Research

A macro-ecological perspective on crassulacean acid metabolism

(CAM) photosynthesis evolution in Afro-Madagascan drylands:
Eulophiinae orchids as a case study
Ruth E. Bone1, J. Andrew C. Smith2, Nils Arrigo3 and Sven Buerki4
1
Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AE, UK; 2Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK; 3Department of Ecology

and Evolution, University of Lausanne, Lausanne CH-1015, Switzerland; 4Department of Life Sciences, Natural History Museum, Cromwell Road, London, SW7 5BD, UK

Summary
Author for correspondence:  Crassulacean acid metabolism (CAM) photosynthesis is an adaptation to water and atmo-
Ruth E. Bone spheric CO2 deficits that has been linked to diversification in dry-adapted plants. We investi-
Tel: +44 (0)208 332 3723
gated whether CAM evolution can be associated with the availability of new or alternative
Email: r.bone@kew.org
niches, using Eulophiinae orchids as a case study.
Received: 5 February 2015  Carbon isotope ratios, geographical and climate data, fossil records and DNA sequences
Accepted: 18 June 2015
were used to: assess the prevalence of CAM in Eulophiinae orchids; characterize the ecological
niche of extant taxa; infer divergence times; and estimate whether CAM is associated with
New Phytologist (2015) 208: 469–481 niche shifts.
doi: 10.1111/nph.13572  CAM evolved in four terrestrial lineages during the late Miocene/Pliocene, which have
uneven diversification patterns. These lineages originated in humid habitats and colonized
Key words: Africa, climate change, dry/seasonally dry environments in Africa and Madagascar. Additional key features (variega-
crassulacean acid metabolism (CAM) tion, heterophylly) evolved in the most species-rich CAM lineages. Dry habitats were also col-
photosynthesis, Eulophiinae, Madagascar, onized by a lineage that includes putative mycoheterotrophic taxa.
Orchidaceae, shift of niche.  These findings indicate that the switch to CAM is associated with environmental change.
With its suite of adaptive traits, this group of orchids represents a unique opportunity to study
the adaptations to dry environments, especially in the face of projected global aridification.

and stored in enlarged cell vacuoles (mostly as the C4 acid

Introduction
Climate change has shaped the evolution, composition and distri-
bution of ecosystems. Following mid-Miocene climate change
(Zachos et al., 2001) the origin and expansion of dryland biomes
have been linked to the increasingly arid conditions of the late
Miocene and Pliocene (Cerling et al., 1997; Tipple & Pagani,
2007), that are contemporaneous with the diversification of dry-
adapted plant lineages, and often associated with the evolution of
key innovations (Hern
andez-Hern
andez et al., 2014; Horn et al.,
2014).
These features may include leaf pigmentation by anthocyanins
(Rozema et al., 1997; Jansen et al., 1998), epidermis thickening
or epicuticular wax (Mulroy, 1979) that provide photoprotection
against increased exposure to UV insolation, leaf hairs that reduce
evapotranspiration (Ehleringer et al., 1976), or carbon-concen-
trating forms of photosynthesis when photorespiration is high
(Ehleringer et al., 1997; Edwards & Ogburn, 2012).
Crassulacean acid metabolism (CAM) is one such evolutionary
adaptation to environmental stress, in water-limited conditions.
By enabling nocturnal (rather than diurnal) opening of stomata
for transpiration and CO2 accumulation (Winter et al., 2008),
CAM plants reduce water loss. During the night, carbon is fixed
malate), and photosynthetically reduced in the Calvin cycle dur-
ing the following daylight period, when stomata are closed (Win-
ter & Smith, 1996). Phenotypic traits associated with CAM
include leaf or stem thickness or some degree of succulence due
to the increased number of mesophyll cell layers required for
long-term water storage, and increased mesophyll cell size to
accommodate enlarged cell vacuoles (L€ uttge, 2012; Silvera et al.,
2014).
The numerous records of CAM photosynthesis, across a diver-
sity of distantly related plant lineages, indicate that selective pres-
sures towards this photosynthetic pathway must have occurred
multiple times throughout evolutionary history (Winter &
Smith, 1996; Edwards & Ogburn, 2012; Keeley et al., 2012). Its
early origins in terrestrial environments have been linked to sea-
sonal extremes of drought stress and high temperatures in the
early to mid-Cretaceous in Gnetophyta (including Welwitschia
mirabilis). Modern CAM lineages (comprising c. 6% of vascular
plants in 35 angiosperm families (Winter & Smith, 1996; Silvera
et al., 2014)), however, are thought to have evolved from the late
Eocene onwards (Keeley et al., 2012), and are particularly associ-
ated with a decline in atmospheric CO2. Major diversificat-
ion events in Aloe and Euphorbia and in the other terrestrial

