Journal of Experimental Botany, Vol. 68, No. 2 pp. e11–e28, 2017
doi:10.1093/jxb/erx005 DARWIN REVIEW
A portrait of the C4 photosynthetic family on the 50th
anniversary of its discovery: species number, evolutionary
lineages, and Hall of Fame
Rowan F. Sage*
Department of Ecology and Evolutionary Biology, University of Toronto, 25 Willcocks Street, Toronto, ON M5R3C6
* Correspondence: [email protected]
Received 5 February 2016; Accepted 1 March 2016
Editor: Donald Ort, University of Illinois This article is was originally published in JXB volume 67 issue 14, pages 4039-4056.
Please cite as: Rowan F. Sage; A portrait of the C 4 photosynthetic family on the 50th anniversary of its discovery: species number, evolutionary
lineages, and Hall of Fame. J Exp Bot 2016; 67 (14): 4039-4056. doi: 10.1093/jxb/erw156
Abstract
Fifty years ago, the C4 photosynthetic pathway was first characterized. In the subsequent five decades, much has
been learned about C4 plants, such that it is now possible to place nearly all C4 species into their respective evolutionary lineages. Sixty-one independent lineages of C4 photosynthesis are identified, with additional, ancillary C4 origins
possible in 12 of these principal lineages. The lineages produced ~8100 C4 species (5044 grasses, 1322 sedges, and
1777 eudicots). Using midpoints of stem and crown node dates in their respective phylogenies, the oldest and most
speciose C4 lineage is the grass lineage Chloridoideae, estimated to be near 30 million years old. Most C4 lineages are
estimated to be younger than 15 million years. Older C4 lineages tend to be more speciose, while those younger than
7 million years have <43 species each. To further highlight C4 photosynthesis for a 50th anniversary snapshot, a Hall of
Fame comprised of the 40 most significant C4 species is presented. Over the next 50 years, preservation of the Earth’s
C4 diversity is a concern, largely because of habitat loss due to elevated CO2 effects, invasive species, and expanded
agricultural activities. Ironically, some members of the C4 Hall of Fame are leading threats to the natural C4 flora due
to their association with human activities on landscapes where most C4 plants occur.
Key words: Biodiversity, C4 Hall of Fame, C4 plants, convergent evolution, Cyperaceae, photosynthetic evolution, Poaceae.
Introduction
Fifty years ago, Hatch and Slack (1966) published the first report
of a four-carbon CO2 fixation pathway identified in sugarcane.
Within a year, Osmond (1967) reported a similar pathway in
the herbaceous eudicot Atriplex spongiosa. These reports galvanized a global effort to understand patterns long associated
with plants of warm habitats (Hatch et al., 1971; Hatch, 2002;
Berry, 2012). These patterns included an association between
Kranz vascular sheath anatomy and dimorphic chloroplasts
from grasses and weedy dicots of warm climates (Haberlandt,
1914; Moser, 1934; Hodge et al., 1955; Laetsch, 1974). In forage sciences, Kranz grasses were known as ‘sour’ grasses of less
nutritional content than the Kranz-free ‘sweet’ grasses, and,
in the dry tropics, Kranz grasses dominated lowland agriculture while Kranz-free grasses were grown at higher elevations
(Sage and Pearcy, 2000). Gas exchange patterns in Kranz species differed from that of most plants, with photosynthetic CO2
© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
For permissions, please email: [email protected]
e12 | Sage
compensation points near 0 µmol CO2 mol−1 air, minimal stimulation of photosynthesis following O2 reduction, and low CO2
saturation points of carbon assimilation (Moss, 1962; Hesketh,
1963; El-Sharkawy and Hesketh, 1965; Bjorkman, 1966;
Tregunna et al., 1964, 1966). With the elucidation of the C4
biochemistry in 1966 and 1967, these disparate patterns finally
made sense, and, within a few years, they had been synthesized
into the textbook paradigm of C4 photosynthesis, where structure and function are modified to create a CO2-concentrating
mechanism that suppresses photorespiration, thereby improving photosynthesis in warm climates (see Table 1 for the main
requirements of C4 photosynthesis; Downton and Tregunna,
1968; Black et al., 1969; Downton et al., 1969; Berry et al., 1970;
and contributions in Hatch et al., 1971).
These early accomplishments formed the foundation for
subsequent research achievements, which now, 50 years on,
provide a deep understanding of C4 photosynthesis that resonates well beyond the plant sciences, to influence developments
in paleontology (de Menocal, 2004; Stromberg, 2011; Cerling,
et al. 2015), anthropology (van der Merwe and Tschauner,
1999; Cerling et al., 2013; Levin, 2015), global change science
(Leakey, 2009; Bond and Midgely, 2012; Hoetzel et al., 2013),
and energy policy (Samson et al., 2005; Bush, 2006; Heaton
et al., 2008). Today, C4 photosynthesis is recognized as one
of the most dynamic examples of convergent evolution, with
dozens of independent origins across the plant Kingdom
(Sage et al., 2011a). This is surprising, because C4 photosynthesis is a complex trait whose evolution requires changes to
hundreds if not thousands of genes (Hibberd and Covshoff,
2010; Gowik et al., 2011). Why, when, and how C4 photosynthesis evolved has been a major topic since the C4 synthesis of
the late 1960s (Evans, 1971; Monson et al., 1984; Ehleringer
et al., 1991, 1997; Beerling and Osborne, 2006; Edwards et al.,
2010; Sage et al., 2012). Through concerted efforts in many
disciplines, a consensus has arisen that the C4 lineages evolved
recently in geological time, beginning 30–35 million years ago
(Mya) and continuing until recent millennia (Kellogg, 1999;
Christin et al., 2008, 2011a; Vincentini et al., 2008; Edwards
et al., 2010; Stromberg, 2011). This was a time when atmospheric CO2 levels declined to near current levels, and global
climate change was creating dry, seasonally variable landscapes in the subtropics and temperate zones (Stromberg,
2011; Zhang et al., 2013). Physiological arguments linked the
decline in CO2 and water availability to enhanced photorespiration, which then created the selection pressure favouring C4 evolution (Ehleringer et al., 1991, 1997; Rawsthorne,
1992; Osborne and Sack, 2012; Sage et al., 2012). With the
discovery of C3–C4 intermediate species beginning in the
mid-1970s (Kennedy and Laetsch, 1974; Edwards and Ku,
1987), genotypes became available to test the physiological
predictions, and to build conceptual and theoretical models
explaining evolutionary origins of C4 plants (Monson et al.,
1984; Rawsthorne, 1992; Sage, 2004; Heckmann et al., 2013).
While the physiological advantage of the C4 pathway
explains the general pattern of C4 abundance in warm but not
cool environments, and allows niche broadening (Lundgren
et al., 2015), it is too simplistic to explain many biogeographic
patterns across the Earth, such as the frequent prevalence of
C3-dominated forests over C4 grasslands at low to mid latitudes (Sage et al., 1999b; Bond, 2008). When incorporated into
ecological studies, however, C4 physiology improves ecological
understanding and the ability to explain vegetation patterns.
As a case in point, photosynthetic performance and the efficiency of water and nitrogen use of woody C3 species are generally inferior to those of C4 grasses in tropical and subtropical
climates; this leads to slower growth and establishment of the
C3 vegetation (Medina and Klinge, 1983; Jones et al., 1992;
Knapp and Medina, 1999; Wedin, 2004). For reasons that
are not fully understood, the C4 pathway is absent in trees,
with the exception of a few rare species in Hawaii (Table 2).
C4 shrubs largely occur in dry or salinized landscapes, such as
the Sahara and Central Asian deserts, so they contribute little if at all to the woody flora of moist landscapes (Sage et al,
1999b). As a result, outside of dry regions, the C4 pathway is
restricted to the herbaceous layer, such that C4 plants will be
shaded and displaced if a woody C3 flora can establish (Wedin,
2004; Bond, 2008). While growth and photosynthetic potentials are typically less in the woody species relative to the C4
plants, the woody seedlings often tolerate low light within the
C4 canopy, and will steadily grow until they overtop the C4
Table 1. Requirements for C4 photosynthesis, an active CO2-concentrating system via PEP carboxylation (1) A CO2 collection capacity via an active, photosynthetically driven metabolic pump
(A) High activity of PEP carboxylase to fix bicarbonate to PEP, forming oxaloacetate.
(B) Conversion of oxaloacetate to a transportable form (malate or aspartate).
(C) Use of light energy to regenerate PEP, the substrate for PEP carboxylase.
(D) A means to convert CO2 to bicarbonate, the substrate of PEP carboxylase.
(2) The ability to trap CO2 and allow its accumulation around Rubisco.
(A) Localization of Rubisco to a compartment where C4 acids are decarboxylated.
(B) Decarboxylation of the C4 metabolite in the cellular region containing Rubisco.
(C) A large diffusive resistance between the Rubisco compartment and the intercellular air spaces, to slow leakage of CO2.
