciation in Pachycladon – The Big Picture
Part I – Achievements to Date
1. So, What’s the Big Deal with these Plants, Again?
1.1 DNA Hybridizations
1.2 RNA Hybridization
1.3 Glucosinolate (GLS) Analysis
1.4 Summary and First Conclusions
2. Collaborations and Acquired Skills
Part II – Future Plans
1. Extension of the P. enysii – P. fastigiata Comparison
1.1 Secondary Metabolite Screening
1.2 Candidate Genes
2. Transcript, Protein and Metabolite Profiling Across the Entire
Pachycladon Radiation
2.1 Microarray vs. Sequencing-Based Expression Profiling
3. Academic Career Development
4. Tentative Schedule for Year 2
Presentations and Manuscripts
Literature
“Explain New Zealand and the rest
of the world falls into place.”
- Gareth Nelson
Overlooking the Tasman River valley while climbing Mt.
Hodgkinson in search for P. fastigiata
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Signatures of Speciation in Pachycladon – The Big Picture
What processes contributed to the evolution of new species and thus the biodiversity that
our own species is surrounded with?
During my Feodor Lynen Studies at the Allan Wilson Centre for Molecular Ecology and
Evolution I am addressing this question by studying speciation in New Zealand-endemic
alpine cress - the genus Pachycladon (1). In the Lockhart Lab, we seek to identify the
ecological traits that evolved differentially and the genes that most likely have been under
natural selection during the radiation of Pachycladon (2). Towards this goal, I conducted a
comparative gene expression and glucosinolate analysis in two closely related, ecologically
and morphologically very similar Pachycladon species – P. enysii and P. fastigiata which
differ in altitude preference and leaf hairiness. From this field study which integrates
transcriptional and biochemical data a suite of candidate genes emerged, which I would like
to investigate further in detailed expression and population genetic studies during a potential
year 2 under the Feodor Lynen Funding Scheme.
Moreover, I propose to extend the comparative gene expression and secondary metabolite
profiling to the entire Pachycladon radiation (3-6 species), eventually replacing array
technology with a new generation, sequencing-based expression profiling technique and
adding another level of analysis – the missing link between transcriptome and metabolome –
the proteome.
Both, the narrow (P. enysii-P. fastigiata) and the wider (Pachycladon radiation) comparison
will provide insights into ecological and molecular processes that drove speciation in
Pachycladon. This, in turn, will allow exciting comparisons with adaptive plant radiations
elsewhere in the world (e.g. Aquilegia and Mimulus) which are assumed to have been
pollinator- and habitat-driven (3-5). But unlike Aquilegia and Mimulus, all Pachycladon
species have unspecialized and inconspicuous flowers, leaving adaptation to different habitats
(possibly different geological substrates and associated soils) as a more likely ecological
driver for speciation than adaptations to different pollinators. By comparing adaptive
radiations across the plant kingdom we can identify the factors which enhanced or constrained
plant diversity in the past. Moreover, these past evolutionary lessons can teach us something
about how plants may diversify and adapt during future periods of climate change. The more
we understand which traits and genes enabled a particular lineage to conquer new habitats and
diversify during past environmental change the better we can assess its future evolutionary
potential.
Part I – Achievements to Date
1. So, What’s the Big Deal with these Plants, Again?
P. enysii and P. fastigiata evolved from an alpine ancestor less than 1Mya. Except for
altitude and leaf hairs they are very much alike. Hybrids between them are fertile. Then,
what’s driving them apart? Would gene expression reveal what else is different between
them, indiscernible by eye? What follows is a summary on gene expression and,
subsequently, glucosinolate profiling across natural populations of P. fastigiata and P. enysii
and some first thoughts on genes and traits under selection along two separate evolutionary
paths.
Figure 1
Rosettes and SEMs
of leaf surfaces of
P. fastigiata (PF)
and P. ensyii (PE).
