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The availability of genetic data from wild populations limits our understanding of primate evolution and conservation, particularly for small nocturnal species such as lorisiforms (galagos, lorises, angwantibos, and pottos). Emerging methods for recovering genomic DNA from historical museum specimens have been rarely used in primate studies. We aimed to optimize extraction and bioinformatics protocols to maximize the recovery of historical DNA to fill important geographic and taxonomic gaps, improve phylogenetic resolution, and inform conservation of Lorisiform primates. First, we compared the performance of two DNA extraction methods by using 238 specimens up to a hundred years old. We then selected 96 samples with the highest DNA yields for shotgun sequencing. To evaluate the impact of phylogenetic divergence in bioinformatic read mapping, we compared coverage depths when using human and three lorisiform reference mitogenomes. Based on whole genomic data, we performed metagenomics and microbial diversity analyses to assess the composition of potentially exogenous content. Lastly, based on the most geographically and taxonomically comprehensive sampling for the West African lorisiforms to date (19/32 currently recognized species), we performed phylogenetic inference using Maximum Likelihood. The results showed that older samples yield lower DNA concentration, with an optimized phenol-chloroform protocol outperforming a commercial kit. However, both extraction methods generated DNA in sufficient amount and quality for phylogenetic inference. Our reference bias comparisons showed that higher phylogenetic proximity between focal species and reference mitogenome increases coverage depth. The metagenomic analysis found human contamination in only one of 96 samples (1%), whereas ten of 96 (11%) samples showed nonnegligible levels of other exogenous contents, among which are certain blood parasites. We inferred low support for the monophyly of Asian and African Lorisids but confirmed the monophyly and previously suggested relationships among Galagid genera. Lastly, we found evidence of cryptic species diversity within the western dwarf galagos (genus Galagoides). Taken together, these results attest to the enormous potential of museomics to advance our understanding of galago evolution, ecology, and conservation, an approach that can be extended to other primate clades.
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More than skin and bones: Comparing extraction methods and alternative sources of DNA from avian museum specimens
Abstract Next‐generation sequencing has greatly expanded the utility and value of museum collections by revealing specimens as genomic resources. As the field of museum genomics grows, so does the need for extraction methods that maximize DNA yields. For avian museum specimens, the established method of extracting DNA from toe pads works well for most specimens. However, for some specimens, especially those of birds that are very small or very large, toe pads can be a poor source of DNA. In this study, we apply two DNA extraction methods (phenol–chloroform and silica column) to three different sources of DNA (toe pad, skin punch and bone) from 10 historical avian museum specimens. We show that a modified phenol–chloroform protocol yielded significantly more DNA than a silica column protocol (e.g., Qiagen DNeasy Blood & Tissue Kit) across all tissue types. However, extractions using the silica column protocol contained longer fragments on average than those using the phenol–chloroform protocol, probably as a result of loss of small fragments through the silica column. While toe pads yielded more DNA than skin punches and bone fragments, skin punches proved to be a reliable alternative source of DNA and might be especially appealing when toe pad extractions are impractical. Overall, we found that historical bird museum specimens contain substantial amounts of DNA for genomic studies under most extraction scenarios, but that a phenol–chloroform protocol consistently provides the high quantities of DNA required for most current genomic protocols.
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- Award ID(s):
- 1652979
- PAR ID:
- 10454036
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Molecular Ecology Resources
- Volume:
- 20
- Issue:
- 5
- ISSN:
- 1755-098X
- Format(s):
- Medium: X Size: p. 1220-1227
- Size(s):
- p. 1220-1227
- Sponsoring Org:
- National Science Foundation
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