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CAM-dominated groups (Asparagaceae subfamily Agavoideae,
Aizoaceae and Cactaceae) occurred 10–5 Myr ago (Ma) in the
late Miocene/Pliocene, broadly coincident with the expansion of
terrestrial dryland ecosystems (Arakaki et al., 2011; Horn et al.,
2014; Grace et al., 2015) and C4 grasslands (Osborne, 2008;
Spriggs et al., 2014).
The transition from C3 to CAM photosynthesis, and diversifi-
cation of CAM lineages, has taken place not only in taxa confined
to arid terrestrial environments, but also in epiphytic plants of
more humid biomes that survive in water-limited microhabitats
such as forest canopies (Bromeliaceae (Crayn et al., 2004) and
Orchidaceae (e.g. Silvera et al., 2005, 2010)).
The Orchidaceae is the largest family of flowering plants, com-
prising over 27 000 species (of which 69% are epiphytic; Zotz,
2013) in 736 genera (Chase et al., 2015). The transition from ter-
restrial to epiphytic habit (Gravendeel et al., 2004) and the preva-
lence of CAM in epiphytic lineages (e.g. 26–100% of taxa in a
range of orchid groups; Silvera et al., 2009 and references therein)
have been suggested as triggers for adaptive radiations into new
niche space, that may help to explain the remarkable diversity of
the family (Silvera et al., 2009).
The Eulophiinae is a species-rich orchid subtribe (c. 270
species in nine genera; Bone et al., 2015) of Epidendroideae; a
subfamily comprising almost entirely epiphytes (90%; Zotz,
2013). Eulophiinae is unusual for being dominated by terrestrial
rather than epiphytic species (Freudenstein & Chase, 2015), with
species-poor epiphytic genera resolved as basal (Bone et al.,
2015). Previous studies have shown that at least six of these ter-
restrial taxa (belonging to Eulophia and Oeceoclades) evolved
towards CAM photosynthesis (Kluge et al., 1995; Silvera et al.,
2010). A recent phylogenetic study found evidence for the inde-
pendent evolution of Eulophia and Oeceoclades (Bone et al.,
2015), thus suggesting multiple origins of CAM photosynthesis
in this group. Unlike other CAM orchid lineages, that are mostly
adapted to more exposed microhabitats in forest canopies,
Eulophiinae are also unique because they underwent high diversi-
fication in dryland ecosystems.
Finally, in addition to exhibiting CAM photosynthesis, several
Eulophiinae species in the genus Eulophia are achlorophyllous or
have reduced or absent foliage leaves, and are assumed to have
evolved towards mycoheterotrophy (Cribb, 1977; La Croix &
Cribb, 1998). The only available study of mycoheterotrophic
(MH) Eulophiinae is for the tropical Asian species Eulophia
zollingeri, that occurs in humid forest in association with
saprophytic fungi (Ogura-Tsujita & Yukawa, 2008). Two other
studies of fungal symbionts in Eulophiinae include terrestrial
Afro-Madagascan taxa, but not those suspected to be MH
(Ochora et al., 2001; Yokoya et al., 2015). In south-central
Africa, putative MH Eulophia species grow in dry, deciduous
miombo woodlands that are dominated by Brachystegia (a genus
of ectomycorrhizal Leguminosae; H€ ogberg & Nylund, 1981).
We suggest that, in addition to CAM, this key innovation may
have provided an adaptation for this largely terrestrial subtribe to
survive in Afro-Madagascan drylands.
CAM has been investigated from biochemical, ecophysiologi-
cal (e.g. Borland et al., 1992; Winter & Smith, 1996; L€ uttge,
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2012) and genomic perspectives (e.g. Borland et al., 2014; Silvera
et al., 2014), and there is a growing literature on the evolutionary
histories of CAM plant lineages (such as Crayn et al., 2004;
Silvera et al., 2009; Arakaki et al., 2011; Horn et al., 2014). How-
ever, few studies deal with the evolution of CAM from an ecolog-
ical perspective (Hern
andez-Hern
andez et al., 2014 and
references therein).
Eulophiinae orchids provide a novel framework in which to
investigate the evolution of CAM photosynthesis in response to
climate change from a macro-ecological perspective. We identify
photosynthetic types across this lineage by using carbon isotope
ratios and leaf titratable acidity as measurable metabolic proxies
(Silvera et al., 2005). With the addition of a temporal scale (in-
ferred from the first dated phylogeny for the group) we recon-
struct CAM evolution along the phylogeny and discuss its origin
(s) in light of past climate change and aridification. We also
extract ecological data of extant species-distribution to character-
ize and compare the niches of CAM and C3 species. This study is
part of a bigger project aiming towards a mechanistic hypothesis
on the key climatic and biological features enabling CAM evolu-
tion in plants.
The following specific questions are investigated here:
 Did CAM photosynthesis evolve multiple times in Eulophiinae
and if so, when?
 Do Eulophiinae species exhibiting CAM photosynthesis
occupy a specific niche?
 Is there evidence for Eulophiinae species switching photosyn-
thetic pathway? If so, is this event associated with a shift of niche?
Materials and Methods
Carbon isotope analysis
Nocturnal fixation of atmospheric CO2 into malate (via oxaloac-
etate) is characteristic of CAM photosynthesis, and involves an
additional carboxylase step by phosphoenolpyruvate (PEPC) that
results in greater accumulation of 13C in CAM plants than in
those that perform C3 photosynthesis (O’Leary, 1988). Carbon
isotope ratios (13C/12C) can therefore be interpreted as a proxy
for photosynthetic pathway (Osmond et al., 1973; Silvera et al.,
2005). We used carbon isotope measurements from herbarium
specimens (dried leaf samples) to infer whether each taxon in our
study was CAM or C3.
Taxon sampling was based on the phylogenetic study by Bone
et al. (2015) and availability of herbarium material for carbon iso-
tope screening (Supporting Information Table S1). Small frag-
ments (≤ 3 mg) of dried leaf tissue were collected from one
