Introduction

The Musaceae, a typical Old-World tropical family with great economic importance, includes three genera Musa, Ensete, and Musella. Musa (ca. 37 spp.) and Ensete (ca. 7 spp.) mainly occur in Asia and Africa respectively (1). The third genus has only one species, Musella lasiocarpa (Fr.) Wu ex H. W. Li, and is endemic to southwestern China, primarily in central and western Yunnan, occurring inside the confier-oak mixed forest domain at elevations of 1500-2500 m (2, 3, 4, 5, 6, 7). The genus Musella has been placed first in Musa and than in Ensete and only recently it was recognized as a separate genus (2, 5, 7).

Musella has been used by local people for many centuries. It is used as medicinal plant, for food, fodder, to weave, to alleviate soil erosion, and also as a ornamental plant.

Musella lasiocarpa is a large perennial herb with rhizomatous growth. It is pollinated by insects, such as the bumblebees (Bombus eximius and B. montivolans), honeybees (Apis cerana and A. florae) and wasps (Vespa mandarinia) (6).

Isozymes are one of two or more enzymes that have a common substrate, but are encoded by different loci and have different electrophoretic mobility. Enzyme polymorphism can be used to estimate population genetic diversity; taxonomic affiliations; and clonal identification. Several varieties of Musa species have been identified by isozyme analysis (9). This is a preliminary study of genetic variation in Musella using isozymes as genetic markers.

Materials and Methods

Seeds from five different populations of Musella were collected at the province of Yunnan, southwestern China in the year 2000. The seeds were germinated and the plants have been cultivated in the Smithsonian Institution Museum Support Center. Additionally, other populations were included in this study although only one individual per population was available. The total number of individuals sampled was 48.

Tender leaf tissue from each one of the different individuals was used for extracts, which were prepared by powdering the fresh tissue in an iced mortar using liquid nitrogen. Proteins were extracted by addition of 0.5 ml of the Phosphate grinding buffer of Soltis et al. (1983). Extracts were absorbed onto 0.5 X 1.0 cm paper wicks and the wicks were frozen at -80°C until loaded onto horizontal 10% potato starch gel and electrophoresed. Heliconia stricta (Kress voucher 78-1043) was used as a standard on all gels. Thirteen enzymes in four different systems were screened. Stains and buffers were used according to Soltis et al. (1983) and Morden et al. 1987. After scoring genotypes the data was analyzed using POPGENE software package (13). The laboratory procedures are described in detail in the Botany Department Electrophoresis Laboratory Manual (12).

Results

Six of the thirteen loci screened for isozymes produced clear patterns of bands (Pgm-1, Pgm-2, Skdh-1, Gdh-1, Aat-1, Idh-1). The locus for Gdh-1 was monomorphic in all populations. Considering only the populations one to five, the mean percent of polymorphic loci (Pp) was 53.3, mean number of alleles per polymorphic locus (App) was 2.70, and the mean observed heterozygosity (Ho) was 0.325 and the expected heterozygosity (Hep) was 0.253 (Table 2). Mean F-statistics (Fst) value for all loci was 0.1575, and estimate number of migrants per generation (Nm) was 1.337. In spite of having only one individual, the analysis done including population six to eleven corroborates to the same pattern of result. The same occurs when we group all the 48 individuals as a single population.

Table 2. Genetic variability within populations of Musella lasiocarpa. Ap, mean number of alleles per locus; APp mean number of alleles per polymorphic locus; Pp percent of polymorphic locus; Ho observed heterozygosity; Hep expected heterozygosity.

Population Ap APp Pp Ho Hep
1 (00-6780) 1.50 2.50 33.3 0.208 0.184
2 (00-6781) 1.83 2.25 66.7 0.344 0.243
3 (00-6784) 1.50 3.00 50.0 0.389 0.249
4 (00-6785) 1.50 3.00 50.0 0.258 0.310
5 (00-6789) 1.83 2.75 66.7 0.428 0.275
Means 1.63 2.70 53.3 0.325 0.252

Table 3. Nei's (1978) genetic identities (above diagonal) and genetic distances (below diagonal) between populations of Musella lasiocarpa.

Population 1 2 3 4 5
1 (00-6780) *** 0.977 0.922 0.856 0.968
2 (00-6781) 0.023 *** 0.976 0.890 0.988
3 (00-6784) 0.080 0.023 *** 0.861 0.985
4 (00-6785) 0.154 0.115 0.149 *** 0.862
5 (00-6789) 0.032 0.011 0.015 0.147 ***

Figure 1. Dendrogram based on Nei's (1978) genetic identities between populations of Musella lasiocarpa.

