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Analysis of Nuclear Markers in Two Species with Highly Divergent Mtdna Lineages in Iceland

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Analysis of Nuclear Markers in Two Species with Highly Divergent Mtdna Lineages in Iceland Analysis of nuclear markers in two species with highly divergent mtDNA lineages in Iceland ITS in Crangonyx islandicus EF alpha in Apatania zonella May 2010 Sonia Sanz Muñoz LÍF016M Research project in biology for foreign students Teacher: Snæbjörn Pálsson University of Iceland Life and Environmental Sciences 1. Abstract This project is a study of variation in two nuclear markers in two arthropods (Crangonyx islandicus and Apatania zonella). Both species have been found to have highly divergent mtDNA lineages within Iceland. Crangonyx islandicus is an endemic groundwater amphipod species recently discovered in Iceland. Based on variation in mtDNA genes, COI and 16S RNA, Kornobis et al, (2010) concluded that this species had survived glaciations periods in sub-glacial refugia. The mtDNA variation defined several monophyletic groups, restricted to different geographic regions in Iceland and which have diverged in Iceland for up to 4 – 5 Million years. This was supported by a correlation between genetic and geographic distances among species. In this study we look at the internal transcribed spacer (ITS) gene, a piece of non-functional RNA situated between structural ribosomal RNAs (rRNA). The results show different patterns from the mtDNA results in Kornobis et al. (2010), with a major split between two populations, between north and southern Iceland, and different partition among samples in southern populations. The main difference is characterized by a large size difference of the ITS1 region due to insertion or deletion, a highly variable microsatellite was funs within this region. The second part of the project is based in the study of Apatania zonella, a caddisflie (Trichoptera), a circumpolar species which lives at high latitudes, in cold-clear water, streams, lakes and marshes. This study is a continuation of a previous study “Mitochondrial variation of the caddisflies Apatania zonella and Potamphylax cingulatus” (Sanz, 2010). The former study showed that Iceland acts as a zone of admixture, where two populations of A. zonella with distinct mtDNA types have arrived, from both ends of its range distribution, one from North America and the other from Europe. In this study we use a nuclear marker, EF alpha, in order to know whether the structure obtained by mtDNA in North America and Europe, is reflected in a nuclear marker and also to verify whether individuals in Iceland of different geographic origin as defined by mtDNA have interbred in Iceland. The results show less clear structure with EF alpha than found in mtDNA. Populations could be sexual or asexual depending on the country. Moreover, results show the rate of evolution of EF alpha is slower than COI’s rate. i 2. Table of contents 1. Abstract .............................................................................................................. i 2. List of contents .................................................................................................. ii 3. List of figures ................................................................................................... iv 4. List of tables ..................................................................................................... vi ITS in Crangonyx islandicus ................................................................................... 1 5. Introduction ....................................................................................................... 2 6. Material and methods ........................................................................................ 6 6.1. Samples .......................................................................................................... 6 6.2.Laboratory protocols ....................................................................................... 7 6.2.1. DNA extraction.................................................................................... 7 6.2.2. Polymerase Chain Reaction ................................................................. 7 6.2.3. Cloning ................................................................................................ 7 6.2.4. Sequencing........................................................................................... 9 6.2.5. Ethanol precipitation ............................................................................ 9 6.3.Sequence variation ........................................................................................ 10 7. Results ............................................................................................................. 11 7.1.Crangonyx islandicus ................................................................................... 11 7.2.Crymostygius thingvallensis ......................................................................... 22 7.3.Stygobromus stegerorum .............................................................................. 23 7.4.Unknow sequences ....................................................................................... 24 8. Discussion ....................................................................................................... 30 8.1.Crangonyx islandicus ................................................................................... 30 8.2.Crymostygius thingvallensis ......................................................................... 33 8.3.Unknow sequences ....................................................................................... 34 EF alpha in Apatania zonella ................................................................................ 35 9. Introduction ..................................................................................................... 36 10. Material and methods ...................................................................................... 37 10.1. Samples ............................................................................................... 37 10.2. Laboratory protocols .......................................................................... 39 10.2.1. DNA extraction.................................................................................. 39 10.2.2. Polymerase Chain Reaction ............................................................... 39 10.2.3. Exosap reaction .................................................................................. 39 ii 10.2.4. Cloning .............................................................................................. 39 10.2.5. Sequencing......................................................................................... 40 10.2.6. Ethanol precipitation .......................................................................... 40 10.3. Sequence variation .............................................................................. 41 11. Results ............................................................................................................. 42 12. Discussion ....................................................................................................... 45 13. Acknowledgements ......................................................................................... 47 14. References ....................................................................................................... 48 15. Appendix ......................................................................................................... 50 iii 3. List of figures ITS1 in Crangonyx islandicus Figure 1. Amphipods anatomy ................................................................................ 3 Figure 2. Sampling locations of groundwater amphipods in Iceland ...................... 4 Figure 3. Organization of the rRNA gene ............................................................... 4 Figure 4. Samples sites of C. islandicus in Iceland ................................................. 6 Figure 5. Map of pNEB206A vector. ...................................................................... 8 Figure 6. Location of the primers used in PCR and sequencing ............................ 9 Figure 7. Agarose gel electrophoresis of DNA fragment of C. islandicus ........... 11 Figure 8. Schematic figure of the 18S rRNA gene, ITS1, 5.8S rRNA gene, ITS2 and 28S rRNA gene ............................................................................... 12 Figure 9. Part of the sequence where number 1, 2 and 3 are shown the insert and the original sequences ................................................................... 12 Figure 10. Network based on 300bp insert within South population ................... 15 Figure 11. Geographical patterns according to the number of repeats ................. 16 Figure 12. ITS1 allele’s network .......................................................................... 18 Figure 13. Geographic distribution of the alleles groups ...................................... 18 Figure 14. ITS2 allele’s network ........................................................................... 19 Figure 15. Allele’s netwrol based on the complete sequence .............................. 20 Figure 16. Network based on part of 28S rRNA gene ......................................... 23 Figure 17. Agarose electrophoresis gel of DNA fragment using ITSF/28SR ...... 24 Figure 18. Network of phylogeny based on 5.8S rRNA gene sequence ............... 28 Figure 19. Schematic figure of how could appear the duplication ........................ 30 Figure 20. Hypothesis of how duplication appears in lineage ............................. 31 Figure 21. Bayesian phylogenetic
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