DNA Barcoding and Phylogeny of Ambystoma mexicanum Cultivated as a Model Organism in Regenerative Medicine Research at Mersin University Aquaculture Units of the Faculty of Fisheries

Öz

Ambystoma mexicanum belongs to the family Ambystomatidae. It is one of the 30 species of the genus Ambystoma, which lives in a wide geography from southern Mexico to southern Alaska. It is accepted as a model organism in evolutionary biology, developmental biology, and regenerative medicine research. It can regenerate the brain, heart, and kidney organs as well as limb regeneration. Accurate identification of the model organism is important for the reproducibility and comparability of experiments. We aimed to confirm the species identification of axolotls using integrated taxonomic methods that were grown at Mersin University Aquaculture Units of the Faculty of Fisheries. Cytochrome oxidase subunit 1 (COI) and cytochrome b (Cytb) gene fragments of mtDNA sequences were used as molecular markers for phylogenetic analyses and species delimitation methods and compared with the sequences that were submitted to NCBI GenBank as species of Ambystoma. In the analyses that were conducted with different data sets, the individuals in question were grouped as a candidate species with the A. mexicanum species whose sequences were given in previous studies. All sequences obtained in this study and A. mexicanum sequences obtained from NCBI GenBank were grouped as haplotypes and their genetic distances were found to be 0 and it was determined that the individuals which were the subject of this study definitely belong to the A. mexicanum species. The results revealed that some species within the genus Ambystoma, especially A. barbouri and A. texanum, may be species complexes. On the other hand, A. mexicanum was grouped together with A. andersoni as candidate species in all analyses performed with the combined datasets of COI, Cytb, and COI+Cytb. These results revealed that the taxa in question are paraphyletic and should be assigned to the A. mexicanum species.

Anahtar Kelimeler

• Regenerative Medicine
• Axolotl
• phylogeny
• species delimitation
• Cytochromes b
• Cytochrome Oxidase Subunit I.

Mersin Üniversitesi Su Ürünleri Fakültesi Uygulama Birimleri’nde Rejeneratif Tıp Araştırmalarında Model Organizma Olarak Yetiştirilen Ambystoma mexicanum’un DNA Barkodlaması ve Filogenisi

Öz

Ambystoma mexicanum Ambystomatidae familyasında yer alır. Güney Meksika’dan Güney Alaska’ya kadar geniş bir coğrafyada yaşayan Ambystoma cinsinin 30 türünden biridir. Limb rejenerasyonunun yanında beyin, kalp, böbrek organlarını rejenere edebilmeleri nedeniyle, evrimsel biyoloji, gelişim biyolojisi ve rejeneratif tıp araştırmalarında model organizma olarak kabul edilir. Model organizmaların kullanıldığı araştırmalarda canlının tür teşhisinin doğru yapılması deneylerin tekrarlanabilirliği ve karşılaştırılabilirliği açısından önemlidir. Bu çalışmada; Mersin Üniversitesi Su Ürünleri Fakültesi Uygulama Birimleri’nde yetiştirilen aksolotlların tür teşhisini bütünleşik taksonomik yöntemler kullanarak kesinleştirmek amacıyla mtDNA sitokrom oksidaz alt ünite 1 (COI) ve sitokrom b (Cytb) gen fragmentleri moleküler belirteç olarak kullanılmış ve NCBI GenBank’ta daha önce dizisi verilmiş olan Ambsytoma türleri ile filogenetik analizler ve tür sınırlarını belirleme yöntemleri ile karşılaştırılmıştır. Farklı veri setlerinin kullanıldığı analizlerin tamamında söz konusu bireyler daha önceki çalışmalarda elde edilen A. mexicanum türü ile aday tür olarak gruplanmıştır. Bu çalışmada elde edilen tüm diziler ile NCBI GenBank’tan elde edilen A. mexicanum dizileri haplotip olarak gruplanmış olup genetik uzaklıkları 0 bulunmuş ve bu çalışmanın konusu olan bireylerin kesin olarak A. mexicanum türüne ait olduğu belirlenmiştir. Sonuçlar Ambystoma cinsi içerisinde bazı türlerin özellikle A. barbouri ve A. texanum ‘un tür kompleksi olabileceğini ortaya koymuştur. Diğer yandan A. mexicanum, COI, Cytb ve COI+Cytb birleştirilmiş veri setleri ile yapılan tüm analizlerde A. andersoni ile aday tür olarak gruplanmıştır. Bu sonuçlar söz konusu taksonların parafiletik olduğunu ve A. mexicanum türüne atanması gerektiğini ortaya koymuştur.

