Rusa deer microbiota: the importance of preliminary data analysis for meaningful diversity comparisons.

The microbiome is an important consideration for the conservation of endangered species. Studies provided evidence of the effect of behavior and habitat change on the microbiota of wild animals and reported various inferences. It indicates the complexity of factors influencing microbiota diversity, including incomplete sampling procedures. Data abnormality may arise due to the procedures warranting preliminary analysis, such as rarefaction, before downstream analysis. This present study demonstrated the effect of data rarefaction and aggregation on the comparison of wild rusa deer's gut microbial diversity. Eighty-five feces samples were collected from 11 deer populations inhabiting three national parks in Java and Bali islands. Using the Illumina Nova-Seq platform, fragments of 16s rRNA gene were sequenced, and raw data of 51,389 reads corresponding to 2 domains, 22 phyla, 45 classes, 83 orders, 182 families, and 460 genera of bacteria were obtained. Data rarefaction was applied at two different library sizes (minimum and fixed) and aggregation (11 populations into 3 research sites) to investigate its effect on the microbial diversity comparison. There are significant differences in alpha diversity between populations, but not research sites, at all library sizes of rarefaction. A similar finding is also found in beta diversity. Moreover, data rarefaction and aggregation result in different values of the diversity metrics. This present study shows that statistical analysis remains a substantial concern in microbiome studies applied to conservation biology. It suggests reporting a more detailed data normalization in microbiome studies as an inherent control of suboptimal sampling, particularly when involving feces.

The Asian songbird crisis.

Alexander Lees and Pramana Yuda introduce the songbird trade of Southern and Eastern Asia that is a major threat to regional bird populations.

Using historical genome-wide DNA to unravel the confused taxonomy in a songbird lineage that is extinct in the wild.

Urgent conservation action for terminally endangered species is sometimes hampered by taxonomic uncertainty, especially in illegally traded animals that are often cross-bred in captivity. To overcome these problems, we used a genomic approach to analyze historical DNA from museum samples across the Asian Pied Starling (Gracupica contra) complex in tropical Asia, a popular victim of the ongoing songbird crisis whose distinct Javan population ("Javan Pied Starling") is extinct in the wild and subject to admixture in captivity. Comparing genomic profiles across the entire distribution, we detected three deeply diverged lineages at the species level characterized by a lack of genomic intermediacy near areas of contact. Our study demonstrates that the use of historical DNA can be instrumental in delimiting species in situations of taxonomic uncertainty, especially when modern admixture may obfuscate species boundaries. Results of our research will enable conservationists to commence a dedicated ex situ breeding program for the Javan Pied Starling, and serve as a blueprint for similar conservation problems involving terminally endangered species subject to allelic infiltration from close congeners.

The conservation value of admixed phenotypes in a critically endangered species complex.

In today's environmental crisis, conservationists are increasingly confronted with terminally endangered species whose last few surviving populations may be affected by allelic introgression from closely related species. Yet there is a worrying lack of evidence-based recommendations and solutions for this emerging problem. We analyzed genome-wide DNA markers and plumage variability in a critically endangered insular songbird, the Black-winged Myna (BWM, Acridotheres melanopterus). This species is highly threatened by the illegal wildlife trade, with its wild population numbering in the low hundreds, and its continued survival urgently depending on ex-situ breeding. Its three subspecies occur along a geographic gradient of melanism and are variably interpreted as three species. However, our integrative approach revealed that melanism poorly reflects the pattern of limited genomic differentiation across BWM subspecies. We also uncovered allelic introgression into the most melanistic subspecies, tertius, from the all-black congeneric Javan Myna (A. javanicus), which is native to the same islands. Based on our results, we recommend the establishment of three separate breeding programs to maintain subspecific traits that may confer local adaptation, but with the option of occasional cross-breeding between insurance populations in order to boost genetic diversity and increase overall viability prospects of each breeding program. Our results underscore the importance of evidence-based integrative approaches when determining appropriate conservation units. Given the rapid increase of terminally endangered organisms in need of ex-situ conservation, this study provides an important blueprint for similar programs dealing with phenotypically variable species.

Eggshell membrane for DNA sexing of the endangered Maleo ( Macrocephalon maleo).

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 gSYNC TM 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.

Detection of avian malaria in wild birds at Trisik Beach of Yogyakarta, Java (Indonesia)

Avian haemosporidian (Plasmodium, Haemoproteus and Leucocytozoon) are abundant and widespread vector-borne parasites in birds. However, our knowledge is very limited. This study used a nested-PCR to detect the prevalence level of haematozoan parasites in wild bird at coastal area at Tisik Beach of Yogyakarta, Java Island, Indonesia. In total, 112 DNA samples of 22 species were used. Amplification of cyt-b mtDNA of birds at Trisik beach detected 11 out of 112 samples (9.8 %) of all the blood parasites. Only 5 species out of 22 wild bird species were infected by the avian malaria parasites. Meanwhile, only one out of 20 samples of domestic birds was infected. All positive samples sequenced consistently generated around 450 base pair nucleotides. Alignments of 12 sequences have revealed six parasite lineages in the wild bird at Trisik Beach of Yogyakarta, consist of five lineages for Plasmodium sp. and the rest respectively one lineages for Haemoproteus sp. and Leucocytozoon sp. The results of this study provide additional evidences for Plasmodium lineages that uniquely were only infected the Pintail Snipe, Javan Munia, and domestic duck, respectively one lineage, and two lineages in the Yellow Bittern. Meanwhile, Haemoproteus and Leucocytozoon were not uniquely infecting specific host.