November 8–9, 2017, Brno, Czech Republic 24 years NUCLEAR GENES CARBAMOYL PHOSPHATE SYNTHETASE AND ELONGATION FACTOR-1α AS TOOL FOR IDENTIFICATION OF INTRASPECIFIC GENE VARIATION IN CASE OF LIME HAWK-MOTH (MIMAS TILIAE) TAMARA MIFKOVA, ALES KNOLL, JAN WIJACKI Department of Morphology, Physiology and Animal Genetics Mendel University in Brno Zemedelska 1, 613 00 Brno CZECH REPUBLIC [email protected] Abstract: Within the distributional areal of Lime Haw-Moth (Mimas tiliae) probably exist several subspecies, some of them are already described (M. t. kitchingi). The aim of this pilot study was to verify usability of the nuclear genes CAD and EF-1α by the DNA barcoding method to distinguish between morphologically different subpopulations and that the DNA sequence of this genes corresponds to its geographic distribution. We have demonstrated that these CAD and EF-1α gene fragments are suitable for species identification, but not for subspecies differentiation. Relation to geographical distribution was not confirmed for CAD gene, but in case of EF-1α gene such a relationship can be seen. Key Words: sphingidae, DNA barcoding, biodiversity, hawk-moth, nuclear genes INTRODUCTION Biodiversity is an essential element to protect life on the Earth. We know three main categories of biodiversity: gene diversity, species diversity and the complex ecosystem diversity. There are estimated 10 million plant and animal species, but only about 1.5 million are described (and more than 1 million are insects) in the World. DNA barcoding is taxonomical method using a very short DNA sequences from standard part of the genome and alignmenting them with sequencing databases. Can be easily be used even for species description (e.g. first vertebrate discovered via DNA barcoding was Coryphopterus kuna Victor 2007). This is method can be useful in cases where species are cryptic and morphologically undistinguishable. For these cases, there are now procedures and methods of molecular analysis to help scientists simplify, accelerate and refine the determination of individual species and possibly discover new species. Molecular taxonomy is a species classification system based on molecular analyses. These are primarily based on the testing of nuclear and mitochondrial DNA sequences in the case of animals and the comparison of sequences of individual genes among the species studied (Mayer et al. 2007). The main goal of molecular taxonomy is to elucidate the molecular relationships between organisms, while DNA barcoding deals primarily with the identification of unknown specimens (Kress et al. 2005). In animals, mitochondrial gene fragments for the cytochrome c oxidase (COI) subunit 1 (Krishnamurthy and Francis 2012) are most commonly used for identification, but the purpose of this study was to investigate CAD (carbamoyl phosphate synthetase) and EF-1α (elongation factor-1α) nuclear genes. To analyse the sequence results of these genes, online accessible bioinformatical tool such as BLAST (Basic Local Alignment Search Tool – available at http://blast.ncbi.nlm.nih.gov/Blast.cgi) were used. We choose Mimas tiliae (Linaeus 1758) as a model species for our study because it very large distributional areal (whole Western Europe, most Russia, Turkey, part of Iran) with strange distributional pattern in the east (see Pittaway 1997). There were described one species from the Far East (Mimas christophi Staudinger 1887) and one species from Elburz, Iran (Mimas kitchingi Melichar and Řezáč 2015), which is by some authors considered as a subspecies of M. tiliae. Our aim was to see how looks the genetic diversity within the species distributional areal and make an outlook to the subspecies taxonomy. Although there were published morphological differences, these could be because the species variability within the areal. 718 November 8–9, 2017, Brno, Czech Republic 24 years MATERIAL AND METHODS Species All samples for analysis were provided by staff at the Sphingidae Museum (Orlov, Czech Republic). Samples were divided into group based on morphological structure of the male genitalia: group 1 (F.24, F.27, F.28, F.46, F.82, F.83) and group 2 (F.16, F.31, F.35, F. 37, F.39, F.74, F.75, F.76, F.77, F.78, F.79, F.80, F.81). The distribution of individuals is listed in Table 1. These were the butterfly limbs, supplied with the exact species designation, the sample number, and the capture point of the butterfly that belonged to the limb. Isolation was performed using the Geneaid Genomic DNA Mini Kit designed to isolate DNA from the tissues and proceed according to the enclosed instructions. The amplification of the CAD and EF-1α nuclear genes was standardized according to protocols available online (www.top-bio.cz/files/1166_pl.pdf). Table 