A Study On Molecular Phylogeny And Molecular Clock Of Cucurbita

The New world genus Cucurbita, one of the most important genus in family Cucurbitaceae, composed by12-14annual or perennial species naturally distributed from tropical to semitropical zone and cultivated in temperate zone. There are5main domesticated species in this genus:C. moschata Duch., C. maxima Duch., C. pepo L. C.ficifolia B. and C. argyrosperma Huber, of which the first3were imported to china and cultivated as major vegetables with important economic value. They are easy to grow and of very high yield. The main international pumpkin producing regions include United States, Mexico, India, and China. China’s annual pumpkin production comprises over40%of the world’s total production with the amount of40million tons. The pumpkin products are nutritious and healthy, and have medical values. Pumpkin also has ornamental value as work of art, mainly in Americas. The genus Cucurbita is one of the earliest domesticated plant groups. Archeological record indicated the domestication happened10000years ago, and different cultivated species have independent domestication histories. The phylogenetic study of the Cucurbita species will help to elucidate evolution history and their corresponding wild progenitors, to explore genetic breeding and speciation mechanism, and also to contribute to evolutionary theory and plant DNA barcoding system. In this study, we collected25taxa of Cucurbita, amplified and sequenced the chloroplast coding region matK and rbcL, non-coding chloroplast trnL intron, adjacent trnL-F intergenic region and rpl20-rps12intergenic region. The mitochondrial atp4-ccmc intergenic region was also included. Previous published phylogenetic studies were consisted by chloroplast sites, internal transcribed spacer nuclear region and mitochondrial intron. After sequence analysis and integration with morphologic and geographic distribution, we discussed the phylogeny relationship of species in genus Cucurbita and domestication history and their geographic distributions. In addition, we did the molecular clock research based on molecular phylogeny tree composed from4concatenated regions of chloroplast segment, and estimated divergence time of main clades in the genus. We have the following conclusions:1. Phylogenetic relationship of CucurbitaBased on DNA fragment analysis, two groups can be recognized in Cucurbita:(1) Xerophytic group:C. digitata, C palmata, C. cordata, C foetidissima, C. pedatifolia and C. ficifolia. Taxa in this group are mainly perennials and with storage roots. The basal clade with ancient morphology is composed of3species of narrow palmate leaves with five fingerlike lobes:C. digitata, C. palmata, and C. cordata. Another clade in this group includes C. foetidissima, C. pedatifolia and C. ficifolia, which have broad leaves and diverged after the first clade. It is worth noting that C. ficifolia is the most ancient domesticated species, and diverged before the other two species in this clade. C. ficifolia is annual, adapted to high altitude and humid environment.(2) Mesophytic group:C. pepo clade, C. maxima clade, C. argyrosperma clade and C. okeechobeensis clade. They were under parallel evolution. In C. pepo clade, both C. pepo ssp.fraterna and C. pepo var. texana are wild species to C. pepo complex. Among this clade, C. pepo ssp.fraterna is in the basal position and probably the ancient progenitor of C. pepo ssp. pepo. C. maxima clade including endemic species C. ecuadorensis, one domesticated species C. maxima and its wild species C. maxima ssp. andreana, was originated from South America with high support value. Our results support that C. ecuadorensis diverged independently from common ancestor and earlier than C. andreana. The monophyletic C. argyrosperma clade has two domesticated species." C. argyrosperma and C. moschata. C. argyrosperma ssp. sororia is the progenitor of C. argyrosperma. C. okeechobeensis clade is monophyletic with high bootstrap value and includes3wild taxa:C. okeechobeensis ssp. martinezii, C. okeechobeensis ssp. okeechobeensis and C. lundelliana.2. Molecular clock study on CucurbitaThe origin of Cucurbita is comparatively late and no fossil record was collected. Therefore we used fossil record from outgroup species to calibrate the molecular clock of Cucurbita using phylogeny tree constructed from4concatenated chloroplast fragments. The results suggest that Cucurbita genus diverged from Peponopsis genus in the middle of Miocene, about15.65Million years ago, and the speciation of xerophytic taxa were around the same time. The speciation of mesophytic taxa happened in Pliocene, and the main five clades diverged into species and subspecies in Quaternary, which is also the time of independent domestications.