Gourds afloat: A dated phylogeny reveals an Asian origin of the gourd family (Cucurbitaceae) and numerous oversea dispersal events

Abstract

Knowing the geographical origin of economically important plants is important for genetic improvement and conservation, but has been slowed by uneven geographical sampling where relatives occur in remote areas of difficult access. Less biased species sampling can be achieved when herbarium collections are included as DNA sources. Here, we address the history of Cucurbitaceae, one of the most economically important families of plants, using a multigene phylogeny for 114 of the 115 genera and 25 per cent of the 960 species. Worldwide sampling was achieved by using specimens from 30 herbaria. Results reveal an Asian origin of Cucurbitaceae in the Late Cretaceous, followed by the repeated spread of lineages into the African, American and Australian continents via transoceanic long-distance dispersal (LDD). North American cucurbits stem from at least seven range expansions of Central and South American lineages; Madagascar was colonized 13 times, always from Africa; Australia was reached 12 times, apparently always from Southeast Asia. Overall, Cucurbitaceae underwent at least 43 successful LDD events over the past 60Myr, which would translate into an average of seven LDDs every 10Myr. These and similar findings from other angiosperms stress the need for an increased tapping of museum collections to achieve extensive geographical sampling in plant phylogenetics.

Full text

Gourds afloat: a dated phylogeny reveals an
Asian origin of the gourd family (Cucurbitaceae)
and numerous oversea dispersal events
Hanno Schaefer*, Christoph Heibl and Susanne S. Renner
Systematic Botany, University of Munich, Menzinger Strasse 67, 80638 Munich, Germany
Knowing the geographical origin of economically important plants is important for genetic improvement
and conservation, but has been slowed by uneven geographical sampling where relatives occur in remote
areas of difficult access. Less biased species sampling can be achieved when herbarium collections are
included as DNA sources. Here, we address the history of Cucurbitaceae, one of the most economically
important families of plants, using a multigene phylogeny for 114 of the 115 genera and 25 per cent of the
960 species. Worldwide sampling was achieved by using specimens from 30 herbaria. Results reveal an
Asian origin of Cucurbitaceae in the Late Cretaceous, followed by the repeated spread of lineages into the
African, American and Australian continents via transoceanic long-distance dispersal (LDD). North
American cucurbits stem from at least seven range expansions of Central and South American lineages;
Madagascar was colonized 13 times, always from Africa; Australia was reached 12 times, apparently always
from Southeast Asia. Overall, Cucurbitaceae underwent at least 43 successful LDD events over the past
60 Myr, which would translate into an average of seven LDDs every 10 Myr. These and similar findings
from other angiosperms stress the need for an increased tapping of museum collections to achieve extensive
geographical sampling in plant phylogenetics.
Keywords: Bayesian molecular clock; biogeography; dispersal; economic plants; museomics;
test of monophyly
1. INTRODUCTION
Molecular clock analyses suggest that the majority of
lineages of legumes that occur on islands are younger than
30 Myr (Lavin & Beyra Matos 2008) and that plant
diaspores from source areas hundreds or thousands of
kilometres away regularly reach isolated Arctic islands
(Alsos et al. 2007), island-like mountains in Eastern Africa
and mountain ranges in the Northern Cape, South Africa
(Galley et al. 2007). Striking dispersal events have also been
documented for the flora of Hawaii ( Wagner et al. 1990),
the montane region of New Zealand ( Winkworth et al.
2005) and many other island systems. Such frequent long-
distance dispersal (LDD) implies that long-established
views on the origin of economically important plants may
need to be re-evaluated based on drastically enlarged
geographical sampling. An example is the origin of
Cucumis sativus, the cucumber. Cucumber ranks among
the top 10 vegetables in world production (Chen et al.
2004). Until 2006, it was thought that the genus Cucumis
had 32 species and was essentially African. Only C. sativus
and C. hystrix were thought to occur naturally in India,
China, Burma and Thailand (Ghebretinsae & Barber
2006). However, broader geographical species sampling
revealed that C. sativus is closer to 13 species from
Australia, India, Yunnan and Indochina than to any
African species (Renner & Schaefer 2008).
Biogeographic inference for economically important
plants is complicated by human transport of seeds
between continents for at least 10 000 years (Smith
1997;Sanjur et al. 2002;Dillehay et al. 2007). The extent
of the anthropogenic transfer, however, is difficult to work
out without comprehensive phylogenetic frameworks,
which can be prohibitively expensive if worldwide
collecting of material is required. In the economically
important plant family Cucurbitaceae, these difficulties
have led to the geography of the closest relatives of
watermelon (Citrullus lanatus), cucumber (Cucumis sati-
vus), loofah (Luffa acutangula), bitter gourd (Momordica
charantia), chayote (Sechium edule), ivy gourd (Coccinia
grandis), snake gourd (Trichosanthes cucumerina)and
creeping cucumber (Melothria pendula) remaining ambig-
uous. Natural LDD of cucurbit diaspores may be frequent
because many are adapted for transport by birds or wind,
or they can withstand long periods in water (Cayaponia,
Fevillea,Hodgsonia,Lagenaria,Luffa and Sicana;Ridley
1930;Whitaker & Carter 1954).
Here, we use worldwide sampling, based on museum
specimens, to infer the biogeographic history of Cucurbi-
taceae, a family consisting of climbers or trailers of tropical
and subtropical regions that are typically strongly
seasonal, lacking aboveground parts during part of the
year. These traits have caused cucurbits to be under-
collected (Gentry 1991), resulting in dozens of species still
known from only one or two collections even in the world’s
leading herbaria (e.g. De Wilde & Duyfjes 2007). Of the
approximately 960 accepted species of Cucurbitaceae,
approximately 40 per cent are endemic in the American
Proc. R. Soc. B (2009) 276, 843–851
doi:10.1098/rspb.2008.1447
Published online 25 November 2008
Electronic supplementary material is available at http://dx.doi.org/10.
1098/rspb.2008.1447 or via http://journals.royalsociety.org.
*Author and address for correspondence: Imperial College London,
Silwood Park campus, Ecology and Evolutionary Biology, Buckhurst
Road,Ascot,BerkshireSL57PY,UK(hanno.schaefer@imperial.ac.uk).
