Why are there so many angiosperms?

With over 300,000 extant species, flowering plants (angiosperms) form the clade Angiospermae, comprising ca. 90% of terrestrial plant biodiversity (de Boer et al., 2012). Their abrupt appearance and rapid diversification in early to mid-Cretaceous (135-100 Mya) evident in paleontological records is famously known as Darwin’s “abominable mystery,” as it challenges Darwin's belief that speciation is the result of gradual accumulated changes, rather than saltational (Friedman, 2009). To this day, controversies surround angiosperms’ time of origin. While the earliest unambiguous angiosperm fossil discovered was dated 130 Mya (Salomo et al., 2017), molecular analyses using 2694 plastosomes of angiosperms and 187 from gymnosperms (outgroup) date angiosperms’ origination to more than 200 Mya (Li et al., 2019). Despite this ambiguity, angiosperms are newcomers relative to other major clades of terrestrial plants like gymnosperms (Crepet, 2014). Mechanisms behind their rapid evolutionary radiation and gain of ecological dominance (Figure 2A) remain enigmatic and are still under ongoing research. Combining paleontological evidence and molecular analyses, this paper discusses three proposed explanations for angiosperms’ early radiation and ecological success: zoophily, increased vein density, and genome-downsizing following polyploidization. 

Zoophily 

A popular, yet potentially problematic, hypothesis ascribes angiosperms’ accelerated diversification and dominance to their shift from anemophily (employed by gymnosperms) to zoophily. Ca. 130 Mya, pollinators were insects (entomophily), which diversified along with angiosperms during mid-Cretaceous as evident in fossils (Hu et al., 2008), indicating coevolution and mutualism. Entomophily soon became the dominant pollination mode, allowing for more efficient reproduction, as pollen grains are directed to receptive stigmas with higher precision via insects than being blown by wind at random directions. Indeed, an examination of pollen fossils from angiosperms in mid-Cretaceous Dakota Formation showed clear clumping patterns, a feature characteristic of entomophilous plants (Hu et al., 2008). Pollen fossilizes well compared to entire plants (Odgaard, 1999), and is therefore used extensively for research. As angiosperm-insect relationships grew more intimate, angiosperms specialized in different animal pollinators (ethological isolation), which partly explains their ecological and morphological variations (Armbruster, 2014). For example, different orchid species within genus Gongora produce distinct scents that attract specific euglossine bees (Hetherington-Rauth & Ramírez, 2016). Both benefit: angiosperms from higher precision in pollen destinations and animals from reduced handling time and search efficiency.  

However, using morphological interpretations to infer pollination mode is prone to inaccuracies. For example, the argument that pollen-clumping denotes entomophily is based on pollen-clumping patterns in extant species, then extrapolating this data to deduce pollination modes of fossilized angiosperms (Hu et al., 2008). This method is a reductionist approach, categorizing pollination syndromes as mutually exclusive and assuming pollen-clumping is the sole determinator of pollination vectors used. In fact, insects also visit some anemophilous plants, and pollen of entomophilous plants can be dispersed by wind, highlighting that pollination syndromes should be placed on a continuum (Martin et al., 2009). Furthermore, pollen clusters are rare, yet not absent, in anemophilous plants. Ragweed (Ambrosia), for instance, has highly clumped pollen, yet is a typical anemophilous plant genus (Sabban et al., 2012).  

Moreover, recent studies suggest extinct gymnosperms that were abundant before mid-Cretaceous, such as Bennettitales, were also entomophilous (Peñalver et al., 2015). For example, pollen from Williamsoniella lignieri (under the Bennettitales order) resemble those attached to abdomen of Zhangsolvidae (family of true flies) in fossils (Peñalver et al., 2015). Additionally, pollen clumps were found on Exesipollenites, a genus under Bennettitales, highlighting that pollen-clumping was not exclusively to angiosperms (Peñalver et al., 2015). These suggest that while zoophily contributed to angiosperm diversity and specialization, angiosperms’ success in initially outcompeting previously dominant gymnosperms likely did not rely on entomophily alone. 

Leaf Vein Density (Dv) 

Recent studies focused on another innovation that conferred an evolutionary advantage to angiosperms: increase in Dv (Feild et al., 2011; de Boer et al., 2012; Brodribb & Feild, 2010). Falling atmospheric CO2 levels during angiosperm radiation (Figure 2B) imposed an evolutionary pressure on all plants, as RuBisCO functions poorly in low CO2 concentrations, limiting photosynthetic rate. Natural selection therefore favored leaves with higher surface conductance efficiency to maintain the level of CO2 in carboxylation sites, which is enabled by increased Dv, as suggested by fossil records (de Boer et al., 2012; Brodribb et al., 2007; Théroux-Rancourt et al., 2021) (Figure 2C & 2D). 

