Molecular phylogenies disprove a hypothesized C4reversion in
Eragrostis walteri (Poaceae)
Amanda L. Ingram1,*, Pascal-Antoine Christin2,3and Colin P. Osborne4
1Department of Biology, Wabash College, Crawfordsville, IN 47933, USA,2Department of Ecology and Evolution, University of
Lausanne, 1015 Lausanne, Switzerland,3Department of Ecology and Evolutionary Biology, Brown University, Providence, RI
02912, USA and4Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK
* For correspondence. E-mail email@example.com
Received: 18 August 2010Returned for revision: 28 September 2010 Accepted: 25 October 2010Published electronically: 23 November 2010
†Background and Aims The main assemblage of the grass subfamily Chloridoideae is the largest known clade of
C4plant species, with the notable exception of Eragrostis walteri Pilg., whose leaf anatomy has been described as
typical of C3plants. Eragrostis walteri is therefore classically hypothesized to represent an exceptional example
of evolutionary reversion from C4to C3photosynthesis. Here this hypothesis is tested by verifying the photosyn-
thetic type of E. walteri and its classification.
†Methods Carbon isotope analyses were used to determine the photosynthetic pathway of several E. walteri
accessions, and phylogenetic analyses of plastid rbcL and ndhF and nuclear internal transcribed spacer DNA
sequences were used to establish the phylogenetic position of the species.
†Results Carbon isotope analyses confirmed that E. walteri is a C3plant. However, phylogenetic analyses
demonstrate that this species has been misclassified, showing that E. walteri is positioned outside
Chloridoideae in Arundinoideae, a subfamily comprised entirely of C3species.
†Conclusions The long-standing hypothesis of C4to C3reversion in E. walteri is rejected, and the classification
of this species needs to be re-evaluated.
Key words: C4photosynthesis, evolution, reversion, Eragrostis, Chloridoideae, Arundinoideae, Poaceae, Africa,
Complex traits have received a great deal of attention by evol-
tionality of transitions between their states and, in particular,
about their reversibility (e.g. Collin and Miglietta, 2008; Tripp
and Manos, 2008; Lynch and Wagner, 2009). C4photosynthesis
is a prime example of a complex trait due to the numerous mor-
phological, anatomical and biochemical adaptations relative to
ancestral C3photosynthesisthat are required for proper function.
These adaptations are thought to involve hundreds of genetic
changes (Bra ¨utigam et al., 2010), but nonetheless have been
demonstrated to be evolutionarily labile. C4photosynthesis has
evolved numerous times independently in distantly related
plant families during the past 30 million years, with .50 inde-
pendent origins inferred in the angiosperms (Giussani et al.,
2001; Kadereit et al., 2003; Sage, 2004; Muhaidat et al., 2007;
Christin et al., 2008; Besnard et al., 2009). The majority of C4
plant species belong to the grass family, in which C4taxa form
a minimum of 17 different lineages separated in the phylogeny
taxa within the PACMAD clade (Panicoideae,
Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae
and Danthonioideae; Sage, 2004; Christin et al., 2008).
Comparisons of the phenotype and genotype of the C4traits
used by these different C4 phylogenetic groups in grasses
suggest that most derive from independent C4origins (Christin
et al., 2010), ranking C4photosynthesis amongst the most con-
vergent of complex traits (Conway-Morris, 2006). Surprisingly,
very few putative losses of the C4pathway and recovery of the
ancestral C3 trait have been identified. Two exceptions are
Alloteropsis semialata (R.Br.) Hitchc. subsp. eckloniana (Nees)
Gibbs Russ., a C3 subspecies nested in a C4 clade of
Panicoideae (Ibrahim et al., 2009), and Eragrostis walteri Pilg.
Eragrostis walteri is a grass endemic to Namibia that was
described in 1941 (Pilger, 1941) and that possesses many mor-
phological features typical of Eragrostis species, including
multifloreted spikelets, paniculate inflorescences and ciliate
ligules. This placement within Eragrostis was confirmed by
phylogenetic analyses of morphological data (van den Borre
and Watson, 1994). Eragrostis contains about 400 species dis-
tributed worldwide and is placed in the main assemblage of
Chloridoideae, which contains .1400 species. Numerous
Chloridoideae species have been studied for leaf anatomy
and/or carbon isotope composition, and all have been found
to be C4, making this group the largest wholly C4clade in
plants. This fact made the report of non-C4leaf anatomy in
E. walteri by Ellis (1984) particularly striking. The photosyn-
thetic pathway employed by Ellis’ E. walteri specimens was
subsequently confirmed by d13C analysis (Schulze et al.,
1996). The presence of a C3plant in an otherwise C4clade
was strongly suggestive of an evolutionary loss of C4photo-
synthesis. Consequently, E. walteri has been repeatedly cited
as the best candidate for C4to C3reversion for the last 25
years (e.g. Renvoize, 1987; Morrone and Zuloaga, 1991;
Kellogg, 1999; Kubien et al., 2008; Ibrahim et al., 2009;
Edwards and Smith, 2010; Roalson, 2011).
