1
NIPs Analysis of unsequenced holometabolic
groups

University Leipzig, Department of Genetics

• Bled, Slovenia, Feb 18, 2008
• Carina Eisenhardt

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt
2
INTRODUCTION
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

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

1/19
3
INTRODUCTION
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

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

2/19
4
INTRODUCTION
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)
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

3/19
5
INTRODUCTION
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
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

4/19
6
INTRODUCTION
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 ColeopteraNeuropterida
• 
  ?
  

• 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

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

5/19
7
INTRODUCTION
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 ColeopteraNeuropterida
• 
  ?
  

• 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

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

5/19
8
METHODS
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
• ?

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

6/19
9
METHODS
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
133-1
146-2
Part of alignment (MacVector)
3024-4

• Degenerate primer - 5 clamp region Tribolium
  sequence
• - 3 degenerate
  core region

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

7/19
10
METHODS
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

NIP
NIP
3882-3
3882-2
3882-1
2088-4
2088-5
2088-3
2088-1
2088-2
NIP
NIP
3024-3
3024-1
3024-2
3024-4
NIP
4441-3
4441-1
4441-4
4441-2
NIP
6763-1
6763-3
6763-2
Degenerate primers for each gene fragment
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

8/19
11
RESULTS
University Leipzig, Department of Genetics

• Results gene fragment 66526442 (eIF2?)
• Intron positions Clytus 295-0
  (synapomorphic) / Apis289-0 (plesiomorphic)

Coleoptera
Neuro- pterida
Meco-pteroidea
?
?
x
?
?
?
?
?
?
?
?
x
x
support Holometabola (inclusive of
Neuropterida) exclusive Hymenoptera
Intron 295-0 synapomorphic character
Insertion of 295-0 ? 300-320 Mya
? no sequence available x no intron exist
Phylogenetic tree with intron positions
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

9/19
12
RESULTS
University Leipzig, Department of Genetics

• Results gene fragment 66526442 (eIF2?)
• Intron positions Tribolium 160-1
  (synapomorphic) / Apis 159-1 (plesiomorphic)

Coleoptera
Neuro- pterida
Meco-pteroidea
?
?
?
?
?
?
?
?
?
?
?
?
?
Intron 160-1 might have originated by sliding of
the intron 159-1
Insertion of 160-1 ? 300-320 Mya
support Coleoptera Mecopteroidea
? no sequence available x no intron exist
Phylogenetic tree with intron positions
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

10/19
13
RESULTS
University Leipzig, Department of Genetics

• Results gene fragment 66563882 (TFIIH)
• Intron positions Tribolium 110-0 /
  Apis 119-2 (plesiomorphic)

Coleoptera
Neuro- pterida
Meco-pteroidea
?
?
?
?
?
?
x
x
x
x
?
?
?
Insertion of 110-0 occur after splitting
up (Coleoptera Neuropterida) from Mecopteroidea
Insertion of 110-0 ? 290 Mya
support the sister group relationship
Coleoptera Neuropterida
? no sequence available x no intron exist
Phylogenetic tree with intron positions
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

11/19
14
RESULTS
University Leipzig, Department of Genetics

• Results gene fragment 66563882 (TFIIH)
• Intron positions Tribolium 165-1
  (synapomorphic) / Apis 172-0 (plesiomorphic)

Coleoptera
Neuro- pterida
Meco-pteroidea
?
?
?
?
?
x
x
x
?
?
?
?
x
x
Confirmation Intron 165-1 synapomorphic
character
Insertion of 165-1 ? 300-320 Mya
support Coleoptera Mecopteroidea
? no sequence available x no intron exist
Phylogenetic tree with intron positions
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

12/19
15
RESULTS
University Leipzig, Department of Genetics

• Results gene fragment 66553024
• Intron positions Tribolium 146-2 /
  Apis 133-1 (plesiomorphic)

Coleoptera
Neuro- pterida
Meco-pteroidea
?
x
x
x
?
?
?
?
?
?
?
x
x
x
x
?