Received 6 October 2008
Accepted 4 November 2008 843 This journal is q2008 The Royal Society

continent, and the remainder occur in Africa (28%),
Asia (26%), Australia (2%) and Europe (1%; Schaefer &
Renner in press).
Based on chloroplast sequences from all but one of
the 115 genera and 25 per cent of the 960 species, and
employing specimens from 30 herbaria and up to 172 years
old, we address here the following questions: did Cucurbi-
taceae initially diversify in Asia, in America, or in Africa and
Madagascar? What are the geographical sources of the
world’s major Cucurbitaceae floras? Are the transoceanic
geographical ranges in the genera Cayaponia,Lagenaria,
Luffa and Sicyos anthropogenic or the result of natural
LDD? We also use Cucurbitaceae to illustrate the still
barely tapped potential of museum collections to achieve
less biased geographical sampling than has traditionally
been employed in tropical plant phylogenetics.
2. MATERIAL AND METHODS
(a)Taxon sampling
We generated 126 sequences, representing 32 species from
seven genera not sampled in previous studies (Anangia,
Cucumeropsis,Gomphogyne,Hodgsonia,Papuasicyos,Pseudosicy-
dium and Zanonia). GenBank accession numbers (EU436320–
EU436422) and vouchers for newly sequenced taxa are listed in
table 1 in the electronic supplementary material. Accession
numbers and voucher information for additional Cucurbita-
ceae sequences from our earlier studies are given in Zhang et al.
(2006),Kocyan et al. (2007),Schaefer et al. (2008a)and Nee
et al. (submitted). Fourteen sequences from Genbank were
included to represent Indomelothria (EF065456), Neoachman-
dra (EF065484–86), Urceodiscus (EF065464) and Zehneria
(EF065485, EF065489, EF065491–493, EF065497,
EF065499–500 and EF065502). This resulted in a sampling
of 114 of the 115 genera currently recognized in Cucurbitaceae
(Schaefer & Renner in press).The only genus of Cucurbitaceae
not yet sequenced is Khmeriosicyos W. J. de Wilde & B. Duyfjes,
which is only known from the Cambodian type collection.
Judging from morphology, it is expected to group with other
Asian Benincaseae. As outgroups, we used 15 species of the
Cucurbitales families Anisophyllaceae, Begoniaceae, Coriar-
iaceae, Corynocarpaceae, Datiscaceae and Tetramelaceae,
based on Zhang et al.(2006). Of these, the Begoniaceae, Dati-
scaceae and Tetramelaceae, with the Cucurbitaceae, constitute
a morphologically and molecularly well-defined clade,
traditionally called the core Cucurbitales (Zhang et al. 2006).
(b)DNA extraction, amplification, sequencing and
alignments
Total genomic DNA was isolated from herbarium specimens
or, more rarely, silica-dried leaves with a commercial plant
DNA extraction kit (NucleoSpin, MACHEREY-NAGEL,
Du
¨ren, Germany), following the manufacturer’s manual. We
amplified the rbcL and matK genes, the trnL intron and the
trnL-F and rpl20–rps12 intergenic spacers. Polymerase chain
reactions (PCRs) were performed with the standard protocol
and primers described in Kocyan et al. (2007), and products
were purified with the Wizard SV PCR clean-up kit ( Promega
GmbH, Mannheim, Germany). Cycle sequencing was
performed with BigDye Terminator cycle sequencing kits
on an ABI Prism 3100 Avant automated sequencer (Applied
Biosystems, Foster City, California, USA). Sequences were
edited with SEQUENCHER v. 4.6 (Gene Codes, Ann Arbor,
Michigan, USA) and aligned by eye, using MACCLADE v. 4.06
(Maddison & Maddison 2003).
The data matrices comprised 245 ingroup species plus 15
outgroup species. The lengths of the individual loci were
1356 aligned nucleotides for the rbcL gene, 1195 for the matK
gene, 667 for the tRNA-Leu (trnL) intron (after exclusion of
a poly A run and a highly variable microsatellite region), 803
for the tRNA-Leu–tRNA-Phe (trnL-F) intergenic spacer and
1010 for the rpl20–rps12 intergenic spacer. The combined
dataset comprised 5031 aligned nucleotides.
(c)Phylogenetic analysis
Maximum-likelihood (ML) tree searches and ML bootstrap
searches were performed using RAxML v. 7.0.3 (Stamatakis
et al.2008;availableathttp://phylobench.vital-it.ch/raxml-bb/)
and GARLI v. 0.951 (Zwickl 2006;availableatwww.bio.utexas.
edu/faculty/antisense/garli/Garli.html). RAxML and GARLI
searches relied on the GTRCGCI model (six general time-
reversible substitution rates, assuming gamma rate hetero-
geneity and a proportion of invariable sites), with model
parameters estimated over the duration of specified runs.
Analyses in RAxML were run both with the combined
unpartitioned data and with a model that partitioned the rbcL
gene from the remaining non-coding regions. GARLI does
not allow data partitioning. The data matrix and trees have
been deposited in TREEBASE (www.treebase.org;study
number S2210).
(d)Molecular clock analysis
Estimation of divergence times relied either on a strict clock
or on a Bayesian relaxed clock with autocorrelated rates
(Thorne et al. 1998). Very short (‘zero-length’) branches are
known to cause problems for time estimation algorithms, and
we therefore reduced their number by using the best-scoring
ML tree for 147 taxa instead of the full 260-taxon tree. The
clock tree was rooted on Coriariaceae and Corynocarpaceae,
instead of Anisophyllaceae, because the latter are extremely
rich in autapomorphies, contributing towards rate hetero-
geneity near the base.
For the Bayesian approach,we used BASEML from the PAML
package (Yang 2007) and MULTIDIVTIME (Thorne et al. 1998;
Thorne & Kishino 2002) in LAGOPUS, an R package written
by Heibl & Cusimano (2008). LAGOPUS checks the input
data for consistency, automates the assignment of constraints
to nodes and connects the executables of the mentioned
software packages in a pipeline. Model parameters for the 147-
taxon matrix were estimated in BASEML, and branch lengths
and their variance then calculated in ESTBRANCHES, all under
the F84CG model (the only model implemented in MULTI-
DIVTIME). Priors for MULTIDIVTIME were as follows: based on
outgroup fossils (below), the prior on the mean age of the
root node was set to 84 Myr, with an equally large standard
deviation. The prior on the substitution rate at the root was
set to the value obtained by dividing the median distance
between the root and the tips in the ESTBRANCHES phylogram by
84 Myr. This yielded a rate of 0.0009 substitutions per site and
million years [S/(S!Myr)].The prior for the Brownian motion
parameter, which controls the magnitude of autocorrelation
along the descending branches ofthe tree, was set to 1.11, with
a standard deviation of the same size. Markov chain Monte
Carlo (MCMC) samples were drawn for every 100th gene-
ration up to one million generations, with a burn-in of 100 000
cycles. Confidence in node ages was assessed using the
95 per cent credibility intervals calculated by MULTIDIVTIME.