specimen per species available at the Royal Botanic Gardens
(RBG) Kew (UK). Carbon stable isotopic composition (d13C)
was measured with an isotope ratio mass spectrometer at the
Research Laboratory for Archaeology and the History of Art,
School of Archaeology, University of Oxford. 13C/12C ratios
were measured against a secondary standard calibrated against
Vienna Pee Dee Belemnite (VPDB; Winter & Smith, 1996). We
categorized species into C3 or CAM based on leaf d 13C values
commonly observed for C3 plants ranging from –33& to –22&
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472 Research
so that the prior encompassed the lower (older) bounds of the
fossil age. DNA substitution, molecular clock and speciation
model settings were assigned to partitions following published
methods for these data (Guo et al., 2012; Bone et al., 2015). Five
runs of 100 million generations were computed in BEAST v1.8
(Drummond et al., 2012) on the CiPRES phylocluster (Miller
et al., 2010), sampling one tree every 10 000 generations. Log
files were viewed in Tracer (Rambaut et al., 2013) to check for
convergence between runs and adequate sampling of tree space
(ESS values were all > 200). Tree files generated by the first two
runs to reach convergence were combined using LogCombiner
(part of the BEAST v1.8 package), with burn-in set to 25%. Aver-
age branch lengths and 95% confidence intervals on nodes were
calculated using TreeAnnotator (part of the BEAST v1.8 package)
on the Maximum Clade Credibility tree.
For the second analysis we estimated divergence times within
the Eulophiinae subtribe, by using three plastid markers (matK,
rpL32-trnL and trnQ-rps16) from the data matrix of Bone et al.
(2015). The estimated crown age of Eulophiinae (from the fam-
ily-level analysis) was used as a secondary calibration point with a
normal distribution on the prior, the mean based on the crown
age, and the standard deviation based on the highest posterior
density (HPD) interval. Run settings and model parameters were
as before, and output files were summarized as previously
described.
The availability of DNA sequence data was uneven among taxa
(i.e. some taxa were represented by multiple accessions in the
phylogeny, whereas only a single accession was available for
others). Because this bias could influence our subsequent analyses
of diversification, ecological niche and ancestral character estima-
tion, we followed the procedure employed by Buerki et al. (2013)
and pruned the phylogeny to one accession per species by using
the drop.tip function in the R package ape (Paradis et al., 2004).
In order to investigate diversification processes in Eulophiinae
and putative shifts of diversification rates in light of either cli-
mate change or the origin of CAM photosynthesis, two types of
analyses were conducted. First, we inferred lineage-through-time
(LTT) plots using the R package ape on 100 randomly selected
BEAST trees after burn-in as well as on the Maximum Clade
Credibility tree. Second, shifts in diversification rates were
inferred using a birth–death model and a maximum-likelihood
criterion as implemented in the R package TreePar (Stadler,
2011) on the same set of trees as the LTT analyses. We took
advantage of the function implemented in TreePar and
accounted for taxon sampling bias (53% of the taxa were
sampled).
C3/CAM photosynthesis evolution
For each Eulophiinae species, we scored photosynthetic pathway
(binary scoring as C3 or CAM; see the carbon isotope and titra-
tion sections above) and reconstructed the ancestral character-
state using the maximum-likelihood method implemented in the
R package ape and the function ace by setting the type argument
to discrete (Paradis et al., 2004). To be conservative in our ances-
tral-state reconstruction (i.e. to minimize the number of shifts),
New Phytologist (2015) 208: 469–481
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Phytologist
species with intermediate carbon-isotope values were scored as
C3, although we recognize that this does not preclude the possi-
bility that such species might have the potential to perform a low
level of nocturnal CO2 fixation under certain conditions (Silvera
et al., 2010). The ancestral reconstruction was performed on the
BEAST Maximum Clade Credibility tree.
Results
Carbon-isotope ratios and titratable acidity
Carbon-isotope ratios in the range of 33& to 10& were
recorded (Table S1), confirming that both CAM and C3 photo-
synthetic pathways occur in Eulophiinae orchids. A frequency
plot of these values revealed a bimodal distribution (Fig. 1) repre-
senting 58 C3 and 19 CAM species, with four species exhibiting
intermediate values between 22& and 18&. These four
species do not show typical CAM traits (see Introduction for
more details), and it is probable that they are C3 or C3–CAM
intermediates; however, further work is needed to characterize
the photosynthetic pathway of these taxa.
Among taxa with d13C values indicative of CAM photosynthe-
sis, two taxa were represented in the living collections at RBG
Kew (Eulophia petersii and Oeceoclades calcarata; 14.57& and
14.44&, respectively). A third taxon with a C3-type d13C value
(E. guineensis, 27.08&) was available for comparison. Leaf
titratable acidity tests revealed a substantial nocturnal acid accu-
mulation in E. petersii (DH+ 39.5 mmol kg 1 fresh mass) and
O. calcarata (DH+ 59.2 mmol kg 1 fresh mass), confirming these
taxa as obligate CAM species (Table 1). Acid accumulation in
20
Eulophia guineensis
C3
15
Number of taxa
10
CAM
Intermediate Eulophia petersii
Oeceoclades calcarata
5
0
−35 −30 −25 −20 −15 −10
Carbon-isotope ratios (δ 13C, ‰)
Fig. 1 Histogram of frequency of observed carbon-isotope ratios plotted
using class intervals of 2&. Downward pointing arrows indicate d13C
values observed for each species sampled for leaf titratable acidity. CAM,
crassulacean acid metabolism.
Ó 2015 The Authors
New Phytologist Ó 2015 New Phytologist Trust

