Adaptive responses to directional trait selection in the Miocene enabled Cape proteas to colonize the savanna grasslands
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Evol Ecol
DOI 10.1007/s10682-013-9645-z
ORIGINAL PAPER
Adaptive responses to directional trait selection
in the Miocene enabled Cape proteas to colonize
the savanna grasslands
Byron B. Lamont • Tianhua He • Katherine S. Downes
Received: 14 November 2012 / Accepted: 2 April 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract Directional selection occurs when the agent of selection changes direction or
strength such that fitness of a dominant trait is relaxed or even annulled, and simulta-
neously the fitness of a rare opposing trait is intensified or even becomes essential. The
value of this concept in evolutionary ecology was demonstrated by mapping fire- and
growth-related traits and regional affinity onto a molecular-based chronogram for 91
species of Protea that is widespread in the shrubland and grassland biomes of southern
Africa. The crown clade arose 22–34 million years ago (Oligocene) in the Cape shrublands
that was increasingly winter wet, nutrient and water-limited, and moderately fireprone.
This environment favoured nonsprouting and resprouting shrubs, on-plant seed storage
(serotiny) and strong sclerophylly. Adjoining grasslands developed 7–19 million years ago
(mid-late Miocene) that were summer wet, carbon-limited and highly fireprone. This
favoured resprouting only, seed release at maturity, and taller plants with large leaves and
weak sclerophylly. Thus, for successful migration from the shrublands to grasslands, the
dominant ancestral condition of serotiny was replaced by almost universal nonserotiny in
response to a change in fire type, and the dominant ancestral condition of nonsprouting by
universal (lignotuberous) resprouting in response to more frequent fire. Taller plants with
epicormic resprouting and larger, softer leaves were also promoted, due to the change in
fire type, growing season and declining pCO2, but appeared 4–6 million years later. Thus,
adaptive radiation via directional selection in the novel grassland environment required a
suite of adaptive responses to various selection pressures that led to species radiation in the
vast habitat available now constrained by stabilizing selection. The biology of grasses in
Electronic supplementary material The online version of this article (doi:10.1007/s10682-013-9645-z)
contains supplementary material, which is available to authorized users.
B. B. Lamont T. He K. S. Downes
Department of Environment and Agriculture, Curtin University, PO Box U1987, Perth, WA 6845,
Australia
e-mail: Tianhua.He@curtin.edu.au
B. B. Lamont (&)
School of Environmental Science, Murdoch University, Murdoch, WA 6150, Australia
e-mail: B.Lamont@curtin.edu.au
123Evol Ecol
savanna grasslands may well have changed during the Miocene/Pliocene but so did the
woody plants that invaded them.
Keywords Resprouter Sclerophylly Serotiny Species diversification Trait
proliferation Winter/summer rainfall
Introduction
A basic tenet of evolutionary theory is that the successful self-perpetuation of a population is
determined by the presence of key traits adapted to the particular constraints of that envi-
ronment (Stanton et al. 2000). But a given agent of trait selection can change markedly in
intensity or direction over time as well as space, leading to directional selection (Lemey et al.
2009). ‘Relaxed’ selection occurs when the agent of selection changes strength/direction
such that fitness of the current dominant trait is reduced or even annulled (Lahti et al. 2009).
At the same time, selection for the opposing trait, currently poorly represented or absent, is
intensified and may even become essential for survival. These are different sides of the same
coin: relaxed selection for one (currently dominant) trait implies intensified selection for the
opposing (currently rare) trait. For example, fire frequency can increase to such an extent that
the previously dominant woody species that were adapted to infrequent fire now have
insufficient time to complete their life cycle and only herbs and grasses remain (Higgins et al.
2000; Scheiter et al. 2012). Thus, identifying directional selection requires knowledge of the
evolutionary trajectory of the trait under consideration during changing selection regimes in
space and/or time. Here we explore the value of this concept by using the genus Protea to
show how changes in selection pressure can lead to adaptive radiation due to directional
selection followed later by stabilizing selection in the new environment.
It was traditionally believed that proteas, woody shrubs that currently dominate the winter-
rainfall shrublands of South Africa, arose from the adjoining summer-rainfall savanna/
grassland proteas (Rourke 1998). However, Valente et al. (2010) showed that the apparent
‘primitiveness’ of the summer-rainfall group is illusory. The stem of the Cape shrubland clade
was dated to the Oligocene, mean of 27.8 million years ago (Ma), and its crown to the early
Miocene, 17.7 Ma. The stem of the grassland clade arose in the mid-Miocene, 12.7 Ma,
similar to that for the grassland clade of the geophytic orchid, Disa, with obligate fire-stim-
ulated flowering (Bytebier et al. 2011; Lamont and Downes 2011). C4 grasslands began to
replace the C3 grasslands in Africa 14–16 Ma, with a marked expansion at 4–9 Ma, apparently
in response to the onset of summer rain, falling CO2 concentrations, increasing drought
associated with declining temperatures, more frequent fire and intensified herbivory (Retal-
lack 1992; Jacobs et al. 1999; Kürschner et al. 2008; Osborne 2008, Scheiter et al. 2012).
The agents of trait selection associated with the formation of the subtropical grasslands
at this time changed markedly in intensity and direction. Fire frequency increased to such
an extent (currently 1–5-year intervals, Bond et al. 2005; Midgley et al. 2010) that woody
plants were excluded; in addition, the wet season switched to the warm time of the year,
and carbon supply gradually became limiting rather than water and nutrients (Scheiter et al.