844 H. Schaefer et al. Biogeography of the Cucurbitaceae
Proc. R. Soc. B (2009)

Cucurbitaceae
outgroup
families
Zanonieae
Fevilleeae
Gomphogyneae
Begoniaceae
Corynocarpaceae
Coriariaceae
Datiscaceae
Anisophylleaceae
Tetramelaceae
10 changes
10 changes
Actinostemmateae
Sicyeae
Cucurbiteae
Coniandreae
Schizopeponeae
Telfairieae
Momodiceae
Thladiantheae
Siraitieae
Indofevilleeae
Bryonieae
Benincaseae
Cucurbitoideae
Sicyos hillebrandii
Sicyos pachycarpus
Sicyos baderoa
Sicyos angulatus
Microsechium helleri
Sechiopsis tetraptera
Sicyosperma gracile
Sechium edule
Parasicyos dieterleae
Echinopepon wrightii
Echinopepon racemosus
Echinopepon paniculatus
Frantzia tacaco
Apatzingania arachoidea
Vaseyanthus insularis
Brandegea bigelovii
Pseudocyclanthera australis
Rytidostylis ciliata
Cyclanthera brachystachya
Hanburia mexicana
Elateriopsis oerstedii
Marah fabaceus
Marah macrocarpus
Echinocystis lobata
Linnaeosicyos amara
Trichosanthes montana
Trichosanthes kinabaluensis
Trichosanthes bracteata
Trichosanthes schlechteri
Trichosanthes pentaphylla
Trichosanthes ovigera
Trichosanthes pendula
Trichosanthes villosa
Trichosanthes kirilowii
Trichosanthes postari
Trichosanthes reticulinervis
Trichosanthes cucumerina
Luffa operculata Mexico
Luffa operculata Senegal
Luffa echinata
Luffa aegyptiaca
Luffa graveolens
Luffa acutangula
Nothoalsomitra suberosa Cucumis sativus
Cucumis hystrix
Cucumis maderaspatanus
Cucumis ritchiei
Cucumis javanicus
Cucumis melo
Cucumis zeyheri
Cucumis messorius
Cucumis sagittatus
Cucumis metuliferus
Cucumis asper
Cucumis oreosyce
Cucumis bryoniifolius
Cucumis humifructus
Cucumis hirsutus
Muellerargia jeffreyana
Muellerargia timorensis
Coccinia grandis
Coccinia sessilifolia
Coccinia rehmannii
Diplocyclos palmatus
Urceodiscus belensis
Papuasicyos papuana
Scopellaria marginata
Cucumeropsis mannii
Posadaea sphaerocarpa
Mslancium campestre
Melothria dulcis
Melothria pendula
Ruthalicia eglandulosa
Ruthalicia longipes
Trochomeria polymorpha
Trochomeria macrocarpa
Dactyliandra welwitschii
Ctenolepis cerasiformis
Zombitsia lucorum
Benincasa hispida
Praecitrullus fistulosus
Borneosicyos simplex
Solena heterophylla
Lemurosicyos variegatus
Cephalopentandra ecirrhosa
Raphidiocystis chrysocoma
Raphidiocystis phyllocalyx
Acanthosicyos horridus
Lagenaria breviflora
Lagenaria siceraria
Peponium caledonicum
Peponium vogelii
Citrullus colocynthis
Citrullus lanatus
Acanthosicyos naudinianus
Neoachmandra sphaerosperma
Neoachmandra japonica
Zehneria neocaledonica
Neoachmandra deltoidea
Neoachmandra samoensis
Zehneria baueriana
Zehneria grayana
Zehneria erythrobacca
Neoachmandra cunninghamii
Anangia macrosepala
Zehneria pallidinervia
Zehneria scabra
Zehneria bodinieri
Zehneria pisifera
Zehneria keayana
Zehneria minutiflora
Zehneria anomala
Cayaponia africana
Cayaponia americana
Cayaponia podantha
Selysia prunifera
Abobra tenuifolia
Cionosicys macranthus
Schizocarpum filiforme
Schizocarpum palmeri
Schizocarpum reflexum
Tecunumania quetzalteca
Sicana odorifera
Bolbostemma paniculatum
Actinostemma tenerum
Xerosicyos perrieri
Xerosicyos danguyi
Xerosicyos pubescens
Siolmatra brasiliensis
Zanonia indica Gerrardanthus macrorhizus
Gerrardanthus paniculatus
Gerrardanthus grandiflorus
Cyclantheropsis parviflora
Sicydium diffusum
Sicydium tamnifolium
Chalema synanthera
Pseudosicydium acariianthum
Pteropepon parodii
Fevillea trilobata
Fevillea pedatifolia
Fevillea anomalosperma
Fevillea pergamentacea
Neoalsomitra clavigera
Neoalsomitra stephensiana
Neoalsomitra trifoliolata
Neoalsomitra capricornica
Gynostemma pentaphyllum New Guinea
Gynostemma pentaphyllum
Hemsleya amabilis
Gomphogyne cirromitrata
Gomphogyne cissiformis
Bayabusua clarkei
Alsomitra macrocarpa
Tetrameles nudiflora
Octomeles sumatrana
Datisca cannabina
Datisca glomerata Coriaria myrtifolia
Coriaria nepalensis
Coriaria ruscifolia
Coriaria sarmentosa
Corynocarpus laevigatus
Anisophyllea fallax
Anisophyllea corneri
Combretoccarpus rotundatus
Begonia herbacea
Begonia oxyloba
Hillebrandia sandwichensis
Neoalsomitra integrifoliola
Neoalsomitra plena
Neoalsomitra angustipetala
Neoalsomitra podagrica
Neoalsomitra sarcophylla
Zygosicyos tripartitus
Calycophysum pedunculatum
Anacaona sphaerica
Penelopeia suburceolata
Cucurbita ficifolia
Cucurbita pepo
Cucurbita okeechobeensis subsp. martinezii
Cucurbita digitata subsp. palmata
Peponopsis adhaerens
Polyclathra cucumerina
Dieterlea aff. maxima
Dieterlea maxima
Dieterlea fusiformis
Ibervillea lindheimeri
Ibervillea millspaughii
Tumamoca macdougalii
Guraniopsis longipedicellata
Apodanthera mandonii
Ceratosanthes palmata
Halosicyos ragonesei
Kedrostis nana
Kedrostis africana
Melothrianthus smilacifolius
Gurania makoyana
Gurania spinulosa
Gurania tubulosa
Psiguria racemosa
Helmontia leptantha
Psiguria umbrosa
Wilbrandia verticillata
Doyerea emetocathartica
Apodanthera sagittifolia
Cucurbitella asperata
Corallocarpus triangularis
Corallocarpus boehmii
Corallocarpus bainesii
Seyrigia humbertii
Trochomeriopsis diversifolia
Dendrosicyos socotranus
Eureiandra formosa
Bambekea racemosa
Edgaria darjeelingensis
Herpetospermum pedunculosum
Biswarea tonglensis
Schizopepon bryoniifolius
Austrobryonia pilbarensis