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Given that the leaf anatomy of E. walteri shows no evidence
of partially C4characters, confirmation of this putative C4to
C3reversion in Eragrostis would demonstrate that C4evol-
ution is reversible. This case is particularly remarkable
because the reversion would probably have occurred tens of
millions of years after the initial C4origin in Chloridoideae,
which is estimated to have occurred between 25 and 32
million years ago (Christin et al., 2008). Furthermore, the
Chloridoideae encompasses numerous species with well-
optimized C4 characters that confer ecological success in
many of the world’s biomes. If the hypothesis of reversion
from C4to C3photosynthesis was proven, E. walteri would
represent an outstanding system in which to investigate the
(Christin et al., 2010). However, it is necessary first to
confirm that E. walteri does in fact use C3photosynthesis
Additionally, the assumption that E. walteri is nested in a C4
clade relies solely on morphological evidence that has not
been confirmed with genetic markers. This is crucial, since
polyphyly has been demonstrated for several grass genera
upon phylogenetic analysis of genetic data (e.g. Aliscioni
et al., 2003; Peterson et al., 2010).
In this study, we investigated the likelihood of an evolution-
ary reversion from C4to C3photosynthesis in E. walteri to
gain insights into the reversibility of C4evolution. To test
the hypothesis of reversion, our work aimed to: (a) verify
the photosynthetic type of several E. walteri specimens using
unambiguous methods; and (b) determine the phylogenetic
position of E. walteri using genetic markers from the plastid
and nuclear genomes.
MATERIALS AND METHODS
Carbon isotope ratio
The C4pathway is defined by the fixation of atmospheric CO2
through a coupling of carbonic anhydrase and phosphoenol-
pyruvate carboxylase, whereas in C3plants this fixation is per-
formed by ribulose-1,5-bisphosphate carboxylase. These
enzymes differentially discriminate between the carbon iso-
topes naturally present in the atmosphere, resulting in different
ratios of carbon isotopes in the plants that can be determined
by mass spectrometry. Values of d13C between –21 ‰ and
–32 ‰ are indicative of C3photosynthesis, while C4plants
have d13C between –9 ‰ and –16 ‰. Some well-developed
C3–C4intermediates can have d13C values between –16 ‰
and –19 ‰, but there is no overlap between the d13C values
of wholly C3and C4species (von Caemmerer, 1992).
Foliar d13C values were determined on six herbarium
samples whose identification as Eragrostis walteri had been
verified by T. A. Cope and M. Vorontsova by reference to
the specimen collection held at RBG Kew. A 20 mg sub-
sample from each was analysed using an ANCA GSL prep-
aration module coupled to a 20–20 stable isotope analyser
(PDZ Europa, Cheshire, UK). Measurements on the same
sample had a reproducibility of 0.5 ‰, and the isotopic com-
position of each (d13C) was calculated as the sample13C/12C
ratio relative to the PDB standard (‰).
Two herbarium specimens of E. walteri (K:Kolberg &
Tholkes 695 and PRE:Hines 262) were selected for genetic
analyses. The Kolberg & Tholkes 695 sample was analysed
in Lausanne, while the Hines 262 sample was analysed at
Wabash College. No laboratory products or PCR primers
were shared between these laboratories, excluding the possi-
bility of contamination. At Lausanne, two plastid markers
(rbcL and ndhF) were amplified and sequenced using a pre-
viously published methodology (Christin et al., 2008). At
Wabash College, rbcL was amplified and sequenced with
new primers designedspecifically
(5′-GGGACTTATGTCACCACAAAC-3′) and 1433R (5′-AC
carried out as described in Ingram and Doyle (2003) with an
annealing temperature of 558C. DNA sequencing was com-
pleted by the Cornell BioResource Center. All plastid
sequences were added to a previously published data set
with representatives of all Poaceae subfamilies (Christin
et al., 2008). Phylogenetic methods are as previously
described (Christin et al., 2008). An independent estimate of
phylogeny was obtained with sequences from the nuclear
ribosomal DNA internal transcribed spacer (ITS). New grass-
specific ITS primers were used for amplification and sequen-
G-3′) and ITS 26S-R-grass (5′-GACGCCTCTCCAGACTAC
AA-3′)]. PCR was as described in Ingram and Doyle (2003)
with annealing temperatures of 56 8C. ITS PCR products
were sequenced directly. An ITS data set was assembled
from sequences deposited in GenBank, which contains
several thousand ITS sequences for grasses. The selected
PACMAD families, with Pooideae outgroups. Details on the
species and GenBank accession numbers can be found in the
Supplementary Data (available online). The sequences were
aligned with ClustalX (Larkin et al., 2007), and a phylogenetic
tree was inferred as described for the plastid markers (Christin
et al., 2008). All E. walteri sequences were deposited in
GenBank (accession numbers HQ329788–HQ329791).
