Background

Melon,Cucumis melo, and cucumber,C. sativus, are among the most widely cultivated crops worldwide.Cucumis, as traditionally conceived, is geographically centered in Africa, withC. sativusandC. hystrixthought to be the onlyCucumisspecies in Asia. This taxonomy forms the basis for all ongoingCucumisbreeding and genomics efforts. We tested relationships amongCucumisand related genera based on DNA sequences from chloroplast gene, intron, and spacer regions (rbcL,matK,rpl20-rps12,trnL, andtrnL-F), adding nuclear internal transcribed spacer sequences to resolve relationships withinCucumis.

Results

Analyses of combined chloroplast sequences (4,375 aligned nucleotides) for 123 of the 130 genera of Cucurbitaceae indicate that the generaCucumella,Dicaelospermum,Mukia,Myrmecosicyos, andOreosyceare embedded withinCucumis. Phylogenetic trees from nuclear sequences for these taxa are congruent, and the combined data yield a well-supported phylogeny. The nesting of the five genera inCucumisgreatly changes the natural geographic range of the genus, extending it throughout the Malesian region and into Australia. The closest relative ofCucumisisMuellerargia, with one species in Australia and Indonesia, the other in Madagascar. Cucumber and its sister species,C. hystrix, are nested among Australian, Malaysian, and Western Indian species placed inMukiaorDicaelospermumand in one case not yet formally described.Cucumis melois sister to this Australian/Asian clade, rather than being close to African species as previously thought. Molecular clocks indicate that the deepest divergences inCucumis, including the split betweenC. meloand its Australian/Asian sister clade, go back to the mid-Eocene.

Conclusion

Based on congruent nuclear and chloroplast phylogenies we conclude thatCucumiscomprises an old Australian/Asian component that was heretofore unsuspected.Cucumis sativusevolved within this Australian/Asian clade and is phylogenetically far more distant fromC. melothan implied by the current morphological classification.

The non-monophyly ofCucumisand why it remained undiscovered; comparison with earlier molecular phylogenies

Parsimony (MP) and maximum likelihood (ML) analyses of combined sequences from the chloroplast genesrbcLandmatK, the chloroplast introntrnL, and the spacersrpl20-rps12 andtrnL-F, under the GTR + G + I model yielded a topology (Fig.1) that was congruent with that obtained from the nuclear internal transcribed spacer region (Fig.2). Chloroplast and nuclear data were therefore combined, and a parsimony tree from the combined data with MP and ML bootstrap support is shown as Fig.3 (seven of the species lack ITS sequences, Table1). In the family-wide analysis (with 123 of 130 genera of Cucurbitaceae sequenced),Cucumisis sister toMuellerargia(Fig.4). The generaCucumella,Dicaelospermum,Mukia,Myrmecosicyos, andOreosyceare embedded among species ofCucumis(Fig.3). The remaining genera of Cucumerinae sensu C. Jeffrey [11],CucumeropsisNaudin,MelanciumNaudin,MelothriaL.,PosadaeaCogn., andZehneriaEndl. (plusNeoachmandraandScopellaria[24]) group far fromCucumis(Fig.4). This fits with their geographic concentration in the New World (whereCucumisis absent):Melanciumis a monotypic genus from Brazil,Posadaeaa monotypic genus from tropical America, andMelothriahas ten species in Central America and South America. However,Cucumeropsis, with a single species from tropical Africa, andZehneria,Neoachmandra, andScopellaria, with 66 species in tropical and subtropical Africa, Madagascar, Asia, New Guinea, and Australia [24] overlap with the natural range ofCucumis.

