Hybridization and Genetic Distances Suggest One Large Monobaramin in the Gourd Family Timothy R. Brophy, Jack R. Gregory, and Brigitte Townsend Center for Creation Studies and Department of Biology & Chemistry Bates, D.M., R.W. Robinson, and C. Jeffrey (editors). 1990. Biology and Utilization of the Cucurbitaceae. Cornell University Press, Ithaca, NY. Dane, F., D.W. Denna, and T. Tsuchiya. 1980. Evolutionary studies of wild species in the genus Cucumis. Zeitschrift fur Pflanzenzucht (Journal of Plant Breeding) 85: 89-109. Dutt, B., and R. P. Roy. 1971. Cytogenetic investigations in Cucurbitaceae. I. Interspecific hybridization in Luffa. Genetica 42:139-156. Jobst, J., K. King, and V. Hemleben. 1998. Molecular Evolution of the Internal Transcribed Spacers (ITS1 and ITS2) and Phylogenetic Relationships among Species of the Family Cucurbitaceae. Molecular Phylogenetics and Evolution 9:204–219. Singh, A. K., and R.P. Roy. 1979. Analysis of interspecific hybrids in Trichosanthes spp. Caryologia 32:239-344. Skalova. D., A. Lebeda, and B. Navratilova. 2004. Embyro and ovule cultures in Cucumis species and their utilization in interspecfic hybridization. In A. Lebeda and H.S. Paris (editors), Proceedings of Cucurbitaceae 2004, pp. 415-430. Olomouc, Czech Republic: Eur. Assoc. Research on Plant Breeding. Van Eseltine, G. P. 1937. Cucurbitahybrids. Proceedings of the American Society of Horticultural Science 34:577-581. Whitaker, T. W. 1933. Cytological and phylogenetic studies in the Cucurbitaceae. Botanical Gazette 94:780-790. The gourds form a primarily tropical and subtropical family (Cucurbitales: Cucurbitaceae) of herbaceous climbers and woody lianas composed of approximately 960 species in 95 genera and 15 tribes. Many of these are globally important food crops and ornamentals including cucumbers, gourds, squashes, melons, pumpkins, and luffas. Utilizing published literature, there is evidence of interspecific hybridization (natural, experimental cross-pollination, and embryo/ovule culture) in five of the 15 tribes. These consist of successful crosses between 131 unique species pairs, including two intergeneric and one intertribal cross, forming eight monobaramins. In addition, species that do not hybridize directly, but hybridize with the same third species are added to the various monobaramins. This results in an additional 227 species pairs, including two intergeneric and two intertribal. A hybridogram, constructed from these direct and indirect hybridization results, reveals the following monobaramins: 1) 23 species from genus Cucumis; 2) 18 species from genus Cucurbita; 3) nine species from genus Luffa; 4) four species from genera Trichosanthes and Momordica(includes intertribal crosses); 5) three species from genus Citrullus; 6) two species from genus Lagenaria; 7) two species from genus Bryonia; and 8) two species from genera Cocciniaand Diplocyclos. Next, using genetic distances (ITS regions of nuclear ribosomal RNA genes) from 26 cucurbit species, several monobaramins are enlarged and some are even connected by adding an additional 113 species pairs, including 82 intergeneric and 58 intertribal. A revised hybridogram, utilizing all of the above methods, reveals the following: 1) the original [Trichosanthes + Momordica] monobaramin connects to the original Luffaand Bryonia monobaramins as well as a new monobaramin, composed of the genera [Echinopepon+ Cyclanthera+ Sicyos + Ecballium], to form one large monobaramin consisting of 20 species from eight genera and three tribes (Sicyoeae, Bryonieae, and Momordiceae); 2) the original Citrullus monobaramin