Appendix S2: Phylogenetic Analyses
Phylogeny reconstruction
As our target species represent a wide phylogenetic range across Eudicots, we estimated
phylogenetic relationships among the species to enable testing for phylogenetic bias in the
statistical analyses of the traits data (see below). Partial plastid DNA encoded matK nucleotide
sequences (Kress et al. 2005; Shaw et al. 2005) were derived from individuals of most of our
species (data to be submitted to GenBank). Fresh young leaves were collected and DNA was
isolated with the KingFisher Flex (Thermo Scientific), using the Agowa Sbeadex Maxi Plant kit
according to the manufacturer’s protocol (Agowa genomics, Berlin, Germany). The DNA
concentration was estimated by gel electrophoresis. For the amplification of the matK fragment,
the primers 3F_KIM (5’-CGTACAGTACTTTTGTGTTTACGAG-3’) and 1R_KIM (5’ACCCAGTCCATCTGGAAATCTTGGTTC-3’) were used (Ki-Joong Kim, School of Life
Sciences and Biotechnology, Korea University, Seoul, Korea, unpublished). PCR reaction mix: 1
ng/µl DNA, 100 µM dNTPs, 2 mM MgCl2, 0,2 µM primers, 0.5 U Taq polymerase (Supertaq,
Sphaero Q) in a final volume of 20 µl. PCR cycling conditions: 94°C 1 min; (35 cycles : 94°C 30
s; 52°C 20 s; 72°C 50 s); 72°C 5 min. The PCR reaction was performed on a Bio-Rad Dyad
thermocycler (Bio-Rad Laboratories, Hercules, California, USA). After PCR, 100 ng of the
fragment was used for sequencing. The PCR fragment was sequenced from boths ends in
separate reactions by Sanger sequencing. The sequencing was performed by Greenomics
(Wageningen, The Netherlands). The sequence mix contained 100 ng PCR product, 2 µl of
DETT (Amersham, GE Healthcare, UK) mix, 2 µl of DETT buffer (5x), 10 ng of primer
(3F_KIM or 1R_KIM) and was filled up to 10 µl with sterile water. The sequencing reaction was
performed on the Biorad Dyad thermocycler under the following conditions: 94°C 20 s; 50°C 15
s; 60°C 60 s for 25 cycles.
In order to avoid possible long-branch attraction artefacts in inferences among our study
species, we constructed a much larger matK tree using sequences from GenBank that represented
most core Eudicot APGIII orders (Stevens 2001). Tracers were edited and assembled using
STADEN (Staden 1996) and aligned using Mesquite v. 2.74 (Maddison & Maddison 2010).
Nucleotides were aligned at the codon-level, avoiding the occurrence of stop-codons as much as
possible. The final alignment included 849 aligned nucleotide positions over 301 terminals
(taxa). We ran the phylogenetic analysis using RAxML (Stamatakis et al. 2008) implemented on
Abe (National Center for Supercomputing Applications, Illinois, USA) via the CIPRES
(Cyberinfrastructure for Phylogenetic Research) Portal (Miller et al. 2010). The analysis
included 100 rapid bootstrap inferences followed by a thorough maximum likelihood search
using the GTRCAT model approximation (Stamatakis 2006) and using Ranunculaceae as the
outgroup. Subsequently, we pruned the derived 301-terminal tree down to a 51-terminal tree of
our target species only (Fig. S2, Fig. S3) that was used in the statistical analyses. We checked
whether this tree topology differed from that of a tree derived from only the 51 terminals of our
species using RAxML. The latter incorrectly positioned two subclades of Rosid I and Rosid II
that were weakly represented in our species sample (Atalaya/Eriotheca and
Gymnosporia/Kielmeyera/Petalostigma).