PF rosette
PE rosette
PF leaves
PE leaves
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1.1 DNA Hybridizations
One of the reasons to study speciation in Pachycladon was its close phylogenetic proximity
to the model plant Arabidopsis thaliana for which a wealth of molecular resources is
available and possibly transferable to close relatives. A particular goal was to use 70mer oligo
microarrays spotted with the entire Arabidopsis genome (27,648 unigenes) for RNA
hybridizations. Cross-species hybridizations however bear the risk of misinterpreting
differences as expression differences when in fact they are caused by sequence divergence
between samples of interest. By hybridizing DNA of three Pachycladon species (P. enysii, P.
fastigiata, P. novae-zealandiae) to the array it could be demonstrated that neither species’
DNA hybridized differentially from the others and thus the probability of false positives due
to sequence divergence was negligible. Moreover, for 23,494 unigene probes the DNA
hybridization signal was above background, leading to an expected 85% of informative spots.
1.2 RNA Hybridizations
Sampling/Data Analysis. Young leaves of 15 plants from three P. enysii (PE) sites and
three P. fastigiata (PF) sites (Fig 2) were sampled, flash-frozen in liquid nitrogen and their
RNA was isolated. RNA from each site was pooled and RNA from each PE site was
competitively hybridized with RNA from at least two PF sites and vice versa according to the
scheme in Fig. 3a using a total of 12 microarrays. After raw data acquisition, background
corrections, filtering and normalization, a linear model was fitted for each gene and
differential expression was decided using a combination of 1.5fold change, adjusted p-values
and posterior probabilities as implemented in the R-based package limma
(www.bioconductor.org). Linear models were fitted twice, once to the ratio data to obtain
species differences and once to separate channel data to obtain expression values for each of
the six sites and model both within- and between-species differences.
1700m
E1
1640m
F2
1200m
E2
1900m
F3
1400m
E3
1780m
F1
Figure 2 Sampling locations of P. fastigiata (F1-3) and P. enysii (E1-E3). Both species occur on
South facing cliffs with P. fastigiata growing at lower elevations than P. enysii.
Results - Differentially Expressed Genes (DEGs), Gene Ontology (GO) & Global
Expression Patterns. Two lists of DEGs were obtained and conservatively intersected into
one (Fig. 3b). The results are robust to applying both the DNA and the RNA filter (23,494 vs
14,825 informative spots, respectively). A GO analysis was performed to determine which
biological processes were represented by the DEGs (Fig. 3c). PF-specific genes were
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particularly enriched in “plasticity genes” (genes responsive to the environment), while PEspecific genes were enriched in cell wall synthesis, glucosinolate synthesis, leucine synthesis
and translation genes. Transcription factors were equally frequent among PF- and PE-specific
genes, trichome differentiation genes may have been missed because the leaves were sampled
long after trichome initiation. Interestingly, high-altitude P. enysii differentially expresses
UV-B response genes. A complete list of DEGs (and more array data) can be found under
http://tur-www1.massey.ac.nz/~cvoelcke. A principal components analysis of 14,825
informative spots revealed higher heterogeneity among expression profiles of the PE sites
than the PF sites (Fig. 3d). Interestingly, the two most geographically close populations - F2
and F3 -were most similar in their expression profiles.
A
B
F1
F1
F2
F2
F3
F3
E1
E2
E2
E3
E3
E1
Analysis of
A) ratio data
B) channel data
A∩B
PF
285
285
240
PE
320
367
286
total
605
652
526
GO Term
response to stimulus
response to cold
response to stress
response to temperature stimulus
response to other organism
response to water deprivation
response to endogenous stimulus
cell wall organization & biogenesis
glucosinolate metabolic process
leucine biosynthetic process
translation
microtubule-based process
regulation of transcription
response to UV-B
trichome differentiation
Total
1901
129
741
210
267
82
645
137
19
5
436
76
1284
23
28
PF
40*
8*
19*
8*
9*
5*
15*
3
1
0
3
1
8
0
0
PE
22
2
10
3
0
0
8
8*
4*
2*
13*
5*
8
2
0
PC2
D
C
PC1
Figure 3. A Hybridization scheme B Number of differentially expressed genes for P. fastigiata (PF)
and P. enysii (PE) calculated from ratio data and channel data, respectively. C Categories of
significantly (*) enriched genes in PF and PE. D Principal components analysis of expression data as
obtained from 14,825 informative spots for the three PE and the three PF sites.