2012). The study by Simon et al. (2009) on the savanna grasslands of South America (the
cerrado), which appeared from 10 Ma, provides a clue to the selective forces at work.
Woody clades from the surrounding non-fireprone floras were unable to invade the
grasslands in the Miocene/Pliocene until they had developed some form of resprouting
(xylopodia, rhizomes) in response to the frequent fires characteristic of grasslands. In
some, even successful, cases the delay in acquiring the necessary traits to enable
123Evol Ecol
colonization of the grasslands exceeded 8 million years (My). The adaptive advantages of
resprouting in these circumstances seem clear: resprouting avoids the risks of prematur-
ation death or insufficient seed production for self-replacement caused by short-interval
fires, or postfire conditions unsuitable for germination or recruitment (Groeneveld et al.
2002; Lamont et al. 2011). In addition, resprouting allows for rapid return to vegetative
growth and flowering (Hoffmann and Solbrig 2003; Lamont and Downes 2011). Thus,
frequent fire should relax the selection pressure for nonsprouting and intensify selection for
resprouting (Lamont et al. 2011).
Almost all shrubland proteas store their indehiscent, wind-dispersed, single-seeded
fruits (hereafter called seeds) in ‘cones’ for one or more years (Rebelo 2001), termed
serotiny (Lamont et al. 1991). Serotinous species evolved from nonserotinous ancestors in
non-fireprone ecosystems (He et al. 2011, 2012; Lamont and He 2012). Central to
understanding the adaptive advantages of serotiny is the role of fire heat in opening the
cones for seed release onto an optimal postfire seedbed. Should the flames fail to reach the
cones, as can be expected with the ground-surface fires associated with grasslands, then the
seeds are not released (Schwilk and Ackerly 2001; Tapias et al. 2001). Further, there are no
advantages in on-plant seed storage if the variation around the extremely short mean fire
intervals is high, including successive annual fires, as occur with grassland fires. There may
even be fitness disadvantages if serotiny requires additional resources (Lamont and Enright
2000; Groom and Lamont 2010). Thus, frequent fire should relax the selection pressure for
serotiny and intensify selection for nonserotiny (Enright et al. 1998a, b).
The change in fire frequency and seasonality also affected other components of growth
at this time (4–9 Ma). While there is little time to reach maturity in frequently burnt
savannas, there is also little time to develop fire-resistant structures and insulated stored
buds. On the other hand, there are advantages in attaining a greater stature, so that apical
buds escape fire heat and vegetative and reproductive growth are not interrupted after fire
so that new seeds may be produced quickly and released onto the postfire seedbed (Lawes
et al. 2011). Also, the time constraint to growth was exacerbated by the decline in pCO2 in
the late Miocene (Kürschner et al. 2008). Carbon acquisition for stem growth and bud
storage structures is promoted by the presence of large leaves and low leaf mass area
(LMA) (Wright et al. 2004). This was facilitated by the change in season of greatest water
availability to the warmest time of the year and associated greater nutrient availability
(Meier and Leuschner 2008; Way and Oren 2010; A. Milewski and A. Mills, pers. comm.).
Thus, the selection pressure for short stature, small leaves and high sclerophylly that exists
in the Cape (Lamont et al. 2002; Yates et al. 2010) should be relaxed in the grasslands and
selection for the opposite traits intensified.
Our objective was to test the proposition that it was a simultaneous change in multiple
traits in response to a change in the critical agents of selection that enabled shrubland
proteas to colonize the adjoining grasslands after they appeared in the mid-Miocene. Once
this act of adaptive radiation was achieved, species radiation into the vast new habitat
available could follow. Specifically, we tested the following hypotheses, that:
(a) On-plant seed storage (serotiny) was the ancestral state in the shrublands whereas its
loss was a prerequisite for successful speciation in the grasslands in the mid-Miocene
as an adaptive response to more frequent, less intense fire;
(b) Nonsprouting was the ancestral state in the shrublands whereas resprouting was a
prerequisite for successful speciation in the grasslands in the mid-Miocene as an
adaptive response to more frequent fire;
123Evol Ecol
(c) Short stature, small leaves and high sclerophylly were ancestral in the shrublands
whereas the evolution of taller plants with larger leaves and low sclerophylly was a
prerequisite for successful speciation in the grasslands in the mid-Miocene as an
adaptive response to less intense fire, summer rain and declining pCO2.
Note that this is a three-stage process: knowledge is required of (a) the properties of the
critical agents of selection that existed over the time frame of interest, (b) the type of traits
that existed before the purported change in direction/strength of the agents of selection, and
(c) the type of traits that existed after the purported change in direction/strength of the
agents of selection.
Materials and methods
Our starting point was the molecular phylogeny of Valente et al. (2010) created for all 70
Protea species in the winter-rainfall Cape shrublands (S) and 17 of the 21 species in the
summer-rainfall grassland/savannas (G) east of the Cape, with one (P. subvestita) occur-
ring in both regions. Valente et al. (2010) combined two kinds of DNA markers and used
Bayesian analyses to reconstruct the Protea phylogeny. They estimated divergence times
of Protea using a relaxed-clock Bayesian Markov Chain Monte–Carlo approach, with the
root node age of Protea constrained to a mean of 28.4 My (based on the fossil calibration
of Proteaceae in Sauquet et al. 2009). For each of the 87 species (plus another four G
species that we added), we assigned each species to its region of occurrence (S, G), fire-
response type (killed, survive), type of resprouting (epicormic, lignotuber, rhizome—
assigned to all creeping plants that resprout), serotiny (yes, no) and plant height (taller, or
shorter than median) (Chisumpa and Brummitt 1987; Rebelo 2001, 2009).