(3) Rapid metabolite transport between mesophyll and bundle sheath cells.
(A) Close spacing of compartments where PEP carboxylase and Rubisco are active (generally means close spacing of mesophyll and bundle sheath cells).
(B) Large metabolite concentration gradients between mesophyll and bundle sheath cells to allow for rapid diffusion.
(C) High capacity for metabolite movement across membranes via plasmsodesmata, membrane channels, and transport enzymes.
After Edwards et al. (2001) and Leegood (2002).
The C4 plant family portrait | e13
Table 2. The principal life forms of C4 photosynthesis
Life form
Family
Examples and notes
Trees
Euphorbiaceae
Shrubs (larger woody perennials)
Chenopodiaceae
Euphorbiaceae
Polygonacae
Zygophyllaceae (Tribuloideae)
(all in arid to semi-arid environments)
Herbaceous and small woody
eudicots (forbs to subshrubs)
Includes C4 plants from all 16 eudicot
families, except Polygonaceae
Graminoids (grasses and sedges)
Cyperaceae
Poaceae
Aquatic herbs
Hydrocharitaceae
Vines
Nyctaginaceae
Zygophyllaceae (Tribuloideae)
Four species from Hawaii, including Euphorbia
olowaluana (to 10 m) and E. herbstii (to 8 m).
Euphorbia olowaluana occurs in dry forests on
Hawaii, but does not form a dense forest canopy.
E. herbstii is largely an understorey tree with
excellent shade tolerance
About 400 species, mainly in Chenopodiaceae
lineages Atriplex and Salsoleae; some Euphorbia
spp.; and most species in the Polygonaceae genus
Calligonum. All shrubs are xerophytic or halophytic,
except for the Hawaiian Euphorbia species.
Haloxylon species can become arborescent
with age (to 8 m).
1300+ species of annuals and perennials, includes
herbaceous forbs, small woody perennials, sand
specialists, halophytes, and numerous weeds.
A few C4 species grow as alpine cushion plants
6400 species of grasses and sedges.
All herbaceous, although many form tough,
fibrous stalks and some grow to 6 m height.
One species of Hydrilla and one known
species of Egeria.
All are prostrate herbaceous vines. No C4 plant
forms woody vines that climb up stems or cliffs.
From Sage (2001) and Sage et al. (1999b).
plants, unless they are reduced or killed by an episodic disturbance (Bond and Midgley, 2000; Wedin, 2004; Bond, 2008). In
C4 grasslands, fire is the principle disturbance that hinders the
woody flora, although flooding, severe drought, and browsing
herbivores also kill woody seedlings and saplings (Sage et al.,
1999b; Bond et al., 2005). Once woody species grow above a
C4 canopy, their height advantage allows them to shade the C4
plants, directly reducing their vigour. Shading also weakens
fire cycles that favour C4 grass vegetation (Wedin, 2004). In the
event of fire, the superior C4 photosynthetic capacity supports
rapid regeneration by the grasses and initial dominance of the
landscape. C4 photosynthesis will also promote subsequent
burning by contributing to a rapid build-up of biomass that
readily burns during dry episodes (Knapp and Medina, 1999;
Bond, 2008). In this way, the C4 grasses enhance fire cycles
which perpetuate their dominance of a landscape. Should
something interrupt fire or other disturbance cycles, then
the advantage is lost and a C4 system can succeed to C3 forest (Bond and Parr, 2010). For this reason, wet, inflammable
regions of the tropics are forested, whereas in seasonally dry
regions, where fires are common, C4 grasslands and savannas
often predominate (Bond et al., 2005; Bond, 2008).
By suppressing photorespiration, the C4 pathway is
described as supercharging photosynthesis, thus enabling
plants to exploit fully the superior growth potential that is
possible in warm environments (Mitchell and Sheehy, 2006).
Theoretically, C4 photosynthesis has a 40% greater ability to
convert sunshine into biomass than C3 photosynthesis, and
maximum growth rates and yields of the most productive
C4 species are 40-50% greater than those of the most productive C3 species (Monteith, 1978; Long, 1999). A desire
to exploit this supercharging potential is behind numerous
initiatives now underway, including research to (i) engineer
the C4 pathway into C3 crops, notably rice (www.C4rice.irri.
org, www.3to4.org; Furbank et al., 2015); (ii) develop novel
bioenergy plants with record productivity (Heaton et al.,
2008; Jones, 2011); and (iii) to improve the efficiency of
existing C4 crops, three of which (maize, sugarcane, and sorghum) are among the world’s top 10 crops (Sage and Zhu,
2011; von Caemmerer and Furbank, 2016). While maize,
sorghum, and sugarcane are leading players in global agriculture, no other C4 row crop has global significance. Of the
top 150 crops directly consumed by humans, only 10 are C4
(FAOSTAT,
http://faostat.fao.org/site/339/default.aspx).
These include maize, sorghum, and sugarcane, which are
mostly grown for animal feed, sugar, or bioethanol, while
the others (four millet species, tef, Amaranth, and fonio)
largely have regional significance (Sage and Zhu, 2011). In
contrast, C4 photosynthesis is common in forage grasses,
which contribute to human nutrition through the grazing
animals they support. It could thus be argued that the C4
contribution to the human food supply is largely as animal
products and sugar, and not through our major fruit, vegetable, oil, grain, and starch crops. Even our major fibre
crops, including plantation trees, are largely C3. Therefore,
to ensure a diverse and abundant food and fibre supply well
into the future, the long-term goal of C4 engineering is to
introduce the C4 engine into many C3 crops. While rice may
e14 | Sage
become the first C3 crop to gain the C4 pathway, the technology developed in its engineering could quickly be used to
engineer any C3 crop of warm climates, such as soybean, citrus, cotton, and tomatoes. Assuming a 50% enhancement in
the yield of each crop, the contribution to global agriculture
from C4 engineering could be trillions of dollars per decade,
far exceeding the original engineering costs (Sheehy et al.,
2007; Sage and Zhu, 2011).
Whatever the ultimate benefit, engineering C4 photosynthesis into C3 crops will be daunting, as shown by genomics surveys indicating that large numbers of genes were
modified during C4 evolution (Bräutigam et al., 2011, 2014;
Gowik et al., 2011). Moreover, essential aspects of C4 plant
biology remain to be discovered before C4 engineering can
proceed, such as the genetic control over Kranz anatomy
(Fouracre et al., 2014). While these challenges are formidable, the frequent evolution of C4 photosynthesis suggests
that its engineering is feasible, and studies of how C4 photosynthesis repeatedly evolved could guide the effort (Gowik
and Westhoff, 2011). Even in failure, the knowledge gained
in attempting C3 to C4 engineering would assist efforts to
improve existing C4 crops.
Given all that we have learned about C4 plants since 1966,
there is much to commemorate on this 50th anniversary of
the discovery of C4. Readers are encouraged to examine
recent reviews that detail the current state of C4 knowledge
(e.g.Hibberd and Covshoff, 2010; Sage et al., 2012; Long and
Spence, 2013; contributions in Ragavendra and Sage, 2011;
and the Journal of Experimental Botany special issues on C4
photosynthesis in 2011, 2014, and this special issue). Instead
of attempting another detailed review, I present a figurative
portrait of the C4 plant family to mark the 50th anniversary
of its discovery. This portrait consists of a listing of all life
forms (Table 2) and evolutionary lineages where C4 photosynthesis independently arose in the plant kingdom (Table 3).
I then assign each C4 genus to its respective lineage (Table 3;
Supplementary Tables S1, S2 at JXB online). Such an effort is
possible due to recent progress in reconstructing phylogenies,
coupled with taxonomic revisions and carbon isotope screens
that identify C3 and C4 members in clades where C4 arose
(e.g. Brown 1977; Hattersley, 1987; Kellogg, 1999; Bruhl and
Wilson, 2007; Sage et al., 2007; Christin et al., 2008, 2011b;
2012; Kadereit et al., 2012; Grass Phylogeny Working Group
II, 2012; Osborne et al., 2014). Using C4 species counts for
each genus, I estimate species number per lineage, providing
for the first time an estimate of the species diversity within
each C4 phylogenetic line. The age of each C4 lineage is then
dated, largely following work by Besnard, Christin, and coworkers (Besnard et al., 2009, 2014; Christin et al., 2008;
2011a, 2012, 2014b; Horn et al., 2014). This effort examines
whether larger, more diverse lineages are also older lineages.
To highlight the stars (and problem species) of the C4 family,
I next present the C4 photosynthesis Hall of Fame, which can
serve as a learning tool that introduces the 40 most important
C4 plants to current and future generations (Table 4). I then
close this commemoration by discussing the status of the
world’s C4 flora on the 100th anniversary of its discovery.