1 Samples of Mimas tiliae divided into two groups Group Sample number Geographical origin 1 F.24 Greece 1 F.27 Italy 1 F.28 Greece 1 F.46 Switzerland 1 F.82 Greece 1 F.83 Greece 2 F.16 Greece 2 F.31 Russia (Altai) 2 F.35 Kazakhstan 2 F.37 Finland 2 F.39 Russia 2 F.74 Czech republic 2 F.75 Czech republic 2 F.76 Czech republic 2 F.77 Czech republic 2 F.78 Czech republic 2 F.79 Northern Ural 2 F.80 Northern Ural 2 F.81 Northern Ural DNA amplification All PCRs were performed in the thermal cycler ABI Verity 96 Well (Applied Biosystems Inc., Foster City, CA, USA) with the following protocol: initial denaturation at 95 °C for 3 min; 30 cycles of denaturation at 95 °C for 40 s, annealing at 59 °C for 40 s and elongation at 72 °C for 1 min; final elongation at 72 °C for 10 min and holding at 4 °C. DNA Sequencing and Data Analysis The sequencing reaction mixture was mixed with a commercially available BigDye Terminator v3.1 Cycle Sequencing Kit from Applied Biosystems, and the mixture was mixed to a volume of 10 μl. The temperature cycling profile was 96 °C for 1 min, 25 cycles of 96 °C for 10 s, 50 °C for 5 s, 60 °C for 4 min. We then purified using DNA BigDye® Xterminator ™ Purification Kit from Applied Biosystems according to the instructions from the manufacturer. The data were analysed using and SeqScape Software v2.7 (Applied Biosystems Inc., Foster City, CA, USA) and MEGA7 to compare different bases and phylogenetic trees, which is freely available at http://www.megasoftware.net/. The evolutionary history was inferred using the Minimum Evolution method (Rzhetsky and Nei 1992). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al. 2004) and are in the units of the number of base substitutions per site. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm (Nei and Kumar 2000) at a search level of 1. The Neighbor-joining algorithm (Saitou and Nei 1987) was used to generate the initial tree. The analysis of CAD and EF-1α genes involved 13 and 19 nucleotide sequences, resp. All positions containing gaps and missing data were eliminated. There were in CAD and EF-1α genes a total of 675 and 563 positions in the final dataset, resp. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2015). 719 November 8–9, 2017, Brno, Czech Republic 24 years RESULTS AND DISCUSSION Amplification of the CAD and EF-1α gene fragments Primers were designed using the OLIGO v4.0 software. For CAD amplification, CAD_F1 + CAD_R1 primer combinations proved best. This combination of primers amplified a fragment of 708 bp in size. In the case of the EF-1α gene, the most suitable combinations of EF1α-F2 + EF-1α-R2 primers were shown to amplify 602 bp fragments. Table 2 Primers used for detection of CAD and EF-1α genes PCR Lenght GC Gene Primers Sequence 5'-3' fragment [bp] [%] [bp] CAD_F1 TGG AAG TTC TAT GAA RAG TGT CG CAD 23 43.5 708 CAD_R1 AAG TAC AAT CTG TCR CTC ATG TC EF-1α_F2 CTC CTG GAC ACA GAG ATT TCA TCA A 44 EF-1α 25 602 EF-1α_R2 CAC AGA CTT GAC TTC AGT GGT GAT G 48 Phylogenetic analyses Phylogenetic trees were created using the MEGA7 program and was used a minimal evolution method that divided individual samples into groups according to differences in individual nucleotides. Figure 1 Evolutionary relationships of studied taxa using the Minimum Evolution method by CAD gene (The optimal tree with the sum of branch length = 0.10648939 is shown). In the case of the CAD gene, groups 1 and 2 were not demonstrably split. From this we can assume that nuclear CAD gene is not suitable for the type of distinction representatives Mimas tiliae. Eastern and Western subpopulation also failed to distinguish. This result, however, is in contradiction with the work of Friedlander et al. which in 1992 introduced this nuclear gene as a candidate and Moulton and Wiegmann 2004, which states that the CAD gene (CPS) is suitable for phylogenetic examination of the Lepidoptera order. For this reason, it would be advisable to carry out an extended study that would include more species of the genus Mimas. EF-1α gene There is little bit different situation in case of EF-1α. The division into two different morphological groups has not been confirmed by EF-1α DNA analysis too, as in the case of CAD gene Although we have been able to divide all samples to two clades, but the position of the particular species 720 November 8–9, 2017, Brno, Czech Republic 24 years is questionable, because we have no this information (if samples from the Czech Republic are from west of east of the country). This is very important information because the position of the country on the boundary of two main populations – eastern and western, according the postglacial re-colonization history confirmed for many other species (e.g.