Sister species and clades in S and G were assessed for maximum height, leaf length and
width on a per species basis using the resources above. Maxima were chosen rather than
means as only ranges were available and maxima represent potential size in response to the
environment and so are not confounded by measurements on juvenile or depauperate plants
due to say frequent burning or end of growing season effects. To further explore the third
hypothesis, the literature was searched for data in the two regions on leaf mass area (as an
index of sclerophylly, Groom and Lamont 1999) and its inverse, specific leaf area; soil and
plant P and N contents; and d13C as an index of water conservation (Lamont et al. 2002)
and collated tables and graphs prepared. The possible role of soil texture, considered
critical at the sister species level of speciation in Protea by Schnitzler et al. (2011), was
also assessed and is presented under S3.
Reconstruction of ancestral states
A time-based phylogeny for Protea was used to determine the ancestral states (details of
DNA markers used, and procedures for constructing the phylogeny and estimating
divergence times, are provided in Valente et al. (2010)). When stating the estimated dates
at a node, we added the difference of the range/2 (y) on either side of the median (x) of the
95 % highest posterior density (i.e., x ± y). To reconstruct the evolutionary history of
traits we used Bayesian MCMC methods to derive posterior distributions of the states and
log-likelihoods of the traits at the nodes/stems of the phylogeny. We used a continuous-
time Markov model, which presumes that traits can evolve repeatedly between their two
possible states at any branch of the phylogenetic tree (Pagel et al. 2004), to construct the
123Evol Ecol
ancestral state at each internal node. See He et al. (2011) for further details. Analysis of
reconstructed ancestral states and state transition rates were implemented in BayesMulti-
state, in software package BayesTrait (Pagel and Meade 2007;
http://www.evolution.rdg.ac.uk). The tree file contained 1000 plausible trees with branch
length (node age) and trait files contained the discrete state for each species (including all
Protea species and five Faurea species as outgroup in the chronogram).
Correlated evolution of traits
Correlated evolution between pairs of binary traits was analyzed in BayesDiscrete (Pagel
and Meade 2007). BayesDiscrete tests for correlated evolution of two binary traits by
comparing the fit (log-likelihood) of two continuous-time Markov models. One is a model
in which the two traits evolve independently on the tree (independent model). The other
allows the traits to evolve in a correlated fashion (dependent model). RJ-MCMC was
adopted following Pagel and Meade (2007). Ten test runs each with 100000 chains were
implemented to choose a ratedev value that produced an acceptable rate of 0.20 to 0.40.
Using the chosen ratedev value and a hyperprior that seeds the exponential form of a
uniform 0–30 distribution, a reversible jump dependent model was first tested. The analysis
was repeated by confining the RJ chain to the independent model. In each model run,
5 9 106 iterations were implemented and the results were sampled every 2000th iteration.
The overall results were summarized as the harmonic mean. The log-Bayes factor is twice
the difference between the two harmonic means derived from the dependent and inde-
pendent models of evolution and is nominally distributed as a v2 with degrees of freedom
equal to the difference in the number of parameters between the two models, which is four:
the independent model requires two parameters per each of two traits and the dependent
model has eight parameters (Pagel and Meade 2007). A difference of [9.49 indicates
strong support (P \ 0.05) for correlation between the two traits.
Species diversification and trait proliferation rates
In order to identify the temporal pattern of speciation, the net species diversification rate
(SDR) was calculated as SDR = (1/t) 9 log (N (1 - e) ? e), where t is the age of the
clade from the chosen tree, N is the number of crown taxa, and e is the hypothetical
extinction rate (Magallón and Sanderson 2001). Logging corrects for the usual escalating
increase in lineages with time (Barraclough et al. 2003) and was adopted here. The SDR
was calculated for every lineage (clade) overall and for the two regions separately. Net
proliferation rate of a lineage with a particular trait state (TPR) was calculated as
TPR = (1/t) 9 (Ni ? t - Ni)/Ni, where N is the number of lineages at the start time i, and
end, i ? t, of the time interval. TPR was applied to the five periods/epochs in which Protea
has been recorded. i was set at the time the trait first appeared in the period if it was not
present at the start of the period.
Results
Arising 27.8 ± 6.1 (± range/2 of 95 % highest posterior density, HPD) Ma, the stem of the
Protea clade was located in the Cape (Valente et al. 2010). Our analysis shows that the
ancestral proteas were most probably nonsprouters (fire-killed) and serotinous (Fig. 1). The
stem of the grassland (G) proteas arose 16.1 ± 7.4 Ma and was fire-killed and serotinous
123Evol Ecol
(i.e. still in sclerophyll shrublands, S). At 12.7 ± 6.1 Ma, it split into an S clade [fire-killed
(P = 0.72), serotinous (P = 0.84), species confined to S] and a G clade [resprouting
(P = 0.72), nonserotinous (P [ 0.99), species confined to G]. The ancestor of one species
(P. sulphurea, fire-killed, serotinous) appears to have migrated back to S at 8.5 ± 5.1 Ma
as its parent stem occurs in G. Net speciation of the grassland clade was delayed until
7.0 ± 3.7 Ma (i.e., the stem lasted 5.7 My). Independently at 4.1 ± 3.0 Ma, the G P.
roupelliae (resprouter, nonserotinous) split from its S sister P. aurea (nonsprouter, serot-
inous) in an otherwise S clade (nonsprouting, serotinous).