The C4 flora of planet Earth
An ability to construct a reasonably accurate listing of the
world’s C4 flora is a testament to the concerted efforts of
many systematicians, physiologists, and ecologists who have
identified C4 plants and classified them into phylogenetic
taxa over the past 50 years (reviewed by Kellogg, 1999; Bruhl
and Wilson, 2007; Christin et al., 2010; Roalson, 2011; Grass
Phylogeny Working Group II, 2012; Kadereit et al., 2012;
Osborne et al., 2014; Soreng et al., 2014). The C4 flora of the
Earth consists of ~418 genera and 8145 species distributed
in 19 families of angiosperms (Table 3). No gymnosperms,
lower vascular plants, or bryophytes are C4. Monocots comprise 80% of the C4 species, with grasses accounting for 5044
species in 321 genera and sedges with 1322 species in 16
genera (Table 3). Two lineages of one species each are present in the Hydrocharitaceae family of monocots; these are
inducible single-celled C4 species of aquatic plants that lack
Kranz anatomy (Bowes, 2011; Chen et al., 2012). Eudicots
have >1750 C4 species in 16 families and 67 genera. The
genus with the most C4 species is Cyperus (Cyperaceae), with
an estimated 500 species, followed by Eragrostis (Poaceae,
427 species), Paspalum (Poaceae, 340 species), Panicum
sensu stricto (in the Panicinae subtribe of the Poaceae with
~300 species), and Fimbrystylis (Cyperaceae 300 species;
Supplementary Tables S1, S2). Two additional monocot genera have 200–300 species (Aristida, Digitaria). In the eudicots,
in contrast, only Euphorbia section Chamaesyce has >200 C4
species (see Supplementary Table S1). The next most speciose eudicot clade is Atriplex, with ~180 C4 species, followed
by Heliotropium section Orthostachys with ~130 species,
Gomphrena with 109 species, and Portulaca with 100 species
(Table 3; Supplementary Table S2). The remaining 74 C4 eudicot genera have <100 species each.
These numbers will change with phylogenetic clarification and taxonomic revision. For example, the C4 Cyperus
clade will be split into multiple genera as it is polyphyletic
(Larridon et al., 2013). Revisions are ongoing in Panicum
(Poaceae) and Salsola (Chenopodiaceae), which are large
polyphyletic genera (Akhani et al., 2007; Chemisquy et al.
2010; Zuloaga et al., 2010; Grass Phylogeny Working
Group II, 2012; Kadereit et al., 2012; Soreng et al., 2014).
Despite these efforts, the final tally of ~8100 C4 species may
remain. In the compilation by Sage et al. (1999a), almost
7600 C4 species were estimated (4600 grasses, 1330 sedges,
and 1600 dicots) which is close to the estimates in Table 3.
This is surprising, given that there has been much taxonomic revision and clarification of photosynthetic pathway
distribution since 1999. Notable changes in the C4 listing
are removal of numerous C3 genera (Celosia, Glinus, and
Anacampseros) that were erroneously listed as having C4
species by Sage et al. (1999a) (Sage et al, 2007; Guralnick
et al., 2008; Christin et al., 2011b), and revising the C4 list to
reflect accepted classification and names. For example, the
weedy genus Kochia (Chenopodiaceae) has been subsumed
into Bassia (Kadereit and Freitag, 2011), Chamaesyce
(Euphorbiaceae) is once again in Euphorbia (Yang and
The C4 plant family portrait | e15
Table 3. Evolutionary lineages of C4 photosynthesis in the kingdom Plantae, showing the number and names of the postulated de novo
lineages, the total number of possible origins for which there is evidence (de novo lineages plus ancillary and uncertain possibilities),
genera and C4 species number per lineage, and postulated evolutionary age for each de novo lineage Family
De novo
lineages
Total no. of possible
C4 origins (ancillary
plus uncertain)
No. of
genera
No. of C4
species
19
Eudicots
(16 families)
Acanthaceae
Aizoaceae
Amaranthaceae s.s.
61
34
65–84
37–53
418
79
8145
1777
(1) Blepharis
(2) Sesuvioideae
5
(3) Aerva
(4) Alternanthera
(5) Amaranthus
(6) Gomphrenoids
(7) Tidestromia
3
(8) Flaveria
(9) Coreopsideae
(10) Pectis
(11) Euploca
(=Heliotropium sect.
orthostachys)
3
(12) Cleome gynandra
(13) C. angustifolia
(14) C. oxalidea
(15) Polycarpaea
9
(16) Atriplex
(17) Bienertia
1–4
1–6
5
1
1
1
1
1
4–5
2-3a
1
1
1–3
1
4
10
1
1
1
6
1
8
1
6
1
1
15
30
257
4
17
90
138
8
138
7
41
90
130
3
1
1
1
1
10–13
1
1
1
1
40
1
1
3
1
1
1
20
558
~180
3
(18) Camphorosmeae
1–2
2
24
14.9 ± 1.8
(19) Tecticornia
1
1
2
6.5b
(20) Caroxylonae
(21) Salsoleae s.s.
1
2–4
13
21
157
153
20.9 ± 2.5
23.4 ± 1.3
3
1
1
1
1
1
2a
2
1
1
1
1–2
1
Polygonaceae
Portulaceae
Suaeda
(22) sect. Borszczowia
(23) sect. Salsina
(24) sect. Schoberia
(25) Euphorbia
(26) Gisekia
(27) Mollugo
2
(28) Allionia
(29) Boerhavia
(30) Calligonum
(31) Portulaca
1
1
1
3
1
2
1
1
40
1
30
9
350
1
2
44
2
42
80
100
Scrophulariaceae
Zygophyllaceae
(32) Anticharis
(33) Tribuloideae
1
1–2
1
3
4
37
NA
NA
1
28–31
6
1
1
339
16
2
1
6368
1322
211
8.6b
Monocots
Cyperaceae
(34) Tetraena simplex
27
6
(35) Bulbostylis
Sage et al. (2011a)
Ocampo et al. (2013);
Christin et al. (2014)
Khoshravesh et al. (2012)
Lauterbach, Kaedereit, Ludwig,
and Sage (unpublished data)
Lauterbach et al. (2016)
14.9 ± 4.7
Besnard et al. (2009)
Asteraceae
Boraginaceae
Capparidaceae
Caryophyllaceae
Chenopodiaceae
Euphorbiaceae
Gisekiaceae
Molluginaceae
Nyctaginaceae
Age of lineage,
million years
(±range)
References
6.8 ± 0.9
21 ± 1.0
Fisher et al. (2015)
Bohley et al. (2015)
Sage et al. (2007)
8.5 ± 6.0
6.7 ± 0.8
8.8 ± 6.8
7.8 ± 0.8
8.1 ± 4.9
2.0 ± 1.0
NA
10.2 ± 1.0
NA
3.8 ± 3.2
0.4 ± 0.2
6.5b
8 ± 2
15.3 ± 0.4
11.7b
7.7b
8.4 ± 1.5
5.1 ± 0.6
19.3 ± 4.0
14.9c
3.8 ± 3.3
6.1b
3.1 ± 0.9
10.6 ± 9.3
23.0d
McKown et al. (2005); Lyu et al. (2015)
Kellogg (1999)
Hansen (2012)
Hilger and Diane, (2002);
Sage et al. (2011a); Sage
(unpublished)
Feodorova et al. (2010)
Kool (2012)
Kadereit et al. (2010)
Schütze et al. (2003);
Kapralov et al. (2006)
Kadereit and Freitag (2011); Freitag and
Kadereit (2014); Kadereit et al.2014)
Shepard et al., 2005; Voznesenskaya
et al. (2008); Kadereit et al., (2012)
Akhani et al. (2007); Kadereit et al. (2012)
Akhani et al. (2007); Wen et al. (2010);
Kadereit et al. (2012)
Schütze et al. (2003);
Kapralov et al. (2006)
Yang and Berry (2011)
Bissinger et al. (2014)
Christin et al. (2011b)
Douglas and Manos (2007);
Sage et al. (2011a)
e16 | Sage
Table 3. Continued
Family
Hydrocharitaceae
Poaceae
De novo
lineages
Total no. of possible
C4 origins (ancillary
plus uncertain)
No. of
genera
No. of C4
species
Age of lineage,
million years
(±range)
References
(36) Cyperus
1
9
757
9.7 ± 1.3
(37) Eleocharis ser.