1
Species for which matK sequences were used for phylogenetic reconstruction from
GenBank, along with accession numbers
Acacia aulacocarpa AF274214; Acacia bidwilli AF274130; Acacia boliviana AF274144; Acacia
bonariensis AF274146; Acacia catechu AF274141; Acacia caven AF274131; Acacia
cochliacantha AY574094; Acacia colei AF274215; Acacia collinsii AY574092; Acacia
constricta AF274135; Acacia cornigera AY574105; Acacia dolichostachya AF523190; Acacia
elata AF274149; Acacia fasciculifera AF274154; Acacia galpinii AF523098; Acacia glomerosa
AF274147; Acacia karroo GQ872230; Acacia ligulata AF274155; Acacia lineata AF274161;
Acacia lysiphloia AF274151; Acacia melanoxylon FJ711538; Acacia microbotrya AF274157;
Acacia modesta AF274142; Acacia monticola AF274152; Acacia myrtifolia AF274160; Acacia
nilotica GQ872238; Acacia notabilis AF274158; Acacia nuperrima AF274164; Acacia
pachycarpa AF274153; Acacia parramattensis AF274150; Acacia platycarpa AF274223;
Acacia rossei AF274162; Acacia schaffneri AF274132; Acacia schottii AF274136; Acacia
schweinfurthii EU812002; Acacia senegal AF274143; Acacia seyal AF274138; Acacia tortilis
AF274140; Acacia translucens AF523087; Acacia tumida AF523111; Acacia wrightii
AF274148; Acer saccharum AY724265; Adansonia digitata AY321168; Albizia bermudiana
EU812001; Albizia harveyi EU812003; Albizia julibrissin HQ619810; Albizia plurijuga
AF523080; Albizia sinaloensis AF274121; Albizia tomentosa AF523093; Albizia versicolor
AF523112; Anacardium excelsum GQ981932; Antidesma alexiteria AY552416; Antidesma
pentandrum AB233810; Arabidopsis lyrata XM 002886827; Aspidosperma australe DQ660502;
Aspidosperma cruentum FJ514713; Aspidosperma cylindrocarpon DQ660503; Aspidosperma
marcgravianum FJ514763; Aspidosperma spruceanum GQ981941; Aspidosperma triternatum
AM295077; Atalaya alata EU720593; Atalaya angustifolia EU720569; Atalaya
salicifoliaAY724272; Aucuba japonica Q997060; Barringtonia asiatica DQ924095; Bauhinia
galpinii EU361875; Bauhinia tomentosa AY386893; Bixa orellana FM179929; Bombax
buonopozense AY321171; Brachylaena discolor AY785090; Brachylaena elliptica EU385330;
Brachylaena huillensis EU214220; Brachystegia boehmii EU361886; Brachystegia bussei
EU361887; Brachystegia spiciformis EU361888; Brassica oleracea AY541618; Bridelia
angolensis FJ439964; Bridelia cathartica FJ439970; Bridelia exaltata FJ439978; Bridelia
glauca FJ439977; Bridelia micrantha FJ439960; Bridelia mollis FJ439965; Caesalpinia
cacalaco EU361898; Caesalpinia calycina EU361899; Caesalpinia ferrea EU361901;
Caesalpinia gracilis EU361902; Caesalpinia paraguariensis EU361905; Caesalpinia
pulcherrima EU361906; Callophyllum antillanum GU135089; Campanula scouleri EU713345;
Capparis frondosa GQ981949; Carica papaya AY483221; Casuarina cunninghamiana
AY033836; Casuarina equisetifolia AB015462; Casuarina glauca AY191705; Casuarina obesa
AY191708; Casuarina parapotamia AY191712; Casuarina pauper AY191711; Casuarina
riparia AY191713; Ceiba pentandra HQ696701; Chrysobalanus icaco EF135519;
Chrysophyllum sanguinolentum FJ037935; Citrus trifoliata FJ716730; Cleome rutidosperma
EU371804; Clusia gundlachii EF135520; Cneorum tricoccon EU042839; Colophospermum
mopane EU361915; Convolvulus arvensis FJ395388; Croton gratissimus EU214230; Croton
kongensis GQ434077; Croton megalobotrys EU214233; Croton pseudopulchellus EU214236;
Dalbergia congestiflora AF142696; Dalbergia sissoo AF203582; Dalbergia sp. AF203580;
Daphne bholua FM179927; Delphinium grandiflorum AY515249; Dichapetalum macrocarpum
