Can DNA Barcode Study be Done from a Museum Specimen Fixed in a Formaldehyde Solution? A Case of Emys orbicularis (Linnaeus, 1758)

Abstract

DNA barcoding, a molecular taxonomy technique, has been increasingly used by herptile taxonomists in recent years. In DNA barcoding studies with museum specimens, there are difficulties in achieving success in specimens that have been exposed to formaldehyde, which is usually used as a fixative, for a long time and intensively. Here we studied the effect of formaldehyde on the application of the DNA barcode method in Emys orbicularis specimens stored in 4% formaldehyde and 70% ethanol solution since 2008 and 2014. Sanger sequence analysis of tissues taken from samples stored in both ethanol and formaldehyde solution successfully yielded sequences of 623 bp. In conclusion, the use of ethanol solutions should be preferred for mid or long-term sample storage, especially in the context of molecular studies. In cases where the use of formaldehyde is unavoidable, it may be advisable to use extremely low concentrations to increase success in molecular research.

Keywords

• Cytochrome oxidase I
• European pond turtle
• DNA barcoding
• Collection
• Türkiye

Formaldehit Çözeltisindeki Bir Müze Örneğinden DNA Barkod Çalışması Yapılabilir mi? Emys orbicularis (Linnaeus, 1758) Örneği

Abstract

Bir moleküler taksonomi tekniği olan DNA barkodlama, son yıllarda herptil taksonomistleri tarafından giderek daha fazla kullanılmaktadır. Müze örnekleri ile yapılan DNA barkodlama çalışmalarında, genellikle fiksatif olarak kullanılan formaldehite uzun süre ve yoğun bir şekilde maruz kalan örneklerde başarı elde etmekte zorluklar yaşanmaktadır. Bu çalışmada, herptil koleksiyon materyali olarak 2008 ve 2014 yıllarından beri %4 formaldehit ve %70 etanol çözeltisinde saklanan Emys orbicularis örneklerinde formaldehitin DNA barkod yönteminin uygulanması üzerindeki etkisi araştırılmıştır. Hem etanol hem de formaldehit çözeltisinde saklanan örneklerden alınan dokuların Sanger dizi analizi sonucunda 623 baz çiftinden oluşan dizileri başarıyla elde edilmiştir. Sonuç olarak, özellikle moleküler çalışmalar bağlamında orta ve uzun süreli numune saklama için alkol solüsyonlarının kullanılmasının tercih edilmesi gerekmektedir. Formaldehit kullanımının kaçınılmaz olduğu durumlarda ise, moleküler araştırmalarda başarıyı artırmak için son derece düşük konsantrasyonların kullanılması tavsiye edilebilir.

Keywords

• Sitokrom oksidaz I
• Benekli kaplumbağa
• DNA bakrodlama
• Biyolojik koleksiyon
• Türkiye

References

1. Bayrakcı, Y., & Ayaz, D. (2014). Dynamics of a Central Anatolian population of Emys orbicularis (Linnaeus, 1758). Herpetozoa, 27(1/2), 29-37.
2. Bayrakcı, Y., Ayaz, D., & Çiçek, K. (2015). Data on the population of syntopic turtles Emys orbicularis (L., 1758) and Mauremys rivulata (Valenciennes, 1883) from Great Menderes Delta (western Anatolia, Turkey). Russian Journal of Herpetology, 22(2), 79-83.
3. Beebee, T. C., & Griffiths, R. (2005). The amphibian decline crisis: A watershed for conservation biology?. Biological Conservation, 125(3), 271‑285. https://doi.org/10.1016/j.biocon.2005.04.009
4. Blum, F. (1894). Notiz über die Anwendung des Formaldehyds (Formol) als Härtungs- und Conservirungsmittel. Anatomischer Anzeiger (Annals of Anatomy), 9, 229–231
5. Bramwell, N. H., & Burns, B. F. (1988). The effects of fixative type and fixation time on the quantity and quality of extractable DNA for hybridization studies on lymphoid tissue. Experimental Hematology, 16(8), 730-732.
6. Broggi, M. F. (2023). Occurrence and status of the European Pond Turtle, Emys orbicularis hellenica (Valenciennes, 1833), on Aegean and Ionian Islands (Greece, Turkey). Herpetozoa, 36, 249-257.
7. Brooke, M. (2000). Why museums matter. Trends in Ecology & Evolution, 15(4), 136-137.
8. Ceríaco, L. M. P., Marques, M. P., de Sousa, A. C. A., Veríssimo, J., Beja, P., & Ferreira, S. (2023). Illustrated keys and a DNA barcode reference library of the amphibians and terrestrial reptiles (Amphibia, Reptilia) of São Tomé and Príncipe (Gulf of Guinea, West Africa). ZooKeys, 1168, 41.