Tenuissimae
(38) Eleocharis vivipara
(39) Fimbrystylis
(40) Rhynchospora
2
(41) Hydrilla
(42) Egeria
19
1
1
10
6e
Besnard et al. (2009);
Larridon et al. (2013)
Roalson et al. (2010)
1
1
1
2
1
1
20–23
1
3
1
2
1
1
321
1
303
40
2
1
1
5044
4.4b
9.1 ± 3.2
5.8 ± 1.6
(43) Aristida
1
1
288
16 ± 3f
(44) Stipagrostis
1
1
56
15 ± 3f
(45) Chloridoideae
(46) Centropodia
(47) Eriachne
(48) Tristachyideae
(49) Andropogoneae
(50) Reynaudia
(51) Axonopus
(52) Paspalum
(53) Anthaenantia
(54) Arthropoginae/
Mesosetum clade
(55) Arthropoginae/
Onchorachis clade
(56) Arthropoginae/
Colaeteania clade
(57) Anthephorinae
(58) Echinochloa
(59) Neurachne +
Paraneurachne
(60) MPC
(Melinidinae/Paninicae/
Cenchrinae)
(61) Alloteropsis
1
1
1
1
1
1
1
1
1
1–2
145
1
2
8
85
1
3
9
2
6
1596
4
50
87
1228
1
90
379
4
35
28.5 ± 3.5
16.1 ± 5.9
9.0 ± 2.5
NA
19.5 ± 2.5
NA
7.7 ± 5.8
11.3 ± 0.6
7.2
11.8 ± 0.5
Grass Phylogeny Working Group II (2012);
Osborne et al. (2014); Soreng et al. (2014)
Grass Phylogeny Working Group II (2012);
Besnard et al. (2014)
Grass Phylogeny Working Group II (2012);
Besnard et al. (2014)
Grass Phylogeny Working Group II (2012)
Grass Phylogeny Working Group II (2012)
Grass Phylogeny Working Goup II (2012)
Grass Phylogeny Working Group II (2012)
Grass Phylogeny Working Group II (2012)
Grass Phylogeny Working Group II (2012)
Grass Phylogeny Working Group II (2012)
Grass Phylogeny Working Group II (2012)
Grass Phylogeny Working Group II (2012)
Grass Phylogeny Working Group II (2012)
1
1
2
7.8
Grass Phylogeny Working Group II (2012)
1–2
1
7
8.4 ± 2.0
Grass Phylogeny Working Group II (2012)
1
1
2a
8
1
2
286
35
2
14.7 ± 6.5
9.1 ± 4.8
2 ± 2
Grass Phylogeny Working Group II (2012)
Grass Phylogeny Working Group II (2012)
Christin et al. (2012)
1
43
889
17.5 ± 1.1
GPWG II; Washburn et al., 2015
1–2
1
5
12.0 ± 2.5
Grass Phylogeny Working Group II (2012);
Lundgren et al. (2015)
Roalson et al. (2010)
Besnard et al. (2009)
Besnard et al. (2009)
Bowes et al. (2011); Chen et al. (2012)
NA
5.9 ± 0.3
See Supplementary Tables S1 and S2 for a listing of the genera in each lineage. Lineage estimates follow Sage et al. (2011a) and references
provided. Estimates of species number are based on Osborne et al. (2014) for grasses, Bruhl and Wilson (2007) for sedges, online databases
for eudicots (http://www.mobot.org/MOBOT/research/APweb/), and references listed. Lineage age estimates are generally the mid-point
between phylogenetic crown and stem node ages (with range between these nodes in parentheses) unless otherwise noted (Christin et al.,
2008, 2011a, 2012; Besnard et al., 2009, 2014; Bissinger et al., 2014; Bohley et al., 2014; Fisher et al., 2015). sect. section; ser., series, s.s., sensu stricto. Numbers in bold font show summed values for the clade.
a
Ancillary lineages are supported in the clade. b
Stem node ages only as crown age not available. c
Crown age used due to high clade diversification. d
Approximation given in text of Ocampo et al. (2013). e
Assumed to be 60% of 10.5 Mya stem age in Christin et al. (2011b).
f
After Roalson et al. (2010) and Besnard et al. (2014).
Berry, 2011), and the large grass genus Pennisetum is now in
Cenchrus (Chemisquy et al., 2010). Not all name consolidations in the grass family (Soreng et al, 2014) are incorporated in the treatment here, because of phylogenetic fluidity
and familiarity with certain genus names. Offsetting these
deletions are dozens of new names, such as Kali for the
Salsola kali clade (Akhani et al., 2007, 2014) and multiple
names for former Panicum clades (e.g. Coleatainia in the
Arthropogoninae, or Sorengia, depending on the authority; Sede et al., 2009; Zuloaga et al., 2010; Grass Phylogeny
The C4 plant family portrait | e17
Table 4. The C4 photosynthesis Hall of Fame. Membership is limited to 0.5% of all C4 species (40 species in total). Criteria for inclusion
are importance to humanity, dominance of major biomes, or some unique, record-setting aspect of performance
Species
Cultivated species—row crops
(1) Zea mays (maize)
(2) Sorghum officinare (sorghum)
(3) Saccharum officinarum (sugarcane)
4) Cenchrus americanus (pearl millet)
(5) Miscanthus×giganteus
(6) Panicum virgatum (switchgrass)
Cultivated species—turf and forage crops
(7) Chloris guyana (Rhodes grass)
(8) Cynodon dactylon (Bermuda grass)
(9) Melinis minutifolia (molasses grass)
(10) Panicum maximum (also known as Megathyrsus
maximus and Urochloa maximum; Guinea grass)
(11) Cenchrus purpureus (Napier grass)
(12) Zoysia japonica
Invasives and weeds
(13) Cyperus rotundus (purple nutsedge)
(14) Digitaria sanguinalis (crabgrass)
(15) Echinochloa crus-galli (barnyard grass)
(16) Eleusine indica (goose grass)
(17) Imperata cylindrica (cogon grass)
(18) Sorghum halepense (Johnson grass)
(19) Salsola kali (tumbleweed)
(20) Tribulus terrestris (puncture vine)
Wild species of note
Biome dominants—grasses and sedges
(21) Andropogon gerardii (big bluestem)
(22) Bouteloua gracilis (blue grama)
(23) Cyperus papyrus (papyrus)
(24) Spartina anglica
(25) Themada triandra
Biome dominants—eudicots
(26) Atriplex confertifolia (four-wing saltbush)
(27) Haloxylon ammodendrum (black saxual)
Record holders (d, dicot; g, grass; s, sedge)
(28) Echinochloa polystachya (g)
(29) Euphorbia olowaluana (d)
(30) Euphorbia herbstii (d)
(31) Amaranthus palmerii (d)
(32) Portulaca oleracea (d)
(33) Orinus thoroldii (g)
Research stars
(34) Alloteropsis semialata (g)
(35) Atriplex rosea (d)
(36) Bienertia sinuspersici (d)
(37) Cleome gynandra (d)
(38) Eleocharis vivipara (s)
(39) Flaveria brownii (d)
(40) Setaria viridis (g)
Notes
World’s most widely grown C4 crop with important. historical, social, and economic impact.
Second most important C4 crop for grain; important bioenergy candidate. First C4 genome sequenced.
Leading source of sugar and bioethanol.
Fourth leading C4 crop; most productive of the millets.
Leading second-generation bioenergy crop.
Leading second-generation energy cane, major forage.
A widespread forage species of low latitude.
Major forage and turf grass, also one of the world’s worst weeds.
Fast-growing pasture grass, also a major invasive species.
Widely grown, productive pasture and weedy grass of low latitude.
Most productive cultivated plant in terms of biomass, in development as a bioenergy crop; can be invasive.
Popular turf grass of southern lawns and golf courses.
Often noted as the world’s worst weed of crops
Crabgrass; one of the top 10 worst weeds of the world, and a well-known pest to gardeners below 45°
latitude.
Top five of world’s worst crop weeds, one of the rice mimics; includes Japanese millet, a minor crop.
Rated fifth most severe C4 weed (Brown, 1999)
Major threat to native species due to aggressive growth, promotes severe wildfire
Very aggressive weed, often rated in top 5 of world’s worst weeds; produces cyanide and is toxic to cattle.
A C4 star in movies and song (‘Drifting along with the tumbling tumbleweeds’)
Also called caltrop, a bane of tyres, paws, and feet
Tall-grass prairie dominant in North America.
Short-grass prairie dominant in North America.
African marsh dominant; source of papyrus, the paper of the Ancients.
Dominant in European coastal salt marshes; arose as a hybrid between American and European parents.
The food of the Serengeti grazers, widespread in tropical grasslands.
Halophytic shrub locally dominant in semi-arid steppes of western North America and representative of
many Atriplex shrubs from dry and saline regions (e.g. A. nummularia in Australia).
Major shrub species of Central Asia and North Africa. Its dense thickets provided fuel to the Silk Road
caravans across Asia.
Greatest productivity rate of all plants.
Largest C4 tree (to 10 m), from Hawaii.
Understorey tree from Hawaii that demonstrates C4 photosynthesis can adapt to shady forest interiors.
Holds the world record photosynthesis rate in ambient air (80 µmol m−2 s−1 at 325 µmol mol−1 CO2);
important genus of weeds and minor C4 crops.
The only C4 lineage with CAM photosynthesis, a widespread weed, and a vegetable in developing nations.
Its Kranz anatomy is visible to the naked eye. Probably the most pulled C4 weed by non-farmers.
World’s highest elevation C4 plant (5200 m)
Contains C3, C3–C4 intermediate, and C4 genotypes.
A C4 plant known to hybridize with a C3 plant.
Single-celled C4 plant, the best studied of six known
A C4 genetic model close to Arabidopsis
C4 above water; C3 below water; best studied example of inducible C4
The most primitive C4 species known, and a member of the leading model lineage for studying C4 evolution
The C4 genetic model grass and a major weed.