Anahtar Kelimeler

• Rejeneratif Tıp
• Aksolotl
• Filogeni
• Tür sınırlarını belirleme
• Sitokrom b
• Sitokrom oksidaz alt ünite 1.

Kaynakça

1. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., & Lipman, D.J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403-410. https://doi.org/10.1016/S0022-2836(05)80360-2
2. Arnason, U., Gullberg, A., Janke, A., Joss, J., & Elmerot, C. (2004). Mitogenomic analyses of deep gnathostome divergences: a fish is a fish. Gene, 333, 61-70. https://doi.org/10.1016/j.gene.2004.02.014
3. Bi, K., & Bogart, J.P. (2010). Time and time again: unisexual salamanders (genus Ambystoma) are the oldest unisexual vertebrates. BMC Evolutionary Biology, 10(1), 1-14. https://doi.org/10.1186/1471-2148-10-238
4. Bortolus, A. (2008). Error cascades in the biological sciences: the unwanted consequences of using bad taxonomy in ecology. AMBIO: A Journal of The Human Environment, 37(2), 114-118. https://doi.org/10.1579/0044-7447(2008)37[114:ECITBS]2.0.CO;2
5. Bouckaert, R.R., & Drummond, A.J. (2017). bModelTest: Bayesian phylogenetic site model averaging and model comparison. BMC Evolutionary Biology, 17(1), 1-11.
6. Bouckaert, R., Heled, J., Kühnert, D., Vaughan, T., Wu, C.H., Xie, D., ... & Drummond, A.J. (2014). BEAST 2: a software platform for Bayesian evolutionary analysis. PLoS Computational Biology, 10(4), e1003537.
7. Chambers, E.A., & Hebert, P.D. (2016). Assessing DNA barcodes for species identification in North American reptiles and amphibians in natural history collections. PLoS One, 11(4), e0154363. https://doi.org/10.1371/journal.pone.0154363
8. Chippindale, P. T., Bonett, R. M., Baldwin, A. S., & Wiens, J. J. (2004). Phylogenetic evidence for a major reversal of life‐history evolution in plethodontid salamanders. Evolution, 58(12), 2809-2822.