RESULTS AND DISCUSSION
The d13C values (Table 1) ranged between –24.3 ‰ and
–29.1 ‰ for the six E. walteri accessions, which unambigu-
ously indicates that these plants assimilated carbon via C3
photosynthesis. This confirms previous conclusions (Ellis,
1984; Schulze et al., 1996) with independent samples of
The rbcL sequences for E. walteri obtained independently in
the two different laboratories were identical. In the phylogeny,
however, they did not group with Chloridoideae as expected
from morphology (van den Borre and Watson, 1994).
Instead, E. walteri was placed within the Arundinoideae,
sister to a clade composed of Molinia and Phragmites
(Fig. 1). This position within Arundinoideae was strongly sup-
ported (Bayesian support values
E. walteri ITS sequence showed the highest similarity with
Molinia caerulea (94%) followed by Phragmites spp. (90–
91 %).The Bayesian inference
.0.95). Blasting the
Ingram et al. — Disproving a C4reversion in Eragrostis walteri322
relationship between E. walteri ITS and those of Molinia and
Phragmites (Fig. 2), congruent with the results from the plastid
markers. This relationship was also highly supported (Bayesian
support value of 1.0). Therefore, both nuclear and plastid
markers show that E. walteri does not belong to Eragrostis,
nor to Chloridoideae, but is unambiguously a member of
Eragrostis is not entirely surprising when morphological fea-
tures are more carefully examined. Eragrostis is a highly het-
erogeneous group, but E. walteri is an outlier in some
otherwise invariable traits. For example, Eragrostis lemmas
are consistently three-nerved, but Pilger (1941) noted in his
original description of E. walteri that this species has three
prominent and two inconspicuous nerves on the lemmas. In
addition, E. walteri lemma apices have been described as
‘nearly awned’ (Watson and Dallwitz, 1992 onwards), in con-
trast to the acute lemma apices found in most other species in
the genus. The discrepancy between morphological classifi-
cation and molecular phylogenies mirrors the numerous
cases of polyphyletic genera in grasses (e.g. Giussani et al.,
2001; Aliscioni et al., 2003; Peterson et al., 2010).
Because molecular data indicate that E. walteri belongs to
Arundinoideae, its C3 type is no longer surprising, as all
other species of Arundinoideae are also C3. Therefore, the
hypothesis that this species is a C4to C3revertant should be
abandoned. With our current understanding of grass phyloge-
netics and photosynthetic pathways, Alloteropsis semialata
subsp. eckloniana is the only plausible C4 revertant in
grasses and should now be more closely investigated to
detect traces of C4loss (Christin et al., 2010). However, this
new discovery regarding the misclassification of E. walteri
clearly demonstrates the dominance of C3to C4transitions
over reversions, suggesting that C4 evolution is almost
always a one-way event. The resolution of the enigma
created by the peculiar foliar anatomy of E. walteri also high-
lights the importance of working with species or even acces-
sions as evolutionary units, and the risks of extrapolating
phylogenetic positions from congenerics. While grass phylo-
geny is far from being resolved at the species level, efforts
should be put into incorporating as many of the evolutionarily
interesting taxa as possible, until an exhaustive phylogeny is
obtained. This could reveal other surprises, including the non-
monophyly of numerous morphological taxonomic units, even
at the subfamily level, as for E. walteri.
TABLE 1. Stable carbon isotope ratio (d13C) for leaf material of
Eragrostis walteri (Pilg.)
Collector and collection number
Giess, W. 8977
Giess, W. 8104A
Kolberg, H. and Tholkes, T. 695
Giess, W. 10413
Giess, W. and Mu ¨ller, M. 14316
Range, P. 14831
All specimens were collected in southern Africa and were identified and
archived in the herbarium of the Royal Botanic Gardens, Kew.
Eragrostis walteri Hines 262
Eragrostis walteri Kolberg & Tholkes 695
FIG. 1. Phylogenetic position of E. walteri inferred from plastid markers. This tree was obtained through Bayesian inference based on ndhF and rbcL sequences.
Bayesian support values are indicated near nodes. The main groups are compressed. Clades containing only C3taxa are in yellow, those containing only C4taxa
are in red, and those containing both C3and C4taxa are in orange. For further details on the data set see Christin et al. (2008).
Ingram et al. — Disproving a C4reversion in Eragrostis walteri323
Supplementary data are available online at www.aob.oxford
journals.org and give the GenBank accession numbers of the
species used in the phylogenetic analysis of ITS data.
We thank David Simpson, Rosa Cerros and other herbarium
staff at Royal Botanic Gardens, Kew (K) and the South
African National Biodiversity Institute (PRE) for assistance
in acquiring E. walteri material. We also thank J. Travis
Columbus for designing the rbcL and ITS primers. This
work was supported by the US National Science Foundation
[grant number DEB-0921203 to A.L.I.]; the Swiss National
Science Foundation [grant number PBLAP3-129423 to
P.A.C.]; and a Royal Society University Research Fellowship
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