• 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
Insertion of 146-2 ? ? -290 Mya
? no sequence available x no intron exist
Phylogenetic tree with intron positions
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

13/19
16
RESULTS
University Leipzig, Department of Genetics

• Results gene fragment 66542088
• Intron positions Tribolium 590-1 /
  Apis 604-2 (plesiomorphic)

Coleoptera
Neuro- pterida
Meco-pteroidea
?
?
?
x
x
x
?
?
?
?
?
?
x
Insertion of 590-1 occur after splitting
up Coleoptera from Neuropterida
Insertion of 590-1 ? 210 -285 Mya
Intron 591-1
Case of intron migration?
? no sequence available x no intron exist
Phylogenetic tree with intron positions
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

14/19
17
RESULTS
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?

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

15/19
18
RESULTS
University Leipzig, Department of Genetics

• Results gene fragment 48096763
• Intron positions Tribolium 490-0 /
  Apis 505-2 (plesiomorphic)

Coleoptera
Neuro- pterida
Meco-pteroidea
?
?
?
?
?
?
x
Insertion of 490-0 ? 220 -255 Mya
Intron position 490-0 Intron of the Polyphaga
? no sequence available x no intron exist
Phylogenetic tree with intron positions
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

16/19
19
DISCUSSION
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

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

17/19
20
DISCUSSION
University Leipzig, Department of Genetics
Summary

• Results support Büning (2005)

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

18/19
21
DISCUSSION
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
  
  

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

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
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
• ?

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

24
INTRODUCTION
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

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

25
INTRODUCTION
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 ColeopteraNeuropterida
• 
  ?
  

• 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 ColeopteraNeuropterida group
• Neuropterida will show apomorphic intron
  positions of Tribolium
• Evidence against the ColeopteraNeuropterida
  group
• Neuropterida will show pleiotrophic intron
  positions of hymenopterans
• 
  ?
• Inclusion of Apocrita-specific intron
  positions to investigate the alternative grouping

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

26
RESULTS
Universität Leipzig, Lehrstuhl für Genetik

• Results gene fragment 66526442 (eIF2?)
• Intron positions 295-0 (synapomorphic) /
  Apis289-0 (plesiomorphic)

Order Genus Intron
Coleoptera Tenebrio ?
Leptinotarsa ?
Harmonia ?
Clytus 295-0
Phyllopertha ?
Phosphuga 295-0
Hydaticus -
Amara ?
Neuroptera Chrysoperla 295-0
Megaloptera Sialis 295-0
Mecoptera Panorpa -

• Intron 295-0
• synapomorphic character
• support
• Holometabola (inclusive Neuropteroidea)
• exclusive Hymenoptera

? no sequence available - no intron exist
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

27
RESULTS
Universität Leipzig, Lehrstuhl für Genetik
Results gene fragment 66526442 (eIF2?)
Intron positions Tribolium 160-1 / Apis
159-1 (plesiomorphic)
Order Genus Intron
Coleoptera Tenebrio 160-1
Leptinotarsa 160-1
Harmonia ?
Clytus 160-1
Phyllopertha ?
Phosphuga ?
Hydaticus ?
Amara ?
Neuroptera Chrysoperla ?
Megaloptera Sialis ?
Mecoptera Panorpa ?

• Intron 160-1 support Coleoptera Mecopteroidea
• Intron 160-1 might have
• originated by sliding
• of the 159-1 intron

? no sequence available - no intron exist
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

28
RESULTS
Universität Leipzig, Lehrstuhl für Genetik

• Results gene fragment 66563882 (TFIIH)
• Intron positions Tribolium 110-0 /
  Apis 119-2 (plesiomorphic)

• support the sister group relationship
  ColeopteraNeuropteroidea
• Insertion of the intron 110-0
• occur after splitting up
• (Coleoptera Neuropteroidea)
• from Mecopteroidea

Order Genus Intron
Coleoptera Tenebrio ?
Agelastica -
Coccinella 110-1
Rhagonycha ?
Phyllopertha ?
Phosphuga ?
Hydaticus ?
Amara, Harpalus -
Neuroptera Chrysoperla 110-1
Megaloptera Sialis 110-1
Mecoptera Panorpa ?
? no sequence available - no intron exist
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

29
RESULTS
Universität Leipzig, Lehrstuhl für Genetik

• Results gene fragment 66563882 (TFIIH)
• Intron positions Tribolium 165-1
  (synapomorphic) / Apis 172-0 (plesiomorphic)

Order Genus Intron
Coleoptera Tenebrio ?
Agelastica -
Coccinella 165-1
Rhagonycha ?
Phyllopertha ?
Phosphuga ?
Hydaticus ?
Amara, Harpalus -
Neuroptera Chrysoperla -
Megaloptera Sialis -
Mecoptera Panorpa ?

