Dear all, I'm happy to post the replies I got to my question about mt-tRNAs as markers for population genetics. Thanks to all for your help! Chiara Papetti chiara.papetti@unipd.it ----- My original question: Has anyone ever used mitochondrial tRNA sequences to investigate population differentiation or species delimitation? I have some tRNA sequences resulting from the sequencing of a mitochondrial gene and I was wondering if they could be informative in terms of population structure and species difference. What strategies and methods have worked (or not) in your experience? Replies I got: I do not think that mtDNA is very wise for population genetics analyses, for different good reasons: Bazin, E., Glemin, S., Galtier, N., 2006. Population size does not influence mitochondrial genetic diversity in animals. Science 312, 570-572. Galtier, N., Jobson, R.W., Nabholz, B., Glémin, S., Blier, P.U., 2009. Mitochondrial whims: metabolic rate, longevity and the rate of molecular evolution. Biol. Let. 5, 413-416. Gerber, A.S., Loggins, R., Kumar, S., Dowling, T.E., 2001. Does nonneutral evolution shape observed patterns of DNA variation in animal mitochondrial genomes? Annu. Rev. Genet. 35, 539-566. Papadopoulou, A., Anastasiou, I., Vogler, A.P., 2010. Revisiting the insect mitochondrial molecular clock: the mid-Aegean trench calibration. Mol. Biol. Evol. 27, 1659'1672. Microsatellite markers will be much more informative and accurate ----- Mitochondrial tRNAs tend to be highly conserved within species and between closely related species (they're less variable than mitochondrial protein-coding genes, for example), but you might get lucky and find a few variable sites that could be informative. ----- To my knowledge the mitochondrial tRNA are not showing polymorphism. I sequenced one tRNA between COI and COII for hundreds of individuals and did not find any polymorphism in this region. If you find some, I will be interested by this. ----- One suggested reference: Stauffer C. A molecular method for differentiating sibling species within the genus /Ips/. In: Gregoire J.C, Liebhold A.M, Stephen F.M, Day K.R, Salom S.M, editors. Proceedings: integrating cultural tactics into the management of bark beetle and reforestation pests. USDA Forest Service General Technical Report NE-237. USDA North East Research station; Syracuse, NY: 1997 ----- In my opinion, you should use your tRNA sequences just like any other (haploid) marker, preferably keeping it attached to the neighboring mitochondrial gene you were targeting for sequencing (since these markers are linked, it makes no sense to analyze them separately). See Fontaneto et al. (2015) Guidelines for DNA taxonomy, with a focus on the meiofauna. Marine Biodiversity, 45:433'451 (https://doi.org/10%2Ff7sds6) for a review of the different approaches you could use for species delimitation. ----- tRNAs and tRNA "pseudogenes" can be very useful, as different regions are subjected to very different selection pressures. So, when using them, you should make very thorough investigations of the sequence blocks you end up using and the secondary structures. Check out my paper on myctophid Met tRNAs genes and their duplicates - a trait of myctophids in particular. They proved rather useful for phylogeny and species delimitation. Ref: Poulsen JY, Byrkjedal I, Willassen E, et al. Mitogenomic sequences and evidence from unique gene rearrangements corroborate evolutionary relationships of myctophiformes (Neoteleostei). /BMC Evol Biol/. 2013;13:111. Published 2013 Jun 3. doi:10.1186/1471-2148-13-111 ----- I used chloroplast DNA for population genetic and phylogenetic studies in horsetails (genus /Equisetum/). In these species, intra-population polymorphism seems very small, likely the consequence of clonal reproduction. For phylogenetic purpose, I was able to use intergenic non coding chloroplast DNA sequences which gave good results.  See attached papers. The primers used for chloroplast DNA amplification are often anchored within the tRNA genes, because these sequences are quite conserved. You will find some sequences of tRNA genes in this paper : Des Marais DL, Smith AR, Britton DM, Pryer KM (2003) Phylogenetic relationships and evolution of extant horsetails,  Equisetum, based on chloroplast DNA sequence data (rbcL  and  trnL-F). Int J Plant Sci 164:737'751 You will also find interesting literature about chloroplast DNA variations and their use for population genetic studies in trees in late 90's papers by Remy J Petit at Bordeaux, France : https://www6.bordeaux-aquitaine.inra.fr/biogeco/Personnel/Annuaire/M-P/Petit-Remy/Liste-complete ----- Chiara UniPD