472 Research
so that the prior encompassed the lower (older) bounds of the
fossil age. DNA substitution, molecular clock and speciation
model settings were assigned to partitions following published
methods for these data (Guo et al., 2012; Bone et al., 2015). Five
runs of 100 million generations were computed in BEAST v1.8
(Drummond et al., 2012) on the CiPRES phylocluster (Miller
et al., 2010), sampling one tree every 10 000 generations. Log
files were viewed in Tracer (Rambaut et al., 2013) to check for
convergence between runs and adequate sampling of tree space
(ESS values were all > 200). Tree files generated by the first two
runs to reach convergence were combined using LogCombiner
(part of the BEAST v1.8 package), with burn-in set to 25%. Aver-
age branch lengths and 95% confidence intervals on nodes were
calculated using TreeAnnotator (part of the BEAST v1.8 package)
on the Maximum Clade Credibility tree.
For the second analysis we estimated divergence times within
the Eulophiinae subtribe, by using three plastid markers (matK,
rpL32-trnL and trnQ-rps16) from the data matrix of Bone et al.
(2015). The estimated crown age of Eulophiinae (from the fam-
ily-level analysis) was used as a secondary calibration point with a
normal distribution on the prior, the mean based on the crown
age, and the standard deviation based on the highest posterior
density (HPD) interval. Run settings and model parameters were
as before, and output files were summarized as previously
described.
The availability of DNA sequence data was uneven among taxa
(i.e. some taxa were represented by multiple accessions in the
phylogeny, whereas only a single accession was available for
others). Because this bias could influence our subsequent analyses
of diversification, ecological niche and ancestral character estima-
tion, we followed the procedure employed by Buerki et al. (2013)
and pruned the phylogeny to one accession per species by using
the drop.tip function in the R package ape (Paradis et al., 2004).
In order to investigate diversification processes in Eulophiinae
and putative shifts of diversification rates in light of either cli-
mate change or the origin of CAM photosynthesis, two types of
analyses were conducted. First, we inferred lineage-through-time
(LTT) plots using the R package ape on 100 randomly selected
BEAST trees after burn-in as well as on the Maximum Clade
Credibility tree. Second, shifts in diversification rates were
inferred using a birth–death model and a maximum-likelihood
criterion as implemented in the R package TreePar (Stadler,
2011) on the same set of trees as the LTT analyses. We took
advantage of the function implemented in TreePar and
accounted for taxon sampling bias (53% of the taxa were
sampled).
C3/CAM photosynthesis evolution
For each Eulophiinae species, we scored photosynthetic pathway
(binary scoring as C3 or CAM; see the carbon isotope and titra-
tion sections above) and reconstructed the ancestral character-
state using the maximum-likelihood method implemented in the
R package ape and the function ace by setting the type argument
to discrete (Paradis et al., 2004). To be conservative in our ances-
tral-state reconstruction (i.e. to minimize the number of shifts),
New Phytologist (2015) 208: 469–481
www.newphytologist.com
New
Phytologist
species with intermediate carbon-isotope values were scored as
C3, although we recognize that this does not preclude the possi-
bility that such species might have the potential to perform a low
level of nocturnal CO2 fixation under certain conditions (Silvera
et al., 2010). The ancestral reconstruction was performed on the
BEAST Maximum Clade Credibility tree.
Results
Carbon-isotope ratios and titratable acidity
Carbon-isotope ratios in the range of 33& to 10& were
recorded (Table S1), confirming that both CAM and C3 photo-
synthetic pathways occur in Eulophiinae orchids. A frequency
plot of these values revealed a bimodal distribution (Fig. 1) repre-
senting 58 C3 and 19 CAM species, with four species exhibiting
intermediate values between 22& and 18&. These four
species do not show typical CAM traits (see Introduction for
more details), and it is probable that they are C3 or C3–CAM
intermediates; however, further work is needed to characterize
the photosynthetic pathway of these taxa.
Among taxa with d13C values indicative of CAM photosynthe-
sis, two taxa were represented in the living collections at RBG
Kew (Eulophia petersii and Oeceoclades calcarata; 14.57& and
14.44&, respectively). A third taxon with a C3-type d13C value
(E. guineensis, 27.08&) was available for comparison. Leaf
titratable acidity tests revealed a substantial nocturnal acid accu-
mulation in E. petersii (DH+ 39.5 mmol kg 1 fresh mass) and
O. calcarata (DH+ 59.2 mmol kg 1 fresh mass), confirming these
taxa as obligate CAM species (Table 1). Acid accumulation in
20
Eulophia guineensis
C3
15
Number of taxa
10
CAM
Intermediate Eulophia petersii
Oeceoclades calcarata
5
0
−35 −30 −25 −20 −15 −10
Carbon-isotope ratios (δ 13C, ‰)
Fig. 1 Histogram of frequency of observed carbon-isotope ratios plotted
using class intervals of 2&. Downward pointing arrows indicate d13C
values observed for each species sampled for leaf titratable acidity. CAM,
crassulacean acid metabolism.
Ó 2015 The Authors
New Phytologist Ó 2015 New Phytologist Trust
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Phytologist
Table 1 Leaf titratable acidity results for three Eulophiinae taxa, showing ‘dusk’ values (PM), ‘dawn’ values (AM), and overnight acid accumulation (DH+)
Accession
Sample Species numbers Date (d/month/yr)
PM Eulophia petersii 19811982 11/09/2014
Oeceoclades calcarata 19752487 11/09/2014
Eulophia guineensis 19991738 11/09/2014
AM Eulophia petersii 19811982 12/09/2014
Oeceoclades calcarata 19752487 12/09/2014
Eulophia guineensis 19991738 12/09/2014
Values of titratable acidity for ‘dawn’ and ‘dusk’ samples are means  SE of three replicates.
E. guineensis was considerably lower (DH+ 14.2 mmol kg 1 fresh
mass), indicating that although this species had a C3-type d13C
value, it appeared to have the capacity for a very small amount of
nocturnal CO2 fixation.
Ecological niche
Data from a total of 5969 georeferenced herbarium specimen
records were gathered, with an average of 73.7 specimens per
species (outliers were one specimen each for Eulophia callichroma
and E. eylesii, and 782 for Geodorum densiflorum).
In the PCA of climatic descriptors (BioClim variables; Fig. S1)
the first two Principal Components (PC) axes explained a com-
bined 66% of variance in the data (Figs 2a, S1). The first PC axis
accounted for 44% of the variance and discriminated habitats by
temperature and seasonality (BioClim variables 2, 4 and 7 vs all
the others; Figs 2a, S1); PC axis 2 (accounting for 22% of the
variance) generally discriminated dry/hot habitats environments
from cold/humid ones (Figs 2a, S1).
Comparison of generalized habitat types (Fig. 2a) against
species-locations (Fig. 2b) revealed that species occurring in the
wettest/most humid habitats included the four epiphytic
Eulophiinae genera Cymbidiella, Eulophiella, Grammangis and
Paralophia (circled in black in Fig. 2b). Species resolved as
drought/heat-tolerant species (PCA values smaller than zero) are
mostly in the genus Oeceoclades, whereas species occurring in
more seasonal habitats are mostly Eulophia, with one or two
species of Orthochilus. By contrast, ecological niches were not
adequately described for several taxa from the mostly South
African genera Acrolophia and Orthochilus (that were plotted in
‘niche space’ with an absence of BioClim variables; Fig. S1).
These genera predominantly occur in dry grasslands or Mediter-
ranean-type habitats of the Cape (Martos et al., 2014) (Fig. 2a),
and their ecological niche does not seem to be explained by the
best fit of the BioClim variables (first two PC axes).
Phylogeny, dating and diversification analyses
Results from the orchid family-level analysis (not shown but
available upon request) suggest a Late Cretaceous origin for
Orchidaceae (at c. 100 Ma, with 95% HPD: 76–126 Ma), con-
gruent with previous studies (Ramirez et al., 2007; Gustafsson
et al., 2010; Guo et al., 2012). The crown-age of Eulophiinae was
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Titratable acidity DH+ (mmol kg 1
Time (mmol kg 1 fresh mass) fresh mass)
20:04 83.3  1.4
20:02 53.3  0
20:00 58.9  1.4
06:56 123.0  0 39.6
06:59 112.5  0 59.2
06:58 73.1  1.5 14.2
estimated at 20 Ma in the early Miocene (95% HPD: 16–26 Ma,
encompassing the Oligocene–Miocene boundary at 23 Ma). This
date was used as a secondary calibration point for further analy-
sis.
The dated Eulophiinae phylogeny (Fig. S2) reveals that basal
lineages comprise C3, mostly epiphytic taxa that occur in humid
environments (Figs 2, 3a, S2). Strong support (1.0 Bayesian pos-
terior probability, hereafter bpp) was recovered for the mono-
phyly of all terrestrial genera, except Eulophia which is resolved as
polyphyletic (in agreement with Bone et al., 2015). Among the
terrestrial genera, estimated divergence times range from the old-
est at 12 Ma (95% HPD: 7.4–17 Ma) for the predominantly
southern African genus Orthochilus, to the most recent at 5.4 Ma
(95% HPD: 2.7–8.6 Ma) for the Cape endemic genus
Acrolophia. Within Eulophia, a group that contains putative
mycoheterotrophic taxa is also resolved in a well-supported clade
(1.0 bpp), with an estimated crown age of 5.3 Ma (95% HPD:
2.8–8.8 Ma).
Although not statistically supported, the diversification analy-
ses concurred in estimating a shift in diversification rate (average
rate over the 100 trees of 0.28–0.63 and 0.08 and 0.15 in the case
of the Maximum Credibility tree) during the late Miocene (at an
average of 7.9 Ma for the 100 randomly selected trees and 6.0 Ma
for the Maximum Credibility tree) (Fig. 3b). As will be high-
lighted in the next section, this shift corresponds with the first
origin of CAM photosynthesis in Eulophiinae. Because these
results are not statistically significant, we will only consider them
as complementary information to support the discussion.
C3/CAM photosynthesis evolution
The relative probability for the two alternative ancestral states
(CAM (scored as 1) or C3 (scored as 0)) was > 95% in all cases;
therefore branches are coloured red (CAM) and black (C3), to
represent > 95% probability values. Maximum-likelihood esti-
mates suggested that C3 was the ancestral state and CAM the
derived state in Eulophiinae, and that CAM photosynthesis
evolved up to four times independently across the subtribe
(branches and/or tips coloured red; CAM 1–4 in Fig. 3a). In each
of these lineages, the transition from C3 to CAM may have
occurred at any time between the inferred node ages of the ances-
tral C3 stem group and the CAM crown group (Christin et al.,
2008).
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(a)
Precipitation