The trait transition rates for resprouting and nonsprouting were equal (0.058 changes per
lineage per million years in either direction), and also presence/absence of serotiny (0.015
changes in either direction) indicating great flexibility in the origin and loss of these traits.
The Bayesian analysis showed that type of fire response was correlated with region
(nonsprouting with S, resprouting with G, log-Bayes factor = 14.9, P \ 0.01). Type of
seed storage was also correlated with region (serotiny with S, nonserotiny with G, log-
Bayes factor = 30.3, P \ 0.001). Eleven of the 13 epicormic species (i.e. recover from
elevated buds rather than underground buds) are confined to G, while 12 of the 14 creeping
(rhizomatous) species are confined to S (collated from Beard 1993; Rebelo 2001).
Overall species diversification rate of Protea in the Miocene (23–5.4 Ma) was about 10
times that in the Pliocene (5.3–2.7 Ma) and 20 times that in the Quaternary (2.6 Ma to
present) (Table 1). This difference remained when S species were considered separately,
but the diversification rate was much more evenly spread over these three periods for G
species, with rates exceeding those for S species in the Pliocene/Quaternary. The trait
proliferation rate (TPR) of both resprouting and nonsprouting in the S during the Miocene
was over four times that in the Pliocene and seven or more times that in the Quaternary.
The TPR for resprouting in G in the Pliocene was over three times that in the Miocene and
Quaternary. Nonsprouting did not develop at all in G. The oldest extant species in S (P.
repens) is estimated to have arisen 14.0 ± 6.5 Ma (it is also the most widespread) while
the oldest in G (P. enervis) arose 5.4 ± 3.1 Ma.
Net species diversification rate of nonsprouters in S was 19–25 % greater than resp-
routers, depending on the assumed extinction rates (Table 2). Only resprouters diversified
in G, at a rate 36–56 % higher than nonsprouters and 56–69 % higher than resprouters in S.
Among resprouting types, lignotubers appear first at 12–13 Ma in both S and G (Table 1).
This was followed by the rhizome habit (5 Ma earlier in S than G). Epicormic resprouting
was the most recent at 3–4 Ma in both S and G. The TPR for epicormic resprouting was
highest in the Pliocene in both regions though it continued strongly in the Quaternary in G
but not at all in S (Table 1). Lignotuberous resprouting evolved in both regions in the three
periods but the TPR was higher in the Miocene-Pliocene for S and the highest among all
periods/regions in the Pliocene for G. Rhizome TPR was strongest in the Pliocene for S and
only occurred in the Quaternary for G.
Figure 2 shows that nonsprouting in S is the starting point for all species and 41 of the
55 nonsprouters there are derived from other nonsprouters. Nonsprouting has given rise to
all other life/growth-forms among the 87 extant species, seven directly, 30 indirectly (by
one nonsprouting-derived growth form evolving into another type or remaining the same)
and eight by resprouters reverting back to nonsprouting. Lignotuberous species gave rise to
the same or other resprouting types 17 times, rhizomatous species nine times, and lig-
notuberous/epicormic species twice. The lignotuberous growth form is the starting point
for all G species except for the epicormic P. roupelliae that arises directly from a nons-
prouting S ancestor. Five pathways seem incomplete, especially the S to G step, as they
123Evol Ecol
Fig. 1 Evolution of reproductive mode [resprouts (green lines or values) or killed (red lines or values) in
response to fire] and aerial seedbank (serotinous [?] or nonserotinous [-]) in Protea (P.). Actual probability
(P) values for reproductive mode (above line) and presence of serotiny (below line) are stated if \0.95;
*P [ 0.95, B0.99; **P [ 0.99. Protea taxa in bold occur in grassland (summer-rainfall region) and arrows
indicate the origin of their clade or lineage; the rest occur in shrubland (winter-rainfall region), though P.
subvestita extends into grassland. F. = Faurea (outgroup). Chronogram based on Valente et al. (2010)
123Evol Ecol
Table 1 Time of species and trait origins and oldest ages, and net species diversification and trait pro-
liferation rates of extant Protea species through geological time in shrubland/grassland (italics) regions
Attribute Time of origin (Ma) Origin of oldest Miocene Pliocene Quaternary
species (Ma) 23–5.4 Ma 5.3–2.7 Ma 2.6–0 Ma
Species diversification rate
Species 27.8/12.7 14.0/5.4 1.932/0.227 0.224/0.357 0.088/0.231
(2.216) (0.241) (0.109)
Trait proliferation rate
Fire response
Killed 27.8/- 14.0/- 1.136/- 0.255/- 0.107/-
Resprout 18.4/12.7 12.1/5.4 0.682/0.170 0.165/0.534 0.102/0.192
Resprouting type
Epicormic 3.3/4.1 3.3/4.0 -/- 0.185/0.741 0.000/0.641
Lignotuberous 12.1*/12.7 12.1/3.9 0.226/0.113 0.247/0.494 0.038/0.220
Rhizomatous 10.6*/5.4 10.6/5.4 0.169/- 0.463/0.000 0.085/0.385
Serotiny
Yes 27.8/2.3 14.0/2.3 1.648/- 0.274/- 0.102/0.384
No 5.9/12.7 3.3/5.4 0.000/0.170 0.357/0.536 0.000/0.154
In parentheses are overall net species diversification rates. - Trait not present. * The P. cynaroides clade is
designated as resprouting but the stem is not significantly rhizomatous (P. lorea, P. scolopendrifolia) or
lignotuberous (P. cynaroides), so maximum ages are based on the individual species
Table 2 Diversification rate of Protea species with different fire responses in the winter and summer
rainfall regions using three hypothetical extinction rates
Region Fire response Diversification rates at different extinction rates
0.05 0.5 0.95
Winter rainfall (S) Killed 0.174 ± 0.075 0.124 – 0.047 0.085 ± 0.030
Resprout 0.146 ± 0.045 0.102 – 0.028 0.068 ± 0.021
Summer rainfall (G) Killed 0 0 0
Resprout 0.246 – 0.115 0.169 – 0.071 0.106 – 0.029
Bold values significantly different at P \ 0.05 for t test
appear to require intermediary steps (i.e. intermediate growth forms or mixed distributions)
lacking among extant species.