Results - Hypotheses Generated by Array Data. Among the DEGs were genes involved in
flavonoid and glucosinolate metabolism which led to several predictions regarding
biochemical differences between P. enysii and P. fastigiata, three of which are summarized in
Table 1.
Table 1
Process
Gene Regulation
TT7, FAH1
up in P. enysii
Response to Herbivory
AOP2, MAM1
Glucosinolate
up in P. enysii
biosynthesis
Response to UVB
Glucosinolate
breakdown
ESP up in P. enysii
ESM1 up in P. fastigiata
Predicted Biochemical Profiles
1.
Quercetin and sinapates
up in P. enysii
2.
C4 and Alkenyl glucosinolates
up in P. enysii
3.
P. enysii makes nitriles
P. fastigiata makes isothiocyanates
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1.3 Glucosinolate (GLS) Analysis
Freeze-dried material from the same samples used for gene expression profiling was
subjected to a GLS analysis using an Arabidopsis protocol (6). A total of 12 methioninederived GLSs were quantified in ninety plants. As predicted from differential gene
expression, PE was found to produce significantly more C4 and Alkenyl GLS than PF (Fig.
4a). Clustering identified 5 GLS chemotypes, three of which were specific to PE, two to PF
(Fig. 4b). Interestingly, these 5 chemotypes were not randomly distributed across sites, but
most individuals at any given site had the same chemotype except for F3, where half of the
individuals had chemotype 2 and the other half chemotype 5.
A
Proportion of total GLS
C3
Methylsulfinyl
C4
1
Alkenyl
1
0.5
0.5
0
0
E3 E2 E1 F3 F1 F2
E
F
E3 E2 E1 F3 F1 F2
E
F
Sites (E1-E3, F1-F3) and species (E, F)
B
Chemotype
#
Distribution
(Site, # Individ.)
12 Glucosinolates
E2, 1
1
E1, 11
2
F3, 8
F1, 14
3
E3, 13
E1, 5
4
E3, 2
E2, 14
5
F3, 6
Allyl
8MSOO
3 Butenyl
4MTB
3MTP
6MSOH
4MOI3M
5MSOP
5 Pentenyl
7MSOH
4MSOB
3MSOP
F2, 16
Figure 4. A Average C3, C4, Methylsulfinyl and Alkenyl Glucosinolates levels at the three PE and PF
sites and across species (F, E). PE produces significantly more C4 and Alkenyl glucosinolates
than PF. B 12 glucosinolate compounds cluster into 5 chemotypes, 3 of which are specific to PE and
2 to PF. Except for F3, each site is dominated by one chemotype.
1.4 Summary and First Conclusions
During the first few months of Pachycladon research many exciting discoveries were made.
First, Arabidopsis resources such as the genome-wide microarray and a high throughput GLS
HPLC assay were successfully applied to NZ alpine Pachycladon, making the system
amenable to molecular studies. Second, expression and GLS profiling revealed P. enysii
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populations to be more variable than P. fastigiata populations (Figs. 3d, 4b), which is
consistent with the differential glacial refugia hypothesis for both species (PE is assumed to
have survived the last ice age in mountain top refugia while PF is assumed to have been
extirpated from the central alps, leading to longer geographical isolation of PE populations
but continued gene flow between PF populations). Third, just 3.6% (526 of 14,825
informative spots) of the genes were differentially expressed between PE and PF rosette
leaves of natural populations, which is in line with given the short divergence time between
species. Differential gene expression patterns nonetheless generated several hypotheses, some
with general implications, and some immediately testable on the biochemical level. Given,
that many “environmental response genes” were more frequently regulated than expected by
chance in P. fastigiata, the general question arises if genes that are known to mediate plastic
responses are also prime candidates for genetic divergence during adaptive radiations.
Specific hypotheses arose from the differential expression of glucosinolate metabolism and
UVB response genes predicting inter-specific biochemical differences. Consistency of AOP2
and MAM1 expression and C4 and Alkenyl GSL production in P. enysii not only reveals
interesting species-specific patterns but also provides a link between transcriptional and
biochemical data.