0.3
Axis 2 (22% variance explained)
Humid forest

0.2
0.1
Swamps Mediterranean

0.0
Dry grasslands

−0.3 −0.2 −0.1

Dry forest Seasonal
grasslands

J M J M

Temperature Seasonality

−0.2 0.0 0.2 0.4

Axis 1 (44% variance explained)

(b) Eulophiella elisabethae

Oeceoclades ugandae

Oeceoclades pulchra
Paralophia epiphytica
Cymbidiella flabellata
0.3

Cymbidiella falcigera

Eulophiella roempleriana

Grammangis ellisii
0.2

Eulophia plantaginea
Acrolophia lunata
Acrolophia bolusii Acrolophia ustulata
Axis 2 (22% variance explained)

Eulophia eylesii

Acrolophia lamellata
Orthochilus rutenbergianus
Eulophia horsfallii Acrolophia cochlearis

Eulophia stachyodes Acrolophia micrantha

Eulophia ibityensis
0.1

Eulophia macowanii Eulophia parvilabris

Oeceoclades saundersianaEulophia euglossa Eulophia macra
Orthochilus aculeatus
Eulophia callichroma Eulophia tenella Eulophia parviflora

Orthochilus odontoglossus Orthochilus ensatus Eulophia calanthoides Eulophia zeyheriana

Eulophia streptopetala Orthochilus foliosus
0.0

Eulophia acutilabra
Orthochilus leontoglossusEulophia meleagris

CAM 4 Eulophia clitellifera

Eulophia longisepala
Eulophia reticulata
Orthochilus welwitschii
Orthochilus milnei Eulophia livingstoneana
Oeceoclades spathulifera Geodorum densiflorum
Eulophia angolensis
Eulophia flavopurpurea
−0.1

Eulophia cucullata
Eulophia andamanensis Orthochilus trilamellatus
Oeceoclades gracillima
Eulophia coeloglossa Eulophia petersii
Oeceoclades maculata
Eulophia guineensis Eulophia speciosa
CAM 2
Eulophia schweinfurthii
Eulophia malangana Eulophia fridericii
Orthochilus
Eulophia euanthus
euantha
Eulophia latilabris
Eulophia seleensis
Eulophia graminea
−0.2

Eulophia arenicola
Oeceoclades calcarata
CAM 1 Eulophia venulosa
Eulophia calantha Eulophia ramosa

Eulophia nyasae Eulophia hereroensis

Eulophia gonychila
Oeceoclades petiolata Oeceoclades angustifolia
Oeceoclades Oeceoclades antsingyensis
Oeceoclades beravensis Oeceoclades decaryana
cordylinophylla
−0.3

Eulophia monile
CAM 3
Eulophia leachii

−0.2 0.0 0.2 0.4

Axis 1 (44% variance explained)
Fig. 2 (a) Generalized habitat types overlaid on the principal component analysis (PCA) plot (b). Inset: cartoons showing a comparison of the generalized
leaf morphology of crassulacean acid metabolism (CAM) 1 taxa, and the CAM 4 taxa with putative heterophylly (J, juvenile to M, mature). (b) PCA plot
showing species-locations in ecological niche space inferred from the 19 BioClim variables. The niche of each species was summarized on the PCA by
computing the median eigenaxes for each species. Species are colour-coded according to whether C3 (black), CAM, (red), or intermediate (grey); taxa in
the four CAM lineages are highlighted in grey, with red text; a black line encircles taxa from the four epiphytic genera that occur in the wettest regions;
taxa included in leaf titratable acidity tests are highlighted in bold underlined text.

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Miocene Pliocene Pleistocene Holocene

(a)
Grammangis ellisii
Cymbidiella falcigera
Cymbidiella flabellata
Eulophiella roempleriana
Eulophiella elisabethae
Orthochilus leontoglossus
Orthochilus aculeatus
Orthochilus euanthus
Orthochilus holubii
Orthochilus foliosus
Orthochilus ensatus
Orthochilus rutenbergianus
Orthochilus welwitschii
Orthochilus trilamellatus
Orthochilus odontoglossus
Orthochilus milnei
Paralophia epiphytica
Oeceoclades gracillima
Oeceoclades saundersiana
Oeceoclades spathulifera
1 Oeceoclades pulchra
Oeceoclades ugandae
CAM 1 Oeceoclades maculata
Oeceoclades antsingyensis
Oeceoclades beravensis
Oeceoclades petiolata
Oeceoclades cordylinophylla
Oeceoclades calcarata
Oeceoclades angustifolia
Oeceoclades decaryana
Eulophia macra
Eulophia graminea
Acrolophia lunata
Acrolophia lamellata
Acrolophia ustulata
Acrolophia micrantha
Acrolophia bolusii
Acrolophia cochlearis
Eulophia ramosa
Eulophia callichroma
p
Eulophia y
ibityensis
E l hi livingstoneana
Eulophia li i t
E l phi plantaginea
Eulophia pl t gi
Eulophia cucullata
E l hi calantha
Eulophia l th
E l hi flavopurpurea
Eulophia flavopu
v r
rpurea
E l hi coeloglossa
Eulophia l l
1 E l hi latilabris
Eulophia latilab
l til bris
ri
E l hi horsfallii
Eulophia hors
h ffallii
E l hi angolensis
Eulophia
Eulophia
E l phi malangana
l g
E l hi acutilabra
Eulophia til b
Eulophia seleensis
1 Eulophia nyasae
Eulophia eylesii
Eulophia venulosa
CAM 2 Eulophia monile
Eulophia longisepala
Eulophia petersii
Eulophia andamanensis
Eulophia stachyodes
Eulophia euglossa
Eulophia hereroensis
0.79 Eulophia parviflora
Eulophia streptopetala
Eulophia parvilabris
Eulophia macowanii
Eulophia calanthoides
Eulophia meleagris
CAM 3 Eulophia tenella
Eulophia zeyheriana
Eulophia leachii
Geodorum densiflorum
Eulophia guineensis
Eulophia gonychila
0.84 Eulophia fridericii
Eulophia schweinfurthii
Eulophia speciosa
1 Eulophia arenicola
Eulophia reticulata
CAM 4 Eulophia clitellifera