The ability to release seeds at maturity (nonserotiny) in S appeared 22 Ma after the
origin of the genus (via P. nitida and P. glabra) while the G clade started with this trait at
12.7 Ma and not until 3.4 ± 1.9 Ma was the ability for on-plant seed storage (serotiny)
regained (via P. rubropilosa, 1–2 Y storage only) (Table 1). The TPR for serotiny of S
species in the Miocene was six times that in the Pliocene and 16 times that in the Qua-
ternary while only in the Pliocene was there any proliferation of nonserotiny (Table 1).
Nonserotiny proliferated in all three periods in G but most strongly in the Pliocene while
the TPR for serotiny was zero until the Quaternary when one species became weakly
serotinous.
123Evol Ecol
Shrubland Grassland
41 * to grassland 5
Major pathway
(common ancestor migrated 12.7 Ma)
N P. P. sulphurea L
3 r ou (at 8.5 Ma)
(a
2
t4 pe
.1 llia
4 4 5
1
M
2 a) e
2
8 R L 5 E 2 LE R
N nonsprouting
2 R rhizomatous
L lignotuberous
E epicormic
LE LE lignotuberous/epicormic
* P. subvestita has spread to the
grassland but remains N
Fig. 2 Evolutionary pathways (arrows from immediate putative ancestral lineage) for types of resprouting
among shrubland (winter rainfall) and grassland (summer rainfall) proteas based on the chronogram of
Valente et al. (2010) for 87 species and fire-response types [nonsprouting or resprouting (lignotuberous,
rhizomatous, epicormic)] from Chisumpa and Brummitt (1987) and Rebelo (2001, 2009) that were mapped
on separate chronograms (not shown but summarized in Fig. S1 in ESM). Numbers are extant species.
Broken lines indicate that intermediate species (in morphology or distribution) appear necessary but none is
extant. If lineage is significant for resprouting then the type of resprouter with highest probability was
accepted as ancestral (except for P. cynaroides—see Table 1)
Table 3 shows for sister species and sister clades confined to each of the two regions,
that species in G are much taller on average (2–3 9) and have larger leaves (mean of
2.3 9 total area) than those in S. Treating values lower than the medians for S as absent
and values larger than the medians as present showed that taller plants arose in the G clades
7.0 ± 3.7 Ma (one of two subclades) and larger leaves arose 8.4 ± 5.1 Ma (results not
presented), i.e., 4–6 My later than resprouting and nonserotiny in the S region. The studies
for two subregions in the Cape and that in Botswana identified in the literature search
showed that their annual rainfalls overlapped but that they had opposing wet seasons
(Table S1). LMA and d13C for Botswana trees were less than half that for Cape shrubs
Table 3 Mean maximum stature and leaf dimensions of related Protea species (1. pair of sister species in
an essentially shrubland (S) clade and 2. pair of sister clades) in the S and grassland (G) rainfall regions
Maximum height (m) Max leaf length (mm) Max leaf width (mm)
1. Sister sp in S (P. aurea) 5 90 40
Sister sp in G (P. roupelliae) 8 170 50
2. Sister clade in S (21 spp) 1.64 129 23.5
Sister clade in G (18 spp) 4.59 164 42.7
P (t test, log [data]) 0.015 \0.001 0.002
123Evol Ecol
100 A 8 (max)
C
Leaf size, SLA, stature (woody spp.)
Serotiny (% woody species)
sclerophyll shrubland
grassland sclerophyll
/savanna forest
grassland
shrubland
/savanna
forest
0 0
1 5 45 10 12 15 20 26
Fire interval (y) Temperature wettest month (°C)
100
15
B Leaf size, SLA, stature (woody sp)
D
Resprouting (% woody species)
sclerophyll grassland
forest
sclerophyll shrubland /savanna
grassland shrubland
/savanna forest
50
0 0
1 5 45 0.02 0.09 0.16
Fire interval (y) Leaf P (% dry mass)
Fig. 3 Models of plant trait values in the sclerophyll shrubland (winter-rainfall) and grassland/savanna
(summer-rainfall) regions with mean values for sister Protea clades in each region from this study added as
dots. a Percentage of woody species in flora that are serotinous in relation to fire interval (overall relationship
based on Lamont and Enright 2000; He et al. 2011) with arrow indicating responses to directional selection
from shorter interval fire (Fig. 1); b percentage of woody species that resprouts in relation to fire interval (based
on Lamont et al. 2011) with arrow indicating responses to directional selection from shorter interval fire
(Fig. 1); c leaf size, specific leaf area (SLA) and plant stature in relation to mean temperature of the wettest
month (http://www.weather-and-climate.com) (based on Meier and Leuschner 2008; Way and Oren 2010) with
arrow indicating responses to directional selection from warmer temperatures when water is most available, the
data points are for plant stature (m, Table 3); d leaf size, SLA and plant stature in relation to leaf P concentration
as a surrogate for available soil P (based on Lamont et al. 2002; Hartshorn et al. 2009) with arrow indicating
responses to directional selection from greater P availability with more frequent fire, the data points are for SLA
(mm2 mg-1, left scale on Y axis, Table S1 in ESM), closed symbol and line connecting them to show range, and
leaf width (mm, right scale, Table 3), open symbol. (max) = maximum value possible
(though they were not matched taxonomically) (Fig. 3). The possible role of soil texture in
speciation is dismissed as an unreliable ecological trait in S3.