Numbers do not refer to order of importance. After Brown (1999), Long, (1999), and Sage et al. (1999a, b, 2011a). See discussion in text for
additional sources.
e18 | Sage
Working Group II, 2012). Given the offsetting nature of
the additions and deletions, the numbers in Table 3 and
Supplementary Tables S1 and S2 are probably close to the
eventual, definitive tally. A final listing should be feasible
within a couple of decades, when taxonomic revisions,
physiological characterizations, and phylogenetic analyses
have clarified uncertainties, particularly in large, difficult
groups such as the grasses and Chenopods.
The distinct C4 evolutionary lineages
In previous listings of C4 evolutionary lineages, Kellogg
(1999) hypothesized 31 independent lineages, Sage (2004)
listed 45, and Sage et al. (2011a) identified 62 distinct origins of C4 photosynthesis. This growth in lineage estimates
reflects improvements in phylogenetic coverage and isotopic
data sets. Since the latest tally by Sage et al. (2011a), additional lineages have been identified and some postulated lineages have been downgraded given phylogenetic uncertainty.
An update is therefore necessary.
In compiling lists of distinct lineages, issues of evolutionary
independence and reversion from C4 to C3 have to be considered (Kellogg, 1999; Christin et al., 2010; Sage et al., 2011a).
Distinct C4 lineages from closely related clades may not be
truly independent, given that common ancestors may have
been well along the evolutionary path from C3 to C4 photosynthesis. This is apparent in Flaveria, where three independent
origins are postulated from common ancestors that are C3–C4
intermediates (McKown et al., 2005; Lyu et al., 2015). The
potential for reversions from C4 to C3 species is a particular
challenge for identifying C4 origins, as reversions produce the
appearance of multiple origins within a phylogeny, when in
fact there may be only one C4 origin with C3 branches arising
as reversions from the C4 state (Kellogg, 1999; Christin et al.,
2010). Uncertainties regarding reversions are most apparent in
the Poaceae and Chenopodiaceae (Kellogg, 1999; Sage et al.,
2011a; Kadereit et al., 2012). To deal with these uncertainties, I have delineated two categories of origins in Table 3. The
first, in column 2 of Table 3, are ‘de novo’ origins which arose
from a functional C3 state, regardless of whether the C3 ancestors have genetic or anatomical traits that might facilitate C4
evolution (e.g. close vein spacing, enlarged bundle sheaths, or
gene duplication; Sage, 2001; Christin et al., 2013, 2015). The
de novo designation applies when all of the principal traits
defining C4 photosynthesis (Table 1) must be independently
acquired. This is indicated by separation of C4 clades by welldefined C3 clades, as is evident in the Amaranthaceae sensu
stricto, where distinct C3 clades separate five C4 clades (Sage
et al., 2007). This contrasts with the Salsoloideae tribe of the
Chenopodiaceae where C4 clades arise from C3 clades embedded in C4 clades, which could indicate reversals or multiple
origins (Akhani et al., 2007; Wen et al., 2010; Kadereit et al.,
2012). In a few instances, I accept an author’s opinion of C4
lineage independence without this separation criterion having
been met, because physiological, genetic, and structural characters indicate independent C4 origin (Kadereit et al., 2014).
A second category of C4 origins is delineated as ‘ancillary/
uncertain’. Ancillary refers to situations where C4 may arise
multiple times from common ancestors in a C3–C4 intermediate state, in which case the distinct origins are not completely
de novo (as occurs in Flaveria and Mollugo, and possibly in
Blepharis, Portulaca, and Neurachne; McKown et al., 2005;
Christin et al., 2011b, 2012, 2014a). ‘Uncertain’ refers to situations where inadequate phylogenetic resolution and reversal
possibilities prevent identification of independent lineages.
Ancillary origins and uncertainties are pooled and then
added to the de novo origins within a clade in order to estimate the potential number of distinct C4 origins (see column
4, Table 3). Future research will need to clarify these uncertainties to reveal the true number of de novo C4 origins, ancillary origins, and reversals to the C3 state.
With this approach, 61 de novo C4 origins are identified from C3 ancestors (Table 3). The grasses are the most
prolific family, with 19 de novo C4 origins, which combined with six de novo origins in the sedge family and two
in the Hydrocharitaceae yields 27 de novo C4 origins in the
monocots. Thirty-four de novo origins are estimated for C4
photosynthesis in the eudicots, with the Chenopodiaceae/
Amaranthaceae alliance accounting for 14 of these origins.
An additional 4–23 distinct origins are possible in 12 of the de
novo clades, either as ancillary origins or as multiple de novo
origins depending on reversion possibilities. If the degree
of independence is ignored, a total of 65–84 origins of C4
photosynthesis from non-C4 ancestors are postulated. These
estimates further demonstrate that C4 photosynthesis is one
of the most prolific examples of complex trait evolution in
the living world. Other examples of extreme convergence
(>60 independent origins) are Crassulacean acid metabolism
(CAM) photosynthesis and seed dispersal by ants (Lengyel
et al., 2010; Edwards and Ogburn, 2012).
The age of C4 photosynthesis
The time when C4 photosynthesis evolved within a lineage can
be evaluated through molecular clock approaches that use
rates of change in DNA sequences to estimate when C4 nodes
diverged on phylogenetic trees. While calibration assumptions and DNA divergence rates used in molecular dating can
be controversial, the method overall is the best available for
estimating origination events in evolutionary time (Vincentini
et al., 2008; Christin et al, 2014b). Divergence dates can then
be compared with complementary fossil and carbon isotopic
evidence from the geological record to build a more comprehensive picture of C4 origins and expansion (Edwards
et al., 2010). In well-resolved phylogenies, origin estimates
using molecular clocks consist of two time points—the age
of the stem node which is the phylogenetic branch between
a C4 and non-C4 clade, and the age of the crown node where
multiple C4 species branch (Christin et al., 2008). Christin
and co-workers have analysed C4 divergence times for >50 C4
lineages (Besnard and Christin, 2009; Christin et al., 2008,
2011a, 2011b, 2012, 2014b), and new estimates are in recent
work (Ocampo et al., 2013; Besnard et al., 2014; Bissinger
et al., 2014; Horn et al., 2014; Bohley et al., 2015; Fisher
et al., 2015). In total, the ages of 54 de novo origins of C4 are
The C4 plant family portrait | e19
25
Chl
And
1200
900
20
Cyp
MPC
750
15
600
450
10
Euph
300
150
Sal
5
Car
Port
0
0
5
10
15
20
Number of lineages
Species per lineage
1500
25
30
Lineage age, million years
0
0
5
10
15
20
25
30
Million years before present
Fig. 1. (A) The number of species in a C4 lineage plotted against the age of the lineage. Ages are estimated as described in Table 3 for 54 lineages.
Grasses, red triangles; sedges and Hydrocharitaceae, blue diamonds; and eudicots are shown as black circles. (B) A frequency distribution of the data
in panel A. The range between stem and crown nodes ages are shown for four speciose lineages to indicate variation in age estimates; see Table 3 for
all range estimates. Abbreviations: And, Andropogoneae; Car, Caroxyloneae; Chl, Chloridoideae; Cyp, Cyperus; Euph, Euphorbia; MPC, Melinidinae/
Paninicae/Cenchrinae; Port, Portulaca; Sal, Salsoleae s.s.; Sev, Sesuvioideae.
now available (Table 3). With these estimates, I examine the
relationship between species richness within a lineage and age
(Fig. 1; Table 3)
All of the estimated C4 origins occur after 30 Mya (Fig. 1A).
Between 30 Mya and 25 Mya, atmospheric CO2 levels are predicted to have declined from 800 µmol mol-1 to near presentday levels (400 µmol mol−1) in the Oligocene epoch (33 to 24
mya; Zhang et al., 2013). During this time, Earth’s climate
cooled, leading to greater levels of aridity in warm, lowlatitude regions (Prothero, 1994; Zachos et al., 2001). The
appearance of most C4 origins after the Oligocene climate and
atmospheric change is considered strong evidence supporting
low CO2, coupled with increased seasonality, aridification,
and/or salinity, as leading environmental agents favouring C4
evolution (Sage, 2001; Christin et al., 2008, 2011a; Vincentini
et al., 2008; Osborne and Freckleton, 2009; Kadereit et al.,
2012). However, if calibration dates are older than assumed
by Christin et al. (2008, 2011a), the oldest C4 lineages could
have evolved before atmospheric CO2 reduction and climate
decay of the late-Oligocene epoch (Vincentini et al., 2008;
Stromberg, 2011; Christin et al., 2014b).