15 10 5 0
Time (Myr)
(b)
80

Late Miocene reduction in atmos-

pheric CO2 and the expansion of
dryland ecosystems
60
Number of taxa
40
20

CAM clades
0

15 10 5 0
Time (Myr)

Fig. 3 (a) Ancestral character-state estimation based on the BEAST Maximum Clade Credibility tree. Numbers below branches are support values (Bayesian
posterior probabilities; see Supporting Information Fig. S2 for details). Branches/tips are coloured red (crassulacean acid metabolism (CAM)) and black
(C3), to represent > 95% support values for the relative probability for the two alternative ancestral states (CAM (scored as 1) or C3 (scored as 0)), which
was > 95% in all cases. Black-filled diamonds indicate epiphytic species; the mushroom symbol indicates a clade of putative mycoheterotrophic taxa; the
green box is referred to in the text and highlights an example of grassland Eulophia species. (b) Lineage through time plot (LTT). In black, LTT of Maximum
Clade Credibility tree. Vertical black line, time of diversification rate shift detected by TreePar analysis. In grey: LTT of 100 randomly selected trees from the
BEAST analysis. Vertical grey line, time of mean diversification rate shift for 100 randomly selected trees. Grey box, Miocene–Pliocene aridification and
expansion of dryland ecosystems.

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Table 2 Age estimates of the four Eulophiinae crassulacean acid
metabolism (CAM) lineages
Lineage Stem group age Crown group age
CAM 1 11.5 (5.6–18.1) 6.2 (3.3–10.1)
CAM 2 9.5 (5.8–13.7) – these lineages occupy different niches along a climatic gradient
CAM 3 7.8 (4.7–11.5) – associated with dry and seasonally dry habitats (Figs 1–3). This
CAM 4 6.5 (3.7–9.6) 4.3 (2.3–6.9)
Lineage names correspond to Fig. 3(a). For each lineage, the age of the
origin of CAM is interpreted as falling between the stem group node age
(maximum age) and the crown group node age (minimum age). Due to
the presence of a single taxon in CAM 2 and CAM 3, the crown group
node age could not be inferred for these lineages. Node ages are reported
in Myr with the highest posterior density (HPD) interval in parentheses.
CAM first arose in the Eulophiinae 11.5–6.2 Ma (CAM 1;
Table 2) in the late Miocene (Walker et al., 2012). The CAM 1
clade comprises all Oeceoclades taxa sampled (1.0 bpp) and repre-
sents the largest diversification of CAM species in the subtribe
with c. 12 species (66% of CAM taxa in our study). It is the only
clade that includes a mixture of CAM and C3 species (Oeceoclades
pulchra); however, our DNA regions do not provide adequate
phylogenetic resolution to assess the position of O. pulchra within
this clade with confidence (namely sister to CAM species or
nested among CAM species).
More recent diversification is inferred in CAM 4, compris-
ing five Eulophia species (1.0 bpp; 6.5–4.3 Ma; Table 2).
Other CAM lineages comprise single taxa: CAM 2 (E. petersii)
and CAM 3 (E. leachii), for which CAM is estimated to have
evolved at a maximum of 9.5 and 7.8 Ma, respectively (inferred
from stem node ages; Table 2). Species with intermediate d13C
values occur in several clades and do not appear to be clus-
tered, with the exception of E. acutilabra and E. nyasae that are
resolved in the clade of suspected partial mycoheterotrophs
(Fig. 3a).
CAM lineages overlaid on the PCA (Fig. 2b) reveal that the
CAM 1 clade has the greatest niche breadth, with the majority of
CAM species occurring in dry habitats and a single CAM species
in humid environments (Oeceoclades ugandae), sharing a similar
niche to the only C3 species in this clade, O. pulchra. (CAM 1;
Figs 2b, 3a). The remaining CAM lineages are all within the
genus Eulophia and occur in niches that are characterized by pro-
nounced seasonality (PCA axis 1; Figs S1, 2a).
Discussion
The Eulophiinae (Epidendroideae) subtribe provides a rare
opportunity to investigate CAM photosynthesis in terrestrial,
rather than epiphytic orchids. This study aims at providing for
the first time a macro-ecological hypothesis on the evolution of
CAM photosynthesis in this group using a combination of car-
bon-isotope screening, leaf titratable acidity tests, a dated molec-
ular phylogeny and a suite of environmental variables. Unlike
other groups of plants that underwent large diversifications in dry
environments and are almost entirely CAM (e.g. Aloe (Grace
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et al., 2015), Asparagaceae subfamily Agavoideae (Good-Avila
et al., 2006), Aizoaceae subfamily Ruschioideae (Klak et al.,
2004) and Cactaceae (Arakaki et al., 2011)), here we have
inferred at least four independent origins of CAM photosynthesis
within Eulophiinae in the late Miocene, and demonstrated that
framework provides an opportunity to investigate the ecological
and morphological features associated with the evolution of
CAM photosynthesis and will also provide a strong basis upon
which to conduct further genomic analyses (e.g. to identify the
specific ppc genes involved in the evolution of CAM photosyn-
thesis, following Christin et al. (2014) and Silvera et al. (2014)).
Finally, we also show that CAM photosynthesis is not the only
adaptive trait to facilitate diversification in dry environments, as
shown by one lineage of Eulophiinae that evolved towards myco-
heterotrophy. Below we discuss each question posed in the
Introduction.
Did CAM photosynthesis evolve multiple times in
Eulophiinae – and if so, when?
Unlike most CAM epidendroid orchids, that grow in humid
cloud forest (e.g. Silvera et al., 2009, 2010), our study has
demonstrated that CAM Eulophiinae species are terrestrial and
associated with dry to seasonally dry ecosystems (Fig. 3). The
transition from epiphytic to terrestrial habit in Eulophiinae
appears to have coincided with the progressive aridification
worldwide that followed the mid-Miocene climatic optimum
(Zachos et al., 2001; Fig. 3). This period may have triggered
evolution of the terrestrial habit in these ancestrally epiphytic
orchid lineages due to expansion of open habitats and grasslands
in Africa and Madagascar (Beerling & Osborne, 2006; Tipple
& Pagani, 2007; Horn et al., 2014). Although the transition
from C3 to CAM photosynthesis was apparently not directly
associated with a shift of growth habit, our data show that the
transition from epiphytism to the terrestrial habit at the end of
the Miocene was strongly associated with colonization of dry
environments (see PCA; Fig. 2), and several key innovations
evolved subsequently in response to aridity. These key innova-
tions are CAM photosynthesis (in four independent lineages)
and putative mycoheterotrophy (in one lineage; Fig. 3a). The
latter feature may provide an efficient way of reducing water
loss in arid environments (Rasmussen, 1995) and will be further
discussed below.
The origins of Eulophiinae CAM photosynthesis lineages from
the late Miocene onwards resulted in three contrasting species
diversification patterns: One ‘species-rich’ clade (CAM 1; 12
species sampled, but the genus has 39 species, most of these
expected to be CAM), one ‘species-poor’ clade (CAM 4; five
species) and two single-species lineages (CAM 2 and CAM 3)
(Fig. 3a). The late Miocene also corresponds to the expansion of
arid environments in the southern hemisphere, especially in
Africa (Jacobs, 2004; Dupont et al., 2011). We will further dis-
cuss the patterns of diversification and distribution ranges of each
of the CAM lineages below.
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seasonality (Fig. 2). Colonization of xeric and seasonally dry