Discussion
Serotiny and past climates and fire regimes
Our analysis traced fireprone habitats, on-plant seed storage (serotiny) and the fire-killed
life-form (nonsprouting) back to the origin of Protea, 28 ± 6 Ma. Serotiny is also
123Evol Ecol
ancestral in Banksia (Proteaceae) although arising 34 My earlier (He et al. 2011); it is
derived in Pinaceae arising 89 Ma (He et al. 2012). Interestingly, the stem of the Disa/
Monadenia complex, all extant species confined to the highly seasonal, fireprone shrub-
lands or grasslands of southern Africa, also arose about 30 Ma (Gustafsson et al. 2010).
These dates for the Cape correspond to the Oligocene where there was an initial marked
drop in global temperatures and rainfall following the Eocene, but average temperatures,
burn probabilities and rainfall stayed well above present-day levels until 13 Ma (Zachos
et al. 2001; Pekar and Christie-Blick 2008; He et al. 2012). It was therefore probably
warmer and wetter throughout the year (maritime) in the Cape than now with periodic (not
necessarily every year) summer droughts and reliable postfire rains, necessary for the
evolution of serotiny (Lamont and Enright 2000; Cowling et al. 2005). Nevertheless, it was
a further 10 My before there was any net speciation among Protea (Fig. 1) when perhaps
the fitness advantages of this sclerophyllous, fire-dependent genus were finally entrenched
in the now well-established mediterranean climate (Cowling et al. 1996).
While the first proteas were serotinous, their sister clade, Faurea, were essentially
rainforest trees with long bifacial leaves; they were not prone to fire and lacked serotiny
(Rebelo 2001; Fig. 1). In addition, Beauprea, the immediate sister clade to Faurea/Protea
(Sauquet et al. 2009), is rare in fireprone vegetation, and resprouting and serotiny are
unknown among the 13 extant species (Jaffré et al. 1998; Wilcox and Platt 2002). The
seeds of Protea are enclosed by a cone of bracts that fold back on drying (or are burnt
back), usually caused by heat-induced death of the peduncle, which allows the seeds to be
released, whereas Faurea lacks an involucre of bracts. Thus, there was directional selection
on Protea associated with the presence of fire in shrublands that favoured the evolution of
serotiny from nonserotinous ancestors in the Oligocene.
Clearly, serotiny is adaptive in the shrublands, centred on the restricted opportunities for
seed production in a water- and nutrient-limited environment and the presence of periodic,
crown-reaching fires occurring within the lifespan of the plants that stimulates synchro-
nized seed release onto an optimal postfire seed bed (Lamont and Enright 2000). However,
the only (extant) proteas to have successfully established in the adjoining grasslands are
nonserotinous, while their sister shrubland clades are 93 % serotinous and the rest of the
genus is 100 % serotinous (Fig. 1). This loss of serotiny can be traced to 12.7 Ma at the
time Protea first colonized the grasslands. The ultimate fine-tuning of this adaptation is P.
rupestris in savanna whose bracts drop off at the time of anthesis (Beard 1993) making
seed retention impossible. Thus, the hypothesis that loss of serotiny was a prerequisite for
successful speciation in the grasslands is strongly supported (Fig. 3a).
It is at first counter-intuitive that a system (savanna grasslands) experiencing the con-
straints of an agent of selection more frequently (fire) can be interpreted as a reduction in
selection pressure for a fire-related trait. The explanation is that serotiny is promoted only
at moderate fire intervals of low stochasticity, with restricted opportunities for seedling
recruitment and involving minimal resource costs (Enright et al. 1998a, b; Lamont and
Enright 2000). Besides, mild grass-fires may not reach the crown for ensuring seed release
(Schwilk and Ackerly 2001). Where a seedbed conducive to seedling recruitment exists
almost every year, or the year immediately following fire is not necessarily optimal
(Hoffmann 1996), serotiny is not only redundant but probably maladaptive. In fact,
reduced fitness of the existing trait is an essential component of the concept of directional
selection. This is supported by the fact that in the highly serotinous genus, Banksia, B.
dentata (a tree restricted to savannas in Australia and evolving 9 Ma) is also nonserotinous
(He et al. 2011). Nevertheless, though arising 12.7 Ma, it took a further 5.7 My for net
speciation to occur in the summer-rainfall Protea clade suggesting much refinement of its
123Evol Ecol
biology (diversification, selection, extinction) in the meantime. In addition, while C3
grasslands began to appear 15 Ma in central Africa, C4 grasslands only became widespread
4–6 Ma (Jacobs et al. 1999), creating many more migration opportunities (Scheiter et al.
2012), when speciation of the grassland proteas peaked (Table 1).
Resprouting and past climates and fire regimes
The ability to resprout appeared in the Miocene, 6 My later in the grasslands than the
shrublands. This is the same epoch as for the origin of resprouting among Pinaceae and
whose appearance was also correlated with fireprone habitats (He et al. 2012). Resprouting
arose concurrently with the loss of serotiny, twice independently, in the grasslands (Fig. 1).