connects to a member of the genus Lagenariaand the resulting monobaramin is slightly larger with six species; 3) the original Cucumis monobaramin has additional internal continuity and may connect to the contribal [Coccinia+ Diplocyclos] and [Citrullus + Lagenaria] monobaramins to form one large monobaramin consisting of 31 species from five genera in Tribe Benincaseae; 4) the original Cucurbita monobaramin connects to one additional species and has 19 species overall, all from genus Cucurbitaand Tribe Cucurbiteae. Finally, the [Sicyoeae + Bryonieae + Momordiceae] monobaramin connects directly to both the Cucurbiteae and Benincaseae monobaramins. Connections between these last two tribes are also suggested by grafting and somatic hybridization experiments which are generally most successful between closely related species. In conclusion, these data suggest that members of all five tribes (70 species and 14 genera) may form one large monobaramin in the Family Cucurbitaceae. Additional research is required (hybridization, molecular, morphological) to determine the baraminological status of the family as a whole. Abstract • Published literature (see References) surveyed for evidence of interspecific hybridization in gourd family: o Successful natural hybridization o Successful experimental cross-pollination o Successful embryo and/or ovule culture o Results of protoplast fusion (i.e. somatic hybridization) experiments not considered as evidence of interspecific hybridization • Genetic distances calculated for 21 of the 26 gourd/ cucurbit species analyzed by Jobst et al. (1998): o ITS regions of nuclear ribosomal RNA genes o Gaps and missing information, including insertion/ deletion sites, removed from sequences prior to distance calculations • Hybridogram constructed based on direct and indirect hybridization and genetic distance data • Two species considered part of same monobaramin if: o Hybridize directly o Hybridize with same 3rd species o Fall within range of genetic distances of hybridizing species o One hybridizes with and other is genetically similar to same 3rd species Methods & Materials References • These data suggest that members of all five tribes (70 species and 14 genera) may form one large monobaramin in the Family Cucurbitaceae • Additional research is required (hybridization, molecular, morphological) to determine the baraminological status of the family as a whole Conclusions • Evidence of direct and indirect hybridization in five of the 15 gourd/cucurbit tribes (Fig. 2–Black & Gray) o Successful crosses between 131 unique species pairs, including two intergeneric and one intertribal cross o Species that hybridize with same third species result in an additional 227 species pairs, including two intergeneric and two intertribal • Genetic distances for hybridizing species range from 0.0181–0.1421 (Fig. 1–Black) o Using direct and indirect genetic distance data adds an additional 113 species pairs, including 82 intergeneric and 58 intertribal (Fig. 2–Dark & Light Blue) • Hybridogram, utilizing all the above methods, reveals the following (Fig. 2–all colors): o One large monobaramin consisting of 20 species from eight genera in three tribes (Sicyoeae, Bryonieae, and Momordiceae) –Luffa, Bryonia, [Trichosanthes + Momordica], and [Echinopepon +Cyclanthera +Sicyos + Ecballium] monobaramins o One large monobaramin consisting of 31 species from five genera in Tribe Benincaseae –Cucumis monobaramin may connect with contribal [Coccinia +Diplocyclos] and [Citrullus +Lagenaria] monobaramins o One large monobaramin consisting of 19 species