Feild et al. (2011) measured Dv of 307 taxa of angiosperm fossils from the Cretaceous and that of gymnosperm and fern fossils, and reported a two-phase increase in angiosperm Dv, while non-angiosperm fossils exhibited consistently low Dv (Table 1). This increase in Dv was mirrored by a concurrent increase in angiosperm fossils during mid-Cretaceous, confirming the role of high Dv in angiosperms’ success (Figure 2A & 2D). Other analyses of fossils and extant angiosperms and its outgroups demonstrated that angiosperm Dv surpasses all other terrestrial plants (Boyce et al., 2009; de Boer et al., 2012). 

Table 1. Summary of Dv data during angiosperm radiation. All data provided by Feild et al. (2011). 

Connection between Dv and surface conductance 

For CO2 to diffuse into leaves, leaves must expose moist internal surfaces to the dry atmosphere, which inevitably leads to transpiration, whereby water evaporates from mesophyll cell walls and into atmosphere via stomata (Simonin & Roddy, 2018). This puts leaves at risk of desiccation. Hence, to increase surface conductance for CO2, plants need more efficient water transport systems to prevent desiccation (Simonin & Roddy, 2018). Water is drawn from xylem of leaf veins into mesophyll cell walls (Sack & Scoffoni, 2012). Water travels faster in xylem than in apoplast. Denser leaf veins decrease the distance between each stoma and its nearest vein terminal (Figure 1). This increases water transport rate, enabling angiosperms to increase their CO2 uptake as they can tolerate higher transpiration rates. Studies using shoots from angiosperms, gymnosperms, and ferns reported a strong correlation between gas exchange capacity and leaf hydraulic conductance, confirming the interconnection between surface conductance and water transport efficiency (Sack & Scoffoni, 2012; Brodribb et al., 2004). Furthermore, studies sampling plants from mosses to angiosperms repeatedly demonstrated that both hydraulic conductivity and maximum photosynthetic rate are proportional to Dv, highlighting the dependence of maximal CO2 uptake on Dv. (Boyce et al., 2009; Brodribb et al., 2007). 

Figure 1. Schematized cross-sections of conifer leaf (top), early angiosperm leaf (middle) with low Dv and Ds, and modern angiosperm leaf (bottom) with high Dv and Ds. Blue ovals represent vein terminal for water supply; blue arrows show distance of apoplast pathway. (de Boer et al., 2012) 

Increased Dv allowed angiosperms to develop leaves with higher stomatal density (Ds) and reduced stomatal pore size to increase maximal diffusive conductance, with equal water loss (Sack & Buckley, 2016; Franks & Beerling, 2009; de Boer et al., 2012; Simonin & Roddy, 2018) (Figure 2C). According to Fick’s law, rate of diffusion is proportional to surface area available for diffusion, which increases with higher Ds, and inversely proportional to thickness of membrane, which decreases with smaller guard cells (Davis et al., 1995). Smaller stomata also enable faster response times (Franks & Farquhar, 2006; Elliott-Kingston et al., 2016), better preventing unnecessary water loss by closing stomata faster.  

Figure 2. A) Angiosperm evolutionary radiation and rise to dominance; B) Atmospheric CO2 modelled using GEOCARBSULF model; C) Stomatal conductance inferred from fossil Ds (diagram above graph shows relative stomatal size and Ds); D) Rising angiosperm Dv reconstructed from leaf fossils. Red dashed line marks the period of most rapid angiosperm radiation in fossil records. All sketched from data provided by de Boer et al. (2012) and supported by other studies (see main text). 

Another process that enabled reduction of guard cell size is rapid genome-downsizing following polyploidization, which exclusively occurred in angiosperms (Simonin & Roddy, 2018; Théroux-Rancourt et al., 2021).  

Polyploidy and diploidization 

Polyploidy resulting from WGD (Whole Genome Duplication) events played a significant role in the evolution of vascular plants, particularly angiosperms (Soltis et al., 2009). Wood et al. (2009) estimated 15% angiosperm speciation involved polyploidization. WGDs provide more genetic material for mutation-driven diversity that natural selection can act upon, contributing to angiosperms’ radiation during mid-Cretaceous (Dodsworth et al., 2015). While most duplicates become non-functional as deleterious mutations accumulate, some, often transcription factors (TFs), specialize in subsets under their ancestral function (subfunctionalization), and others acquire new functions (neofunctionalization) (Moriyama & Koshiba-Takeuchi, 2018; Conant et al., 2014). Subfunctionalization also creates phenotypic diversity, as the combinatorial interaction and expression patterns of paralogous homeotic genes give rise to distinct morphologies.  