EF135527; Dimorphandra conjugata EU361934; Diospyros maingayi DQ924033; Dipsacus
mitis AF446917; Dipterocarpus palembanicus AB295903; Dodonaea viscosa EU720684;
2
Dombeya spectabilis AY321173; Drypetes littoralis AB233822; Drypetes madagascariensis
AY552458; Drypetesroxburghii EF135530; Enterolobium cyclocarpum AY650277; Erythrina
sousae EU717411; Euclea crispa FJ238116; Euclea divinorum FJ238117; Euclea natalensis
FJ238118; Euclea pseudebenus FJ238119; Euclea racemosa FJ238120; Euclea schimperi
FJ238121; Euclea undulata FJ238122; Eugenia coloradoensis GQ981988; Eugenia sulcata
AM489987; Eugenia uniflora GU135006; Fagus lucida EF057139; Faidherbia albida
EU812008; Garcinia latissima FJ670008; Garcinia subelliptica AB233746; Garrya elliptica
AJ429319; Geranium palmatum EU922315; Grevillea banksii AF542583; Grevillea caleyi
EU642709; Grevillea curviloba EU169629; Grevillea juniperina EU169630; Grevillea robusta
EU169631; Grewia bicolor EU214255; Grewia biloba AY321193; Grewia flavescens
EU214260; Grewia villosa EU214261; Guarea grandifolia GQ982002; Gunnera manicata
EU002179; Gymnosporia haberiana EU328982; Gymnosporia harveyana EU328967;
Gymnosporia linearis EU328973; Gymnosporia mossambicensis DQ217550; Gymnosporia
pyria EU328980; Gymnosporia royleana EU328978; Gymnosporia senegalensis EU328979;
Gymnosporia urbaniana EU328984; Heteropyxis natalensis AF368208; Hymenocardia acida
AY830269; Impatiens noli AF542608; Julbernardia brieyi EU361985; Julbernardia
pellegriniana EU361986; Kiggelaria africana EF135555; Lonicera maackii GU168651;
Lysiphyllum gilvum EU361876; Mammea siamensis FJ670006; Manilkara zapota DQ924092;
Melaleuca aff nesophila AY525135; Melaleuca quinquenervia GU135000; Melaleuca viridiflora
AF184708; Melia azedarach EU042834; Mimosa albida AY944555; Mimosa foetida
EU812006; Mimosa pudica AY177668; Nothofagus solandri AB015464; Olax emirnensis
DQ790173; Olea parvilimba EU409441; Olinia ventosa AY151573; Paullinia pinnata
AY724320; Pelargonium cotyledonis EU922531; Peltophorum dubium AY386846;
Peltophorum pterocarpum EU362023; Penaea dahlgrenii AY151588; Petalostigma pubescens
EF135579; Phyllanthus sauropodoides EU643745; Planchonella ripicola GQ248187;
Plathymenia reticulata AF521858; Polygonum sibiricum EU024771; Psiloxylon mauritianum
AF368215; Qualea grandiflora AF368216; Qualea rosea FJ514694; Quercus tomentella
FJ185058; Quercus trojana FN675331; Ranunculus andersonii AY954238; Rhus gueinzii
EU214277; Rhus transvaalensis EU214283; Saxifraga lyallii GU226267; Shorea smithiana
AB295901; Sideroxylon capiri GQ429074; Solanum lepidotum GQ982099; Spirostachys
africana AB268064; Stachystemon axillaris EF135595; Sterculia tragacantha AY321178;
Strychnos decussata EU214294; Strychnos lucida DQ660545; Strychnos madagascariensis
EU214295; Strychnos minor DQ660546; Strychnos nux Z70193; Strychnos spinosa EU214300;
Stryphnodendron coriaceum AF521863; Stryphnodendron moricolor FJ037919; Stylapterus
micranthus AY151596; Syzygium australe AF368221; Syzygium cordatum DQ088571; Syzygium
cumini DQ088575; Syzygium guineense DQ088581; Syzygium ngoyense DQ088596; Syzygium
pondoense DQ088599; Syzygium samarangense DQ088605; Syzygium tierneyanum DQ088616;
Terminalia catappa GU135057; Terminalia complanata GQ248208; Terminalia muelleri
GU135121; Thalictrum robustum EF437138; Trichilia emetica AY128202; Viola grypoceras
DQ842579; Vochysia ferruginea GQ982128; Vochysia hondurensis AY572446; Ximenia
americana GQ997871; Zanthoxylum sp. EU042838.