Do Eulophiinae species exhibiting CAM photosynthesis
occupy a specific niche?
The ecological analyses showed that the four CAM lineages
occupy different niches along a climatic gradient reflecting sea-
sonality in dry environments (PCA axis 1, Fig. 2). The most
speciose CAM 1 (Oeceoclades) clade is mainly restricted to dry
forests, with the exception of O. ugandae that occurs in humid
habitats while retaining a CAM photosynthesis mode (see ‘Is
there evidence for Eulophiinae species switching photosynthetic
pathway? If so, is this event associated with a shift of niche?’ for
more discussion on the shift of niche observed in this lineage).
The other CAM lineages are found in habitats characterized by
habitats is not always associated with a shift to CAM photosyn-
thesis. For instance, south-central African grassland has been col-
onized by one clade of Eulophia (green box in Fig. 3), yielding at
least 10 species in which CAM has not appeared.
This study also reveals a radiation in a clade of putative partial
mycoheterotrophs that have colonized dry forests in south central
Africa (Fig. 3a), indicative of adaptation following a separate evo-
lutionary path among these terrestrial orchids. By sourcing car-
bon via fungal partners, rather than via photosynthesis directly
from atmospheric CO2 (Bidartondo, 2005), full and partial
mycoheterotrophs reduce the need for a functional photosyn-
thetic apparatus (chlorophyllous leaves (Bidartondo et al., 2004)

(a) (b) (c)

(d) (e) (f)

Fig. 4 Examples of morphological variation

among terrestrial Eulophiinae lineages:
crassulacean acid metabolism (CAM) 1 (a–c),
CAM 2 (d), CAM 3 (e), a typical Eulophia
species (f), CAM 4 (g, h). (a) Leaves of (g) (h)
Oeceoclades cf. maculata and (b)
O. spathulifera showing typical variegation
patterns; (c) leaves and habit of the C3
species O. pulchra; (d) mature thickened
leaves of Eulohpia petersii*; (e) Eulophia
leachii leaf shoot with flower (inset); (f) a
typical Eulophia species, Eulophia
guineensis*; (g) Eulophia speciosa with
broad plicate leaves at maturity; (h) a juvenile
semi-succulent leaf shoot of the same
species. Asterisks indicate taxa that were
included in leaf titratable acidity tests.
Photographs courtesy of: (a–c) Moritz
Grubenmann; (d–g) the first author, R.E.B.;
(h) Lari Merrett.