2. Collaborations and Acquired Skills
For our research we rely on collaborations with groups in and outside New Zealand. For
example, without the support of the botanist team at Landcare Research Lincoln the extensive
Pachycladon sampling in the Southern Alps would not have been possible. Furthermore,
Landcare Research maintains seed supplies and provides growing facilities plus the green
thumb all of which is necessary to grow Pachycladon in future common garden experiments.
During several visits to Lincoln I established an excellent working relationship with the
people at Landcare Research. Microarray facilities were provided by HortResearch Auckland
and during my two visits to their RNA hybridization labs, I initiated a fruitful working
relationship there as well. Last but not least, ongoing ties to my home institute in Jena, the
MPI for Chemical Ecology, enabled the screening of my field samples for GLSs. All
collaborators contributed substantially to the success of my project and their expertise could
be relied upon during year 2 and beyond.
Personally, I acquired many new skills, most importantly, the use of the R programming
language for standard and advanced statistical analyses. I became familiar with R-based
microarray packages and Gene Ontology mining software. Being able to quickly analyze
array data sets using freeware only will make array analyses more transparent and widely
accessible to any researcher interested in gene expression profiling. The use of array freeware
will be of great value for authoring a teaching module paper on microarray analysis for
undergraduate students (see page 8).
Part 2 - Future Plans
Based on the findings during the previous months, several emerging question will be
addressed in parallel during the following year. Future experiments will mainly fall into two
categories: those, which continue to examine within- and between species differences in P.
enysii and P. fastigiata and those, which compare gene expression, protein synthesis, and
secondary metabolite levels across the entire Pachycladon radiation. The toolkit will continue
to contain RNA hybridizations to microarrays and HPLC-based GLS screenings, but will be
extended for two biochemical assays (flavonoid and GLS hydrolysis products screens),
quantitative PCR assays, a new-sequencing based expression profiling technique and massspectrometry based proteomics. I will work with both, natural populations and same-aged
common garden cohorts. The choice between those two will mainly be governed by scientific
reasoning but also by the feasibility of field trips and accessibility of growing facilities.
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1. Extension of the P. enysii – P. fastigiata Comparison
1.1 Secondary Metabolite Screening
I plan to follow up on biochemical differences between P. enysii and P. fastigiata in two
different ways. First, in collaboration with the Plant Physiology Department at Lincoln
University, I will analyze the remainder of my field samples for differences in flavonoids and
thus be able to test the hypothesis that a) higher altitude P. enysii either constitutively
expresses UVB response genes differentially from or b) exhibits a higher level of phenotypic
plasticity in UVB response than lower altitude P. fastigiata. Furthermore, in collaboration
with the MPI for Chemical Ecology, I will test if a previously among Arabidopsis accessions
described polymorphism in glucosinolate hydrolysis products (7-9) separates P. enysii from
P. fastigiata as suggested by my array data (Table 1). The differential production of either
nitriles or isothiocyanates, which influences herbivory in Arabidopsis, may be a wide-spread
defense mechanism in brassicaceous plants and the two underlying loci (ESP and ESM1),
may have evolved differently in P. enysii and P. fastigiata. PE and PF specimens will be
grown and screened for their hydrolysis products by Dr. Krügel and Dr. Reichelt from the
MPICE, respectively.
1.2 Candidate Genes
The study of candidate genes aims at discovering allelic variation that results in phenotypic
variation and understanding of how allelic polymorphisms are distributed across populations
and species (10-12). Ideally, I would like to study expression patterns and sequence variation
to determine a) the extent to which gene expression is influenced by genotype and
environment, b) how gene expression changes over plant development, c) if candidate genes
accumulated deleterious mutations in their coding or promoter regions and show signatures of
selection, and d) the frequency of different alleles across P. enysii and P. fastigiata
populations. An obvious choice for candidate genes are flavonoid genes (TT7, FAH1) and
GLS genes (MAM1, AOP2, AOP3, ESP, ESM1), because their expression and genotypes can
be directly linked to phenotypes. Other candidates are genes that exhibited large differences
in expression between P. enysii and P. fastigiata (http://tur-www1.massey.ac.nz/~cvoelcke).
