Investigation of the utilization of microsatellites for fingerprinting in three endangered southern African crane species.

Abstract

Cranes are large elegant birds that occur on all continents of the world except for South America and Antarctica. Of the fifteen species of crane worldwide, three predominantly occur in southern Africa; the Wattled crane (Bugeranus carunculatus), the Blue crane (Anthropoides paradisea) and the Crowned crane (Balearica regulorum). Crane numbers throughout the world are diminishing, mostly because of the destruction of their habitat and illegal bird trading. Efforts are underway to prevent species extinction, legally and through the compilation of a studbook that contains descriptions of physical attributes, ownership, location and possible kinships of birds in captivity . This investigation, first of its kind, WdS undertaken to assess whether twelve published and unpublished microsatellite primers developed for the related Whooping crane and Red-Crowned crane could be used to fingerprint the southern African crane species using cost effective polyacrylamide gel electrophoresis. The results obtained were then used to determine the extent of genetic variation within species and distance between species. All primer sets amplified heterologous microsatellite loci in the three crane species, however, the unpublished primers produced poorly defined fingerprints even after extensive optimization. Of the twelve microsatellite loci investigated, the Blue crane and the Wattled crane revealed a high level of polymorphism. The Blue crane displayed 76% polymorphism and the Wattled crane 92%. In contrast, for the Crowned crane, that belongs to a different subfamily, Balearicinae, only 50% of the loci were polymorphic. The alleles displayed sizes similar to that of the species for which the primers were developed. Little variation in size, less than 10 bp, was noted for the different alleles of the polymorphic loci. The number of alleles, on the other hand, at each of the polymorphic loci was found to be low. The frequency of the most prevalent allele at most of the loci was generally reasonably high. These results therefore suggest that these primer sets are not suitable for individual identification and differentiation using polyacrylamide gel electrophoresis. Xll The observed heterozygosity of the three crane species was low; 12% in Blue crane; 7% in Crowned crane; and 13% in Wattled crane. Nei's identity further confirmed the high similarity between individuals; 66-100% for Blue crane; 55-100% for Crowned crane and 41-95% for Wattled crane. This low genetic variation is attributed to possible relatedness between birds supplied by aviculturists whom have a limited number of birds in captivity. A Hardy-Weinberg test for equilibrium revealed that most of the microsatellite loci displayed a deficiency of heterozygotes, while a few loci displayed an excess of heterozygotes. In general, the Hardy Weinberg test of equilibrium supported the notion that the individuals within each of the species might have been related. Differentiation between the three crane species ranged from 3-5%, with Blue and Wattled crane displaying a higher degree of genetic similarity when compared to the Crowned crane, known to be the oldest extant crane species. The limited allelic variation within the microsatellite loci tested, as well as the extensive genetic similarity between individuals suggests that a wide-ranging search for additional microsatellite loci that are more polymorphic and contain a larger number of alleles should be undertaken for the southern African crane species.