Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK
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Received: 24 February 2005 Returned for revision: 23 March 2005 Accepted: 12 April 2005 Published electronically: 19 June 2005
• Aims This Botanical Briefing reviews how the integration of palaeontology, geochemistry and developmental biology is providing a new mechanistic framework for interpreting the 40- to 50-million-year gap between the origination of vascular land plants and the advent of large (megaphyll) leaves, a long-standing puzzle in evolutionary biology.
• Scope Molecular genetics indicates that the developmental mechanisms required for leaf production in vascular plants were recruited long before the advent of large megaphylls. According to theory, this morphogenetic potential was only realized as the concentration of atmospheric CO2 declined during the late Palaeozoic. Surprisingly, plants effectively policed their own evolution since the decrease in CO2 was brought about as terrestrial floras evolved accelerating the rate of silicate rock weathering and enhancing sedimentary organic carbon burial, both of which are long-term sinks for CO2.
• Conclusions The recognition that plant evolution responds to and influences CO2 over millions of years reveals the existence of an intricate web of vegetation feedbacks regulating the long-term carbon cycle. Several of these feedbacks destabilized CO2 and climate during the late Palaeozoic but appear to have quickened the pace of terrestrial plant and animal evolution at that time.
Key words: Carbon cycle, feedbacks, fossil plants, genetics, geochemistry, leaves, stomata
Annals of Botany 2005 96(3):345-352. © The Author 2005. Published by Oxford University Press on behalf of the Annals of Botany Company.
Plants evolved leaves on at least two independent occasions and the legacy of these historic evolutionary events is represented in extant floras by microphylls in lycophytes (clubmosses, spikemosses and quillworts) and megaphylls in euphyllophytes (ferns, gymnosperms and angiosperms). Microphylls, with a distinctive vasculature and (usually) unbranched venation, are thought to have evolved from spine-like enations and predate megaphylls in the terrestrial plant fossil record (Gifford and Foster, 1988). Of greater significance, however, was the origin of megaphylls in vascular plants through the developmental modification of lateral branches. Megaphylls altered the evolutionary trajectory of terrestrial plant and animal life, the biogeochemical cycling of nutrients, water and carbon dioxide and the exchange of energy between the land surface and the atmosphere. The vast majority of the estimated 250 000 or so extant species of flowering plants, as well as most gymnosperms and (extinct) pteridosperms, utilize(d) a flat-bladed megaphyll with a network of veins to capture solar energy for photosynthetic carbon assimilation. A true measure of their success in terrestrial environments is the capacity of leaves to endure climatic extremes between the tropics and the tundra whilst simultaneously facilitating the global scale net fixation of approx. 207 billion tonnes of CO2 (56·4 x 1015 g C) year–1 (Field et al., 1998). This primary production provides energy for virtually all forms of terrestrial life on Earth, especially tetrapods and insects, and links many ecosystem and biogeochemical processes.
Evidently, leaves are a global success. However, the advent of large megaphylls took place some 40–50 million years (Myr) after the origination of vascular land plants, suggesting that they were far from an evolutionary inevitability. The earliest ancestral vascular plants, dating to the late Silurian 410 Myr ago (Edwards and Wellman, 2001), were composed of simple or branched axial stems with sporangia but no leaves. Surprisingly, plants continued to remain leafless for the next 40–50 Myr, with megaphylls finally becoming widespread at the close of the Devonian period (360 Myr ago) (Kenrick and Crane, 1997; Boyce and Knoll, 2002; Osborne et al., 2004a,b). Surprise in the delayed appearance of a seemingly simple developmental modification is 3-fold. First, palaeontological evidence shows that the structural framework necessary for assembling a simple laminated leaf blade (meristem, vasculature, cuticle and epidermis) (Kenrick and Crane, 1997) was in place long before the advent of large megaphylls. Secondly, the same interval marks an unparalleled burst of evolutionary innovation in the history of plant life, which witnessed the rise of trees from herbaceous ancestors, and the evolution of complex life cycles, including the seed habit (Kenrick and Crane, 1997). Thirdly, the tiny deeply incised megaphylls of the rare early-Devonian plant Eophyllophyton bellum from Chinese rocks shows that plants had the capacity to produce a simple megaphyll (Hao and Beck, 1993; Hao et al., 2003). Why were plants unable to release this morphogenetic potential?