The sister genus toCucumis,Muellerargia, consists of one species in Madagascar and one in Indonesia and Queensland. Both are herbaceous trailers or climbers with straight or apically reflexed anthers and softly spinose fruits.Muellerargiahas never been recognized as closely related toCucumis[12,25], perhaps because it is extremely poorly collected, with but a few specimens even in major herbaria: The Madagascan species,Muellerargia jeffreyanaKeraudren, is known from three collections (in the Paris herbarium), and permission was not granted to sacrifice material for this study. It is morphologically similar to the Indonesian-Australian speciesM. timorensisCogn. [26]. The poor documentation of the genus in herbaria also led to the Australian species being described at least three times; first asMuellerargia timorensisCogn., then asMelothria subpellucidaCogn., and then asZehneria ejectaBailey (syn.Melothria ejecta(Bailey) Cogn.).

TheCucumisspecies relationships found here differ from those found in earlier studies [1,20-22]. An unrooted nuclear isozyme tree [21] showedC. sativusas the genetically most distant species, whileC. melowas sister to an African clade. The neighbor-joining tree from nuclear ITS sequences of Garcia-Mas et al. [22] was rooted onCitrullus lanatusandCucurbita pepo, and showedC. sativusas the first-branching species in the genus, whileC. melowas sister to a large African clade. Finally, the chloroplast tree of Chung et al. [1] also was rooted onCitrullusand showedC. sativusandC. hystrixas sister toC. melo(as did studies focusing onC. sativus; e.g., [27]). By contrast, the data presented here (Figs.1,2,3,4) indicate that (i) the deepest divergence inCucumisis betweenC. hirsutusandC. humifructuson the one hand and all other species on the other, (ii)C. sativus(cucumber) andC. hystrixare closer toDicaelospermumandMukiathan they are to any species ofCucumis, and (iii)C. melo(melon) is sister to a clade comprisingDicaelospermum,Mukia, C. sativus,C. hystrix, and a new species from Australia (HS414).

There are several possible explanations for the contrasting findings of the earlier phylogenetic studies. First, the use of distant outgroups might have "attracted" the long-branched (i.e., mutation-rich)C. sativus, pulling it to the base of the tree. Garcia-Mas et al. [22] and Chung et al. [1] usedCitrullus lanatusand/orCucurbita pepoas sole outgroups. Both taxa are many clades, and millions of years of evolution, removed from theCucumisclade (Fig.4) and therefore add long branches to neighbor-joining and parsimony analyses [1,22]. The inclusion of these long branches could have caused long-branch attraction between them andC. sativus.

A second reason why previous molecular phylogenetic studies were unable to test the monophyly ofCucumisand to infer the sister clades of cucumber and melon is that they did not include a sufficiently broad sample of taxa. For example, rigidly testing the monophyly ofCucumissectionMelorequired sequencing all of its species,C. melo,C. hirsutus,C. humifructus, andC. sagittatus. Results (Figs.1,2,3) show thatC. hirsutusandC. humifructus, rather than being close toC. melo, are sister to all other species ofCucumissensu lato, that is, including all five genera nested inCucumis.

Another possible reason for apparent differences between earlier topologies and the phylogeny found here is insufficient signal in the data and misidentified material. Comparison of the ITS sequences of Garcia-Mas et al. [22] to our ITS sequences showed that the sequence labeledCucumis membranifoliusin GenBank (AJ488223) andOreosyce africanain the published paper (these names refer to the same species fide [12]), does not representOreosyce africana. The sequence came from a seed provided by the North Central Regional Plant Introduction Station in Ames, Iowa, and since there is no voucher, its identification cannot be verified. We also could not reproduce the topology and bootstrap support obtained in the original paper [22], partly probably because the phylogenetic signal in the data is weak, resulting in many equally likely trees. Garcia-Mas et al. [22] included sequences resulting from direct sequencing as well as sequences obtained by pGEM-T Easy Vector cloning and found sequences from multiple accessions generally grouping by species. OurCucumisITS sequencing confirmed these authors' assessment that ITS lineage sorting is not a problem inCucumis. The twoC. ficifoliussequences obtained by Garcia-Mas et al. [22] that do not group (Fig.2) come from different plants and may simply represent different species; however, since the material is unvouchered, the identifications cannot be checked.