Discussion

Musella lasiocarpa presented great isozyme variation. At this point we should ask what associations could be established between isozyme and life history data? Liu et al. 2002, based on field investigation, discovered that insects such as bumblebees, honeybees, and wasps are the principal visitors and effective pollinators of this plant. During the breeding systems experiments, they also found that in inflorescences that were bagged the fruits were poorly developed and no seed was produced.

They also showed that Musella has female and male flowers separated by time of anthesis and arrangement in the inflorescence, and that the female flowers have a longer life-span and produce more nectar, what could increase the numbers of visits of different insects pollinators leading to effective cross pollination. The great genetic variation found between and within populations indicates that outcrossing has occurred and suggests that a larger number than we initially supposed of sympatric populations exists.

Sampling more populations that are closer to each other would give us a better idea about the gene movement between population groups.

Compared to mean values reported to other taxa (13) with similar life-history characteristics (Pp = 30.6-40.3; Ap = 1.44-1.66; Hep = 0.084-0.144: Hamrick & Godt 1989) the populations two to five showed higher numbers for mean polymorphic loci than expected while population one fits in the expected range. All population's mean numbers of alleles per locus (Ap) fit inside the range reported by Hamrick & Godt (1989).

The dendrogram based on Nei's (1978) genetic distances does not reflect the geographical distribution data. Thus populations that are separated by short geographical distance appear to share few genetic similarities. The actual distribution of Musella has probably been influenced by human transportation and introduction into new areas.

Conclusions

Acknowledgements

The authors gratefully acknowledge the assistance provided by Ida Lopez, Ai Zhong Liu, Leslie Brothers, and Mike Bordelon. This work was completed while the senior author was a participant in the 2002 Research Training Program at the National Museum of Natural History, Smithsonian Institution. Funding for this study was provided by the Smithsonian Women's Committee.

Literature Cited

1. Liu A-Z. 2001. Phylogeny and biogeography of Musaceae. Ph. D. dissertation. Kunming Institute of Botany, the Chinese Academy of Sciences.

2. Li, H-W. 1978. The Musaceae of Yunnan. Acta Phytotax. Sinica 16(3): 54-64.

3. Wu, C-Y. ed. 1979. Flora of Yunnan. 2: 725-733.

4. Li, H-W. 1981. Musaceae in D. L Wu, ed., Flora Reipublicae Populares Sinicae 16(2): 1-14.

5. Wu, D. L. and W. J. Kress. 2001. Musaceae in C. Y. Wu and P. H. Raven eds., Flora of China 24: 314-318.

6. Liu, A-Z. W. J. Kress, H. Wang and D-Z, Li. 2002. Insect pollination of Musella lasiocarpa (Musaceae), a monotypic genus endemic to Yunnan, China. Plant Sys. Evol. In press.

7. Li, H-W. 1979. Musaceae. In: Wu C-Y. (ed.) Flora of Yunnan, Science Press, Beijing, 2: 275-733.

8. Liu, A-Z. W. J. Kress, Long, C-L. 2002. Customary use and conservational attention to Musella lasiocarpa (Musaceae), a monotypic genus endemic to China. Plant Sys. Evol. In press.

9. Jarret, R. L. and R. E. Litz. 1986. Enzyme polimorphism in Musa acuminata Colla. The Journal of Heredity. 77: 183-188.

10. Hedrick, P. W., M. E. Ginevan, E. P. Erwing. 1976. Genetic polymorphism in heterogeneous environments. Ann. Rev. Ecol. Syst. 7: 1-32.

11. Yeh, F. C., Yang, R. 1999. Popgene version 1.31, Microsoft Window-based freeware for Population Genetic Analysis. University of Alberta and Tim Boyle, Centre for International Forestry Research.

12. Wright, S. 1978. Evolution and the Genetics of Population. Vol. 4. Variability within and among natural Populations. University of Chicago Press, Chicago.

13. Hamrick, J. L., M. J. W. Godt. 1989. Allozyme diversity in plant species. In Brown, A. H. D. et al. eds.: Plant population genetics, breeding, and genetic resources, pp. 43-63. Massachusetts, Sianauer.

14. Soltis, D. E., C. H. Haufler, D. C. Darrow, and G. J. Gascony. 1983. Starch gel electrophoresis of ferns: a compilation of grinding buffers, gel and electrode buffers, and staining schedules. American Fern Journal. 73: 9-27.

15. Cardy, B. O., C. W. Stuber, and M. M. Goodman. 1981. Techniques for starch gel electrophoresis of enzymes from maize (Zea mays L.). Instit. Statistical Mimeograph Series No. 1317. North Carolina State Univ., Raleigh, N.C.

16. Morden, C. W., J, F. Doebley, and K. F. Schertz. 1987. A manual of techniques for starch gel electrophoresis of Sorghum Isozymes. Texas Agricultural Experiment Station. MP-1635. College Station, Texas.

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