However, despite the advantages, larger genomes impose constraints on minimum cell size. In guard cells, this prevents plants from reducing stomatal size (Simonin & Roddy, 2018; Théroux-Rancourt et al., 2021). Using data for 400 species of ferns, gymnosperms, and angiosperms, Simonin & Roddy (2018) showed that genome size is proportional to guard cell length. Furthermore, new beneficial recessive mutations will be masked by existing alleles, resulting in inefficient selection (Dodsworth et al., 2015; Otto, 2007). WGD therefore cannot continue indefinitely, and downsizing mechanisms are required to reduce duplicate-gene redundancy. 

Over time, natural selection favors smaller genomes and more beneficial phenotypes, which can be simultaneously achieved through post-polyploidization (Dodsworth et al., 2015). Diploidization is a mechanism to downsize genomes and allows genomic novelties to be unveiled, thus truly initiating phenotypic diversity (Mandáková & Lysak, 2018). While WGD is common among terrestrial plants, rapid genome-downsizing is specific to angiosperms (Simonin & Roddy, 2018; Théroux-Rancourt et al., 2021), conferring an advantage over all non-angiosperms. Time-calibrated phylogenies show that angiosperms’ rapid diversification follows WGD, with a time lag in-between (Tank et al., 2015; Eric Schranz et al., 2012). Meudt et al. (2015) and Franzke et al. (2011) performed genomic analyses on extant species, both polyploid and diploid, from genera Veronica and Brassica, respectively, and, combined with phylogenetic trees, concluded that this time-lag was due to post-polyploid diploidization.  

Diploidization is achieved through chromosomal rearrangements (Edger & Pires, 2009). Many hypotheses exist for rearrangement mechanisms, the best supported one being Gene-Balance Hypothesis (Conant et al., 2014), which argues that preservation of dosage-sensitive genes, such as TFs, are more likely, maintaining stoichiometric balance of interacting proteins (Edger & Pires, 2009; De Smet et al., 2013). An example supporting this is diploidization in Brassica (Xie et al., 2019). Genome analyses comparing topologically associating domains (TADs), regions where genes extensively interact with each other and are therefore dosage-sensitive, in 2987 sequenced B. rapa and 4693 sequenced B. oleracea (which underwent independent diploidization) showed statistically significant TAD-overlaps between the species (64.1% as compared to 13.62% for random) (Xie et al., 2019). This preserves the subfunctionalization from WGD in TFs and other interacting genes, therefore maintaining morphological diversity. 

While some genes show diploidization-resistance, others are mostly present in single copies, such as certain orthologous groups in C. rapa and Musa acuminata (De Smet et al., 2013), demonstrating further biases in diploidization, as frequencies of single-copy groups exceed the random expectation (De Smet et al., 2013). Via deletion of paralogues, recessive beneficial mutations (neofunctionalization) can be unmasked. 

Thus, chromosomal rearrangements involved in diploidization can preferentially retain dosage-sensitive genes, while deleting others, including those in single-copy orthologous groups. While mechanisms behind this remain unclear, they create phenotypic variation, allowing genomic novelties from WGD to be fully realized, while preserving stoichiometric balance. Diploidization therefore explains both periods of angiosperm radiation and their rise to dominance. 

Conclusion 

While zoophily and angiosperm-insect coevolution played a key role in angiosperm diversification and specialization, high vein density and diploidization, which are exclusive to angiosperms, likely enabled angiosperms to outcompete the previously dominant species in the first place. Angiosperms are the dominant primary producers in most ecosystems. Further studies investigating the evolutionary history of angiosperms, from molecular processes to morphological variations and adaptations, and eventually their ecological niches, will help scientists better understand extant angiosperms and predict their future evolutionary patterns, which have implications for identifying conservation priorities. 

Reference list: 

Armbruster, W.S. (2014) Floral specialization and angiosperm diversity: Phenotypic divergence, fitness trade-offs and realized pollination accuracy. AoB PLANTS, 6. https://doi.org/10.1093/aobpla/plu003. 

Brodribb, T.J. & Feild, T.S. (2010) Leaf hydraulic evolution led a surge in leaf photosynthetic capacity during early angiosperm diversification. Ecology Letters, 13(2), 175–183. https://doi.org/10.1111/j.1461-0248.2009.01410.x. 