Older C4 lineages are often species rich, while lineages
younger than 7 million years are species poor (Fig. 1A). Of
the 18 lineages that are younger than 7.1 million years, two
have 40–42 species and all of the remainder <18 species. In
the grasses, the oldest and most species-rich C4 lineages occur
in the tribes Chloridoidae and Andropogonae, while in the
eudicots the four most species-rich C4 lineages are among
the oldest (Fig 1A). There are numerous species-poor lineages older than 7 million years, notably Gisekia, a nearly 15
million-year-old lineage with just one polymorphic C4 species (Bissinger et al., 2014). Grasses and eudicots are similarly
represented in the older clades as previously noted (Christin
et al., 2011a), while the C4 lineages younger than 7 million
years are largely eudicot (Fig 1A; Table 3).
The mid-points between stem and crown nodes for 22
C4 transitions occur in the 5–10 Mya interval in Fig 1B,
indicating increased C4 evolution in the late-Miocene epoch.
This is notable because proliferation of C4 lineages 5–10 Mya
is coincident with the global expansion of C4-dominated
grasslands in the late-Miocene (Cerling, 1999; Edwards et al.,
2010; Stromberg, 2011). Carbon isotope shifts in ancient
paleosols, herbivore teeth, and grass phytoliths indicate
that C4 plants were becoming common, but not dominant,
in the tropics to warm temperate zones between ~22 Mya
and 10 Mya (Stromberg, 2011). After 10 Mya, isotopic values dramatically shift towards C4 values in deposits from
East Africa and South Asia, demonstrating that C4 grasses
expanded to dominate the landscape; similar shifts occur in
deposits from South America, China, and the Great Plains
of North America after 8 Mya (Cerling, 1999; Stromberg,
2011). Together, the molecular clock and paleontological
results present a picture of C4 lineages repeatedly evolving
after the Oligocene, and gradually spreading across Miocene
landscapes of low to mid latitudes until some of them expand
to dominance after 10 Mya. As indicated by species numbers
and age estimates for the Chloridoideae, Andropogoneae,
and MPC grasses (Fig. 1A), species from these clades probably produced the isotopic evidence of C4 presence on earlyto mid-Miocene landscapes. This possibility is consistent
with Chloridoideae leaf materials identified in mid-Miocene
deposits (Cerling, 1999; Stromberg, 2011). The increased
number of origins after 15 Mya in Fig. 1A is also coincident
with a postulated 100 µmol mol−1 decline in atmospheric CO2
below 450 ± 50 µmol mol−1 at 14 Mya (Zhang et al., 2013).
This CO2 drop, coupled with subsequent deterioration in the
global climate, may have set the stage for the increased number and spread of C4 lineages 5–10 Mya (Edwards et al., 2010;
Hoetzel et al., 2013).
While Fig. 1 presents a relatively clear picture, it must be
emphasized that it is based on numerous assumptions and,
in some cases, limited data. Christin et al. (2011a) argue that
molecular dating of C4 origins should be reassessed once
comprehensive phylogenetic data are available for each clade,
e20 | Sage
a point shared here. Given the pace of phylogenetic progress,
reanalysis of C4 divergence times across all angiosperm clades,
using consistent dating methods, should be possible in the
near future. This will be an important contribution, because
advances in paleo-climate and landscape reconstructions are
describing environmental conditions over the past 50 million
years for specific localities across the Earth (Feakins and de
Menocal, 2010; Stromberg, 2011; Hoetzel et al., 2013). Once
centres of C4 diversification can be identified, matching of C4
origin time and environment will be possible, thereby providing clear evidence for the selection environments favouring C4
evolution.
The C4 photosynthesis Hall of Fame
To complete the picture of the C4 family, I now present the
‘C4 Hall of Fame’, a knowledge tool that will introduce current and future generations to the most significant C4 species
that have influenced humanity and the biosphere, for better
or worse, as well as those species that stand out because of
uniquee attributes or contributions to our understanding of
C4 plant biology (Table 4). To establish the C4 Hall of Fame,
I drafted a preliminary list which was then circulated among
leading C4 researchers for comments (Christin, E. Edwards,
G. Edwards, Ehleringer, Furbank, Hibberd, Langdale, Long,
Monson, Osborne, and Vosnesenskaya). Hall of Fame membership is restricted to 40 species, or 0.5% of the world’s 8100
or so C4 species.
Obvious stars in the C4 flora are the three leading C4 crops,
maize, sorghum, and sugarcane (Brown, 1999). Maize in particular stands out, given that it is the most productive food
staple in the world and the second most productive commodity crop worldwide (FAOSTAT, http://faostat.fao.org/site/339/
default.aspx). Maize has also had a major impact on world
history, particularly in supporting the rise of New World civilizations (van der Merwe and Tschauner, 1999). Sugarcane is
the world’s most productive commodity crop, and is the source
of cheap sugar for much of the world (FAOSTAT, http://
faostat.fao.org/site/339/default.aspx). It has a tortured relationship with humanity, given its association with the enslavement of Africans between 1600 and 1800, and its continuing
contribution to global epidemics of tooth decay, diabetes,
and obesity; however, few commodities bring as much pleasure and are as widely used as sugar (Mintz, 1991). Sugarcane
and maize are also the two major bioethanol producers for
automobiles, in what are termed first-generation bioenergy
crops (Jones, 2011). Sorghum is the ninth most productive
food staple in the world, and represents the first C4 genome
to be sequenced in support of efforts to develop sorghum as
a biofuel crop and improve the stress tolerance of C4 plants
(Mullet et al., 2014). Additional C4 crops are Cenchrus glaucum (pearl millet, formerly Pennisetum glaucum) which was
chosen as it is the most productive of the millets, a group of
half a dozen C4 grain crops that are important producers in
tropical nations, often on drought-prone soils (Brown, 1999).
The leading second-generation bioenergy crops Miscanthus
and switchgrass are included given the attention they have
received in recent years for their potential to become major
crops and landscape modifiers when bioenergy production
finally takes off (Heaton et al., 2008).
Most C4 species of economic value are forage grasses in
warm climates, of which there are more than a dozen notable
species (Brown, 1999; Moser et al., 2004); five of the most
notable species have been selected for the Hall of Fame, based
on Brown’s survey. These are noted for very high growth rates
and water use efficiencies, leading to their preferred use in
tropical and subtropical pastures, and, recently, consideration
as biofuel candidates (Monteith, 1978; Samson et al., 2005);
however, the high productivity also contributes to their ability
to become problem weeds (D’Antonio and Vitousek, 1992;
Moser et al., 2004). As a representative of the many C4 turf
grasses used for lawns and golf courses in warm climates,
Zoysia japonica was selected because it produces carpet-like
lawns as few other grasses can. Bermuda grass (Cynodon
dactylon) is a diverse species that has importance as a forage
and turf grass, and is number two on the world’s list of worst
weeds (Brown, 1999; Taliafero et al., 2004).
C4 plants are also recognized as having severe negative
impacts because they are among the world’s most serious
crop weeds and many are bioinvasives, causing great harm
for regional biodiversity (D’Antonio and Vitousek, 1992;
Brown, 1999). In the Infamy section of the Hall of Fame, we
include the worst of these weeds, and some that may not be
terrible in an economic or biodiversity sense, but are widely
known due to frequent interaction with the average person.
Among the worst weeds are purple nutsedge and Johnson
grass; their impacts on crop yield justify their top rankings on
Brown’s (1999) world’s worst weed list. Echinochloa crus-galli
is a widespread weed that is number three on Brown’s (1999)
worst weed list, in part due to its impact on rice production. It
and its sister species E. colona (number four on Brown’s worst
weed list) form the rice mimics, which have been selected
by thousands of years of weeding to closely resemble rice
(Barrett, 1983). Given the widespread cultivation of rice, a
particularly labour-intensive crop, it is possible that weeding
of E. crus-galli has generated more sweat and back pain than
any other weed in human history.
In natural ecosystems, the worst C4 invaders are highly
productive tropical grasses, often of African origin, that have
become invasive in New World, Australian, and Island ecosystems in part through their ability to intensify fire cycles (e.g.
Imperata cylindrica). By doing so, they degrade natural forest
and rangeland ecosystems (D’Antionio and Vitousek, 1992).
These weeds are a particularly severe threat to both C3 and
C4 diversity on natural landscapes, as intensification of fire
cycles and their robust growth often lead to complete replacement of the native vegetation by the invasive aliens (Cochrane
and Schulze, 1999; Sage and Kubien, 2003). For the average
person, however, crabgrass (Digitaria sanguinalis) and puncture vine (Tribulus terrestris) have generated more enmity and
curses as they are widespread lawn and garden weeds, and, in
the case of puncture vine, the source of much pain for hound
and human alike. Another name for puncture vine is caltrop
(foot-trap in Latin), which reflects the resemblance of the
spiny T. terrestris fruits to the dreaded ‘caltrop’ antipersonnel
The C4 plant family portrait | e21
weapons made of four spikes arrayed in a tetrahedron, so that
one always sticks up. One wonders whether T. terrestris fruits
were the inspiration for military caltrops, which have been
scattered along roads and battlefields from ancient to modern
times to impede advancing armies. Caltrops were the landmine equivalent before the 20th century, dating to at least the
Ancient Greeks, who termed the weapon ‘Tribuli’.