Until recently, our understanding of the evolution of megaphylls largely stemmed from Zimmermann's telome theory (Zimmermann, 1930, 1952) describing the sequence of overtopping, planation and webbing leading to appearance of the laminated leaf blade. The ancestral form of a dichotomizing axis branching out in three dimensions and typified by the rhyniophytes (Fig. 1) represents the basal state in the telome theory. Evolutionary ‘overtopping’ followed producing a main axis bearing reduced, lateral, determinate photosynthetic stems, branching out in three dimensions (e.g. trimerophytes). These 3-D lateral branch systems of terete stem segments then became ‘flattened’ into a single plane (e.g. cladoxyaleans). Finally, a webbing of photosynthetic mesophyll tissue joined the flattened segments of the lateral branches to form a laminate leaf blade (e.g. some progymnosperms) (Fig. 1). In this scheme, transformation of a branch into a leaf was achieved by simple modification of existing organs rather than a major change in body plan.
The mechanistic hypothesis of Beerling et al. (2001) links the gap between the earliest vascular plants and the advent of large megaphylls with a dramatic 90 % drop in the atmospheric CO2 concentration during the late Palaeozoic (Berner, 2004). The large fall in CO2 corresponded with a marked rise in the stomatal density of vascular land plants, with densities increasing a 100-fold from 5–10 mm–2 on early vascular plant axes to 800–1000 mm–2 on the cuticles of late Carboniferous megaphylls (McElwain and Chaloner, 1995; Edwards, 1998). These evolutionary shifts in leaf anatomy are consistent with the effects of CO2 on stomatal development observed in modern plants cultivated in controlled conditions under different CO2 concentrations (Woodward, 1987).
According to theoretical calculations with a model of leaf biophysics and physiology, the rise in stomatal density held special significance for the evolution of leaves by permitting greater evaporative cooling and alleviating the requirement for convective heat loss (Beerling et al., 2001). Simulations indicate that archaic land plants with axial stems, few stomata, and low transpiration rates only avoided lethal overheating because they intercepted a minimal quantity of solar energy (Beerling et al., 2001; Roth-Nebelsick, 2001). In contrast, a megaphyll intercepting at least twice as much solar energy (per unit area of the photosynthetic organ) reached temperatures approaching the highly conserved lethal threshold of extant tropical taxa (Beerling et al., 2001) because the same limited evaporative cooling was inadequate to dissipate the absorbed thermal energy. Theoretical arguments therefore indicate that early vascular land plants were prevented from developing large laminate leaves because their low stomatal densities placed a tight constraint on evaporative cooling.
Even if the stomatal density of the early vascular land plants was not under the influence of atmospheric CO2, and megaphylls evolved a high density, the transpiration rates required to maintain cool temperatures (approx. 9–13 mmol H2O m–2 s–1) are calculated to outstrip the capacity of xylem to supply it by factor of ten, assuming the primitive stele of Psilotum nudum is a reasonable analogue for that of the early rhyniophytes (Schulte et al., 1987). In this case, dehydration precluded the evolution of large megaphylls with high stomatal densities in early land plants; no fossils of such an anatomically modified organ have yet been discovered.
By the time large laminate leaves became widespread in late Devonian/early Carboniferous fossil floras, the concentration of atmospheric CO2 had fallen, and stomatal densities had increased by up to a 100 times the value of early vascular land plant axes. Large leaves of late Devonian/early Carboniferous plants attained transpiration rates sufficiently high to maintain temperatures well below the lethal threshold, despite intercepting more solar energy (Beerling et al., 2001). Greater evaporative cooling also meant that large leaves stayed cool despite diminished convective heat loss, a flux that declines with increasing leaf size as friction across the surface slows the passage of air and the transfer of heat. Large-leaved perennials in today's deserts similarly rely on a high transpiration rate to prevent overheating and maintain leaf temperatures 8–18 °C below that of the air (Smith, 1978; Ehleringer, 1988), with some species in southern California even evolving a correspondingly lower temperature optimum for photosynthesis (Smith, 1978). For these large-leaved desert species, summertime precipitation permits high transpiration rates. But in the late Palaeozoic, the evolution of large leaves required the co-evolution of the root and vascular systems for improved water delivery and transport to sustain higher transpiration rates (Knoll et al., 1984; Raven and Edwards, 2001). Deeper roots accessed water and nutrients from a greater volume of soil, whilst xylem conduit enlargement and the appearance of secondary growth of xylem by the end of the Devonian increased the hydraulic conductance through stems and trunks to the leafy canopy, helping to maintain higher transpiration rates (Rowe and Speck, 2005).