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and stomata (Yukawa & Stern, 2002)), thus reducing water loss
via evapotranspiration and increasing tolerance of shaded, low-
light environments. These characteristics may have enabled
Eulophia to colonize dry forest environments in association with
ectomycorrhizal fungi, as has been suggested for Cymbidium
(Motomura et al., 2010). The dominance of the ectomycorrhizal
legume Brachystegia in the dry, deciduous miombo woodlands
(where most of the mycoheterotrophic Eulophia occur) would
support this hypothesis that will need to be formally tested.
Diversification patterns in CAM lineages
CAM 1 and CAM 4 originated relatively recently during the late
Miocene/larly Pliocene climatic oscillations in Madagascar, and
south-central Africa, respectively. The success of the CAM 1
clade might be due to the evolution of CAM photosynthesis in
association with a range of morphological traits of the leaves that
are unique among Eulophiinae: Leaves of the CAM species
O. ugandae resemble the thin, herbaceous leaf morphology of the
C3 species O. pulchra (Fig. 4c), that shares a similar ecological
niche (Figs 2, 3), whereas the majority of CAM Oeceoclades
species grow in dry, deciduous forests and are characterized by
thickened, evergreen leaves, often with distinctive variegation
(white spotting to densely mottled, or ‘snakeskin’ patterns;
Fig. 4a,b). Givnish (1990) interpreted leaf-mottling as camou-
flage against herbivores in ‘sun-dappled’ understorey environ-
ments, particularly among low-growing evergreen plants that
persist during periods of deciduous plant senescence. Empirical
evidence for a reduction in herbivory has been shown for several
plant groups (Soltau et al., 2009; Lev-Yadun, 2015). The reduced
photosynthetic activity of white-mottled leaves (caused by an
absence of chlorophyll) may also reduce photosynthetic activity
during periods of high irradiance, when the deciduous woodland
canopy is absent (Givnish, 1990).
In Oeceoclades gracillima and O. spathulifera (Fig. 4b) purplish
colouration suggests the presence of anthocyanins that may also
provide photoprotection under high UV radiation (Rozema
et al., 2002; Campanella et al., 2014). Anthocyanins have been
associated with the increased success of a CAM species (Agave
striata; in a study by Fondom et al. (2009)), in addition to her-
bivory-defence in several plant groups, particularly for juvenile
leaves (Hansen et al., 2004; Fadzly et al., 2009).
The second lineage of CAM plants that has diversified (CAM
4; a derived clade within Eulophia), occurs in seasonal grassland
habitats and dry forest margins in southern and eastern Africa.
These taxa occur in habitats that fluctuate in seasonal water avail-
ability, with periods of high precipitation and flooding. In con-
trast to the other CAM lineages, these taxa do not exhibit the
typical succulent or thickened leaf morphology, but rather exhibit
a pattern of leaf heterophylly (juvenile–adult leaf dimorphism)
that has been observed by the first author in two species of this
clade (one in Madagascar, one in Africa), in which juvenile leaves
are succulent, whereas mature leaves are grass-like (Figs 2a, 4g,h).
Carbon-isotope ratios were measured from mature leaves for
these taxa (Table S1), suggesting that either CAM photosynthesis
is retained despite an absence of pronounced succulence in the
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mature leaves, or conceivably that there is a shift to C4 (which
would show similar d13C values). Further work is needed to
investigate the leaf morphology of these taxa (including titration
tests on living material which are not currently available) to ascer-
tain whether mature leaves of these taxa are CAM or C4. Both C4
and CAM pathways have evolved in four plant families (Keeley
et al., 2012), although this has not yet been reported for Orchi-
daceae.
The two remaining CAM lineages (CAM 2 and CAM 3) are
each represented by a single species. Eulophia petersii (CAM 2) is
an isolated CAM taxon, sister to a clade containing both C3 and
CAM species (CAM 3 and CAM 4). With varying degrees of suc-
culence to its leaves, serrated leaf margins (Fig. 4d) and branched
inflorescences up to 3 m tall (La Croix & Cribb, 1998), the range
of this aloe-like species encompasses south-central Africa to the
Arabian Peninsula, extending as far east as Oman (WCSP, 2015)
where it occurs on coastal cliffs (D. Lupton, pers. comm.).
Among the few Eulophiinae orchids with broad geographical dis-
tributions, this species appears to have undergone range expan-
sion, tracking aridification across continental Africa (similar to
Aloe).
By contrast Eulophia leachii (CAM 3; Fig. 4e), which has simi-
lar morphological traits to E. petersii (La Croix & Cribb, 1998),
has a narrower geographic and ecological range, being limited to
low-altitude dry and riverine forest margins in southern Africa
(La Croix & Cribb, 1998), suggesting that this species could have
undergone range contraction or have reached the limit of its
available niche.
Is there evidence for Eulophiinae species switching
photosynthetic pathway? If so, is this event associated with
a shift of niche?
CAM 1 is the only clade in Eulophiinae that is dominated by
CAM taxa and that also contains a C3 species: Oeceoclades
pulchra, the species with the broadest geographical distribution of
any Eulophiinae orchid (WCSP, 2015). The ancestral character
estimation suggested CAM as the ancestral state for this clade
with a single reversal to C3 in the case of O. pulchra. However,
further taxon sampling and additional DNA regions are required
to resolve relationships within this clade with confidence, and
determine the ecological origin of CAM photosynthesis in these
taxa (i.e. whether in dry or humid habitats). Moreover, to further
test these hypotheses and provide more precise estimates of how
many times CAM photosynthesis has evolved in this clade, we
aim to sequence genes directly involved in photosynthesis (e.g.
PEPC enzyme encoding genes in the ppc gene family).
The clustering of CAM taxa in hot/dry niche-space vs one
CAM (O. ugandae) and one C3 taxon (O. pulchra) occurring in
the wet/humid niche (associated with epiphytic Eulophiinae
genera), suggests that evolution of photosynthetic pathway may
also be associated with shifts of niche. CAM 1 is therefore a
suitable candidate for further studies of shift in photosynthetic
pathway. Furthermore, investigation of the suite of leaf-mor-
phology traits in Oeceoclades (described above) may have poten-
tial to deepen our understanding of plant evolution in
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conditions that are generally considered unfavourable to plant
survival.
Perspectives
The multiple origins of CAM photosynthesis since the late
Miocene/early Pliocene, and the relationship between photosyn-
thetic types and ecological factors, could lead to future studies on
the response of plants to present-day climate change and habitat
fragmentation (which magnifies the effect of climate change and
drives desertification; Vitousek et al., 1997) using this subtribe as
a case study. Moreover, this group provides a novel framework
within which to investigate a range of functional questions, such
as the underlying genetic architecture of CAM traits across these
four lineages, and whether this shift follows distinct evolutionary
paths, thus shedding light on the link between phenotype and
genotype in the transition from C3 to CAM in terrestrial orchids
(as has been investigated for C3 to C4 transitions in the grasses,
e.g. by Christin et al. (2007, 2009, 2012)).
Acknowledgements
This research was funded by the Swiss Orchid Foundation.
We are grateful to Royal Botanic Gardens (RBG) Kew (UK)
for additional funding for carbon isotope measurements; Iain
Farrell (RBG) for his guidance and expertise with the titra-
tions; and Andre Schuiteman for permission to sample speci-
mens in the Orchid Herbarium (RBG). Thanks to
participants at the New Phytologist Symposium ‘Next Genera-
tion Scientists’ (2014) for discussions that helped to shape the
ideas presented here; to Yan-Yan Guo for providing the
orchid family dataset, and to Moritz Grubenmann and Lari
Merrett for permission to include their photographs. We are
grateful to the reviewers and Editor for their valuable com-
ments that greatly improved the manuscript.
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 BioClim climatic descriptors and their corresponding
coordinates on eigenaxes produced using principal components
analysis.
Fig. S2 BEAST Maximum Clade Credibility tree.
Table S1 Details of herbarium specimens sampled for carbon
stable isotopic composition (d13C) measurements
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