These combinations are clearly critical prerequisites for long-term survival in the grass-
lands and the reverse of those in the shrublands. This is supported by the only species
apparently returning to the shrublands, P. sulphurea, that re-acquired both serotiny and
nonsprouting, and by P. subvestita, which reaches the margin of the grasslands and is a
nonsprouter and weakly serotinous, but its presence there is currently under constant threat
from frequent fire (Rebelo 2001). The only (extant) proteas to have successfully colonized
the adjoining grasslands are 97 % resprouters (allowing half a positive value for P. sub-
vestita) while their sister shrubland clades are 23 % resprouters and the rest of the genus
are 42.5 % resprouters (Fig. 1). Thus, the hypothesis that the adoption of resprouting was a
prerequisite for successful speciation in the grasslands is strongly supported (Fig. 3b).
The explanation for universal resprouting in grasslands must revolve around replacing
the imperative for precocious annual seed production and guaranteed seedling recruitment
following fires at 1–5-year intervals with the apparently more feasible alternative to sur-
vive fire vegetatively within the same time span. Lack of opportunities for establishment of
woody plants may also have been exacerbated by the declining pCO2 levels in the Late
Miocene/Pliocene through its effects on grass demand for surface water (Polley et al.
2002). While there may be sufficient time for young plants to produce insulated buds at
ground level it may be inadequate for them to become fully fertile; there is also an
increased risk that current flowers and/or fruits would be incinerated by the imminent fire.
Both decrease the reliability of sexual reproduction in this new environment. Indeed,
unlike shrubland species that may take many years to attain fire-tolerance (Lamont et al.
2011), savanna species are usually fully fire-tolerant by 1–3 Y (Gignoux et al. 2009). What
facilitated this remarkable ability to develop a large fire-tolerant, bud-storing organ in such
a short time? The answer must lie with the shift to the coincidence of maximum water
availability with the warmest time of year for plant growth. Warm moist conditions
coupled with frequent fire also promote nutrient release from soil particles, especially the
growth-limiting nutrients, nitrogen (N) and phosphorus (P) (Coetsee et al. 2008; Craine
et al. 2008; Hartshorn et al. 2009).
Note that resprouting can also be adaptive in the shrublands as indicated by their
presence in four of five multiple-species clades there (Fig. 1) and that once they become
resprouters, they retained this ability with ongoing speciation (Fig. 2). The lignotuberous
resprouting type was ancestral in both regions, followed later by rhizomes and last by
epicormic resprouting (Table 1). Of particular interest is the early appearance of rhizo-
matous species at a stronger proliferation rate than the lignotuberous form in the shrub-
lands (Fig. S1 in Electronic Supplementary Material, ESM): along with the potential for
clonality, soil gives greater protection to dormant buds than bark (Clarke et al. 2013), as
well as their low stature ensuring that the seed release mechanism operates during fire (He
et al. 2011). This contrasts with their late appearance (5 Ma) and dearth of speciation in the
123Evol Ecol
grasslands where low stature is no longer relevant for ensuring seed release from fire.
Clonality among banksias also arose from lignotuberous ancestors and proliferated in the
Miocene (He et al. 2011; Lamont et al. 2011).
Not until the Pliocene did epicormic resprouters appear, but they only speciated strongly in
the grasslands, possibly coinciding with the rapidly declining temperatures, rainfall and pCO2
(Osborne 2008) and thus reduced fuel loads among the grasses making the ‘escape height’
response a fitter option than the shoot incineration/replacement response of lignotuberous
species (R. Cowling, pers. comm.). Taller plants are less likely to be crown-burnt or die in
savannas, mainly due to their thicker bark (Lawes et al. 2011). This ‘escape’ applies not just to
foliage but also to flowers and immature fruits, whose retention unharmed would promote
early seed release onto bare ground and rapid postfire recovery. Interestingly, the only
nonserotinous species in the winter rainfall region (P. nitida and P. glabra) are typically tall
epicormic resprouters in habitats where fire is rare or mild (Rebelo 2001; R. Cowling, pers.
comm.)—the principle of inability of fire to guarantee seed release and abundant opportu-
nities for seedling recruitment is the same as for the grasslands (He et al. 2011). This tendency
to grow tall in grassland is confirmed at the clade level (Table 3) and supports the hypothesis
that selection for taller stature was favoured in the grasslands (Fig. 3c) but it was not a
prerequisite as it appeared later than the resprouting–nonserotiny combination.
Stature, leaf size and LMA and past climates and fire regimes
Selection for rapid growth rates to build up fire-tolerant organs and taller trunks under the
influence of frequent fire does not lead to adaptation unless (a) a mechanism exists to
enable that to occur, and (b) the resources are available to enable that mechanism to be
expressed. We suggest that the shift to large leaves and low leaf mass area (LMA, index of
sclerophylly) provided the required mechanism (Midgley et al. 2004; Table 3, Table S1 in
ESM). This was only possible because of the switch of the wet season to the warmest time
of year (Fig. 3c) that supported the resource requirement. Leaf area and LMA covary
negatively with water availability (Witkowski and Lamont 1991; Scholes et al. 2004). d13C
is inversely correlated with LMA as a response to drought among proteas and other
members of the Proteaceae, independent of nutrient supply (Groom and Lamont 1997;
Lamont et al. 2002). Since LMA and d13C are much lower among Botswana savanna trees
than among Cape proteas (Table S1) this supports our contention that water availability for
growth is much higher in the grasslands, despite little difference in the range of annual
rainfall in each region (Higgins et al. 2000; Lamont et al. 2002). In support, Midgley et al.
