Eggshell membrane for DNA sexing of the endangered Maleo (Macrocephalon maleo) [version 3; peer review: 2 approved]

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.


Amendments from Version 2 Introduction
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] . Oftentimes a necessary prerequisite for answering evolutionary or ecological questions is access to a good DNA sample. Birds' blood contains nucleated red blood cells with abundant DNA, making it a preferred source of DNA 5 . However, obtaining blood requires the capture of a bird, which can provoke an increased level of stress and might results in unusual behavior or nest desertion. For example, blood sampling has been reported to reduce annual survival in Cliff Swallows (Petrochelidon pyrrhonota) 6 , although the effects of blood collection in free-living adult and immature birds is not thought to have major negative effects on adult survival, reproductive success, body condition, or behavior 7 .
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 . In addition the application of moderately invasive sampling such as buccal swabs 14,15 has also increased.
Megapodes (family Megapodiidae) are a galliform clade, centered in Australasia 16 , that are known for their unique superprecocial behavior 17 . Megapodes are also known as the only bird species that are not incubating their eggs, but make use environmental heat to incubate their eggs 18 . Their ground-living habits, large body size and large egg size make them particularly vulnerable to human persecution, habitat destruction and habitat loss: 11 out of 21 species are now considered endangered or threatened in some form 19. Given this precarious conservation situation, the application of noninvasive DNA sampling techniques is crucial for megapode birds. Yet previous genetic studies on this family have used either blood 20 or fresh tissue 21 .
Previously, all megapodes were assumed to be monogamous.
The mating system is considered to correlate with sexual selection, with sexually dimorphic birds are non-monogamous and monomorphic birds are monogamous. The evolution of non-monogamous systems in birds was believed to be an adaptive solution to an unbalanced sex ratio 22 . The sex-ratio in Maleo (Macrocephalon maleo) is unknown, but based on previous assumptions, it is expected that the Maleo has an evenly balanced sex ratio. Even though Maleo are slightly sexual dimorphic, the available population data only report total population size and never mention sex ratio. A study on the correlation of incubation temperature and sex ratio of chicks has been carried out in the Australian brush-turkey (Alectura lathami), which revealed that at average temperature the hatched chicks in the proportion of 1:1 of male and female chicks 23 .
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. Adult male and female Maleo are morphologically slightly different, but the chicks are not. To determine the sex in Maleo chicks, vent sexing has been conducted. Base on cloaca size and shape, a one-day-old male Maleo chick cloaca is bigger (3.96 ± 0.11 cm) and rounded, than the female cloaca (3.20 ± 0.10 cm), which is more oval in shape. The concentration of estrogen in female birds was also higher 23 . Until recently, no molecular technique has been applied for sex determination of Maleo. As in all non-ratite birds, determination of sex in Maleo are based on heteromorphic Z and W chromosomes. Female birds are heterogametic sex ZW, meanwhile males are homogametic sex ZZ 24,25 . .
Maleo are endemic to Sulawesi, Indonesia 16,26 . The bird is a burrow-nesting megapode that incubates its eggs in communal nesting sites on beaches (coastal nesting grounds) and in soil heated by volcanic activity mostly at inland localities. Due to its small, severely fragmented population and continued rapid decline, the International Union for Conservation of Nature has classified Maleo as an endangered species 27 . Among the major threats are the over-exploitation of eggs and loss of connectivity between forest and nesting grounds 28 . To minimize these threats at some nesting grounds, conservation programs are currently removing eggs and hatching them in safer, semi-natural hatcheries, built close to the nesting grounds. These facilities provide an opportunity to collect non-invasive DNA samples from the eggshell membrane left in the soil or brought to the soil surface by the hatched Maleo. The DNA of the chicks can be obtained from the extraembryonic membranes of fertilized egg, which was attached on the hatched eggshell membrane. The extraembryonic membranes were consisted of amnion, allantois and chorion, and formed to support the life and growth of the bird embryo 29 . During the embryonic development stage, the allantois sac expands, causing the inner shell membrane and chorion being combined and forming a chorioallantois membrane (CAM), which contains lots of blood capillaries 29,30 .

