1. „ NIPs: Analysis of unsequenced holometabolic groups “ Bled, Slovenia, Feb 18, 2008 Carina Eisenhardt „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics

2. INTRODUCTION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Reconstruction of the organismal evolutionary tree based mainly on analysis of molecular sequences  insufficient to reliably resolve in deep branches Novel class of phylogenetic marker:  NIP (near intron pair)  derived by insertion of a novel intron less than 50 nt away from an evolutionary older intron  small distance should exclude the coexistence of both introns  possible to characterize one of the introns as ancient (plesiomorphic) and the other as novel (derived or apomorphic)  gain of the new intron nearly always associated with the loss of the old intron 1/19

3. INTRODUCTION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Suitability of this marker class demonstrated by analysis of the relatedness of holometabolic insects:  Hymenoptera + (Coleoptera + (Diptera + Lepidoptera)) - theory Tribolium has synapomorphic intron positions with other holometabolic orders Apis has plesiomorphic intron positions Resulting phylogenetically informative intron distributions are investigated in representative species of other holometabolic insect orders  expansion of the genome-scaled studies of holometabolic insects 2/19

4. INTRODUCTION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Additional to Hymenoptera and Coleoptera  other groups are involved in the basal evolutionary splitting event of Holometabola:  Neuropteroidea, Strepsiptera, Siphonaptera and Mecoptera Büning (2005) ? ? ? ? 3/19

5. INTRODUCTION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Investigation of the distribution of phylogenetic informative intron position pairs in:  Mecoptera: Panorpa spec.  Megaloptera: Sialis spec.  Neuroptera: Chrysoperla carnea  Coleoptera:- Tenebrio molitor - Leptinotarsa decemlineata, Agelastica alni - Harmonia axyridis, Coccinella septempunctata - Rhagonycha fulva - Phyllopertha horticola - Phosphuga atrata - Hydaticus seminiger - Amara aenea, Harpalus affinis Neuropteroidea 4/19

6. INTRODUCTION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Side result of our analysis: Tribolium-specific introns  Determination of the relative age of the intron position  Investigation: hypothetical sister group relationship Coleoptera+Neuropterida   Alternatively: group consisting of Hymenoptera, Neuropterida, Mecoptera and Siphonaptera as sister group to all other Holometabola (Whiting 2002)   Sister group relationship Coleoptera + Neuropterida relatively weakly supported (Büning 2005)  via Tribolium-specific introns  Evaluation of these hypotheses:  Evidence for the Coleoptera + Neuropterida group:  Neuropterida show apomorphic intron positions of Tribolium  5/19

7. INTRODUCTION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Side result of our analysis: Tribolium-specific introns  Determination of the relative age of the intron position  Investigation: hypothetical sister group relationship Coleoptera+Neuropterida   Alternatively: group consisting of Hymenoptera, Neuropterida, Mecoptera and Siphonaptera as sister group to all other Holometabola (Whiting 2002)  Sister group relationship Coleoptera + Neuropterida relatively weakly supported (Büning 2005)  via Tribolium-specific introns  Evaluation of these hypotheses:  Evidence against the Coleoptera + Neuropterida group:  Neuropterida show always plesiomorphic intron positions of hymenopterans instead of apomorphic positions of Tribolium  Inclusion of Apocrita-specific intron positions to investigate the alternative grouping 5/19

8. METHODS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Methods DNA isolation from representative specimens  PCR analysis with genomic DNA (Investigation of orthologous sequences surrounding the determined NIP which contained apomorphic intron positions of Tribolium) Derivation of degenerate primers  genomic PCR (Touch-Down-PCR) Derivation of nested degenerate primers  genomic PCR (Nested-PCR)  Cloning and sequencing of the resulting PCR fragments  Comparison of genomic DNA and protein  Intron positions for phylogenetic analysis 6/19

