replying to: N. Diaz-Arce; Scientific Reports https://doi.org/10.1038/s41598-022-26720-y (2023).
Diaz-Arce and Rodriguez-Ezpeleta1 discuss the utility of barcoding using best-match criterion for the identification of fish species from commercial samples. Focusing on tuna, the evidence provided is a phylogenetic tree containing six problem mitochondrial control region sequences of Blanco-Fernandez et al.2 and 21 reference sequences taken from databases: 8 Thunnus thynnus, 5T. thynnus with T. alalunga introgression, 7T. alalunga, 1T. albacares. Three problem sequences (MW557512, MW557513 and MW557514) identified as T. thynnus from best BLAST hit2 clustered together with the T. alalunga references and only one T. thynnus reference, while the rest of T. thynnus were in other clades. This tree poses doubts about the species status of those three samples barcoded as T. thynnus, questioning the conclusions obtained from barcoding. However, with a few references per species it is very difficult to capture the variation of the control region in T. thynnus and T. alalunga. The results can vary depending on the references selected from GenBank to reconstruct the tree. As a proof of this we produced a tree adding three more references to the sequences employed by Diaz-Arce and Rodriguez-Ezpeleta1, with the same parameters and testing the best-fit evolutionary model. In the resulting tree (Fig. 1) the cluster that contains the three problem sequences has now four T. thynnus references: AY650502, AY699942, AY699946 and EU562888 (Table 1). AY650502 (haplotype BFT94 in Alvarado Bremer et al.3) comes from a T. thynnus voucher specimen identified as introgressed with T. alalunga3. AY699942 and AY99946 correspond to T. thynnus sampled for population genetics4,5. EU562888 belongs to an individual morphologically identified as T. thynnus used in a study of population genetics in this species6. From this tree the problem sequences MW557512, MW557513 and MW557514, assigned to T. thynnus from barcoding, can be interpreted as belonging to introgressed T. thynnus. Alternatively, we could treat them as T. alalunga, although the best match in BLAST was T. thynnus6 in the three cases2.
Maximum Likelihood tree built from problem sequences from Blanco-Fernandez et al.2 (MW557511-16) and reference tuna sequences from GenBank.
BLAST-based barcoding with mitochondrial markers has been employed alone to identify hakes, monkfish, tunas, catfish, and many other fish species using best-match criterion7,8,9,10. However, the reasonable doubt that arises from Fig. 1 cannot be solved without nuclear markers. Phylogenetic studies show T. thynnus has haplotypes corresponding to T. alalunga mtDNA3, while the reciprocal introgression of T. thynnus mitochondrial DNA in albacore has not been detected3,11. Nuclear markers such as the first internal transcribed spacer (ITS) within the nuclear rDNA could be employed to solve this ambiguity. This marker has been already used to distinguish between T. alalunga and T. thynnus12, and would allow to confirming T. thynnus issued from former hybridization events involving T. alalunga females.
In summary, we recognize that the assignation of problem sequences to Thunnus thynnus in Blanco-Fernandez et al.2, although supported from barcoding and not rejected from phylogenies, should be validated employing nuclear markers. This can be extended to the rest of cases where interspecific introgression occurs. Finally, we have renamed the problem sequences reported in Blanco-Fernandez et al.2 including the Open Nomenclature qualifier cf. (= the identification is not achievable without further comparison with reference material13) before the species name, stating in their description that they were obtained from seafood samples (see Accession numbers MW557511-MW557516). This way we hope to reduce the noise in databases commented by Diaz-Arce and Rodriguez-Ezpeleta1.
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Funding
This study was supported by the Spanish Ministry of Science and Innovation, Grant GLOBALHAKE PID2019-108347RB-I00.
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E.G-V. and A.A. wrote the manuscript; C.F-B. developed the phylogenetic tree; G.M-S. revised and improved the manuscript, that was checked and approved by the four authors.
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Blanco-Fernandez, C., Ardura, A., Machado-Schiaffino, G. et al. Reply to: Best BLAST hit alone cannot be used as evidence of fraud. Sci Rep 13, 914 (2023). https://doi.org/10.1038/s41598-022-26737-3
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DOI: https://doi.org/10.1038/s41598-022-26737-3
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