Squeezing water from a stone: high-throughput sequencing from a 145-year old holotype resolves (barely) a cryptic species problem in flying lizards
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,
Djoko T. Iskandar
Affiliations
Jimmy A. McGuire
Museum of Vertebrate Zoology, University of California, Berkeley, CA, United States of America
Darko D. Cotoras
Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, CA, United States of America
Brendan O’Connell
Department of Biomolecular Engineering and Bioinformatics, Baskin School of Engineering, University of California, Santa Cruz, CA, United States of America
Shobi Z.S. Lawalata
Museum of Vertebrate Zoology, University of California, Berkeley, CA, United States of America
Cynthia Y. Wang-Claypool
Museum of Vertebrate Zoology, University of California, Berkeley, CA, United States of America
Alexander Stubbs
Museum of Vertebrate Zoology, University of California, Berkeley, CA, United States of America
Xiaoting Huang
Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Qingdao, China
Guinevere O.U. Wogan
Department of Environmental Science, Policy, and Management, University of California, Berkeley, CA, United States of America
Sarah M. Hykin
Museum of Vertebrate Zoology, University of California, Berkeley, CA, United States of America
Sean B. Reilly
Museum of Vertebrate Zoology, University of California, Berkeley, CA, United States of America
Ke Bi
Computational Genomics Resource Laboratory, California Institute for Quantitative Biosciences, University of California, Berkeley, CA, United States of America
Awal Riyanto
Museum Zoologicum Bogoriense, Research Center for Biology-The Indonesian Institute of Sciences, Cibinong, Indonesia
Evy Arida
Museum Zoologicum Bogoriense, Research Center for Biology-The Indonesian Institute of Sciences, Cibinong, Indonesia
Lydia L. Smith
Museum of Vertebrate Zoology, University of California, Berkeley, CA, United States of America
Heather Milne
Department of Biomolecular Engineering and Bioinformatics, Baskin School of Engineering, University of California, Santa Cruz, CA, United States of America
Jeffrey W. Streicher
Department of Life Sciences, The Natural History Museum, London, United Kingdom
Djoko T. Iskandar
School of Life Sciences and Technology, Institute of Technology Bandung, Bandung, West Java, Indonesia
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 (721 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.