• Intron 165-1 support Coleoptera Mecopteroidea
• Confirmation Intron 165-1
• synapomorphic character

? no sequence available - no intron exist
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

30
RESULTS
Universität Leipzig, Lehrstuhl für Genetik

• Results gene fragment 66553024
• Intron positions Tribolium 146-2 /
  Apis 133-1 (plesiomorphic)

Order Genus Intron
Coleoptera Tenebrio -
Leptinotarsa ?
Harmonia -
Rhagonycha -
Phyllopertha -
Phosphuga ?
Hydaticus ?
Amara -
Neuroptera Chrysoperla ?
Megaloptera Sialis ?
Mecoptera Panorpa -

• 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

? no sequence available - no intron exist
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

31
RESULTS
Universität Leipzig, Lehrstuhl für Genetik

• Results gene fragment 66534441
• Intron positions Tribolium 67-1 / Apis
  66-0 (plesiomorphic)

Order Genus Intron
Coleoptera Tenebrio 67-1
Leptinotarsa 67-1
Harmonia ?
Rhagonycha ?
Phyllopertha 67-1
Phosphuga ?
Hydaticus -
Amara -
Neuroptera Chrysoperla ?
Megaloptera Sialis -
Mecoptera Panorpa ?

• Insertion of the intron 67-1
• occur after
• splitting up
• (Coleoptera Neuropteroidea)
• from Mecopteroidea

? no sequence available - no intron exist
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

32
RESULTS
Universität Leipzig, Lehrstuhl für Genetik

• Results gene fragment 66542088
• Intron positions Tribolium 590-1 /
  Apis 604-2 (plesiomorphic)

Order Genus Intron
Coleoptera Tenebrio ?
Leptinotarsa 591-1
Harmonia ?
Rhagonycha -
Phyllopertha ?
Phosphuga -
Hydaticus ?
Amara -
Neuroptera Chrysoperla -
Megaloptera Sialis 604-2
Mecoptera Panorpa ?

• Insertion of the intron 590-1
• occur after splitting up
• Coleoptera
• from Neuropteroidea
• Case of intron migration?

? no sequence available - no intron exist
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

33
RESULTS
Universität Leipzig, Lehrstuhl für Genetik

• Results gene fragment 48096763
• Intron positions Tribolium 490-0 /
  Apis 505-2 (plesiomorphic)

Order Genus Intron
Coleoptera Tenebrio 490-0
Leptinotarsa 490-0
Harmonia ?
Rhagonycha 490-0
Phyllopertha 490-0
Phosphuga -
Hydaticus 505-2
Amara 505-2
Neuroptera Chrysoperla ?
Megaloptera Sialis 505-2
Mecoptera Panorpa 505-2

• Intron position 490-0
• Intron of
• the Polyphaga

? no sequence available - no intron exist
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

34
INTRODUCTION
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)

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

35
INTRODUCTION
Universität Leipzig, Lehrstuhl für Genetik

• Side result of our analysis Tribolium-specific
  introns
• Investigation hypothetical sister group
  relationship ColeopteraNeuropterida
• 
  ?
  

• 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

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

36
INTRODUCTION
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 ColeopteraNeuropterida
• 
  ?
  

• 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

NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt

37
RESULTS
University Leipzig, Department of Genetics

• Results gene fragment 66534441
• Intron positions Tribolium 67-1 / Apis
  66-0 (plesiomorphic)

Coleoptera
Neuro- pterida
Meco-pteroidea
?
x
x
x
x
?
?
?
?
?
?
?
?
Insertion of 67-1 occur after splitting
up Coleoptera from Mecopteroidea
Insertion of 67-1 ? 220 -290 Mya
? no sequence available x no intron exist
Phylogenetic tree with intron positions
NIPs Analysis of unsequenced holometabolic
groups Dipl.-Biol. Carina Eisenhardt