Thursday, March 22, 2018

[Herpetology • 2018] Squeezing Water from A Stone: High-throughput Sequencing from A 145-year Old Holotype Resolves (barely) A Cryptic Species Problem in Flying Lizards

Figure 1: Phylogenetic tree for the Draco fimbriatus group including the Dcristatellus holotype.
Phylogenetic tree for the Draco fimbriatus group based on a parsimony analysis of the complete mitochondrial ND2 gene (1,032 bp). The D. cristatellus holotype includes 183 bp of sequence data. Only two of 28 available D. maculatus samples were included to simplify the image. Non-parametric bootstrap values (1,000 replicates) are superimposed on the single most parsimonious phylogram for select clades.
The photo in the bottom left is Draco punctatus. Photo: Jimmy A. McGuire.

in McGuire, Cotoras, O'Connell, et al., 2018.
   DOI: 10.7717/peerj.4470


We used Massively Parallel High-Throughput Sequencing to obtain genetic data from a 145-year old holotype specimen of the flying lizard, Draco cristatellus. Obtaining genetic data from this holotype was necessary to resolve an otherwise intractable taxonomic problem involving the status of this species relative to closely related sympatric Draco species that cannot otherwise be distinguished from one another on the basis of museum specimens. Initial analyses suggested that the DNA present in the holotype sample was so degraded as to be unusable for sequencing. However, we used a specialized extraction procedure developed for highly degraded ancient DNA samples and MiSeq shotgun sequencing to obtain just enough low-coverage mitochondrial DNA (547 base pairs) to conclusively resolve the species status of the holotype as well as a second known specimen of this species. The holotype was prepared before the advent of formalin-fixation and therefore was most likely originally fixed with ethanol and never exposed to formalin. Whereas conventional wisdom suggests that formalin-fixed samples should be the most challenging for DNA sequencing, we propose that evaporation during long-term alcohol storage and consequent water-exposure may subject older ethanol-fixed museum specimens to hydrolytic damage. If so, this may pose an even greater challenge for sequencing efforts involving historical samples.

The development of HTS has revolutionized biological research by making genome-scale data readily available at a reasonable cost, even for non-model organisms. Systematists have fully embraced these advances in data acquisition for freshly sampled specimens, but are just beginning to harness HTS for the millions of fluid-preserved historical samples housed in natural history collections around the world. As we have shown here, acquiring genetic data from old museum specimens will sometimes present special challenges, but the information that can be gleaned from such specimens may be the only way to conclusively resolve previously intractable evolutionary and taxonomic questions.

Jimmy A McGuire, Darko D Cotoras, Brendan O'Connell, Shobi Z S Lawalata, Cynthia Y Wang-Claypool, Alexander Stubbs, Xiaoting Huang, Guinevere O U Wogan, Sarah M Hykin, Sean B Reilly, Ke Bi, Awal Riyanto, Evy Arida, Lydia L Smith, Heather Milne, Jeffrey W Streicher and Djoko T Iskandar. 2018. Squeezing Water from A Stone: High-throughput Sequencing from A 145-year Old Holotype Resolves (barely) A Cryptic Species Problem in Flying Lizards.  PeerJ. 6:e4470.  DOI: 10.7717/peerj.4470

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