9. METHODS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Derivation of degenerate primers Derivation of degenerate primers from nearby conserved coding sequences  using the corresponding protein alignment For example: gene fragment 66553024 3024-3 3024-1 3024-2 3024-4 133-1146-2 Part of alignment (MacVector) Degenerate primer: - 5` clamp region = Tribolium sequence - 3` degenerate core region 7/19

10. METHODS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Up to now we received 12 Tribolium-specific introns by our analysis  Investigation of 5 gene fragments (the rest was too weakly conserved )  66563882:  66542088:  66553024:  66534441:  48096763: 3882-1 3882-3 3882-2 NIP 2088-3 2088-1 2088-4 2088-2 2088-5 NIP 3024-33024-13024-23024-4 4441-34441-1 4441-4 4441-2 6763-2 6763-3 6763-1 Degenerate primers for each gene fragment NIP 8/19

11. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Results: gene fragment 66526442 (eIF2  ) Intron positions: Clytus 295-0 (synapomorphic) / Apis:289-0 (plesiomorphic) Phylogenetic tree with intron positions Insertion of 295-0  300-320 Mya Coleoptera Neuro- pterida Meco- pteroidea support Holometabola (inclusive of Neuropterida) exclusive Hymenoptera Intron 295-0 = synapomorphic character ? ? ? ??? ? ? ? ? ?= no sequence available x= no intron exist x xx 9/19

12. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Results: gene fragment 66526442 (eIF2  ) Intron positions: Tribolium: 160-1 (synapomorphic) / Apis: 159-1 (plesiomorphic) Phylogenetic tree with intron positions Insertion of 160-1  300-320 Mya Coleoptera Neuro- pterida Meco- pteroidea Intron 160-1 might have originated by sliding of the intron 159-1 support Coleoptera + Mecopteroidea ?? ? ? ????? ? ? ? ? ?= no sequence available x= no intron exist 10/19

13. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Results: gene fragment 66563882 (TFIIH) Intron positions: Tribolium: 110-0 / Apis: 119-2 (plesiomorphic) Phylogenetic tree with intron positions Insertion of 110-0  290 Mya Coleoptera Neuro- pterida Meco- pteroidea support the sister group relationship Coleoptera+ Neuropterida Insertion of 110-0 = occur after splitting up (Coleoptera + Neuropterida) from Mecopteroidea ? ? ? ? ? ? ?? ? x x xx ?= no sequence available x= no intron exist 11/19

14. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Results: gene fragment 66563882 (TFIIH) Intron positions: Tribolium: 165-1 (synapomorphic) / Apis: 172-0 (plesiomorphic) Phylogenetic tree with intron positions Insertion of 165-1  300-320 Mya Coleoptera Neuro- pterida Meco- pteroidea Confirmation: Intron 165-1 = synapomorphic character support Coleoptera + Mecopteroidea ? ?? ? ? ? ? ?? x x x x x ?= no sequence available x= no intron exist 12/19

15. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Results: gene fragment 66553024 Intron positions: Tribolium: 146-2 / Apis: 133-1 (plesiomorphic) Insertion of 146-2  ? -290 Mya Phylogenetic tree with intron positions Coleoptera Neuro- pterida Meco- pteroidea Intron 146-2 seems very young  probably long space of time between intron loss and gain Insertion of 146-2 = occur after splitting up (Coleoptera + Neuropterida) from Mecopteroidea ? ?? ? ? ? ? ? ?x x x xxx x ?= no sequence available x= no intron exist 13/19

16. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Results: gene fragment 66542088 Intron positions: Tribolium: 590-1 / Apis: 604-2 (plesiomorphic) Phylogenetic tree with intron positions Insertion of 590-1  210 -285 Mya Coleoptera Neuro- pterida Meco- pteroidea Case of intron migration? Insertion of 590-1 = occur after splitting up Coleoptera from Neuropterida ? ? ? ? ? ? ? ? ? x x xx ?= no sequence available x= no intron exist = Intron 591-1 14/19

17. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Results: gene fragment 66542088  Intron positions: Apis: 604-2 / Tribolium: 590-1 / Leptinotarsa: 591-1   Up to now intron position 590-1 was only found in Tribolium and 591-1 only in Leptinotarsa  One of these introns resulted from insertion of a novel intron  The other might have originated by intron migration (intron sliding) require some convergent base substitutions based on structure of splice sites, intron sliding might have occurred most probably at positions spaced by one or three nucleotides  Question: Which is the older position? 15/19

18. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Results: gene fragment 48096763 Intron positions: Tribolium: 490-0 / Apis: 505-2 (plesiomorphic) Insertion of 490-0  220 -255 Mya Phylogenetic tree with intron positions Coleoptera Neuro- pterida Meco- pteroidea Intron position 490-0 = Intron of the Polyphaga ?? ? ? ?? x ?= no sequence available x= no intron exist 16/19

19. DISCUSSION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Summary Introns not very recent  the youngest introns investigated:  66542088: Intron 590-1 or 591-1 might have originated by intron migration  one of these introns could be younger than 210 Mio years  48096763: intron 490-0 = intron of the Polyphaga  intron could be younger than 255 Mio years In 1 case (TFIIH: 110-0/119-2)  Neuropterida specimens show apomorphic intron positions of Tribolium  evidence for the Coleoptera + Neuropterida group 17/19

20. DISCUSSION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Summary 18/19 Results support: Büning (2005)

21. DISCUSSION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Further investigations Expansion of the genome-scaled studies of holometabolic insects  Inclusion of Strepsiptera and Siphonaptera Bioinformatics Group (Prof. Stadler): development of tools for automatic gene structure annotation and orthologous intron extraction  automated NIP analysis  side result: further Tribolium-specific introns  investigation  Up to now investigation of Tribolium-specific introns, where at least Diptera or Lepidoptera has the plesiomorphic intron  also interesting: Tribolium-specific introns, where Diptera and Lepidoptera are intronless 19/19

22. Acknowledgements Dr. Veiko Krauss Department of Genetics Christian Thümmler Institute of Biology II Franziska Georgi University Leipzig Prof. Peter Stadler Bioinformatics Group Jörg Lehmann Department of Computer Science University Leipzig Thanks for your attention !

23. INTRODUCTION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt Universität Leipzig, Lehrstuhl für Genetik Methods (1) DNA and RNA isolation from representative specimens  PCR analysis with cDNA and genomic DNA (Investigation of orthologous sequences surrounding the determined NIP which contained apomorphic intron positions of Tribolium) Derivation of degenerate primers  RT-PCR Derivation of non-degenerate primers based on the cDNA  genomic PCR (Touch-Down-PCR)  Cloning and sequencing of the resulting PCR fragments  Comparison of genomic and cDNA  Intron positions for phylogenetic analysis

24. INTRODUCTION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt Universität Leipzig, Lehrstuhl für Genetik Investigation of the distribution of phylogenetic informative intron position pairs in:  Mecoptera: Panorpa spec.  Panorpidae  Megaloptera: Sialis spec.  Sialidae  Neuroptera: Chrysoperla carnea  Chrysopidae  Coleoptera:- Tenebrio molitor  Tenebrionidae - Leptinotarsa decemlineata, Agelastica alni  Chrysomelidae - Harmonia axyridis, Coccinella septempunctata  Coccinellidae - Rhagonycha fulva  Cantharidae - Phyllopertha horticola  Scarabaeidae - Phosphuga atrata  Silphidae - Hydaticus seminiger  Dytiscidae - Amara aenea, Harpalus affinis  Carabidae

25. INTRODUCTION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt Universität Leipzig, Lehrstuhl für Genetik Side result of our analysis: Tribolium-specific introns  Determination of the relative age of the intron position  Investigation: hypothetical sister group relationship Coleoptera+Neuropterida   Alternatively: group consisting of Hymenoptera, Neuropterida, Mecoptera and Siphonaptera as sister group to all other Holometabola (Whiting 2002)   Sister group relationship Coleoptera + Neuropterida relatively weakly supported (Büning 2005)  by Tribolium-specific introns  Evaluation of these hypotheses:  Evidence for the Coleoptera+Neuropterida group:  Neuropterida will show apomorphic intron positions of Tribolium  Evidence against the Coleoptera+Neuropterida group:  Neuropterida will show pleiotrophic intron positions of hymenopterans  Inclusion of Apocrita-specific intron positions to investigate the alternative grouping 

26. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt Universität Leipzig, Lehrstuhl für Genetik Results: gene fragment 66526442 (eIF2  ) Intron positions: 295-0 (synapomorphic) / Apis:289-0 (plesiomorphic) OrderGenusIntron Coleoptera:Tenebrio? Leptinotarsa? Harmonia? Clytus295-0 Phyllopertha? Phosphuga295-0 Hydaticus- Amara? Neuroptera:Chrysoperla295-0 Megaloptera:Sialis295-0 Mecoptera:Panorpa- ? = no sequence available - = no intron exist Intron 295-0 = synapomorphic character support Holometabola (inclusive Neuropteroidea) exclusive Hymenoptera

27. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt Universität Leipzig, Lehrstuhl für Genetik Results: gene fragment 66526442 (eIF2  ) Intron positions: Tribolium: 160-1 / Apis: 159-1 (plesiomorphic) OrderGenusIntron Coleoptera:Tenebrio160-1 Leptinotarsa160-1 Harmonia? Clytus160-1 Phyllopertha? Phosphuga? Hydaticus? Amara? Neuroptera:Chrysoperla? Megaloptera:Sialis? Mecoptera:Panorpa? ? = no sequence available - = no intron exist Intron 160-1 = support Coleoptera + Mecopteroidea Intron 160-1 might have originated by sliding of the 159-1 intron

28. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt Universität Leipzig, Lehrstuhl für Genetik Results: gene fragment 66563882 (TFIIH) Intron positions: Tribolium: 110-0 / Apis: 119-2 (plesiomorphic) OrderGenusIntron Coleoptera:Tenebrio? Agelastica- Coccinella110-1 Rhagonycha? Phyllopertha? Phosphuga? Hydaticus? Amara, Harpalus- Neuroptera:Chrysoperla110-1 Megaloptera:Sialis110-1 Mecoptera:Panorpa? ? = no sequence available - = no intron exist support the sister group relationship Coleoptera+Neuropteroidea Insertion of the intron 110-0 = occur after splitting up (Coleoptera + Neuropteroidea) from Mecopteroidea

29. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt Universität Leipzig, Lehrstuhl für Genetik Results: gene fragment 66563882 (TFIIH) Intron positions: Tribolium: 165-1 (synapomorphic) / Apis: 172-0 (plesiomorphic) OrderGenusIntron Coleoptera:Tenebrio? Agelastica- Coccinella165-1 Rhagonycha? Phyllopertha? Phosphuga? Hydaticus? Amara, Harpalus- Neuroptera:Chrysoperla- Megaloptera:Sialis- Mecoptera:Panorpa? ? = no sequence available - = no intron exist Intron 165-1 = support Coleoptera + Mecopteroidea Confirmation: Intron 165-1 = synapomorphic character

30. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt Universität Leipzig, Lehrstuhl für Genetik Results: gene fragment 66553024 Intron positions: Tribolium: 146-2 / Apis: 133-1 (plesiomorphic) OrderGenusIntron Coleoptera:Tenebrio- Leptinotarsa? Harmonia- Rhagonycha- Phyllopertha- Phosphuga? Hydaticus? Amara- Neuroptera:Chrysoperla? Megaloptera:Sialis? Mecoptera:Panorpa- ? = no sequence available - = no intron exist Insertion of the intron 146-2 = occur after splitting up (Coleoptera + Neuropteroidea) from Mecopteroidea Intron seems very young  probably long space of time between intron loss and gain

31. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt Universität Leipzig, Lehrstuhl für Genetik Results: gene fragment 66534441 Intron positions: Tribolium: 67-1 / Apis: 66-0 (plesiomorphic) OrderGenusIntron Coleoptera:Tenebrio67-1 Leptinotarsa67-1 Harmonia? Rhagonycha? Phyllopertha67-1 Phosphuga? Hydaticus- Amara- Neuroptera:Chrysoperla? Megaloptera:Sialis- Mecoptera:Panorpa? ? = no sequence available - = no intron exist Insertion of the intron 67-1 = occur after splitting up (Coleoptera + Neuropteroidea) from Mecopteroidea

32. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt Universität Leipzig, Lehrstuhl für Genetik Results: gene fragment 66542088 Intron positions: Tribolium: 590-1 / Apis: 604-2 (plesiomorphic) OrderGenusIntron Coleoptera:Tenebrio? Leptinotarsa591-1 Harmonia? Rhagonycha- Phyllopertha? Phosphuga- Hydaticus? Amara- Neuroptera:Chrysoperla- Megaloptera:Sialis604-2 Mecoptera:Panorpa? ? = no sequence available - = no intron exist Insertion of the intron 590-1 = occur after splitting up Coleoptera from Neuropteroidea Case of intron migration?

33. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt Universität Leipzig, Lehrstuhl für Genetik Results: gene fragment 48096763 Intron positions: Tribolium: 490-0 / Apis: 505-2 (plesiomorphic) OrderGenusIntron Coleoptera:Tenebrio490-0 Leptinotarsa490-0 Harmonia? Rhagonycha490-0 Phyllopertha490-0 Phosphuga- Hydaticus505-2 Amara505-2 Neuroptera:Chrysoperla? Megaloptera:Sialis505-2 Mecoptera:Panorpa505-2 ? = no sequence available - = no intron exist Intron position 490-0 = Intron of the Polyphaga

34. INTRODUCTION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Reconstruction of the organismal evolutionary tree based mainly on analysis of molecular sequences  insufficient to reliably resolve in deep branches Novel class of phylogenetic marker:  NIP (near intron pair)  derived by insertion of a novel intron less than 50 nt away from an evolutionary older intron  small distance should exclude the coexistence of both introns  possible to characterize one of the introns as ancient (plesiomorphic) and the other as novel (derived or apomorphic)

35. INTRODUCTION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt Universität Leipzig, Lehrstuhl für Genetik Side result of our analysis: Tribolium-specific introns  Investigation: hypothetical sister group relationship Coleoptera+Neuropterida   Alternatively: group consisting of Hymenoptera, Neuropterida, Mecoptera and Siphonaptera as sister group to all other Holometabola (Whiting 2002)   Sister group relationship Coleoptera + Neuropterida relatively weakly supported (Büning 2005)  by Tribolium-specific introns  Evaluation of these hypotheses

36. INTRODUCTION „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Side result of our analysis: Tribolium-specific introns  Determination of the relative age of the intron position  Investigation: hypothetical sister group relationship Coleoptera+Neuropterida   Alternatively: group consisting of Hymenoptera, Neuropterida, Mecoptera and Siphonaptera as sister group to all other Holometabola (Whiting 2002)   Sister group relationship Coleoptera + Neuropterida relatively weakly supported (Büning 2005)  via Tribolium-specific introns  Evaluation of these hypotheses:  Evidence for the Coleoptera + Neuropterida group:  Neuropterida show apomorphic intron positions of Tribolium 

37. RESULTS „ NIPs: Analysis of unsequenced holometabolic groups “ Dipl.-Biol. Carina Eisenhardt University Leipzig, Department of Genetics Results: gene fragment 66534441 Intron positions: Tribolium: 67-1 / Apis: 66-0 (plesiomorphic) Phylogenetic tree with intron positions Insertion of 67-1  220 -290 Mya Coleoptera Neuro- pterida Meco- pteroidea Insertion of 67-1 = occur after splitting up Coleoptera from Mecopteroidea ? ???? ? ?? ? xxxx ?= no sequence available x= no intron exist