Implications for the evolution and biogeography ofCucumis

The phylogeny from the combined nuclear and chloroplast data (Fig.3) implies that the deepest divergence lies between the common ancestor ofC. hirsutusandC. humifructusand the stem lineage of the remainder of the genus. From the geographic ranges of the species ofCucumis sensu lato(i.e., the natural clade identified here) and its sister genusMuellerargia(with one species in tropical Australia and Indonesia, the other in Madagascar), the area whereCucumismay have originated cannot reliably be inferred. Strict and semi-parametric molecular clocks indicated that the deepest divergence inCucumismay date back to 48–45 my and that the split between theC. melolineage and its Australian/Asian sister clade is only slightly younger. The divergence ofC. sativusfromC. hystrixmay be about 8 my old and that of their common ancestor from the ancestor ofDicaelospermumandMukia maderaspatanaabout 19 my. The bulk of the African species appears to have evolved more recently. The absence of a fossil constraint withinCucumis, however, cautions against over-confidence in the molecular clock estimates.

Based on the tree (Fig.3), the earliest divergence events inCucumislikely took place in Africa. However, contrary to the traditional classification [12], which groupsC. melowith the AfricanC. hirsutus,C. humifructus, andC. sagittatus, melon is closest to an Australian/Asian clade (marked in grey-green in Fig.3) that comprises an undescribed Australian species [28], species currently placed inMukia(M. javanica,M. maderaspatana),Dicaelospermum ritchieifrom Western India (recently transferred toMukia[29]), andCucumis sativusandC. hystrixfrom India, China, Burma, and Thailand. In addition to the two species we sequenced,Mukiacomprises three others [29], its overall geographic range extending from Indo-China southeast to Java, Borneo, and the Philippines, and west through India, Pakistan, and the Yemen into sub-Saharan Africa. Given the geographic distribution of its extant closest relatives (Fig.3),C. meloitself could have originated somewhere in Asia and then reached Africa from there, rather than originating in Africa as traditionally assumed [14,15]. Notably, Indian melon landraces exhibit the largest isozyme variation among Asian melons [16] and Australia is a center of complex morphological variation ofC. melo[28].

The evolution of morphological traits relevant forCucumisbreeders, for example fruit type, habit, and sexual system, will need to be reinterpreted based on the phylogenetic relationships presented here. Most of the 52 described species in theCucumisclade are monoecious perennials, and the monoecious sexual system and perennial habit may be the ancestral condition from which an annual habit and dioecy appear to have evolved several times. However, the sexual system and habit of key taxa, such asDicaelospermum, Muellerargia, and the as yet undescribed species from Australia [28] (Fig.3) are not reliably known because species are under-collected and have not been studied in the field. Of the 17 species ofCucumisnot yet sequenced, most are monoecious perennials; onlyC. kalahariensisA. Meeuse andC. rigidusSond. are dioecious and perennial. Species currently placed inMukia[29] andCucumella[13] are mostly monoecious and perennial. The evolution of smooth fruits from spiny fruits, a traditional key character inCucumis, and the mode of fruit opening are much more plastic than formerly thought. For example, inOreosyce africanaandMuellerargia timorensisthe fruits open explosively [[30]; I. Telford, Beadle Herbarium, Armidale, personal communication, Feb. 2007); inC. humifructus, fruits mature below ground and are then dug up and the seeds dispersed by antbears,Orycteropus afer[31]; in the new species from Australia (HS414 in Figs.1 and2), the developing fruit is pushed into rock crevices by the elongating pedicel and also matures below ground; and inMyrmecosicyos messoriusthe fruits are tiny and apparently dispersed by harvesting ants around whose nest entrances the species grows.