(2004) noted that Botswana trees still photosynthesised strongly at [35 °C suggesting
water supply was non-limiting.
These observations cast doubt about the traditional emphasis on the central place of
drought severity for recruitment in savannas (Higgins et al. 2000; Beerling and Osborne
2006), at least compared with mediterranean regions, though it does support conclusions on
the importance of the ability to respond rapidly to early warm season rains (Gignoux et al.
2009). This wet season switch may have been critical for ensuring rapid growth among
woody plants as pCO2 plummeted in the Late Miocene/Pliocene and the C4 photosynthetic
pathway was not an option (Beerling and Osborne 2006). These limits to growth rates
might explain why the onset of much larger, softer leaves was delayed compared with
nonserotiny-resprouting.
It is unclear how the absolute levels of soil nutrients compare between the two regions
but there is strong evidence that available P and N, which limit growth in both regions
(Craine et al. 2008; Groom and Lamont 2010), are enhanced by warm, wet conditions,
123Evol Ecol frequent fire and the presence of (nitrogen-fixing) acacias (Coetsee et al. 2008; Hartshorn et al. 2009; Cech et al. 2010), so that leaf P and N concentrations in the savanna grasslands are much higher than in the shrublands (Table 3d; Singh 1993; Lamont et al. 2002; Hartshorn et al. 2009). Leaf size (positive) and LMA (negative) are also a function of nutrient supply in Proteaceae (Witkowski and Lamont 1991; Lamont and Groom 2002). Herbivory by mammals would not have had such a critical role as (a) proteas are not palatable compared with grasses, (b) the nutritive effect is confined to the forage or indirectly via changes in species composition, and (c) grazing is patchy (Anderson et al. 2007; Cech et al. 2010). Thus, selection for small, dense and thick leaves as a response to both low water and nutrient availability in the shrublands (Lamont et al. 2002; Yates et al. 2010) was reversed in the grasslands (Fig. 3c, d). It is worth noting that these morpho- logical properties in the shrublands also promote plant flammability in contrast to their opposing properties in the grasslands (Dickinson and Kirkpatrick 1985; He et al. 2011). Timing of adaptive changes Frequent fire acts to exclude forbs, shrubs and trees from subtropical grasslands unless they possess special traits (Bond et al. 2005; Lamont and Downes 2011). Adaptive radiation into the grasslands therefore required a radical change in the biology of Protea that was achieved about 12.7 Ma, 2–3 My after the first appearance of grasslands in Africa (Jacobs et al. 1999). While it was another 4 My before the parent grassland clade split into a grassland and shrubland clade, it is unclear if this was because grasslands (initially C3) were not yet established in southern Africa (Jacobs et al. 1999) or this time lag was required for the necessary suite of traits to evolve. The onset of fire-stimulated flowering among summer-rainfall orchids 14 Ma has been interpreted as evidence of grasslands occurring then (Bytebier et al. 2011) but it is also adaptive among winter-rainfall geo- phytes (Lamont and Downes 2011) so its ancestors may still have been under fireprone winter-rainfall conditions. More telling is the 5.5 My delay in speciation of the grassland clade that implies an extended adaptive ‘struggle’ in the new environment, possibly associated with the ongoing decline in CO2 levels and temperature, replacement of C3 by C4 grasses, and increasing fire frequency and seasonal drought (Scheiter et al. 2012). In summary, at least two prerequisites for successful speciation by proteas in the grasslands had to be satisfied: abandonment of on-plant seed storage, and adoption of vegetative recovery via insulated buds, as direct responses to more frequent fire. These were followed later by an increase in plant height and larger and less sclerophyllous leaves, that were prompted by frequent ‘mild’ fire and possibly declining CO2 levels, as mecha- nisms for promoting faster growth rates. Such ultimate responses are consistent with the switch of the wet season to the warmest part of the year. Thus, slow rates of lineage proliferation of lignotuberous and epicormic resprouting and nonserotiny in the late Miocene were followed by fast rates in the Pliocene as the summer-rainfall clade speciated into the vast areas of grasslands that became available during that time. This intensified selection for resprouting and relaxed selection for seed storage in (sub)tropical grasslands worldwide seems universal (Grau and Veblen 2000; Simon et al. 2009; He et al. 2011; Lamont et al. 2011; Lawes et al. 2011) and might also explain the abundance of species with fleshy fruits (i.e., no storage) in some savannas (Ozinga et al. 2004). But the direction of selection for growth rates depends on origin of the source floras—where the parent genera/families are in non-fireprone forests, selection for high growth rates declines, whereas if already fireprone (as here) selection for high growth rates increases (Fig. 3c, d). 123
Evol Ecol
Acknowledgments Without Tim Barraclough giving us access to the Protea chronogram (Valente et al.
2010) this analysis would not have been possible. We thank Tony Rebelo for access to the Protea Atlas;
Mike Crisp, Richard Cowling and four other reviewers for their insightful comments on the manuscript;
Antoni Milewski and Anthony Mills for their advice on relative nutrient levels in savanna; and Kings Park
and Botanic Garden for providing facilities for TH in the early stages of this project. This work was
supported by the Office of Research and Development, Curtin University and the Australian Research
Council (DP120103389). TH acknowledges the support of a Curtin Research Fellowship.
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