Boyce, C.K. et al. (2009) Angiosperm leaf vein evolution was physiologically and environmentally transformative. Proceedings of the Royal Society B: Biological Sciences, 276(1663), 1771–1776. https://doi.org/10.1098/rspb.2008.1919. 

Brodribb, T.J., Feild, T.S. & Jordan, G.J. (2007) Leaf maximum photosynthetic rate and venation are linked by hydraulics. Plant Physiology, 144(4), 1890–1898. https://doi.org/10.1104/pp.107.101352. 

Brodribb, T.J. et al. (2004) Leaf hydraulic capacity in ferns, conifers and angiosperms: Impacts on Photosynthetic Maxima. New Phytologist, 165(3), 839–846. doi: 10.1111/j.1469-8137.2004.01259.x. 

Conant, G.C., Birchler, J.A. & Pires, J.C. (2014) Dosage, duplication, and diploidization: Clarifying the interplay of multiple models for duplicate gene evolution over time. Current Opinion in Plant Biology, 19, 91–98. https://doi.org/10.1016/j.pbi.2014.05.008. 

Crepet, W.L. (2014) Advances in flowering plant evolution. eLS. https://doi.org/10.1002/9780470015902.a0023964. 

Davis, P.D., Parbrook, G.D. & Kenny, G.N.C. (1995) Diffusion and osmosis. Basic Physics and Measurement in Anaesthesia, 89–102. https://doi.org/10.1016/B978-0-7506-1713-0.50012-3. 

de Boer, H.J. et al. (2012) A critical transition in leaf evolution facilitated the Cretaceous Angiosperm Revolution. Nature Communications, 3(1). doi: 10.1038/ncomms2217 (2012). 

De Smet, R. et al. (2013) Convergent gene loss following gene and genome duplications creates single-copy families in Flowering plants. Proceedings of the National Academy of Sciences, 110(8), 2898–2903. https://doi.org/10.1073/pnas.1300127110. 

Dodsworth, S., Chase, M.W. & Leitch, A.R. (2015) Is post-polyploidization diploidization the key to the evolutionary success of angiosperms? Botanical Journal of the Linnean Society, 180(1), 1–5. https://doi.org/10.1111/boj.12357. 

Edger, P.P. & Pires, J.C. (2009) Gene and genome duplications: The impact of dosage-sensitivity on the fate of nuclear genes. Chromosome Research, 17(5), 699–717. https://doi.org/10.1007/s10577-009-9055-9. 

Elliott-Kingston, C. et al. (2016) Does size matter? atmospheric CO2 may be a stronger driver of stomatal closing rate than stomatal size in taxa that diversified under low CO2. Frontiers in Plant Science, 7. doi: 10.3389/fpls.2016.01253. 

Eric Schranz, M., Mohammadin, S. & Edger, P.P., 2012. Ancient whole genome duplications, novelty and diversification: The WGD radiation lag-time model. Current Opinion in Plant Biology, 15(2), 147–153. https://doi.org/10.1016/j.pbi.2012.03.011. 

Feild, T.S. et al. (2011) Fossil evidence for Cretaceous escalation in angiosperm leaf vein evolution. Proceedings of the National Academy of Sciences, 108(20), 8363–8366. https://doi.org/10.1073/pnas.1014456108. 

Franks, P.J. & Beerling, D.J. (2009) Maximum Leaf conductance driven by CO2 effects on stomatal size and density over geologic time. Proceedings of the National Academy of Sciences, 106(25), 10343–10347. https://doi.org/10.1073/pnas.0904209106. 

Franks, P.J. & Farquhar, G.D. (2006) The mechanical diversity of stomata and its significance in gas-exchange control. Plant Physiology, 143(1), 78–87. https://doi.org/10.1104/pp.106.089367. 

Franzke, A. et al. (2011) Cabbage family affairs: The evolutionary history of brassicaceae. Trends in Plant Science, 16(2), 108–116. https://doi.org/10.1016/j.tplants.2010.11.005. 

Friedman, W.E. (2009) The meaning of Darwin's “Abominable mystery.” American Journal of Botany, 96(1), 5–21. https://doi.org/10.3732/ajb.0800150. 

Hetherington-Rauth, M.C. & Ramírez, S.R. (2016) Evolution and diversity of floral scent chemistry in the euglossine bee-pollinated Orchid Genusgongora. Annals of Botany, 118(1), 135–148. https://doi.org/10.1093/aob/mcw072. 

Hu, S. et al. (2008) Early steps of angiosperm–Pollinator Coevolution. Proceedings of the National Academy of Sciences, 105(1), 240–245. https://doi.org/10.1073/pnas.0707989105. 