Taxon Sampling and Data Sets Analyzed

Table1 lists all species sampled with authors, status as generic types where applicable, plant sources, and GenBank accession numbers [TreeBASE: study accession S1604, matrix accession M2887, M3250 and M3251]; 79 chloroplast and 20 ITS sequences were newly generated for this study. Species concepts and generic assignments throughout this study follow recent classifications [11-13,29], although as a result of this study, species in several genera have been transferred intoCucumis([34]; this also provides a morphological key to the 52 described species). To resolve species relationships withinCucumis, we added sequences from the nuclear internal transcribed spacer region (220 nt of ITS 1, 163 nt of the 5.8S gene, and 240 nt of ITS 2) for the same species for which chloroplast data were generated. DNA extraction, purification, and sequencing of the selected loci followed standard procedures [10]. All PCR products were sequenced in both directions. Direct PCR amplification of ITS yielded single bands and unambiguous base calls, except inC. ficifolia, the sequences of which were therefore not used. Sequences were edited and assembled with the Sequencher software (Gene Codes) and aligned by eye, using MacClade [35]. The aligned chloroplast matrix comprised 4,375 positions after exclusion of a poly-T run in thematKgene, a poly-A run in thetrnLintron, a TATATA microsatellite region in thetrnL-Fintergenic spacer and a poly-A run in therpl20-rps12intergenic spacer. The aligned ITS matrix comprised 677 aligned positions, and we excluded a poly-G stretch of 25 nt and a poly-C stretch of 17 nt from the ITS1 and a poly-C stretch of 11 nt from ITS2.

Phylogenetic Analyses

Equally weighted parsimony analyses were conducted using PAUP 4.0b10 [36]. The search strategy involved 100 random taxon addition replicates with tree-bisection-reconnection branch swapping, MulTrees and Steepest Descent in effect, no limit on trees in memory, and saving all optimal trees. For MP analyses, gaps were treated as missing data, while for ML searches (below) they were mostly removed. To assess node support, parsimony bootstrap analyses were performed using 1000 replicate heuristic searches, each with 10 random addition replicates and otherwise the same settings as used for tree searches. More computationally intensive heuristic approaches have been found not to increase the reliability of bootstrapping [37]. Maximum likelihood analyses and bootstrapping were performed using GARLI 0.951 [38]. GARLI searches relied on the GTR + G + P-invar model, which ModelTest 3.06 [39] selected as the best fitting model for the combined data. Parameters were estimated over the duration of specified runs.

Molecular clock dating

Molecular clock dating in Cucurbitaceae is problematic because of the family's scarce fossil record. Without multiple calibrations, such as could come from several securely assigned fossils, relaxed molecular clock methods have been shown to perform poorly [40-42]. We therefore relied on a strict clock approach and compared it with results obtained with the semi-parametric penalized likelihood approach [40] implemented in r8s vs. 1.7). For strict clock dating, we employed the maximum likelihood topology obtained (under GTR + G) with the family data set of Kocyan et al. [10] augmented by theCucumissequences generated for this study for a total of 193 taxa and 5,028 aligned nucleotide positions. The tree was imported into PAUP [36], rooted on Corynocarpaceae, andrbcLbranch lengths were then calculated under a GTR + G + I + strict clock model. Branch lengths were saved and a mutation rate obtained by dividing the distance from the most recent common ancestor (mrca) ofTrichosanthesto the present (0.01416) by 65 my, based on the oldest seeds assigned to this genus [43]. Using the resulting rate of 0.000218 substitutions/site/my, we obtained an age of 47.6 my for the mrca ofCucumisby dividing the distance from the basal divergence inCucumisto the present (0.01037) by 0.000218. The time of the divergence ofC. melofrom its sister clade was calculated accordingly (0.00975 : 0.000218 = 44.7 my). To check this strict clock estimate based onrbcL, we imported the 193-taxon-ML tree with branch lengths from the combined chloroplast data (5,028 nt) into r8s and ran a cross validation analysis, using the following upper and lower temporal constraints. The mrca of the family Cucurbitaceae was constrained to maximally 100 my and minimally 65 my old based on Cucurbitales family relationships and fossil records [9,43]; the mrca ofTrichosantheswas constrained to minimally 65 my [42]; and the mrca of an endemic clade of two species occurring on Hispaniola was constrained to maximally 30 my old based on the oldest ages of Dominican amber [44]. Penalized likelihood yielded an age of 44.9 my for the mrca ofCucumis.

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