Study sites and genetic sampling
Post-hatched egg-shell membranes were collected from seminatural hatcheries of Maleo at two different nesting grounds: an inland geothermal heated nesting ground at Tambun (Bogani Nani Wartabone National Parks and a sun-heated sand beach nesting ground at Tanjung Binerean, North Sulawesi, Indonesia ( Figure 1). All samples were collected from 4 th April until 1 st May 2018. Samples were egg-shell membranes from maleo chicks that hatched less than 24 hours earlier based on records of staff from semi-natural hatcheries. The samples were collected from the surface and inside the substrate (soil and sand) where the eggs are laid. To prevent post-sampling contamination, each sample was placed separately in a zip-lock plastic bag and stored in silica gel for delivery to laboratory. The samples were stored at -40°C until DNA extraction were conducted.

DNA extraction
We used two different DNA extraction methods: the alkaline lysis method and gSYNC™ DNA Extraction Kit (Genaid). For the alkaline lysis method, we followed the recommended procedure for rapid preparation of mouse tails or nail lysates suitable for amplification using DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis (KOD FX Neo 1103; TOYOBO Co. Ltd.). The eggshell membrane used was consisted of dry chorioallantois membrane (CAM), included allantois blood vessels, as DNA materials of the chicks, except five samples from Binerean (MB04, MB09, MB10, MB11 and MB12) with no blood vessels. The membrane (20-25 mg) was grinded using a micro-pestle in a 1.5 mL microcentrifuge tube; next, 180 µL NaOH (50 mM) was added, the suspension mixed thoroughly by vortexing and then incubated at 90°C in water-bath for 10-29 min. Following this, 20 µL Tris-HCl (1 M, pH 8.0) was added and the tube was vortexed thoroughly, then centrifuged at 12,000 RPM for 5 min. Lysate was removed to new 1.5 mL microtube and store at freezer until used for PCR.
Meanwhile the protocol for gSYNC™ DNA Extraction Kit (Genaid) followed the provided user manual with little modifications. The eggshell membrane (25 mg) was grinded using a micro-pestle in a 1.5 mL microcentrifuge tube; 300 µl GST Buffer (Tris, SDS) and 30 µl Proteinase K (10 mg/ml) was added to the sample mixture, mixed thoroughly by vortexing and incubated at 60°C in water-bath overnight or until the tissue was lysed completely. Next, 200 µl GSB Buffer was added to the sample mixture, mixed thoroughly by pulse-vortexing and incubated at 70°C for 10 min. After this, 200 µl ethanol (100%) was added to the sample mixture, which was mixed thoroughly by pulse-vortexing and brief spinning of the tube to remove drops from the inside of the lid. Next, a GS Column was placed in a Collection Tube and the mixture (including any precipitate) was carefully transferred to the GS Column, which was centrifuged at 14,000 RPM for 1 min then the GS Column was placed in a new Collection Tube. Following this, 400 µl W1 Buffer was added to the GS Column and centrifuged at 14,000 RPM for 1 min then flow-through was discarded. Next, 750 µl Wash Buffer was added to the GS Column, centrifuged at full speed for 1 min, then the flow-through was discarded, the tube centrifuged at 14,000 RPM for an additional 3 min to dry the column, 50 µl of preheated Elution Buffer (pH 7.5-9.0) added to the membrane of the GS Column. The GS Column was then left to stand for 3 min, following a final centrifugation at full speed for 2 min to elute the DNA. The eluded DNA (1 µl) was quantified using NanoVue Plus™ (Biochrom, Harvard Bioscience, Inc), at A260 nm. The 260/280 nm absorbance ratio was also measured to give an indication of purity of the DNA. Pure DNA has expected ratios of 1.7-1.9.

