mtActinopterygii: Analysing evolution of mitogenomes belonging to the most dominant class of vertebrates Sevgi Kaynar1, Esra Mine Ünal1, Tuğçe Aygen1, Emre Keskin1 1 Evolutionary Genetics Laboratory (eGL), Department of Fisheries and Aquaculture, Agricultural Faculty, Ankara University, Ankara, Turkey Mitochondrial DNA data has widely been used for phylogenetic and population genetic analyses and becomes a potential tool in our understanding of vertebrate evolution regarding its properties such as maternal inheritance, low effective population size, lack of recombination and high evolutionary rates compared to the nuclear DNA. The main complication underlying in mitochondrial analyses are mostly based on substitution rates of the extreme variations among different sites and difficulties in estimation of pairwise distance related with parallel mutations, which makes phylogenetic inferences controversial. In order to enhance the basic information gathered from mitochondrial DNA studies of vertebrate evolution, we focused on the global mtDNA diversity in Actinopterygii (ray-finned fishes) which is known as the most dominant class of vertebrates compromising nearly 99% of 33.888 fish species. Thirteen protein coding genes and two ribosomal RNA genes of 348 families with diverse origins were analysed to provide a concurrent view on vertebrate evolution. mitochondria & actinopterygii § INTRODUCTION analysing the mitochondrial genome § METHODS Sequences from 2 ribosomal RNA’s and 13 protein coding genes were used to construct the phylogenetic tree of the Actinopterygii. Sequences for all genes for species assigned to the class Actinopterygii were downloaded from Genbank (accessed June 2016). Sequences for all genes were aligned using the MAFFT v1.3 Biomatters Ltd plugin in Geneious v6.1.6. Sequences were used to build a linked gene tree in BEAST v 1.8.0. The appropriate nucleotide substitution model for each gene was determined using jModeltest v2.1.4, using Akaike information criterion model selection. Incomplete sampling was accounted for by a state-dependent sampling factor. We performed model simplification with MuSSE in a maximum likelihood framework based on likelihood ratio tests. We verified these results using MuSSE and our MCC tree. MuSSE allows us to associate changes in speciation with multiple character states and has been developed to incorporate the effects of incomplete taxonomic sampling. MuSSE was implemented using the R package Diversitree. Phylogenetic and statistical analyses were conducted using MEGA 7. results from phylogenetic and statistical analyses § Table 1   12S rRNA 16S rRNA ND1 ND2 COI COII ATP8 ATP6 COIII ND3 ND4L ND4 ND5 ND6 CYTB Average base pairs 950 1688 959 1046 1552 691 168 684 785 349 297 1381 1837 522 1141 Conserved* 232/1493 372/2929 200/1071 120/1156 559/1911 86/979 29/301 96/748 247/792 72/360 34/303 211/1476 113/2154 61/763 275/1180 Variable* 1147/1493 2134/2929 770/1071 1008/1156 1344/1911 678/979 216/301 612/748 541/792 282/360 263/303 1199/1476 2001/2154 644/763 886/1180 Parsimony informative* 921/1493 1740/2929 680/1071 891/1156 934/1911 514/979 181/301 547/748 480/792 248/360 244/303 1065/1476 1746/2154 516/763 775/1180 T 21 21.3 28.1 25.6 29.7 27.4 26 29.3 27.9 30.8 28.3 27.6 27.3 15.3 29.1 C 26.1 24.7 31 33.4 26.9 31.2 31.1 32.9 30.4 30.2 33 30.3 A 33.8 25.4 25 28.7 31.3 25.2 22.7 23.1 27.1 28.5 38.2 G 21.8 20.2 15.5 13.1 18.4 16.4 11.5 13.4 17.3 15.4 15.7 14.9 13.9 13.5 15.2 GC 47.9 44.9 46.5 45.3 43.8 42.7 44.6 47 48.6 44.1 45.5 si 105 191 131 159 182 90 28 98 91 54 42 200 269 82 152 sv 74 164 124 194 144 69 30 80 49 206 283 97 138 R=si/sv 1.4 1.2 1.1 0.8 1.3 0.9 1 Substitution Model GTR+G+I (TN93+G+I) GTR+G+I (TN93+G) GTR+G+I (TN93+G+I) GTR+G (TN93+G+I) TN93+G+I (GTR+G+I) GTR+G+I (HKY+G+I) GTR+G (HKY+G+I) Pairwise Distance 0.148 0.169 0.415 0.624 0.302 0.386 0.728 0.539 0.325 0.523 0.492 0.505 0.627 0.64 0.409 Standard Error 0.013 0.008 0.021 0.035 0.027 0.034 0.188 0.037 0.026 0.055 0.043 0.023 0.044 0.053 0.019 Total Major Insertions 23 7 2 24 3 6 5 11 Total Major Deletions 58 57 32 50 4 16 52 48 Max In+Del per Single Species 15 Porichthys myriaster 1+25 Tetrabrachium ocellatum 0+28 Triacanthus biaculeatus 10+10 Trichomycterus areolatus 10+11 Acanthurus leucosternon 5+10 Zu cristatus 0+14 P. myriaster T. biaculeatus A. leucosternon T. areolatus T. ocellatum Z. cristatus Size of the mitogenomes show a great deal of variance from 15.725 base pairs (Drepaneids; Drepane punctata) to 23.152 base pairs (Protanguillidae; Protanguilla palau) long. Most conserved regions were found out to be 12S and 16S rRNA with a genetic distance of 0.148 and 0.169 for respectively. Cytochrome c oxidase I gene of the four armed frogfish from Tetrabrachiidae family consist 28 major deletions which is highly unexpected for this region known as the barcoding gene. Detailed results of analysed genes were given in Table 1 and phylogenetic patterns were given in Figure 1. The main difference between different levels of pairwise distance could be correlated with the evolutionary mutation rate of the genes; in which protein coding 13 genes found to be much more diverse than two ribosomal RNA’s. The main nucleotide substitution model calculated for the most (14/15) of the genes was General Time Reversible. Non-uniformity of evolutionary rates among sites may be modelled by using a discrete Gamma distribution (+G) and by assuming that a certain fraction of sites are evolutionarily invariable (+I). ATP8 was calculated as the most diversified gene among specimens but this is mainly biased towards having a short (168 bp) nucleotide sequence. Dispersion patterns pointing out a more stable structure in cytochrome oxidase genes, especially in COI which is known as DNA barcode region in animals because of this attribute. Species with the highest number of InDel’s are known to be so called «ancient» species and this occurrence could be synchronised with their molecular clock. evolution of the ray-finned fishes § RESULTS & DISCUSSION ND2 12S rRNA 16S rRNA ND1 COII ATP8 ATP6 COI COIII ND4 ND5 ND3 ND4L ND6 CYTB Diversification patterns of phylogenetic trees derived from mitochondrial genome § Figure 1