Primer design for quantitative PCR and gene sequencing will initially be based on
Arabidopsis ESTs but will be facilitated later by a yet to be produced EST library and
database for Pachycladon. The NZ Genome Consortium, which the Allan Wilson Centre is
part of, has recently purchased both a GS FLX gene 454 sequencer and a Solexa 1G Genetic
Analyzer. These new generation sequencing technologies will be used to establish EST
libraries for a variety of New Zealand native species, among them Pachycladon.
2. Transcript, Protein and Metabolite Profiling Across the Entire Pachycladon
Radiation
Schist
Generalist
Figure 5.
Greywacke
Reconstruction of phylogenetic
relations in the Pachycladon
radiation (S. Joly, unpublished
data)
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One hypothesis on the evolutionary history of Pachycladon is that a P. cheesemanii-like
ancestor colonized NZ in the first place and later diversified by specializing on different
geological substrates (hence a schist and a greywacke subclade). With the uplifting of the
Southern Alps 2 Mya, a multitude of new habitats was created and like other NZ alpine
radiations, Pachycladon diversified by adapting to these new habitats. Extant species differ in
plant architecture (leaf, seed, fruit shape and size, trichomes), life history (polycarpic vs
monocarpic), specialization to geological substrate (generalist, schist, greywacke, limestone),
altitude and other traits (1). I will investigate the changes in gene expression, protein
synthesis and secondary metabolite levels that evolved during this radiation by applying the
tools from year 1 (array technology, GLS screen) and tools to be developed in year 2. The
latter include – aside from additional biochemical screens such as a flavonoid assay and a
GLS hydrolysis product assay – a new sequencing-based expression profiling technique using
the aforementioned Solexa Genome Analyzer. This machine was recently purchased by the
AWC and is scheduled to arrive in August. Moreover, during the “Plant Functional Genomics
Workshop” in Sydney, I discussed a potential collaboration with Dr. Paul Haynes from the
Australian Proteome Analysis Facility (hosted at Sydney’s Macquarie University). Dr.
Haynes is very interested in developing a proteomics assays for Pachycladon and I am very
excited to bridge the gap between expression and metabolite studies by characterizing
proteomes across the Pachycladon radiation. Material for the different screens will come from
synchronously germinated common garden populations of at least three but maximal six
species (1 or 2 representatives of each subclade) that will be harvested at several comparable
developmental stages. A first set of seeds has already been started at Landcare Research and a
second set will be started immediately after the completion of the growth room at the AWC
(September 07). Natural populations may be sampled as a backup (December 07).
By profiling transcripts, proteins and metabolites across the Pachycladon radiation we will
generate many new hypotheses regarding the traits and genes that diverged during the process
of speciation and adaptation.
2.1 Microarray vs. Sequencing-Based Expression Profiling
Over the next year we seek to test the potential of the Solexa Genome Analyzer in replacing
microarrays for gene expression profiling. We will start by comparing the same samples using
both platforms to determine the degree of correlation between results (13). First
pyrosequencing-based expression studies using the GS FLX gene 454 sequencer in
Arabidopsis have been very encouraging (14). Sequencing-based expression profiling may be
superior in many ways. For example, less starting material is required (compare 5 μg total
RNA for one run as compared to 100 μg total RNA for a dye flip hybridization), which
enables sampling from smaller plant parts (seedlings, flowers) and sampling from the same
plants over a time course. Moreover, Solexa sequencing is very likely to pick up any
transcript present in a sample whereas an array can only query transcripts that have been
spotted onto the array in the first place. Lastly, but very importantly, confounding effects due
to cross-species hybridizations will not have to be controlled for in the Solexa method as this
problem simply does not exist. Correct annotation of the short sequence tags generated by the
Solexa profiling will be ensured by aligning the tags to the Pachycladon EST library. Thus,
establishing the EST library and associated databases is crucial not only to the primer design
and cloning of candidate genes but also to the successful switch from array-based to Solexabased expression profiling and will be given priority over the next half year.