The hypothesis of Beerling et al. (2001) makes the clear prediction that larger leaves gradually appeared as CO2 levels declined and stomatal numbers rose to increase evaporative cooling and ease the thermal burden of absorbed solar energy. Osborne et al. (2004a, b) achieved a successful test of this prediction with a morphometric analysis of 300 fossil specimens archived in major European collections. Their results showed a 25-fold enlargement of leaf blades as atmospheric CO2 fell during the late Palaeozoic (Fig. 2B). Leaf enlargement occurred first in the progymnosperms during the mid- to late-Devonian with the initial radiation of Archaeopteris and soon afterwards in the pteridosperms, a group attaining a larger maximum size coincident with a lower atmospheric CO2 concentration.
The consistent pattern of leaf blade enlargement seen in these two phylogenetically independent clades (Boyce and Knoll, 2002; Osborne et al., 2004a,b) is consistent with the argument that CO2 acted as an environmental driver for this aspect of plant evolution. But as the concentration of atmospheric CO2 declined to permit the evolution of leaves, competition for light and space between neighbouring plants intensified. Competition is therefore envisaged as providing a powerful selective force for plants to become leafier and taller. These shifting ecological interactions were most obviously manifested as the well-documented increase in plant size (Chaloner and Sheerin, 1979) that tracked historical patterns of leaf enlargement (Fig. 2C).
Leaves in vascular plants are produced by determinate growth on the flanks of indeterminate shoot apical meristems. Inderminate growth of the shoot apical meristem is controlled by the knotted-like homeobox gene family (KNOX) (reviewed in Hake et al., 2004). KNOX genes are present in some green algae (e.g. Acetabularia), mosses, ferns, gymnosperms and angiosperms and their function may be highly conserved (Sano et al., 2005). Over-expression of fern KNOX-like genes in Arabidopsis thaliana, for example, produces a similar phenotype (altered leaf shape) as over-expression of arabidopsis KNOX-like genes in Arabidopsis (Sano et al., 2005). Proper development of leaves requires permanent negative repression of KNOX genes and several genes have so far been discovered in euphyllophyte species for maintaining the KNOX-off state, including PHANTASTICA (PHAN) isolated from snapdragon (Antirrhinum majus) (Waites and Hudson, 1995) and homologues of PHAN in maize (Timmermans et al., 1999) and Arabidopsis (Byrne et al., 2000). The two sets of genes operate in a co-ordinated manner with KNOX-like transcription factors repressing determinate growth promoted by PHAN, and PHAN-like transcription factors repressing KNOX to promote indeterminacy.
The system is likely controlled by auxin, which determines the site of leaf initiation and is correlated with decreased KNOX and increased PHAN activity. Environmental influences on KNOX and PHAN are not known. However, it is interesting to note that the original PHAN mutation in Antirrhinum was temperature-sensitive so that plant morphology was approximately normal at 25 °C but altered at lower temperatures (Waites and Hudson, 1995), implying an interaction of PHAN-mediated morphogenesis with temperature-sensitive elements of leaf development. Genes such as PHAN may be prime candidates for involvement in the first stage of the telome theory, i.e. the evolution of determinant lateral shoot systems in trimerophytes (Fig. 1) (Cronk, 2001).
Leaf production also requires differentiation between adaxial (upper) and abaxial (lower) surfaces because the former is specialized for the efficient capture of solar energy and the latter for gas exchange. Deriving from the apical vegetative meristem flank, the abaxial surface is as old as land plants themselves, so genes specifying adaxial identity constitute a key innovation in leaf evolution (Cronk, 2001). Plants appear to have evolved a complex hierarchy of transcription factor activation and depression, with the HD-ZIP (homeodomain–leucine zipper) gene family promoting adaxial leaf surfaces and others promoting abaxial differentiation (Cronk, 2001). Interestingly, HD-ZIP gene expression is subject to a novel form of post-transcriptional regulation involving microRNAs found in bryophytes, lycopods, ferns and seed plants suggesting that it may be very ancient, dating back more than 400 Myr (Floyd and Bowman, 2004). Interactions between HD-ZIP gene and vascular tissue polarity have been demonstrated in Arabidopsis (Emery et al., 2003) and, since vascular tissue predates the leaf (Kenrick and Crane, 1997), this suggests that the same developmental unit was recruited for leaf polarity (Emery et al., 2003).