Austrobryonia argillicola
Austrobryonia micrantha
Austrobryonia centralis
Bryonia dioica
Bryonia alba
Bryonia verrucosa
Ecballium elaterium
Tricyclandra leandrii
Odosicyos bosseri
Ampelosicyos scandens
Ampelosicyos humblotii
Telfairia occidentalis
Telfairia pedata
Cogniauxia podolaena
Cogniauxia trilobata
Momordica cochinchinensis
Momordica cissoides
Momordica spinosa
Momordica cymbalaria
Momordica charantia
Momordica foetida
Momordica calantha
Thladiantha davidii
Thladiantha dubia
Thladiantha punctata
Thladiantha nudiflora
Thladiantha pustulata
Thladiantha hookeri
Sinobaijiania yunnanensis
Baijiania borneensis
Sinobaijiania smitinandii
Siraitia siamensis
Siraitia grosvenorii
Microlagenaria africana
Indofevillea khasiana
Indomelothria chlorocarpa
Gymnopetalum integrifolium
Gymnopetalum chinense
Hodgsonia heteroclita
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Figure 1. (Caption overleaf.)
Biogeography of the Cucurbitaceae H. Schaefer et al. 845
Proc. R. Soc. B (2009)

To translate relative times into absolute times, the
Bayesian clock relied on the following simultaneous con-
straints. (i) The age of core Cucurbitales (the root node) was
constrained to maximally 84 Myr, based on the earliest fossils
of the sister group of Cucurbitales, the Fagales (Herendeen
et al. 1995). However, we also performed two runs in which
the root node was unconstrained or constrained to minimally
84 Myr. This yielded age estimates for the Cucurbitaceae
crown group that were older than the oldest angiosperm
fossils (132 Myr). In general, relaxed molecular clocks will
yield reliable ages only with at least one minimal and one
maximal constraint, the latter preferentially at or near the root
(Thorne et al. 1998). (ii) The age of the split between Datisca
and Octomeles/Tetrameles was set to minimally 68 Myr old or,
in an alternative run, to minimally 65.5 Myr old, based on the
fossil wood of Tetrameleoxylon prenudiflora from the Deccan
intertrappean beds at Mohgaonkalan in India ( Lakhanpal &
Verma 1965;Lakhanpal 1970). These beds have been dated
to the Maastrichtian or Late Maastrichtian ( Khajuria et al.
1994;Kar et al. 2003), and we therefore used either the
midpoint of the Maastrichtian (68 Myr) or the Maastrichtian/
Palaeocene border (65.5 Myr). (iii) The crown group of
Cucurbitaceae was set to minimally 65 Myr or, alternatively,
55.8 Myr, based on the seeds from the Palaeocene Felpham
flora (Collinson 1986;Collinson et al. 1993). These dates
span the upper and lower boundary of the Palaeocene.
(iv) The split between Linnaeosicyos, with tetracolpate–
reticulate pollen, and the remaining New World Sicyeae
(Schaefer et al. 2008a), which usually have polycolpate
pollen, was set to minimally 33.9 Myr or, alternatively,
23 Myr, based on Hexacolpites echinatus pollen from the
Oligocene of Cameroon, which is the oldest hexacolpate
Sicyeae-type pollen (Salard-Cheboldaeff 1978;Muller 1985).
Polycolpate pollen is not found in other Cucurbitaceae except
the African Neoachmandra peneyana (Van der Ham &
Pruesapan 2006). The Oligocene epoch ranges from 33.9 to
23 Myr, and the stratum containing Hexacolpites has not been
precisely dated; therefore, in alternative analyses, we used the
upper or lower boundary as minimal constraints. (v) The split
between the Hispaniola endemics Anacaona and Penelopeia
was set to maximally 20 or 15 Myr, based on the age of
Dominican amber, which was produced by tropical trees and
provides a proxy for the presence of tropical forest on that
island; the amber age is estimated as 15–20 Myr ( Iturralde-
Vinent & MacPhee 1996).
To explore the sensitivity of the Bayesian clock to the
various priors, we performed alternative MCMC runs in
which we tested (a) the effect of a more clock-like Brownian
motion parameter of 0.4 instead of 1.11, (b) the effect of
using up to four data partitions, thereby allowing the genes
and spacer regions to have different rates, and (c) the effects of
varying the age constraints. For the latter exploration, we ran
an analysis in which all constraints were set to the lowest age
boundaries, another in which all constraints were set to the
highest age boundaries, and four analyses that used the
minimum age for one of the constraints and the maximum
age for the remaining constraints. All other parameters, such
as root rate and MCMC chain length, were constant between
these six runs. Finally, we performed a run (d) with mean ages
for four constraints, namely 66.8 Myr (constraint ii),
60.4 Myr (iii), 28.5 Myr (iv) and 17.5 Myr (v).
For the strict clock approach, rbcL branch lengths were
calculated under a GTRCGCICclock model on the
preferred ML topology. The tree was imported into PAUP,
rooted on Coriaria (Zhang et al. 2006), and branch lengths
were then calculated under the ‘enforce clock’ option. The
distance between a calibration node and the present was
divided by the age of the calibration node to obtain a
substitution rate, and this rate was then used to calculate
the age of divergence events of interest. As calibration
nodes, we used either the age of the earliest Cucurbitaceae
seeds (constraint iii) or the oldest Sicyeae-type pollen
(constraint iv).
