Abstract
Background: Noninvasive DNA sampling has been applied across many avian genetic studies for a variety of purposes including conservation and management of endangered birds. However, its application in megapodes is still lacking. The previous genetic studies on megapodes used either blood or fresh tissue. Here we present the first demonstration of the use of eggshell membrane for research on endangered Maleo (Macrocephalon maleo). Methods: We used 24 post-hatched eggshell membranes collected from two different sites, Tambun and Tanjung Binerean, in North Sulawesi, 12 samples in each. Two different DNA extraction methods: alkaline lysis method and gSYNCTM DNA Extraction Kit were applied. To determine the sex of Maleo, we utilized PCR-based DNA sexing using CHD genes, with the primer set 2550F/2718R. Results: We successfully extracted all samples; the mean sample concentration was 267.5 ng/µl (range 47–510.5 ng/µl) and samples were of high purity (A260/280 ratio 1.85±0.03). All samples were used to successfully identified sexes, 9 females and 15 males. Conclusions: Our research clearly illustrates that eggshell membranes can be used for DNA sexing and open the possibility to build noninvasive DNA collections over large spatial scales for population study of endangered birds.
Highlights
Studies on molecular ecology have a great impact on our knowledge on ecology and evolution of animals, i.e. the phylogenetic relationships and systematics of organisms, population genetics, mating systems, micro-evolutionary processes and host-parasite interactions[1,2,3,4]
The purpose of our study was to determine whether the eggshell membrane of the endangered Maleo, a monotypic genus within the megapodes, could be successfully extracted and amplified for DNA sexing
The average DNA concentration extracted from eggshell membrane collected from coastal nesting grounds (Tanjung Binerean: 213±179 ng/μl,) was significantly lesser than of that of inland nesting grounds (Tambun: 322±153 ng/μl, p=0.004; Data Supp.1)
Summary
Studies on molecular ecology have a great impact on our knowledge on ecology and evolution of animals, i.e. the phylogenetic relationships and systematics of organisms, population genetics, mating systems, micro-evolutionary processes and host-parasite interactions[1,2,3,4]. As an alternative to invasive sampling, researchers have adopted noninvasive sampling methods such as DNA capture from molted feathers[8,9], feces[10,11], and egg shell membrane[12,13]. Noninvasive DNA sampling has been applied across many avian genetic studies for a variety of purposes including conservation and management of endangered birds. Methods: We used 24 post-hatched eggshell membranes collected from two different sites, Tambun and Tanjung Binerean, in North Sulawesi, 12 samples in each. Conclusions: Our research clearly illustrates that eggshell membranes can be used for DNA sexing and open the possibility to build noninvasive DNA collections over large spatial scales for population study of endangered birds
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