from genus Cucurbitain Tribe Cucurbiteae o [Sicyoeae + Bryonieae + Momordiceae] monobaramin connects directly to both Cucurbiteae and Benincaseae monobaramins via genetic distance data o Connections exist between Cucurbiteae and Benincaseae via grafting and somatic hybridization which are generally most successful between closely related species Results & Discussion Figure 2. Hybridogram based on direct and indirect hybridization and genetic distance data. Black = two species hybridize directly; Gray = two species hybridize with same 3rd species; Dark Blue = two species fall within range of genetic distances of hybridizing species; Light Blue = one species hybridizes with and the other is genetically similar to same 3rd species. Figure 1. Genetic distance matrix, using ITS regions of nuclear ribosomal RNA genes, for 21 of 26 cucurbit species analyzed by Jobst et al. (1998). Gaps and missing information, including insertion/deletion sites, were removed from sequences prior to distance calculations. 1 2 3 4 5 6 7 8 91011121314151617181920 1. Cucurbita pepo 2. Cucurbita pedatifolia 0.0926 3. Ecballium elaterium 0.1532 0.1665 4. Echinopepon wrightii 0.1626 0.2010 0.1451 5. Cyclanthera brachystachia 0.1654 0.1701 0.1578 0.1722 6. Sicyos angulatus 0.1390 0.1567 0.1400 0.1265 0.1212 7. Bryonia cretica 0.1665 0.1850 0.1097 0.1528 0.1999 0.1571 8. Trichosanthes cucumerina 0.1355 0.1485 0.1135 0.1179 0.1713 0.1309 0.0836 9. Momordica charantia 0.2119 0.2212 0.1793 0.1698 0.2593 0.1954 0.1551 0.1419 10. Luffa cylindrica 0.1701 0.1790 0.1250 0.1382 0.1742 0.1567 0.1291 0.0833 0.1686 11. Citrullus lanatus 0.2045 0.2045 0.1949 0.2153 0.1944 0.1854 0.1890 0.1753 0.2293 0.2026 12. Lagenaria vulgaris 0.2123 0.2225 0.1838 0.1944 0.1618 0.1478 0.1922 0.1742 0.2267 0.2065 0.1298 13. Cucumis sativus 0.2483 0.2735 0.2225 0.2362 0.2614 0.2367 0.2016 0.1871 0.2472 0.2155 0.2205 0.2497 14. Cucumis melo 0.1875 0.2319 0.1587 0.1713 0.2199 0.1904 0.1771 0.1674 0.2272 0.1749 0.1742 0.2165 0.1380 15. Cucumis metuliferus 0.1820 0.2153 0.1583 0.1757 0.2031 0.1761 0.1528 0.1390 0.2040 0.1651 0.1518 0.1879 0.1291 0.0638 16. Cucumis anguria 0.2107 0.2565 0.1870 0.1989 0.2430 0.2136 0.1810 0.1753 0.2334 0.1977 0.1690 0.2158 0.1421 0.0795 0.0405 17. Cucumis myriocarpus 0.2010 0.2572 0.1864 0.1895 0.2393 0.2101 0.1717 0.1661 0.2287 0.1883 0.1605 0.2119 0.1291 0.0756 0.0405 0.0181 18. Cucumis dipsaceus 0.2246 0.2712 0.1954 0.2127 0.2630 0.2382 0.2045 0.1985 0.2489 0.2072 0.1925 0.2403 0.1596 0.1081 0.0715 0.0407 0.0521 19. Cucumis zeyherii 0.2246 0.2826 0.2096 0.2127 0.2642 0.2340 0.1944 0.1886 0.2489 0.2115 0.1739 0.2358 0.1417 0.0956 0.0597 0.0330 0.0218 0.0597 20. Cucumis africanus 0.2305 0.2893 0.2153 0.2132 0.2648 0.2293 0.1850 0.1753 0.2472 0.2169 0.1736 0.2310 0.1373 0.0999 0.0636 0.0481 0.0367 0.0793 0.0218 21. Zanonia indica 0.1777 0.2259 0.2162 0.2212 0.2468 0.2016 0.1916 0.1686 0.2103 0.2099 0.2371 0.2507 0.2497 0.2208 0.1916 0.2059 0.1873 0.2455 0.2106 0.2152 Hybrid Hybrid with Same 3rd Species Within Range of Hybridizing Not Within Range TRIBE Genus 1 2 3 4 5 6 7 8 9 10111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596061626364656667686970 CUCURBITEAE Cucurbita 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 BENINCASEAE Citrullus 20 21 22 Cucumis 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Lagenaria 46 47 48 Coccinia 49 Diplocyclos 50 SICYOEAE Trichosanthes 51 52 53 Luffa 54 55 56 57 58 59 60 61 62 Echinopepon 63 Cyclanthera 64 Sicyos 65 BRYONIEAE Bryonia 66 67 68 Ecballium 69 MOMORDICEAE Momordica 70
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