(e)Biogeographic analysis
For a dispersal-vicariance analysis, we used the 147-taxon
dataset also used for the clock runs and coded the distribution
ranges of all species in a binary matrix in MACCLADE. The
species were recorded as present in one of five regions: Asia;
Europe; Africa (including Madagascar); America (including
Caribbean, Galapagos, and Hawaii); and Australia (including
New Guinea and Polynesia). We then used the parsimony-
based approach implemented in DIVA v. 1.1 ( Ronquist 1996,
1997) to infer vicariance and dispersal events. The maximum
number of areas simultaneously occupied by hypothetical
ancestral lineages was experimentally constrained to 4, 3 or 2
because it is unlikely that an ancestral species would have
ranged over several continents.
3. RESULTS
Herbarium material yielded suitable DNA in more
than 95 per cent of the cases, even for 50–100-year-
old collections.
The highest-scoring ML tree obtained for the 260-taxon
dataset (figure 1) shows Cucurbitaceae highly supported
as monophyletic and family relationships similar to those
found by Zhang et al. (2006) with a much larger amount
of sequence data. Within Cucurbitaceae, there are five
main clades (figure 1), namely: (i) a group of approxi-
mately 100 genera traditionally treated as subfamily
Cucurbitoideae (Kosteletzky 1833) and usually subdi-
vided into several tribes (below); (ii) a clade of Asian
genera, including Alsomitra,Bayabusua and Neoalsomitra
that corresponds to the tribe Gomphogyneae of Bentham &
Hooker (1867); (iii) a clade of one African and five
Neotropical genera, including Fevillea and Sicydium,that
corresponds to the tribe Fevilleeae of Bentham & Hooker
(1867); (iv) a clade of a few genera from Madagascar,
continental Africa, Asia and South America corresponding
to the tribe Zanonieae of Blume (1826);and(v)a
clade consisting of the two Asian genera Actinostemma
and Bolbostemma.
Clades (ii–v) have been treated as subfamily Nhandir-
oboideae (an illegitimate name) or Zanonioideae
Figure 1. (Overleaf.). Best ML tree for Cucurbitaceae and relatives found with combined chloroplast gene, spacer and intron
sequences (5031 nucleotides) analysed under a GTRCGCI model with unlinked partitions for coding and non-coding regions.
Likelihood bootstrap values greater than 60% are given at the nodes. Rooting follows Zhang et al. (2006). The geographical
occurrence of genera is colour coded as follows: green, America (including Galapagos, Hawaii and the Caribbean); yellow,
mainland African; brown, Madagascar; red, Asia; blue, Australia/New Guinea/Polynesia; black, Europe.
846 H. Schaefer et al. Biogeography of the Cucurbitaceae
Proc. R. Soc. B (2009)

(a taxonomic synonym of Fevilleoideae), but this sub-
family is not supported as monophyletic by our data.
Clade (i), Cucurbitoideae, can be divided into geographi-
cally or morphologically more homogeneous groups that
correspond to the traditional tribes Herpetospermeae,
Bryonieae,Sicyeae,Coniandreae,Benincaseae and Cucurbi-
teae, plus a few clades of similar phylogenetic depth that
have not traditionally been ranked as tribes, such as the
Asian Thladiantha and Baijiania, the Asian/African Siraitia
and Microlagenaria, the African/Asian Momordica, the
African Telfairia,Cogniauxia, a group of Madagascan
genera and the Himalayan Indofevillea, which is sister to all
remaining Cucurbitoideae (figure 1). Well-known genera
found to be poly- or paraphyletic include Citrullus
(must include Acanthosicyos), Ampelosicyos (must include
Tricyclandra and Odosicyos), Gomphogyne (must include
Hemsleya), Xerosicyos (must include Zygosicyos), Apodanthera,
Psiguria and Trichosanthes.
MULTIDIVTIME dating runs with a Brownian motion
parameter of 0.4 instead of 1.11, yielding barely different
estimates for the ingroup nodes of interest. Runs that
allowed uncoupled rates for the two genes and the
spacers also yielded essentially identical estimates, and
final runs therefore modelled the data under a single
model. The estimates from the run in which all constraints
were set to their lowest boundaries differed significantly
from those obtained when all constraints were set to
their highest boundaries ( Wilcoxon signed-rank test,
pZ0.0085; see fig. 1c,din the electronic supplementary
material). Among the test runs in which one constraint
was set to the minimum age and the others to the
maximum age, only two yielded significantly different
results: the Tetrameleoxylon fossil set to the minimum age
and the Felpham flora seed set to the minimum age
(see fig. 1ein the electronic supplementary material).
However, all results were within the 95 per cent confi-
dence intervals of the estimates obtained when the
constraints were set to mean ages (see fig. 1a,bin the
electronic supplementary material).
The substitution rates obtained under a strict clock
model calibrated with either the seed or the pollen fossil
(§2) were 0.00018 S/(S!Myr) (oldest Sycieae-type
pollen, constraint (iv)) or 0.00030 S/(S!Myr) (earliest
Cucurbitaceae seeds, constraint (iii)). An average rate of
0.00024 S/(S!Myr) yielded absolute times that for the
most part were older than those obtained with the relaxed
clock model (see table 2 in the electronic supplementary
material that lists the ages obtained with the relaxed
clock and with the strict clock model). The following
discussion focuses on the relaxed clock estimates
because they provide 95 per cent confidence intervals as
a measure of uncertainty.