Collectively, C4 plants are responsible for almost a quarter of the primary productivity on the terrestrial Earth (Still
et al., 1993). Most C4 species are relatively minor floristic
elements, but a few dozen have become important contributors to primary productivity in grasslands and shrub biomes
where growing seasons are warm and forests do not dominate (Sage et al., 1999b). Some of the notable dominants are
selected for the Hall of Fame, such as Andropogon gerardii
(big bluestem), the dominant species of the Tall Grass Prairie
biome of North America, which was viewed as an inland sea
of grass by early settlers (Weaver, 1954). Themada triandra
stands out as perhaps the most widespread C4 dominant, as
it is important in grasslands across the tropics and subtropics
of the world (Hatch and White, 2004). For the wider public,
it is notable as a leading grass on the Serengeti landscape,
which is the most famous C4 grassland where natural grazing megafauna and their predators still roam (McNaughton
and Banyikwa, 1995). C4 sedges are important in freshwater
marshes of low latitude, and they are represented by papyrus (Cyperus papyrus), an important dominant of tropical marshes in Africa but which is best known as a source
of parchment for ancient scribes (Jones, 1986). In turn, C4
grasses in the genus Spartina dominate coastal salt marshes
of the warmer world, and they are represented by S. anglica,
a well-studied marsh grass of Europe. Spartina anglica is a
hybrid of European and American salt marsh dominants and
has become a major invasive in European coastal marshes,
growing to near 60°N latitude (Long and Woolhouse, 1979).
Atriplex confertifolia from the American West is representative of many saltbush species that dominate semi-arid landscapes in North America, Central Asia, and Australia, while
Haloxylon ammodendrum (black saxual) is a widespread
Chenopod shrub from Mongolia to North Africa that provided fuel and fodder to Silk Road caravans for centuries; it
is now being considered as a biofuel species for harsh soils in
Central Asia (Caldwell, 1985; Walter and Box, 1986; Pyankov
et al., 1999).
Hall of Fames should include record holders, because they
tell us something about the boundaries of accomplishment
and endurance. C4 plants are recognized as the most prolific producers on the planet, with productive potentials well
above those of the leading C3 crops (Monteith, 1978; Long,
1999). Of these, Echinochloa polystachya holds the world productivity record of any plant species, with annual biomass
yields >100 dry T ha−1 year−1, over double that of the most
productive C3 crops (Monteith, 1978; Piedade et al., 1991;
Long, 1999). It is unique in that it grows along river margins
in Amazonian South America, beginning growth on sandbars and mudflats in the dry season, and then growing as fast
as the rivers rise during the wet season. With an abundance
of water and nutrients, E. polystachya is able to put the C4
engine into high gear in a manner that C3 photosynthesis cannot due to photorespiratory constraints (Long, 1999). Other
record holders include Orinus thoroldii, the world’s highest C4
plant which at 5200 m in the Tibetan plateau demonstrates
that C4 species can persist in cold, alpine environments, contrary to textbook assertions that C4 plants are adapted to hot,
dry conditions (Sage et al., 2011b). The world’s fastest photosynthesis rate in recent atmospheric conditions occurs in
Amaranthus palmerii, a ruderal species of the Mojave desert
region in south-western North America (Ehleringer, 1983). As
a ruderal, A. palmerii is able to invest high amounts of nitrogen into leaves, which, with the higher nitrogen use efficiency
of the C4 pathway, leads to record-setting photosynthesis
rates of 80 µmol m−2 s−1 (Ehleringer, 1983). In comparison,
peak photosynthesis rates in maize are near 60 µmol m−2 s−1
and in wheat and rice they approach 35 µmol m−2 s−1 (Evans,
1989; Dohlmann and Long, 2009; Adachi et al., 2014). Other
notables in the record-holder category include purslane
(Portulaca oleracea), a well-known weed and minor crop that
is the only known plant to conduct both C4 and CAM photosynthesis, and Euphorbia olowaluana, a once common tree on
Hawaii that grows to 10 m in height (Guralnick et al., 2002;
Native Plants Hawaii, http://www.nativeplants.hawaii.edu/
plant/view/Euphorbia_olowaluana). The term ‘tree’ is notable here because the C4 pathway is largely absent from the
arborescent life form, which has great consequences for the
biosphere because it means forests are exclusively C3 dominated (Sage et al., 1999b). Euphorbia olowaluana and its sister
species E. herbstii (formerly E. forbesii), E. remyi var kauaiensis, and E. rockii represent the only known C4 trees, in the traditional sense of the term (Yang, 2012). Numerous Hawaiian
Euphorbia species such as E. herbstii are exceptional for C4
biologists because they grow as understorey trees or shrubs
in Hawaiian forests, demonstrating that the C4 pathway is
not incompatible with low-light habitats (Pearcy and Calkin,
1983; Yang, 2012). Sadly, these Euphorbia species are endangered due to invasive species and habitat degradation (Native
Plants Hawaii, http://www.nativeplants.hawaii.edu/plant/
view/Euphorbia_olowaluana). Each should be cultivated in
arboretums, gardens, and parks to ensure survival of these
unique C4 life forms.
The last major category of the C4 Hall of Fame are the
research stars, which in some way have contributed significantly to our understanding of C4 plant biology. Some of
the research stars are already in the Hall of Fame as crops,
notably maize and sugarcane in which the C4 pathway was
discovered, and sorghum, the first C4 plant to be sequenced.
Those selected just on the basis of their research roles include
the genetic model species—the grass Setaria viridis and the
eudicot Cleome gynandra—which are leading current efforts
to unravel the genetic control of the C4 pathway (Marshall
et al., 2007; Li and Brutnell, 2011). The genus Flaveria has
served as the leading model for studies of C4 evolution given
it has multiple C3, C4, and C3–C4 intermediate species. It is
represented by F. brownii, which is the best example of an
early-stage version of C4 photosynthesis. Flaveria brownii is
commonly termed a C4-like intermediate because it exhibits
residual activity of Rubisco and the C3 cycle in its mesophyll
e22 | Sage
tissue (Monson and Rawsthorne, 2000). In the treatment presented in Table 3, it is considered a C4 plant because it meets
all of the criteria of C4 photosynthesis (Table 1).
While the vast majority of C4 species use some version of
the two-tissue Kranz anatomy to concentrate CO2, there are
a handful of species that do it all within a single cell. The
most spectacular of these are the three species from the
genus Bienertia, which concentrate CO2 into a central cellular region where a ball of Rubisco-containing chloroplasts
are localized (Sharpe and Offermann, 2014). The best studied of the three Bienertia species is B. sinuspersici, which has
been selected for the Hall of Fame as a representative of the
single-celled C4 plants. Other research stars include Atriplex
rosea, which is the only C4 plant to form fertile hybrids with
a C3 species (Atriplex prostrata; Bjorkman et al, 1971; Oakley
et al., 2014). The grass Alloteropsis semialata is the only species with both C3 and C4 genotypes (Lundgren et al., 2015);
a C3–C4 intermediate genotype of A. semialata has just been
reported (Lundgren et al., 2016). Finally, a number of C4 species are known to produce C3 tissues, such as C3 cotyledons in
terrestrial chenopods and C3 tissues in submerged portions of
marsh grasses and sedges (Ueno et al., 1988; Pyankov et al.,
2000). The best studied of these species is Eleocharis vivipara,
a Florida marsh grass whose ability to produce C3 culms when
submerged and C4 culms when emergent is being studied to
understand the genetics of C4 induction (Ueno et al., 1988).
Finally, movie stars are regulars in a Hall of Fame, and
so we need to include tumbleweed (Salsola kali), a star of
many Western-themed films and the 1998 cult favourite The
Big Lebowski. It is the only non-cultivated C4 species to make
it in Hollywood, where it has also inspired numerous songs,
including the popular ballad ‘Tumbling Tumbleweeds’ by Roy
Rodgers and The Sons of the Pioneers. Although tumbleweed
does not have a star on the Hollywood Walk of Fame, it is
well represented there, in sidewalk cracks and old movie sets,
tumbling along with the coastal breezes wafting off of the
blue Pacific Ocean.
The world’s C4 flora in 2066
To finish this 50th anniversary perspective, it is worth considering the future prospects of the C4 family and its status
on the 100th anniversary of Hatch and Slack’s (1966) publication. At a minimum, the picture of the C4 family should
be complete by then, as the current rate of phylogenetic and
physiological progress should fill in the uncertainties within
a few decades. Also, there may be new, engineered C4 species to include in the C4 Hall of Fame. If C4 engineering is
a success, people in 2066 should be able to judge whether
the benefits lived up to the promise and transformed global
agriculture. C4 rice could meet the food demands of a larger
population well into the next century, while higher water
use efficiency of C4 cotton and wheat could extend limiting
water supplies in drought-prone regions where much of their
cultivation occurs. In legumes, the supercharged C4 engine
could be coupled to the nitrogen-fixing symbiosis to create
self-fertilizing crops, thus reducing expensive nitrogen inputs.
