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Squeezing water from a stone: High-Throughput Sequencing from a 145-year old holotype resolves (barely) a cryptic species problem in flying lizards

McGuire J.A.a, Cotoras D.D.b,c, O’Connell B.b, Lawalata S.Z.S.a,d, Wang-Claypool C.Y.a, Stubbs A.a, Huang X., Wogan G.O.U., Hykin S.M.a, Reilly S.B.a, Bi K., Riyanto A., Arida E., Smith L.L.a, Milne H.b, Streicher J.W.i, Iskandar D.T.j

a Museum of Vertebrate Zoology, University of California, Berkeley, 94720, United States
b Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, Santa Cruz, 95064, United States
c Entomology Department, California Academy of Sciences, San Francisco, 94118, United States
d United in Diversity Foundation, Jakarta, 10220, Indonesia
e Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Ministry of Education, Qingdao, 266003, China
f Department of Environmental Science, Policy, and Management, University of California, Berkeley, 94720, United States
g Computational Genomics Resource Laboratory, California Institute for Quantitative Biosciences, 238 Koshland Hall, University of California, Berkeley, 94720, United States
h Museum Zoologicum Bogoriense (MZB), Indonesia Institute of Sciences (LIPI), Cibinong, 16911, Indonesia
i Department of Life Sciences, Natural History Museum, London, SW7 5BD, United Kingdom
j School of Life Sciences and Technology, Istitut Teknologi Bandung, Labtek XI Building, Bandung, 40132, Indonesia

[vc_row][vc_column][vc_row_inner][vc_column_inner][vc_separator css=”.vc_custom_1624529070653{padding-top: 30px !important;padding-bottom: 30px !important;}”][/vc_column_inner][/vc_row_inner][vc_row_inner layout=”boxed”][vc_column_inner width=”3/4″ css=”.vc_custom_1624695412187{border-right-width: 1px !important;border-right-color: #dddddd !important;border-right-style: solid !important;border-radius: 1px !important;}”][vc_empty_space][megatron_heading title=”Abstract” size=”size-sm” text_align=”text-left”][vc_column_text]© 2018 PeerJ Inc.. All rights reserved.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.[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Author keywords” size=”size-sm” text_align=”text-left”][vc_column_text][/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Indexed keywords” size=”size-sm” text_align=”text-left”][vc_column_text][/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Funding details” size=”size-sm” text_align=”text-left”][vc_column_text]Sanger sequence data are available on GenBank (will be submitted if accepted) and a matrix for the mitochondrial ND2 gene for the D. fimbriatus group is included as supplemental materials. This research was undertaken in accordance with UC Berkeley Animal Use Protocol Number AUP-2014-12-6954. Fieldwork was undertaken with research permits issued by the Economic Planning Unit of Malaysia (UPE:40/200/19 SJ.363) and the Indonesia Institute of Sciences (LIPI: No. 2411/FRP/SM/X?2008 and No. 0115/FRP/SM/VI/2009).[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”DOI” size=”size-sm” text_align=”text-left”][vc_column_text]https://doi.org/10.7287/peerj.preprints.26466[/vc_column_text][/vc_column_inner][vc_column_inner width=”1/4″][vc_column_text]Widget Plumx[/vc_column_text][/vc_column_inner][/vc_row_inner][/vc_column][/vc_row][vc_row][vc_column][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][/vc_column][/vc_row]