9. Chambers, E. A., & Hebert, P. N. (2016). Assessing DNA barcodes for species identification in North American reptiles and amphibians in natural history collections. PLOS One, 11(4). https://doi.org/10.1371/journal.pone.0154363
10. Chovanec, A., & Grillitsch, B. (1994). Amphibien als bioindikatoren für die schadstoffbelastung von leingewässern. Umweltbundesamt. Wien. [ISBN 3-85457-188-7]
11. Coombs, N. J., Gough, A. C., & Primrose, J. N. (1999). Optimisation of DNA and RNA extraction from archival formalin-fixed tissue. Nucleic acids research, 27(16), e12-i.
12. Greer, C. E., Lund, J. K., & Manos M. M. (1991). PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective studies. PCR Methods and Applications, 1(1), 46–50. https://doi.org/10.1101/gr.1.1.46 PMID: 1842921
13. Dayrat, B. (2005). Towards integrative taxonomy. Biological Journal of the Linnean Society, 85, 407–415. doi: 10.1111/j.1095-8312.2005.00503.x
14. Do, H., & Dobrovic, A. (2015). Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization. Clinical Chemistry, 61(1), 64-71.
15. Dutta, S., Chowdhury, G., & Gates, K. S. (2007). Interstrand cross-links generated by abasic sites in duplex DNA. Journal of the American Chemical Society, 129(7), 1852–1853. https://doi.org/10.1021/ja067294u PMID: 17253689
16. Escoriza, D., Franch, M., Ramos, S., Sunyer-Sala, P., & Boix, D. A. N. I. (2020). Demographics and survivorship in the European pond turtle (Emys orbicularis): a 31‒year study. Herpetological Conservation and Biology, 15(1), 41-48.
17. Ficetola, G. F., Padoa-Schioppa, E., Monti, A., Massa, R., Bernardi, F. D., & Bottoni, L. (2004). The importance of aquatic and terrestrial habitat for the European pond turtle (Emys orbicularis): implications for conservation planning and management. Canadian Journal of Zoology, 82(11), 1704-1712.
18. Forsthoefel, K. F., Papp, A. C., Snyder, P. J., & Prior, T. W. (1992). Optimization of DNA Extraction from Formalin-fixed Tissue and Its Clinical Application in Duchenne Muscular Dystrophy. American Journal of Clinical Pathology, 98, 98-104.
19. Fox, C. H., Johnson, F. B., Whiting, J., & Roller, P. P. (1985). Formaldehyde fixation. Journal of Histochemistry & Cytochemistry, 33(8), 845–853. https://doi.org/10.1177/33.8.3894502 PMID: 389450
20. Fritz, U., Ayaz, D., Hundsdörfer, A. K., Kotenko, T., Guicking, D., Wink, M., ... & Buschbom, J. (2009). Mitochondrial diversity of European pond turtles (Emys orbicularis) in Anatolia and the Ponto-Caspian Region: Multiple old refuges, hotspot of extant diversification and critically endangered endemics. Organisms Diversity & Evolution, 9(2), 100-114.
21. Gavrilov, A., & Razin, S. V. (2009). Formaldehyde Fixation of Cells Does not Greatly Reduce the Ability to Amplify Cellular DNA. Analytical Biochemistry, 390, 94-96.
22. Goldstein, P. Z., DeSalle, R., (2011). Integrating DNA barcode data and taxonomic practice: Determination, discovery, and description. Bioessays, 33, 135-147. doi: 10.1002/bies.201000036
23. Greer, C. E., Peterson, S. L., Kiviat, N. B., & Manos, N. M. (1991). PCR Amplification from Paraffin-Embedded Tissues. Effects of Fixative and Fixation Time. American Journal of Clinical Pathology, 95, 117-124.
24. Hamazaki, S., Koshiba, M., Habuchi, T., Takahashi, R., & Sugiyama, T. (1993). The Effect of Formalin Fixation on Restriction Endonuclease Digestion of DNA and PCR Amplification. Pathology-Research and Practice, 189, 553-557.
25. Hawlitschek, O., Morinière, J., Dunz, A., Franzen, M., Rödder, D., Glaw, F., & Haszprunar, G. (2016). Comprehensive DNA barcoding of the herpetofauna of Germany. Molecular Ecology Resources, 16(1), 242-253.
26. Hebert, P. D., Cywinska, A., Ball, S. L., & DeWaard, J. R. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society of London. Series B: Biological Sciences, 270(1512), 313-321.
27. IUCN. (2023). The IUCN Red List of Threatened Species. Version 2023-1. Access date: 15 December2023, https://www.iucnredlist.org.
28. Lindahl, T., & Andersson, A. (1972). Rate of chain breakage at apurinic sites in double-stranded deoxyribonucleic acid. Biochemistry, 11(19), 3618–3623. https://doi.org/10.1021/bi00769a019 PMID: 4559796
29. Meyer, C. (2003). Molecular systematics of cowries (Gastropoda: Cypraeidae) and diversification patterns in the tropics. Biological Journal of the Linnean Society, 79(3), 401‑459. https://doi.org/10.1046/j.1095-8312.2003.00197.x