It is possible that rising atmospheric CO2 may boost yields
of C3 crops enough to reduce the need for C4 crops and C4
engineering, but, even if this is the case, there is still room for
a C4 contribution. High CO2 does not harm C4 plants, and
often enhances their performance, particularly in terms of
increased water and nitrogen use efficiency (Sage and Pearcy,
2000; Morgan et al., 2004, 2011; Leakey et al., 2009; Polley
et al., 2014). Elevated CO2 reduces stomatal conductance in
C4 plants as it does in C3 plants, so that when coupled to the
high water use efficiency already present in C4 species, superwater use-efficient plants may result. In regions where water
shortages occur, natural and engineered C4 crops could sustain yields while sparing critical supplies for use by cities, fisheries, hydroelectricity, and natural ecosystems.
Numerous papers have discussed the future of Earth’s
C4 vegetation, which is important given that rising CO2
will eliminate one of the principal drivers that favoured the
diversification of the C4 flora (Henderson et al., 1994; Sage
and Kubien, 2003; Wedin, 2004; Leakey, 2009; Bond and
Midgely, 2012; Polley et al., 2013). The relative effect of rising
CO2 on C3 and C4 vegetation is well studied at the physiological level, and in natural swards of prairie and marsh grasses
(Wand et al., 1999; Polley et al., 2002, 2014; Ainsworth and
Long, 2005; Leakey et al., 2009; Morgan et al., 2011). The
general conclusion is that C4 photosynthesis is modestly
(5–25% on average) enhanced with a doubling of CO2, from
both direct effects on the photosynthetic biochemistry and
indirect effects from improved water status; however, the
stimulation of C3 photosynthesis is 2- to 3-fold greater due
to the suppression of photorespiration (Ghannoum et al.,
2000; Ainsworth and Long, 2005), which can shift biomass
production and some (but not all) competitive outcomes in
favour of C3 species (Ainsworth and Long, 2005; Morgan
et al., 2004, 2011; Polley et al., 2012a, b, 2013, 2014). In
natural ecosystems, CO2 enrichment effects on C3 versus
C4 vegetation vary with many interacting factors such as
growth form, the timing and intensity of precipitation and
disturbance, abiotic stress, biotic interactions, and resource
availability. Regardless of CO2, warmer winters could
favour C3 vegetation, because the cool growing season may
expand as winter severity is reduced; alternatively, warmer
summers could favour C4 grasses, especially if drought and
fire become more common. As a case in point, tall-stature
C4 grasses are stimulated more by rising CO2 than shortstature grasses (Polley et al., 2012a, b). This could be significant where invasive C4 grasses from Africa, which tend to
be tall-statured, compete against native shorter-statured C4
grasses, as has been documented in South America (Baruch
et al., 1985; Knapp and Medina, 1999). Once established,
the tall exotics burn hotter, thereby intensifying fire cycles
that are damaging to native species of all photosynthetic
pathways (D’Antonio and Vitousek, 1992). Woody C3 species appear to be particularly favoured in CO2-enriched
environments, because they can establish more rapidly and
overtop the grass canopy earlier than in lower CO2 conditions, thereby shading the grasses and disrupting fire cycles
(Bond et al., 2003; Davis et al., 2007; Bond and Midgely,
2012). In recent decades, many grassland and savanna areas
The C4 plant family portrait | e23
around the world have been invaded by woody species, leading to suggestions that increased atmospheric CO2 is already
playing a role (Polley et al., 1996; Bond et al., 2003; Bond
and Midgely, 2012). If so, current trends will probably continue if not accelerate in future decades such that by 2066,
the amount of C4 habitat on the Earth will have been substantially reduced by woody encroachment.
The effects of elevated CO2 may not be the greatest threat
to C4 vegetation, however, because the Earth’s landscape
is already dominated by human action, and it is the decisions of people who control landscapes that will determine the future of the natural C4 flora (Sage and Kubien,
2003; Ellis and Ramunkutty, 2008; Parr et al., 2013). With
increasing population and economic growth, and new agricultural developments such as biofuel cultivation, exploitation of landscapes will increase, further threatening natural
diversity (Foley et al., 2005; Field et al., 2008). The largest negative impact on C4 species to date has been from the
conversion of natural grassland and savanna to agricultural
land, because these landscapes have deep soils and relatively flat topography (Hoekstra et al., 2005; Boakes et al.,
2010; Ellis et al., 2010). If not ploughed, C4-dominated
rangelands are used for raising cattle, which often introduces exotic pasture species or degrades the rangeland due
to overgrazing (Hoekstra et al., 2005; Ellis et al. 2010). In
North America, the tall grass prairie biome has been so
heavily converted that it is now considered an ecoregion
in critical crisis (Hoeskstra et al., 2005). The wire-grass
(Aristida stricta) pine savanna in the south-eastern USA is
now mostly farms or timber plantations, while much of the
semi-arid grasslands in south-western North American have
become dense woody scrub, largely due to overgrazing, fire
suppression, and, possibly, effects of CO2 fertilization (van
Auken, 2000; Earley, 2004). C4 grassland conversion has not
received the attention that deforestation has in environmental circles, even though natural grassland loss is proportionally greater than deforestation (Hoeksta et al., 2005; Parr
et al., 2014). A sense that C4 grasslands are already ‘anthropogenic’ explains the relative lack of concern, and allows
them to become targets for further development, including
‘green’ programmes that promote intensively managed biofuel or carbon sequestration plantations as part of efforts
to offset greenhouse gas emissions (Field et al., 2008; Parr
et al., 2014; Bond, 2016). Of great concern, invasive species
can magnify human impacts and complete the collapse of
the native diversity (Vitousek, 1994). In Hawaii, for example, many of the native C4 Euphorbia species, including
the Hall of Fame members E. herbstii and E. olowaluana,
are critically endangered due to habitat degradation from
invasive species such as strawberry guava (Psidium cattleyanum), feral pigs, and pyrophytic C4 grasses(Hueneke and
Vitousek, 1990; Sporck, 2011; Native Plants Hawaii, http://
www.nativeplants.hawaii.edu/plant/view/Euphorbia_olowaluana). Invasive annual grasses, both C3 (Bromus rubrens)
and C4 (Cenchrus ciliaris), similarly threaten native C4 (and
CAM) diversity in the hot desert biomes of the American
Southwest due to enhancement of fire cycles and intense
competition for water (Tellmann, 2002).
Concluding thoughts
In closing, the future health of much of the world’s 8100 C4
species is uncertain. Significant changes in climate, CO2, fire
regime, land use, and exotic invasions are happening in concert, and with such speed that the response capacity of the
native C4 flora is being overwhelmed. This is a common concern for all species on Earth, but, unlike C3 plants or insects,
there is much less redundancy in the C4 flora to withstand
extinction events. For example, an across the board loss of
75% of the world’s species would leave >50 000 C3 species, but
just 2000 C4 species. Certainly, numerous C4 species will do
well, particularly those that are used by humans for food, bioenergy, pastures, and golf courses. The weedy species that are
well adapted to people will also proliferate, given the expected
increase in human activity. Many of these species are C4 Hall
of Fame members, and it is somewhat ironic that they could
be the means by which much of the native C4 diversity is
reduced. C3 weeds will have their share of global impacts, but,
because of similar environmental requirements, the impacts
of C4 weeds and land clearing for C4 crops will be greatest in
landscapes supporting Earth’s natural C4 diversity.
Given these concerns, humanity will need to pay particular
attention to the preservation of the native C4 flora and its habitat over the next 50 years (Bond and Parr, 2010; Parr et al.,
2014). To ensure this happens, it will be the responsibility of
the C4 research and teaching community to bring these concerns to the attention of policy makers, the public, and land
managers, in order to ensure that appropriate conservation
measures are enacted. If we fail at this, then on the occasion
of the 100th anniversary of the C4 discovery, the portrait of
the C4 family in 2066 could be much smaller than it is today.
Supplementary data
Supplementary data are available at JXB online.
Table S1. Evolutionary lineages of C4 photosynthesis in the
eudicots, Cyperaceae, and Hydrocharitaceae.
Table S2. The postulated number of C4 lineages in the
Poaceae, with estimated number of genera and species per
lineage.
Acknowledgements
I send a particularly strong note of thanks to Professor Colin Osborne of
Sheffield University whose years of effort helped create a downloadable list of
the world’s grass genera with species numbers and photosynthetic pathways.
Professor Osborne kindly shared this list and comments during preparation
of the C4 family portrait. I also thank Pascal Christin (Sheffeld), Gudrun
Kadereit (Mainz University), and Erica Edwards (Brown University) for valuable comments on the manuscript, and Professor Jeremy Bruhl (University
of New England, Australia), who shared his insight on sedges and provided
his detailed list of C4 sedge species (Bruhl and Wilson, 2007). I also am grateful to the members of the C4 research community who provided feedback on
the C4 photosynthesis Hall of Fame (see text for names).
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