Li, H.-T. et al. (2019) Origin of angiosperms and the puzzle of the Jurassic Gap. Nature Plants, 5(5), 461–470. doi: 10.1038/s41477-019-0421-0. 

Mandáková, T. & Lysak, M.A. (2018) Post-polyploid diploidization and diversification through dysploid changes. Current Opinion in Plant Biology, 42, 55–65. https://doi.org/10.1016/j.pbi.2018.03.001 

Martin, M.D. et al. (2009) Pollen clumping and wind dispersal in an invasive angiosperm. American Journal of Botany, 96(9), 1703–1711. https://doi.org/10.3732/ajb.0800407. 

Meudt, H.M. et al. (2015) Is genome downsizing associated with diversification in polyploid lineages ofveronica? Botanical Journal of the Linnean Society, 178(2), 243–266. https://doi.org/10.1111/boj.12276. 

Mondragón-Palomino, M. & Theißen, G. (2009) Why are orchid flowers so diverse? reduction of evolutionary constraints by paralogues of class B floral homeotic genes. Annals of Botany, 104(3), 583–594. doi: 10.1093/aob/mcn258. 

Mondragón-Palomino, M. & Theißen, G. (2011) Conserved differential expression of paralogous deficiens- and globosa-like MADS-box genes in the flowers of Orchidaceae: Refining the ‘Orchid Code.’ The Plant Journal, 66(6), 1008–1019. https://doi.org/10.1111/j.1365-313X.2011.04560.x. 

Moriyama, Y. & Koshiba-Takeuchi, K. (2018) Significance of whole-genome duplications on the emergence of evolutionary novelties. Briefings in Functional Genomics, 17(5), 329–338. https://doi.org/10.1093/bfgp/ely007. 

Odgaard, B.V. (1999) Fossil pollen as a record of past biodiversity. Journal of Biogeography, 26(1), 7–17. https://doi.org/10.1046/j.1365-2699.1999.00280.x. 

Otto, S.P. (2007) The evolutionary consequences of polyploidy. Cell, 131(3), 452–462. https://doi.org/10.1016/j.cell.2007.10.022. 

Peñalver, E. et al. (2015) Long-proboscid flies as pollinators of cretaceous gymnosperms. Current Biology, 25(14), 1917–1923. https://doi.org/10.1016/j.cub.2015.05.062. 

Sabban, L., Jacobson, N.-lee & van Hout, R. (2012) Measurement of pollen clump release and breakup in the vicinity of ragweed (A. Confertiflora) staminate flowers. Ecosphere, 3(7). https://doi.org/10.1890/ES12-00054.1. 

Sack, L. & Buckley, T.N. (2016) The developmental basis of stomatal density and Flux. Plant Physiology, 171(4), 2358–2363. https://doi.org/10.1104/pp.16.00476. 

Sack, L. & Scoffoni, C. (2012) Measurement of leaf hydraulic conductance and stomatal conductance and their responses to irradiance and dehydration using the evaporative flux method (EFM). Journal of Visualized Experiments, (70). doi: 10.3791/4179. 

Salomo, K. et al. (2017) The emergence of earliest angiosperms may be earlier than fossil evidence indicates. Systematic Botany, 42(4), 607–619. doi: 10.1600/036364417X696438. 

Simonin, K.A. & Roddy, A.B. (2018) Genome downsizing, physiological novelty, and the global dominance of flowering plants. PLOS Biology, 16(1). https://doi.org/10.1371/journal.pbio.2003706. 

Soltis, D.E. et al. (2009) Polyploidy and angiosperm diversification. American Journal of Botany, 96(1), 336–348. https://doi.org/10.3732/ajb.0800079. 

Tank, D.C. et al. (2015) Nested radiations and the pulse of angiosperm diversification: Increased diversification rates often follow whole genome duplications. New Phytologist, 207(2), 454–467. https://doi.org/10.1111/nph.13491. 

Théroux-Rancourt, G. et al. (2021) Maximum CO2 diffusion inside leaves is limited by the scaling of cell size and genome size. Proceedings of the Royal Society B: Biological Sciences, 288(1945), p.20203145. https://doi.org/10.1098/rspb.2020.3145. 

Wood, T.E. et al. (2009) The frequency of polyploid speciation in vascular plants. Proceedings of the National Academy of Sciences, 106(33), 13875–13879. https://doi.org/10.1073/pnas.0811575106. 

Xie, T. et al. (2019) Biased gene retention during diploidization in brassica linked to three-dimensional genome organization. Nature Plants, 5(8), 822–832. https://doi.org/10.1038/s41477-019-0479-8.