3
Phylogenetic analysis
A first set of ANCOVA models was run on each variable using ordinary least squares regression
without phylogenetic correction. Hereafter these models are termed the Non-Phylogenetic
models. A second and third set of models used a generalised least squares regression to impose a
phylogenetic correction on the data, where the error structure imposed on the model was the
covariance matrix of common distances between species derived directly from a phylogenetic
tree (Grafen 1989; Martins & Hansen 1997; Garland & Ives 2000). The second set of models
used the additive tree derived from the matK sequences (Fig. S2). Hereafter, these models are
termed the Additive Tree models. The third set of models used an ultrametric tree (Fig. S3)
derived from the additive tree using the phylogr() function in APE package of R (Paradis et al.
2004; Pinheiro et al. 2011), which estimates the ultrametric structure using a semi-parametric
method based on penalised likelihood (Sanderson 2002). We assumed maximum possible
difference in evolution rate within separate branches (λ=0). This third set of models was
considered necessary to test because of the very short terminal branches within certain parts of
our additive matK tree, which were potentially points of high influence in the model. Hereafter
these models are termed the Ultrametric Tree models.
All models were analysed using the Regressionv2.m program (Lavin et al. 2008). In addition,
the strength of the phylogenetic signals for the Additive Tree models and Ultrametric Tree
models were estimated simultaneously with the regression coefficients, using an OrnsteinUhlenbeck (OU) evolutionary process along the phylogenetic trees, as applied in
Regressionv2.m (Lavin et al. 2008). Regressionv2.m estimates the OU transform parameter, d,
using REML. When d is close to 0, this indicates that the Non-phylogenetic model best fits the
data, while when d is close to 1, the phylogenetic model best fits the data. Phylogenetic models
for each trait were also compared against non-phylogenetic models using maximum likelihood
(ML) and Akaike information criteria (AIC) estimates for each model. Statistical results of the
non-phylogenetic and phylogenetic analyses are presented in Table S2.
4
Table S2. Statistical tests of ANCOVA models for significant effects of climate type of origin (Clim.), continent of origin (Cont.), their interaction (C × C) and
the covariate seedling mass (Mass) on organ mass fractions (LMF, SMF, RMF), root morphology traits (RER, STRL) and growth rate (RGR) of 51 savanna tree
species grown under common conditions. Model results are presented for a general linear model without phylogenetic adjustment (“Non-phylogenetic model”)
and two linear models with phylogenetic adjustment (“Additive tree model”) and (“Ultrametric tree model”) (see text for complete explanation). Model results are
provided for the full model including the interaction term only when the interaction was significant or the AIC value improved significantly with the inclusion of
the interaction term. Significant effects are emboldened.
Non-phylogenetic model
Model tests§
Trait
ln ML
AIC
Additive tree model
Tests of effects (F.prob.††)
Mass†
Cont.
Clim.
Ultrametric tree model
Model tests
C×C
ln ML
AIC
Tests of effects (F.prob.)
§§
d
Mass
Cont.
Clim.
Model tests
C×C
ln ML
AIC
Tests of effects (F.prob.)
d
Mass
Cont.
Clim.