9. Crandall, M.C.D.P.K., Clement, M., & Posada, D. (2000). TCS: a computer program to estimate gene genealogies. Molecular Ecology, 9, 1657-1660. https://doi.org/10.1046/j.1365-294x.2000.01020.x
10. Demircan, T., Hacıbektaşoğlu, H., Sibai, M., Fesçioğlu, E.C., Altuntaş, E., Öztürk, G., & Süzek, B.E. (2020). Preclinical molecular signatures of spinal cord functional restoration: Optimizing the metamorphic axolotl (Ambystoma mexicanum) model in regenerative medicine. OMICS: A Journal of Integrative Biology, 24(6), 370-378. https://doi.org/10.1089/omi.2020.0024
11. Diaz Quiroz, J.F., & Echeverri, K. (2013). Spinal cord regeneration: where fish, frogs and salamanders lead the way, can we follow?. Biochemical Journal, 451(3), 353-364. https://doi.org/10.1042/BJ20121807
12. Echeverri, K. (2020). The various routes to functional regeneration in the central nervous system. Communications Biology, 3(1), 1-4. https://doi.org/10.1038/s42003-020-0773-z
13. Farkas, J.E., & Monaghan, J.R. (2015). Housing and maintenance of Ambystoma mexicanum, the Mexican axolotl. Salamanders in Regeneration Research (pp. 27-46), Springer. https://doi.org/10.1007/978-1-4939-2495-0_3
14. Flot, J.F. (2015). Species delimitation's coming of age. Systematic Biology, 64(6), 897-899. https://doi.org/10.1093/sysbio/syv071
15. Gehlbach, F.R. (1967). Ambystoma tigrinum. Catalogue of American Amphibians and Reptiles (CAAR).
16. Gresens, J. (2004). An introduction to the Mexican axolotl (Ambystoma mexicanum). Lab Animal, 33(9), 41-47. https://doi.org/10.1038/laban1004-41
17. Hall, T. (2004). BioEdit version 7.0. 0. Distributed by the author, website: www.mbio.ncsu.edu/BioEdit/bioedit.html
18. Hoang, D.T., Chernomor, O., Von Haeseler, A., Minh, B.Q., & Vinh, L.S. (2018). UFBoot2: improving the ultrafast bootstrap approximation. Molecular Biology and Evolution, 35(2), 518-522. https://doi.org/10.1093/molbev/msx281
19. Katoh, K., & Standley, D.M. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution, 30(4), 772-780. https://doi.org/10.1093/molbev/mst010
20. Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16(2), 111-120. https://doi.org/10.1007/BF01731581
21. Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35(6), 1547. https://doi.org/10.1093/molbev/msy096
22. Lanfear, R., Frandsen, P.B., Wright, A.M., Senfeld, T., & Calcott, B. (2017). PartitionFinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution, 34(3), 772-773. https://doi.org/10.1093/molbev/msw260
23. Lévesque, M., Villiard, É., & Roy, S. (2010). Skin wound healing in axolotls: a scarless process. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, 314(8), 684-697. https://doi.org/10.1002/jez.b.21371
24. Lust, K., & Tanaka, E.M. (2019). A comparative perspective on brain regeneration in amphibians and teleost fish. Developmental Neurobiology, 79(5), 424-436. https://doi.org/10.1002/dneu.22665
25. Maddison, W.P. (2021). Mesquite: a modular system for evolutionary analysis. Version 3.6. http://mesquiteproject.org
26. Mashkouli, M., Aghaei, M., & Mofid, M. R. (2020). Purification of Soluble Membrane-Bound Ambystoma mexicanum Epidermal Lipoxygenase from E. coli and Its Growth Effect on Human Fetal Foreskin Fibroblast. The Protein Journal, 39(4), 377-382. https://doi.org/10.1007/s10930-020-09898-w
27. Menger, B., Vogt, P.M., Allmeling, C., Radtke, C., Kuhbier, J.W., & Reimers, K. (2011). AmbLOXe—an epidermal lipoxygenase of the Mexican axolotl in the context of amphibian regeneration and its impact on human wound closure in vitro. Annals Of Surgery, 253(2), 410-418 https://doi.org/10.1097/SLA.0b013e318207f39c
28. Minh, B.Q., Schmidt, H.A., Chernomor, O., Schrempf, D., Woodhams, M. D., Von Haeseler, A., & Lanfear, R. (2020). IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Molecular Biology and Evolution, 37(5), 1530-1534. https://doi.org/10.1093/molbev/msaa015
29. Moritz, C., Schneider, C.J., & Wake, D.B. (1992). Evolutionary relationships within the Ensatina eschscholtzii complex confirm the ring species interpretation. Systematic Biology, 41(3), 273-291. https://doi.org/10.1093/sysbio/41.3.273
30. Mueller, R.L., Macey, J.R., Jaekel, M., Wake, D.B., & Boore, J.L. (2004). Morphological homoplasy, life history evolution, and historical biogeography of plethodontid salamanders inferred from complete mitochondrial genomes. Proceedings of the National Academy of Sciences, 101(38), 13820-13825. https://doi.org/10.1073/pnas.0405785101
31. O'Neill, E.M., Schwartz, R., Bullock, C.T., Williams, J S., Shaffer, H.B., Aguilar‐Miguel, X., ... & Weisrock, D.W. (2013). Parallel tagged amplicon sequencing reveals major lineages and phylogenetic structure in the North American tiger salamander (Ambystoma tigrinum) species complex. Molecular Ecology, 22(1), 111-129. https://doi.org/10.1111/mec.12049