30. Miller, J. A., Beentjes, K. K., van Helsdingen, P., & IJland, S. (2013). Which specimens from a museum collection will yield DNA barcodes? A time series study of spiders in alcohol. ZooKeys, (365), 245.
31. Murphy, R., Crawford, A., Bauer, A., Che, J., Donnellan, S., Fritz, U., Haddad, C. B., Nagy, Z., Poyarkov, N., Vences, M., Wang, W., & Zhang, Y. (2012). Cold code: The global initiative to DNA barcode amphibians and nonavian reptiles. Molecular Ecology Resources, 13(2), 161‑167. https://doi.org/10.1111/1755-0998.12050
32. Perl, R. G. B., Nagy, Z., Sonet, G., Glaw, F., Wollenberg, K., & Vences, M. (2014). DNA barcoding Madagascar’s amphibian fauna. Amphibia-Reptilia, 35(2), 197‑206. https://doi.org/10.1163/15685381-00002942
33. Puillandre, N., Bouchet, P., Boisselier‐Dubayle, M. C., Brisset, J., Buge, B., Castelin, M., ... & Samadi, S. (2012). New taxonomy and old collections: integrating DNA barcoding into the collection curation process. Molecular Ecology Resources, 12(3), 396-402.
34. Ratnasingham, S., & Hebert, P. D. (2007). BOLD: The Barcode of Life Data System (http://www.barcodinglife.org). Molecular Ecology Notes, 7(3), 355‑364. https://doi.org/10.1111/j.1471-8286.2007.01678.x
35. Rhodin, A. G., Stanford, C. B., Van Dijk, P. P., Eisemberg, C., Luiselli, L., Mittermeier, R. A., ... & Vogt, R. C. (2018). Global conservation status of turtles and tortoises (order Testudines). Chelonian Conservation and Biology, 17(2), 135-161.
36. Riedel, A., Sagata, K., Suhardjono, Y. R., Tänzler, R., & Balke, M. (2013). Integrative taxonomy on the fast track – towards more sustainability in biodiversity research. Frontiers in Zoology, 10, 1-9. doi: 10.1186/1742-9994-10-15
37. Rogers, B. B., Alpert, L. C., Hine, E. A. S., & Buffone, G. J. (1990). Analysis of DNA in Fresh and Fixed Tissue by Polymerase Chain Reaction. The American Journal of Pathology, 136(3), 541-548.
38. Schlaepfer, M., Hoover, C., & Dodd, C. K. (2005). Challenges in evaluating the impact of the trade in amphibians and reptiles on wild populations. BioScience, 55(3), 256-264. https://doi.org/10.1641/0006-3568(2005)055[0256:cietio]2.0.co;2
39. Smith, M. A., Poyarkov, N., & Hebert, P. N. (2008). DNA Barcoding: CO1 DNA barcoding amphibians: take the chance, meet the challenge. Molecular Ecology Resources, 8(2), 235‑246. https://doi.org/10.1111/j.1471-8286.2007.01964.x
40. Tamura, K., Stecher, G., & Kumar, S. (2021). MEGA11: Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution, 38(7), 3022‑3027. https://doi.org/10.1093/molbev/msab120
41. Totoiu, C. A., Phillips, J. M., Reese, A. T., Majumdar, S., Girguis, P. R., Raston, C. L., & Weiss, G. A. (2020). Vortex fluidics-mediated DNA rescue from formalin-fixed museum specimens. PloS one, 15(1), e0225807.
42. Vences, M., Nagy, Z., Sonet, G., & Verheyen, E. (2012). DNA barcoding amphibians and reptiles. DNA Barcodes, 79‑107. https://doi.org/10.1007/978-1-61779-591-6_5
43. Whitfield, J. B. (1999). Destructive sampling and information management in molecular systematic research: an entomological perspective. In S. Byers & D. Metsger (Eds.), Managing the Modern Herbarium: An Interdisciplinary Approach (pp. 301–314): Society for Preservation of Natural History Collections, New York and Royal Ontario Museum, Toronto.
44. Will, K. W., Mishler, B. D., Wheeler, Q. D. (2005). The perils of DNA barcoding and the need for integrative taxonomy. Systematic Biology, 54, 844-851. doi: 10.1080/10635150500354878
45. Wong, S. Q., Li, J., Salemi, R., Sheppard, K. E., Do, H., Tothill, R. W., ... & Dobrovic, A. (2013). Targeted-capture massively-parallel sequencing enables robust detection of clinically informative mutations from formalin-fixed tumours. Scientific Reports, 3(1), 3494.
46. Wong, S. Q., Li, J., Tan, A. Y., Vedururu, R., Pang, J. M. B., Do, H., ... & The CANCER 2015 Cohort. (2014). Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing. BMC medical genomics, 7, 1-10.
47. Yagi, N., Satonaka, K., Horio, M., Shimogaki, H., Tokuda, Y., & Maeda, S. (1996). The role of DNase and EDTA on DNA degradation in formaldehyde fixed tissues. Biotechnic & histochemistry, 71(3), 123-129.
48. Zangl, L., Daill, D., Schweiger, S., Gassner, G., & Koblmueller, S. (2020). A reference DNA barcode library for Austrian amphibians and reptiles. PLoS One, 15(3), e0229353.