C×C
Allocation
RMF
37.5
-62.6
0.315
SMF
62.4
112.8
0.004
0.052
-
36.1
-58.3
LMF
42.8
-73.5
-147.3
-41.5
160.9
<0.001
0.001
0.011
0.094
-
39.4
-64.9
0.141
0.218
0.151
0.310
-
<0.001
0.943
<0.001
-
67.0
0.187
<0.001
0.643
-
36.9
-116.1
0.036
0.012
0.326
<0.001
0.028
64.1
-114.3
0.177
0.058
0.313
<0.001
0.003
-59.8
<0.001
0.077
0.007
0.527
-
44.0
-74.0
0.058
0.475
0.022
0.140
-
306.6
<0.001
0.005
<0.001
-
95.1
<0.001
0.042
0.009
-
-160.6
335.2
<0.001
<0.001
0.058
0.013
-
-147.0
308.0
<0.001
<0.001
0.006
0.001
-
-72.5
159.0
<0.001
0.002
0.462
0.162
-
-41.7
97.4
<0.001
<0.001
0.048
0.010
-
309.8
0.202
0.166
0.833
-
130.6
-247.3
<0.001
0.317
0.589
0.983
-
161.2
-308.5
<0.001
0.190
0.137
0.909
-
Morphology
RER
STRL‡
Growth rate
RGR
† The covariate Mass was natural log transformed for the ANCOVA to normalise the distribution. Mean species mass at 20 weeks was used for models of RMF, SMF and LMF, and mean species mass at
5 weeks was used for RER, SRL and RGR.
†† All F ratio tests had 44 error df for models including the interaction term and 46 error df for models excluding the interaction term.
‡ STRL was natural log transformed for the ANCOVA to stabilise variance.
§
Models are compared using maximum likelihood (ML) and Akaike’s Information criterion (AIC) (less is better). Significant LRTs (> 3.8414, χ2 for 0 or 1 df) calculated between the phylogenetic
models and the Non-phylogenetic model are indicated at the 5% level (*)
§§
Best-fit models with phylogenetic adjustment were obtained with the residuals modelled as an Ornstein-Uhlenbeck process (following Lavin et al. 2008). d is the REML estimate of the OrnsteinUhlenbeck transformation parameter. It is also a measure of the strength of the phylogenetic signal.
5
Fig. S1 Target species used (black) and their phylogenetic relationships dispersed across main Angiosperm ‘core
Eudicot’ phylogeny (grey), based on plastid encoded matK DNA sequence comparison (RAxML bootstrap).
Sequence data were collected directly from seedlings of most species, and phylogenetic positions of those species for
which no DNA could be extracted were estimated using sequences taken from GenBank for either the same species
or closely related species. Those species for which other species had to be used instead were: Acacia shirleyi
(substitute: Acacia aulacocarpa), Lysiphyllum hookeri (substitute: Lysiphyllum gilvum) and Kielmeyera coriaceae
(substitute: Garcinia subelliptica). Scale bar indicates number of nucleotide substitutions per site. Files for the
phylogeny (Nexus format) can be obtained from the corresponding author.
6
Fig. S2 Additive tree of phylogenetic relationships of considered species resulting from pruning from the wider 301
terminal Angiosperm eudicot phylogeny (Fig. S1). Sequence data were collected directly from seedlings of most
species, and the placeholder species were used in cases where no DNA could be extracted (GenBank accession
numbers are presented elsewhere in Appendix S2): Acacia shirleyi (substitute: Acacia aulacocarpa), Lysiphyllum
hookeri (substitute: Lysiphyllum gilvum) and Kielmeyera coriaceae (substitute: Garcinia subelliptica). Scale bar
indicates number of nucleotide substitutions per site.
7
Fig. S3 Ultrametric tree structure derived from the additive consensus tree (Fig. S2) using the semi-parametric
method described by Sanderson (2002) as implemented via the phylogr() function in APE (Paradis et al. 2004). The
tree model was derived under the assumption of maximum possible difference in evolution rate among branches
(λ=0). Branch lengths are scaled to a root to tip length of 1 unit.
8
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