Whether megaphylls, which arose independently in four vascular plant lineages (ferns, sphenopsids, progymnosperms and seed plants) (Boyce and Knoll, 2002; Osborne et al., 2004a, b), recruited the same gene systems is open to investigation (Cronk, 2001). However, this does seem a possibility given that a common developmental mechanism for leaf production appears to have been recruited independently at least twice in the evolution of land plants (Harrison et al., 2005).
Current understanding of the genetic controls of stomatal formation in response to environment signals is limited, although it is clear that genetic modification more strongly alters the relationship between stomatal density and pore size, with attendant effects on leaf gas exchange, than short-term changes in environmental conditions (Hetherington and Woodward, 2003). Stomatal research on Arabidopsis has identified the HIC (high carbon dioxide) gene, which responds to CO2 and negatively regulates stomatal formation (Gray et al., 2000). HIC encodes a putative 3-ketoacyl coenzyme-A synthase, an enzyme involved in the synthesis of wax components found in the cuticle. Wax-deficient mutant plants show a 42 % increase in stomatal density with CO2 enrichment to 1000 ppm compared with that at 400 ppm CO2 (Gray et al., 2000). The possible involvement of the HIC gene (or similar) in mediating stomatal formation under different CO2 concentrations in other plant groups remains to be seen.
The realization that aspects of plant evolution may be directed by changes in atmospheric CO2 gains greater significance when considered alongside the impact that plants themselves exert on the long-term carbon cycle. Before plants, Earth's global climate and atmospheric CO2 were regulated on a multi-million year timescale by the inorganic carbon cycle, in which CO2 is supplied to the atmosphere by volcanism and metamorphic degassing, and removed by the chemical weathering of Ca–Mg silicate rocks (Walker et al., 1981; Berner et al., 1983). The closed cycle can be represented as a simple systems diagram with arrows indicating a direct response (no bull's-eyes) or an inverse response (with bull's-eyes) (Fig. 3A). In these diagrams an even number of arrows with or without bull's-eyes defines a positive feedback and an odd number with bull's-eyes a negative feedback. The inorganic carbon cycle (Fig. 3A) is stabilized by a negative feedback loop because silicate-weathering rates increase with temperature (Walker et al., 1981; Berner et al., 1983). Rising CO2 levels, for example, strengthen the greenhouse effect, warm the climate, accelerate the chemical weathering of Ca–Mg silicate rocks, remove CO2 from the atmosphere and lead to a cooler climate (Fig. 3A).
CO2 + (Ca, Mg) SiO3 --> (Ca, Mg) CO3 + SiO2
The four most important PFLs involve the action of CO2 on plant evolution and its feedback on rock weathering rates, and sedimentary organic carbon burial (Fig. 3). In the first PFL (Fig. 3B) a drop in the atmospheric CO2 concentration and a rise in stomatal density permits the evolution of larger leaves through the mechanisms discussed earlier. Higher stomatal densities maximize CO2 diffusion into the leaf under conditions favourable for photosynthesis, and larger leaves capture more solar energy; both traits promote primary production and leafier canopies (Beerling and Berner, 2005). Higher densities are also associated with smaller stomata that can open and close more rapidly helping to protect the xylem water pathway from cavitation and allowing taller plants (Hetherington and Woodward, 2003). Taller, leafier plants require deeper rooting systems and more symbionts, including mycorrhizae, for uptake of water and nutrients (Raven and Edwards, 2001). Deeper roots and more abundant mycorrhizae increase nutrient removal and the surface area of the soil–root interface, accelerating the chemical weathering of silicates and further enhancing the removal of CO2 from the atmosphere (Berner et al., 2003).
In the second PFL, a similar chain of cause and effect follows a drop in atmospheric CO2, but with larger more productive plants enhancing organic carbon burial, both in terrestrial wetlands and marine environments after transport to the sea by rivers. Increasing organic carbon burial with falling atmospheric CO2 reflects a major evolutionary trend towards woody plants containing a high proportion of the relatively non-biodegradable compound lignin (Berner, 2004). An expanding terrestrial biomass, promoting CO2 removal from the atmosphere, is recorded as an enormous increase in sedimentary organic carbon burial on land and in the sea (Fig. 2D), most obviously manifested as carboniferous coal deposits. Two further complementary PFLs to those already described operate through the direct action of CO2 on climate, via the greenhouse effect (Fig. 3D and E).