The split between the two genera of Begoniaceae,
Begonia and Hillebrandia, is estimated as 29 (41–18) Myr
old, roughly the age of the Hawaiian archipelago
(ca 30 Myr), where Hillebrandia is endemic (Clement
et al. 2004). The deepest split in the Cucurbitaceae is ca
63 (69–61) Myr old, while crown group Cucurbitoideae
are 53 (60–48) Myr old, Gomphogyneae 56 (63–51)
Myr, Fevilleeae 46 (55–37) Myr and the Actinostemma/
Bolbostemma clade 52 (59–44) Myr. The Madagascan
Xerosicyos clade (split Zanonia–Xerosicyos) appears to be
49 (57–40) Myr old; the likewise Madagascan Ampelosi-
cyos clade is 29 (39–19) Myr old. The Madagascar/
Southeast Asia disjunction between the two species of
Muellerargia is only ca 12 (18–7) Myr old. The South
America/Asia disjunction in the Zanonia clade (Siolmatra–
Zanonia split), finally, is 24 (38–11) Myr old, and the
South America/Africa disjunction in the Fevillea clade
(Chalema–Cyclantheropsis split) 41 (51–31) Myr. Other
estimates of specific interest (Introduction; see table 2 in the
electronic supplementary material) concern Cayaponia
(stem age 10 (17–5) Myr), Luffa (stem age 35 (41–31)
Myr), Sicyos (stem age 8 (13–4) Myr) and the Lagenaria
crown group (8 (14–3) Myr; figure 2).
The DIVA analysis yielded Asia as the most likely
region of origin of the Cucurbitaceae (figure 3a). From
there, at least five lineages reached Africa, and 12 lineages
reached Australia. No fewer than seven lineages indepen-
dently reached the American continent, and no fewer
than 13 lineages dispersed from Africa to Madagascar.
Some disjunctions are best explained as secondary
dispersals from Africa back to Asia (Coccinia and
Momordica). A few lineages reached Africa via LDD
from America (Cayaponia africana,Cucumeropsis, perhaps
Cyclantheropsis and Kedrostis).
4. DISCUSSION
(a)The geographical origins of the world’s
regional Cucurbitaceae floras
Our results suggest that Cucurbitaceae initially diversified
in Asia (specifically, the region north of the Tethys)
sometime in the Late Cretaceous. This fits with the
observation that India contains more deeply divergent
lineages of Cucurbitaceae than any other similar-sized
geographical area (Chakravarty 1946,1959;thisstudy).Of
the family’s Late Cretaceous radiations, two clades (the
Gomphogyneae and the Actinostemmateae) are now
almost restricted to subtropical Asia (figure 1). A third
clade, Fevilleeae, is mainly Neotropical except for a small
African ‘extension’, Cyclantheropsis. The ancestors of
Fevilleeae were probably more widely distributed in the
Laurasian tropics and reached the American continent by
dispersing across a still-narrow Atlantic (figure 3b;seedsof
Fevilleeae are wind- and water-dispersed). Cyclantheropsis
must result from a back dispersal from South America to
Africa in the Middle Eocene. The ancestors of the fourth
ancient clade, Zanonieae, apparently reached the African
continent early and from there dispersed to Madagascar (the
Early Eocene Xerosicyos lineage; figure 3b). Later, in the
Oligocene, at least two LDD events brought the Siolmatra
lineage to America and the Zanonia lineage back to tropical
Asia (figure 3c). The fifth and last ancient clade, the
Cucurbitoideae, diversified partly in Asia (e.g. Thladiantha,
Siraitia,Trichosanthes), and partly in Africa (e.g.Momordica,
Cucumis,Coccinia,Kedrostis). The cucumber (Cucumis
sativus) and its closest relatives (not all included in the
present study) evolved from a common ancestor ca 3(6–1)
Myr ago. The wax gourd Benincasa and its sister group
Praecitrullus, an important vegetable in parts of India,
apparently split only 5 (10–1) Myr ago. Further dispersals
from Africa back to Asia are present within Momordica,
Coccinia,Kedrostis and Corallocarpus (figure 3c).
The native European cucurbit flora consists only of
Bryonia, with 10 species ( Volz & Renner 2008), and its
monotypic sister Ecballium, which probably represent a
lineage that spread along the Tethys border from Asia to
Biogeography of the Cucurbitaceae H. Schaefer et al. 847
Proc. R. Soc. B (2009)

80 60 40 20 0 a
g
e in m
y
r
Sinobaijiania smitinandii
Sinobaijiania yunnanensis
Baijiania borneensis
Thladiantha dubia
Thladiantha hookeri
Microlagenaria africana
Siraitia grosvenorii
Momordica charantia
Momordica cissoides
Momordica cochinchinensis
Cogniauxia podolaena
Telfairia occidentalis
Telfairia pedata
Odosicyos bosseri
Tricyclandra leandrii
Ampelosicyos scandens
Austrobryonia centralis
Austrobryonia micrantha
Bryonia verrucosa
Bryonia dioica
Ecballium elaterium
Schizopepon bryoniifolius
Herpetospermum pedunculosu
m
Biswarea tonglensis
Dendrosicyos socotranus
Seyrigia humbertii
Trochomeriopsis diversifolia
Ibervillea millspaughii
Dieterlea maxima
Apodanthera mandonii
Guraniopsis longipedicellata
Melothrianthus smilacifolius
Ceratosanthes palmata
Doyerea emetocathartica
Psiguria racemosa
Gurania makoyana
Wilbrandia verticillata
Kedrostis africana
Halosicyos ragonesei
Cucurbitella asperata
Corallocarpus boehmii
Bambekea racemosa
Raphidiocystis chrysocoma
Citrullus colocynthis
Citrullus lanatus
Lagenaria breviflora
Lagenaria siceraria
Peponium caledonicum
Peponium vogelii