32. Pons, J., Barraclough, T.G., Gomez-Zurita, J., Cardoso, A., Duran, D.P., Hazell, S., ... & Vogler, A.P. (2006). Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Systematic Biology, 55(4), 595-609.
33. Puillandre, N., Lambert, A., Brouillet, S., & Achaz, G.J.M.E. (2012). ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Molecular Ecology, 21(8), 1864-1877. https://doi.org/10.1111/j.1365-294X.2011.05239.x
34. Rambaut, A. FigTree v1. 4.4. Institute of Evolutionary Biology, University of Edinburgh, Edinburgh. 2018.
35. Rambaut, A., Drummond, A., Xie, D., Baele, G., & Suchard, M.A. (2018). Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Systematic Biology, 67(5), 901. https://doi.org/10.1093/sysbio/syy032
36. Reiß, C., Olsson, L., & Hoßfeld, U. (2015). The history of the oldest self‐sustaining laboratory animal: 150 years of axolotl research. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, 324(5), 393-404. https://doi.org/10.1002/jez.b.22617
37. Ren, Z.L., Yao, N.N., Liu, L., Wu, Y., & Qian, Z.Q. (2019). Characterization of the complete mitochondrial genome of the mole salamander Ambystoma talpoideum (Caudata: Ambystomatidae). Conservation Genetics Resources, 11(4), 397-400. https://doi.org/10.1007/s12686-018-1031-2
38. Robertson, A. V., Ramsden, C., Niedzwiecki, J., Fu, J., & Bogart, J. P. (2006). An unexpected recent ancestor of unisexual Ambystoma. Molecular Ecology, 15(11), 3339-3351.
39. Ronquist, F., Teslenko, M., Van Der Mark, P., Ayres, D.L., Darling, A., Höhna, S., ... & Huelsenbeck, J.P. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61(3), 539-542. https://doi.org/10.1093/sysbio/sys029
40. Roy, S., & Lévesque, M. (2006). Limb regeneration in axolotl: is it superhealing?. The Scientific World Journal, 6, 12-25. https://doi.org/10.1100/tsw.2006.324
41. Rozas, J., Ferrer-Mata, A., Sánchez-DelBarrio, J.C., Guirao-Rico, S., Librado, P., Ramos-Onsins, S.E., & Sánchez-Gracia, A. (2017). DnaSP 6: DNA sequence polymorphism analysis of large data sets. Molecular Biology and Evolution, 34(12), 3299-3302. https://doi.org/10.1093/molbev/msx248
42. Sabin, K.Z., Jiang, P., Gearhart, M.D., Stewart, R., & Echeverri, K. (2019). AP-1 cFos/JunB/miR-200a regulate the pro-regenerative glial cell response during axolotl spinal cord regeneration. Communications Biology, 2(1), 1-13. https://doi.org/10.1038/s42003-019-0335-4
43. Samuels, A.K., Weisrock, D.W., Smith, J.J., France, K.J., Walker, J.A., Putta, S., & Voss, S.R. (2005). Transcriptional and phylogenetic analysis of five complete ambystomatid salamander mitochondrial genomes. Gene, 349, 43-53. https://doi.org/10.1016/j.gene.2004.12.037
44. Shaffer, H.B., & McKnight, M.L. (1996). The polytypic species revisited: genetic differentiation and molecular phylogenetics of the tiger salamander Ambystoma tigrinum (Amphibia: Caudata) complex. Evolution, 50(1), 417-433. https://doi.org/10.1111/j.1558-5646.1996.tb04503.x
45. Sibai, M., Parlayan, C., Tuğlu, P., Öztürk, G., & Demircan, T. (2019). Integrative analysis of axolotl gene expression data from regenerative and wound healing limb tissues. Scientific Reports, 9(1), 1-15. https://doi.org/10.1038/s41598-019-56829-6
46. Siler, C.D., Freitas, E.S., Yuri, T., Souza, L., & Watters, J.L. (2021). Development and validation of four environmental DNA assays for species of conservation concern in the South-Central United States. Conservation Genetics Resources, 13(1), 35-40. https://doi.org/10.1007/S12686-020-01167-3
47. Sites Jr, J.W., & Marshall, J.C. (2003). Delimiting species: a Renaissance issue in systematic biology. Trends in Ecology & Evolution, 18(9), 462-470. https://doi.org/10.1016/S0169-5347(03)00184-8
48. Smith, M.A., Poyarkov Jr, N.A., & Hebert, P.D. (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
49. Soriano-Lopez, M., Mota-Rojas, D., Iglesias, A.V., Ramirez-Necoechea, R., Olmos-Hernandez, A., Toca-Ramirez, J., & Alonso-Spilsbury, M. (2006). The Axolotl (Ambystoma mexicanum): Factors That Limit its Production and Alternatives for its Conservation. International Journal of Zoological Research 2(4), 362-368. https://doi.org/10.3923/ijzr.2006.362.368
50. Stamm, A., Strauß, S., Vogt, P., Scheper, T., & Pepelanova, I. (2018). Positive in vitro wound healing effects of functional inclusion bodies of a lipoxygenase from the Mexican axolotl. Microbial Cell Factories, 17(1), 1-9. https://doi.org/10.1186/s12934-018-0904-0
51. Zhang, J., Kapli, P., Pavlidis, P., & Stamatakis, A. (2013). A general species delimitation method with applications to phylogenetic placements. Bioinformatics, 29(22), 2869-2876. https://doi.org/10.1093/bioinformatics/btt499
52. Zhang, P., & Wake, D.B. (2009). Higher-level salamander relationships and divergence dates inferred from complete mitochondrial genomes. Molecular Phylogenetics and Evolution, 53(2), 492-508. https://doi.org/10.1016/j.ympev.2009.07.010