The strengthening of this suite of PFLs during the late Palaeozoic evolution of the terrestrial flora, especially rooted forests, strongly amplified the extent and rate of both silicate weathering and sedimentary organic carbon burial. It was by way of these geochemical effects that plants brought about the precipitous decline in atmospheric CO2 that led ultimately to the Permo-Carboniferous glaciation (Berner, 2004; Beerling and Berner, 2005). The accelerated removal of CO2 from the atmosphere was only stabilized by the negative CO2-climate feedback loop of the inorganic carbon cycle (Fig. 3A), as the climate cooled and decelerated rates of silicate weathering.
In the long term, plants brought about a gradual and continual alteration of the global environment that modified selection pressures on subsequent generations, effectively facilitating their own evolution through the process of niche construction (Odling-Smee et al., 2003). Moreover, plant activities appear to have caused rates of evolution in terrestrial animals to accelerate. Late Palaeozoic insect and tetrapod faunas diversified together with terrestrial plants, and enhanced burial of organic carbon raised global oxygen levels (Berner, 2004), fuelling a spectacular radiation of insect gigantism (Graham et al., 1995).
Establishing a framework for understanding the origin and diversification of leaves in the Palaeozoic requires information from a broad range of disciplines that include palaeontology, plant physiology, geochemistry and molecular developmental genetics. Progress in many of these fields of research has seen such a framework begin to emerge and suggests that the exceptionally long 40- to 50-Myr delay in the advent of large megaphylls was a product of environmental opportunity and genetic potential. Once the morphogenetic potential of plants was released by falling atmospheric CO2 concentrations, leaf evolution entrained global consequences not only for the regulation of CO2 and climate but also for terrestrial organisms. Plants themselves effectively policed their own evolution through their influence on the silicate-rock-weathering–CO2-climate feedback cycle.
I thank Robert Berner, Ben Fletcher and Colin Osborne for comments on the manuscript, Andrew Fleming for discussions on the evolutionary developmental biology of leaves, Carl Bowser (University of Wisconsin) for permission to reproduce his photograph in Fig. 4 and Elizabeth and Robert Berner for drawing it to my attention. I am indebted to Bill Chaloner for many stimulating discussions on the evolution of megaphylls.
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FIG. 1. Stages in the evolution of the megaphyll as documented by Zimmermann's telome theory (Zimmermann, 1930), with representative fossil plants for each stage illustrated below. Upper schematic: Osborne et al. (2004b), images of Rhynia, Psilophyton and Archaeopteris after Gifford and Foster (1988), image of Actinoxylon after Matten (1968).
FIG. 2. Evolution of the atmosphere and land plants in the late Palaeozoic. (A) Changes in atmospheric CO2, modelled (open circles) or reconstructed from fossil soils (closed). (B) Observed increase in maximum width of megaphylls. Points indicate average maximum size for 5- or 10-Myr intervals. (C) Maximum plant height calculated from measurements of fossil stem diameter, assuming modern stem diameter–height relationships. (D) Changes in terrestrial carbon burial. Modified after Beerling and Berner (2005).
FIG. 3. Systems analysis diagrams of the long-term carbon cycle with and without geophysiological feedbacks involving land plants. (A) The inorganic geochemical carbon cycle. (B and C) Geophysiological feedbacks introduced by plant evolutionary responses to changes in atmospheric CO2 that result in changes in silicate rock weathering and organic carbon burial, respectively. (D and E) Geophysiological feedbacks as in B and C but including the direct effects of CO2 on climate via the atmospheric greenhouse effect. Arrows originate at causes and end at effects. Blue, inorganic processes; green, organic processes.
FIG. 4. Tropical weathering by deep-rooting trees on Kohala Mountain, Hawaii. The image shows the weathering ‘halo’ around the roots caused by mineral depletion. Depth of roots is approx. 7 m. Photograph courtesy of Carl Bowser. Reprinted from Berner et al. (Treatise on Geochemistry 5, p. 170; ©2003, with permission from Elsevier).
Annals of Botany 2005 96 (3):345-352. © The Author 2005. Published by Oxford University Press on behalf of the Annals of Botany Company.