Papuasicyos papuana
Benincasa hispida
Praecitrullus fistulosus
Trochomeria macrocarpa
Dactyliandra welwitschii
Zombitsia lucorum
Ctenolepis cerasiformis
Cephalopentandra ecirrhosa
Lemurosicyos variegatus
Solena heterophylla
Acanthosicyos horridus
Zehneria anomala
Zehneria bodinieri
Zehneria scabra
Neoachmandra japonica
Neoachmandra cunninghamii
Diplocyclos palmatus
Coccinia grandis
Cucumis hirsutus
Cucumis melo
Cucumis hystrix
Cucumis sativus
Muellerargia jeffreyana
Muellerargia timorensis
Ruthalicia eglandulosa
Indomelothria chlorocarpa GB
Melothria pendula
Melothria dulcis
Melancium campestre
Posadaea sphaerocarpa
Cucumeropsis mannii
Calycophysum pedunculatum
Anacaona sphaerica
Penelopeia suburceolata
Tecunumania quetzalteca
Schizocarpum reflexum
Cayaponia americana
Cayaponia africana
Selysia prunifera
Cionosicys macranthus
Polyclathra cucumerina
Cucurbita pepo
Cucurbita ficifolia
Peponopsis adhaerens
Nothoalsomitra suberosa
Luffa acutangula
Luffa operculata – Mexico
Luffa operculata – Senegal
Linnaeosicyos amara
Marah macrocarpus
Echinocystis lobata
Parasicyos dieterleae
Sechium edule
Sicyosperma gracile
Sechiopsis tetraptera
Sicyos hillebrandii
Sicyos baderoa
Brandegea bigelovii
Vaseyanthus insularis
Echinopepon wrightii
Cyclanthera brachystachya
Pseudocyclanthera australis
Hanburia mexicana
Hodgsonia heteroclita
Trichosanthes schlechteri
Trichosanthes cucumerina
Gymnopetalum chinense
Indofevillea khasiana
Actinostemma tenerum
Bolbostemma paniculatum
Gerrardanthus macrorhizus
Gerrardanthus grandiflorus
Siolmatra brasiliensis
Zanonia indica
Xerosicyos danguyi
Xerosicyos pubescens
Fevillea trilobata
Fevillea pergamentacea
Cyclantheropsis parviflora
Pseudosicydium acariianthum
Pteropepon parodii
Sicydium tamnifolium
Chalema synanthera
Alsomitra macrocarpa
Bayabusua clarkei
Neoalsomitra capricornica
Neoalsomitra podagrica
Gynostemma pentaphyllum
Gomphogyne cissiformis
Hemsleya amabilis
Datisca cannabina
Hillebrandia sandwichensis
Begonia herbacea
Octomeles sumatrana
Tetrameles nudiflora
14 ± 5
27 ± 6
32 ± 6
24 ± 4
v
iv
iii
ii
33 ± 8
31 ± 5
35 ± 5
29 ± 5
32 ± 4
23 ± 4
13 ± 4
7 ± 3
10 ± 55 ± 3
15 ± 5
22 ± 6
29 ± 4
22 ± 4
14 ± 5
15 ± 4
9 ± 3
13 ± 3
14 ± 4
19 ± 4
15 ± 412 ± 3
13 ± 3
11 ± 3
17 ± 46 ± 3
15 ± 4
13 ± 3
11 ± 3
12 ± 4
16 ± 3
18 ± 3
12 ± 3
8 ± 3
2 ± 2
4 ± 2
5 ± 2
13 ± 37 ± 3
10 ± 3
7 ± 3
12 ± 3
14 ± 3
17 ± 3
19 ± 4
16 ± 310 ± 3
14 ± 4
11 ± 39 ± 3
20 ± 4
15 ± 3
14 ± 39 ± 33 ± 2
1 ± 1
12 ± 3
15 ± 47 ± 3
10 ± 3
8 ± 3
13 ± 3
17 ± 3
21 ± 3
30 ± 4
39 ± 3
53 ± 3
63 ± 3
81 ± 2
73 ± 3
63 ± 4
34 ± 3
41 ± 3
38 ± 3
25 ± 4
44 ± 3
46 ± 3
49 ± 3
51 ± 3
45 ± 4
3 ± 3
23 ± 4
19 ± 3
17 ± 3
16 ± 310 ± 33 ± 2
1 ± 1
7 ± 3
16 ± 4
11 ± 5
15 ± 3
31 ± 2
37 ± 3
35 ± 3
33 ± 2
28 ± 3
31 ± 3
28 ± 3
32 ± 6
28 ± 6
24 ± 7
23 ± 7
17 ± 6
18 ± 5
27 ± 5
23 ± 5
29 ± 6
26 ± 7
39 ± 5
44 ± 5
51 ± 4
56 ± 3
59 ± 3
58 ± 3
54 ± 4
52± 4
49 ± 4
46 ± 5
41 ± 5
39 ± 5
34 ± 6
11 ± 3
10 ± 3
9 ± 2
8 ± 2
16 ± 3
17 ± 3
12 ± 3
15 ± 3
13 ± 39 ± 3
4 ± 2
2 ± 2
19 ± 4
16 ± 3
4 ± 2
i
19 ± 4
21 ± 4
Figure 2. Chronogram obtained for Cucurbitaceae under a Bayesian autocorrelated rates relaxed clock model applied to the
combined data (5031 nucleotides) and calibrated with three minimal (yellow) and two maximal (orange) constraints as in run
(d)of§2(d). Age estimates with their 95% confidence ranges shown in purple. Rooting follows Zhang et al. (2006). Green,
America (including Galapagos, Hawaii and the Caribbean); yellow, mainland Africa; brown, Madagascar; red, Asia; blue,
Australia/New Guinea/Polynesia; black, Europe.
848 H. Schaefer et al. Biogeography of the Cucurbitaceae
Proc. R. Soc. B (2009)

the Mediterranean 32 (41–24) Myr ago. The remaining
cucurbit species that occur in Europe are the result of
recent introductions (Echinocystis lobata,Sicyos angulatus
and Thladiantha dubia) or casual escapes from cultivation
(Citrullus lanatus,Cucumis melo,C. sativus and Cucurbita
pepo). The closest extant relative of Bryonia and Ecballium
is the Australian genus Austrobryonia (four species), which
may have reached Australia from Asia ca 13 (21–6) Myr
ago (Schaefer et al. 2008b).
African Cucurbitoideae (25 genera) are the result of
five dispersals from Asia to Africa and two from America
to Africa (in the genera Cucumeropsis and Cayaponia).
The watermelon (Citrullus lanatus) and its sister species
(C. colocynthis) apparently evolved from a common
ancestor as recently as 2 (6–0.1) Myr ago. The lineage
leading to the cucumber tree, Dendrosicyos socotranus,
endemic on Socotra, some 350 km off the Arabian
peninsula, is estimated as 22 (30–14) Myr old, while the
Socotra archipelago is only some 10 Myr old (Ghebreab
1998). Dendrosicyos thus seems to be an island relict of a
progenitor lineage that went extinct on the mainland. This
example of a species that is twice as old as the island on
which it occurs cautions against using geological calibra-
tions in molecular clock dating. Another supposed
example of a species being older than the island on
which it lives, that of the Hawaiian Hillebrandia sandwi-
censis (Clement et al. 2004), is not supported by our data
(see table 2 in the electronic supplementary material).