3. Academic Career Development
During my Lynen studies my priority will be conducting research on the genetic basis of
speciation. However, I will also take advantage of opportunities that have presented
themselves at the AWC to develop other skills essential for a successful academic career,
specifically teaching and mentoring. I am co-authoring a BioQUEST “investigatory module”
(http://www.bioquest.org/) based on my research. This module will be part of an anthology of
evolutionary bioinformatics activities for use in undergraduate teaching (“Evolutionary
Bioinformatics – Making Meaning of Molecular Messages”). I will test the suitability of my
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module by giving guest lectures in Pete Lockhart’s course on bioinformatics in the upcoming
spring semester. Moreover, there is the opportunity to supervise summer students at the
AWC, and also to contribute to BioEd 2009 (http://www.ldes.unige.ch/bioEd/cbe.htm), which
will be hosted by the AWC in February 2009 in celebration of Charles Darwin’s 200 th
birthday. This international meeting will bring together science educators, planners and policy
makers from around the world.
4. Tentative Schedule for Year 2
P. fastigiata vs P.
ensyii
GLS + GLS hydrolysis
Aug-07 screens at MPICE Jena
Pachycladon
Radiation Survey
Resource
Development
Common garden exp with
Delivery of the Solexa
6 species - 1. seed set
Genome Analyzer
started in Lincoln
QPCR assay & primer
Sep-07 development for candidate
Other Activities
Work on Teaching Module
"Testing Ecological &
Evolutionary Ideas with
Microarrays"
genes
Oct-07
Flavonoid screens at
Lincoln University
Common garden exp with
Completion of the growth
6 species - 2. seed set
room at AWC
started at the AWC
Send off samples to APAF
Solexa User training
for intial proteomics
Submit manuscript on
"Transcriptional and
Nov-07 biochemical signatures of
divergence in P. enysii
and P. fastigiata"
Field work: Collections of varios Pachycladon species
Dec-07 for DNA & RNA studies
Guest Lecture(s) in
Bioinformatics Course at
Massey
Work on Manuscript from
previous PostDoc on
"Expression patterns in
floral whorls of Aquilegia
formosa "
Jan-08
Feb-08
Mar-08
Apr-08
May-08
Jun-08
Harvest from common
garden populations or use
field samples for transcript,
Allele frequency and/or
protein and metabolite
expression studies of
profiling across 3-6
candidate genes
Pachycladon species:
depending on available
DNA or RNA material from Sampling scheme and
field collections
logistics of varios profiling
efforts still need to be
worked out
Jul-08
Aug-08
Sep-08 Summarize and publish
Oct-08 results
Development of EST
library and database for
Pachycladon (and other
New Zealand plant taxa)
(Group effort)
Annual New Zealand
Phylogenetics Meeting
"Whitianga08"
Society for Molecular
Biology and Evolution
2008 Annual Meeting,
Barcelona
Summarize and publish
results
Presentations and Manuscripts
Oral Presentations
1. Annual Meeting of the New Zealand Plant Radiation Network, Kaikoura, 14.-15.6.07
(http://awcmee.massey.ac.nz/NZPRN/index.htm)
2. Evolution 2007, Christchurch, 16.-21.6.07
(http://www.evolution2007.com)
3. “Plant Functional Genomics and Comparative Ecology Workshop”, Sydney, 26.-27.6.07
http://www.vegfunction.net/wg/12/12_forward.html
Poster Presentations
4. Annual Meeting of the International Society for Chemical Ecology, Jena, 22.-26.7.07
http://www.gdch.de/isce2007
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Manuscripts in Preparation
1. Voelckel C, Heenan P, Jansen B, Reichelt M, Hoffman R, Lockhart P.
“Transcriptional and biochemical signatures of divergence in P. fastigiata and P. enysii.”
2.
3.
Voelckel C, Borevitz J, Hodges SA.
“Differential gene expression in floral whorls of Aquilegia formosa.”
Voelckel C, Jansen B, Lockhart P.
“Testing ecological and evolutionary ideas with microarrays.”
In: Evolutionary bioinformatics: Making Meaning of Molecular Messages.
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