DNA sexing
To determine the sex of Maleo, we applied PCR based DNA sexing by using CHD genes, with the primer set 2550F/2718R 31 . PCR used a 10 µl total volume containing 1 µl ( We employed egg-shell membranes from the domestic chicken as a positive control. The positive control consistently identified as a female with the same PCR condition in this research.
Amplification of CHD Genes were resolved on a 2% agarose gel. Electrophoresis was conducted using TAE (0.5×) buffer, stained by ethidium bromide (1%), at 100 V for 30 minutes; and 5 µl PCR product was mixed with 1 µl loading dye. After finish, the gel was visualized and analyzed on Gel Logic 200 Imaging System and Kodak Molecular Imaging Software. To confirm that the amplified fragments were the CHD genes, the PCR products of one male and one female sample, respectively, were sequenced. The gels were cut on upper and lower bands for female samples and the single band for male sample, then purified for sequencing. The sequence reactions were carried for both direction in sequencing services laboratory provided by     In total 9 samples were identified as females and 15 were males (   "The DNA of the chicks can be obtained from the extraembryonic membranes of fertilized egg, which was attached on the hatched eggshell membrane. The extraembryonic membranes were consisted of amnion, allantois and chorion, and formed to support the life and growth of the bird embryo". Please rewrite these 2 sentences as follows: ""The DNA of the chicks can be obtained from the extraembryonic membranes that remain attached to eggshell membranes of post-hatched eggs. The extraembryonic membranes consist of the amnion, allantois and chorion, and form to support the life and growth of the bird embryo".

2.
Competing Interests: No competing interests were disclosed.

Jessica Worthington Wilmer
Biodiversity and Geosciences Program, Queensland Museum, South Brisbane, Qld, Australia This paper examines the capacity to genetically sex Maleo (Macrocephalon maleo) chicks from DNA extracted from the membranes of hatched eggs. This is interesting work and potentially very important for understanding hatching sex ratios of Maleo and for future conservation work of this endangered megapode. The paper has already gone through one round of review and is generally well presented, however, there are a couple of fundamental issues that need to be highlighted and then addressed by the authors.
1) Egg-Chick membrane anatomy and biology: It states in the title and throughout the paper that eggshell membranes are being used but the authors must be careful in implying that the eggshell membranes themselves are the source of chick DNA. The shell membranes are produced by the hen when the shell is formed in the oviduct and, therefore, will always be female. Thus, eggshell membranes on their own without the inclusion embryonic tissue will not be useful for DNA sexing of hatched chicks. So, the source of the chick DNA extracted must be from one of the extraembryonic membranes or some other chick tissue left behind in the shell at hatching. This fundamental egg-biology information must be included in the introduction.

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The issue becomes clearer in the methods section where the authors detail the tissue used for DNA extraction "The eggshell membrane (20-25mg; mostly with dry allantois blood vessel ) was grinded..." This is the critical information as these blood vessels comprise the extraembryonic arterial system and therefore are the source of chick DNA in this study. The question then is did the authors specifically target these sections of the eggshell membranes because of the blood vessels (with reference to the fundamental egg-chick biology)? If so, then this should be clearly stated as what needs to be done when dealing with hatched eggs.
○ "Mostly" is not an accurate scientific term. Please provide detail as to how many eggshell membranes included allantois blood vessels out of the total number (24) of samples. This is ○ important because of the 9 females that were detected. How many of the samples that didn't include allantois blood vessels were sexed as being female? Those membranes could still include other extraembryonic tissues however, there needs to be a way for the authors to demonstrate with confidence that it is the chick that was sexed as female and not because they have amplified the sex chromosomes of the hen. (see Arnold et al, 2003 1 ).

2) Maleo and Megapodes
The introduction needs to include the fundamental biological information that megapodes are the only avian species/group that are not brood incubators (Frith 1956 2 ). All megapodes employ environmental heat to incubate their eggs. Temperature dependent sex biased mortality has been shown to occur in the Australian Brush Turkey (Eiby et al. 2008 3 ) and it may well be that further in depth studies of Maleo may reveal a similar mechanistic interaction between their environmental incubation temperature and hatching sex ratios. The current observed skew in hatching ratio is interesting although the low sample numbers may mean it is not biologically / statistically significant.
○ It would also be good to include some basic information regarding sex determination in birds and that in megapodes, as in all other non-ratite birds, sex is determined by heteromorphic Z and W chromosomes (Belter and Deboer, 1984 4 ) and, unlike in mammals, females are the heterogametic sex ZW and Males ZZ.

Reviewer Expertise: Wildlife molecular ecologist
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