Madagascar has 16 native Cucurbitaceae genera with
50 species in total. From our data it appears that
Cucurbitaceae reached Madagascar at least 13 times,
apparently always from the African mainland, and that
these 13 ancestors then underwent local radiations,
giving rise to today’s 50 species. Using Madagascar as a
stepping stone, one of these clades, Pepo nium,later
reached the Seychelles (the endemic species there has
not yet been sequenced).
South America has approximately 350 species of
Cucurbitoideae in 47 genera that all descend from five
LDD events, mostly from Africa to South America. These
involved the ancestors of Cucurbiteae, Sicyinae, a clade of
Coniandreae, the Melothria clade and a subclade of Luffa.
Based on the tree topology (figure 1), Luffa originated in
the Old World or Australia, and one species then reached
the New World by LDD from Africa across the Atlantic as
suggested by Heiser & Schilling (1988). The fruit is dry
with fibrous tissue and probably well adapted to floating
(Ridley 1930). The Neotropical Melothria clade (figure 1)
appears to have crossed the Pacific because the sister
group of Melothria,Indomelothria, is endemic in Southeast
Asia. Today’s pumpkin and squash species (Cucurbita spp.
in the Cucurbiteae) apparently originated in Central or
South America, and the genus Cucurbita split from its
sister clade, Peponopsis, only some 16 (23–9) Myr ago.
North American Cucurbitaceae, finally, all descend from
seven expansions of Central and South American lineages
that occurred at widely different times (figure 2; see table 2
in the electronic supplementary material).
The indigenous Australian Cucurbitaceae flora consists
of 30 species in 12 genera of which two are endemic:
Nothoalsomitra, a liana species of Queensland’s rainforests;
and Austrobryonia, four species of trailers or creepers in
the dry regions of (mostly) Central Australia. This low
Australian species diversity is in marked contrast with the
minimally 12 independent dispersal events into Australia
(figures 1and 3d). The largest Australian ‘radiation’
comprises only four species (Schaefer et al. 2008b), even
1
CucurbitaceaeCucurbitaceae
Fevilleeae
ZanonieaeZanonieae
Xerosicyos
2
3
Luffa
CayaponiaCayaponia
Sicyos 7
8
9
8
11x
12x
AmpelosicyosAmpelosicyos
Siolmatra
Zanonia
4
5
6
MomordicaMomordica
(a)
(b)
(c)
(d)
Figure 3. The biogeographic history of Cucurbitaceae as
inferred from the statistical approach described in the text
(coastlines drawn after Smith et al. 1994). (a) Late Cretaceous,
ca 70 Myr ago; (1) origin of the Cucurbitaceae in Asia.
(b) Palaeocene/Eocene, 60–40 Myr ago; (2) the ancestor of the
Zanonieae reaches Africa, the ancestor of the Xerosicyos
lineage reaches Madagascar, and (3) the ancestor of Fevilleae
reaches South America. (c) Oligocene, ca 30 Myr ago; (4) the
ancestor of the Siolmatra lineage disperses over the Atlantic
into South America, (5) the ancestors of Momordica cochinch-
inensis and Zanonia indica independently disperse from Africa
to Southeast Asia, and (6) the ancestor of the Ampelosicyos
lineage reaches Madagascar. (d) Middle Miocene, ca 10 Myr
ago; (7) the ancestors of Neotropical Luffa disperse from
Africa to the Americas, (8) the ancestor of Cayaponia africana
disperses from South America to West Africa, and (9)
ancestors of several Sicyos species groups spread from South
America to Hawaii, Galapagos, New Zealand and Australia.
Biogeography of the Cucurbitaceae H. Schaefer et al. 849
Proc. R. Soc. B (2009)

though the ecological conditions in Australian rainforests
and bushland are similar to those in Southeast Asian
forests and African bushland, where cucurbit diversity
is much higher.
Overall, Cucurbitaceae underwent at least 43 success-
ful LDD events over the past 60 Myr, which would
translate into an average of seven LDDs every 10 Myr.
These events seem to have occurred throughout the
evolutionary past of the family, rather than being clustered
at a particular geological time (see table 2 in the electronic
supplementary material). The most striking case of a rapid
radiation following LDD is Sicyos, which reached Hawaii
only ca 2 (6–0.1) Myr ago (figure 3d) and now has 15
Hawaiian species. Sicyos species often grow in seabird
colonies (www.botany.hawaii.edu/gradstud/eijzenga/
OIRC/lehua.htm# Vegetation), and the hooked barbs on
the fruits may help external dispersal on bird feathers.
Another extreme case is Muellerargia, the closest relative of
Cucumis (Renner & Schaefer 2008), which comprises just
two species, one endemic in Madagascar, and the other in
northeast Australia and Timor.
(b)Using museomics to achieve less biased taxon
sampling
Results of this study may have implications for the
conservation of wild Cucurbitaceae gene pools for future
crop improvement; this is almost certainly true for Luffa
and Cucumis. They also show that the gourd family, which
appears to be of Asian origin, has undergone numerous
natural LDD events, prior to anthropogenic LDD, and
that these events have played a large role in the build-up of
local cucurbit floras. More generally, this study illustrates
the great potential of herbarium material for generating
broad phylogenetic frameworks at relatively low costs and
in a time-efficient manner (no lengthy permit procedures).
With rapidly improving molecular techniques for DNA
isolation from tiny fragments of ancient collections, it is
now possible to study the phylogeny of worldwide lineages
without time-consuming and difficult field trips. And
broad geographical sampling in turn is the precondition
for assessing the full extent of LDD in the angiosperms at
different geological times and across different ocean
basins. Given that habitat destruction in the centres of
cucurbit diversity (Madagascar, Southeast Asia, West
Africa and Central America) is extremely high, the use
of herbarium material also may soon be the only option for
future research on the phylogenetics of this plant group.
We thank B. Duyfjes and W. J. de Wilde (L) for silica samples
and advice; M. Nee (NY), M. Pignal (P), J. Wieringa (WAG)
and the curators of the herbaria AAU, ASU, B, BKF, BR,
BSC, CHR, CMU, E, EA, F, FTG, G, GIFU, K, KUN, L,
LE, LISC, M, MEXU, MO, NE, RSA, ULM, US and Z for
permission to sample DNA from specimens in their care;
E. Vosyka for help in the laboratory; and the German science
foundation for financial support ( DFG RE603/3-1).
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