„ nips: analysis of unsequenced holometabolic groups “ bled, slovenia, feb 18, 2008 carina...

„ 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

INTRODUCTION



•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

INTRODUCTION



•Suitability of this marker class demonstrated by analysis of the relatednessof holometabolic insects:

Hymenoptera + (Coleoptera + (Diptera + Lepidoptera)) - theory

•Tribolium has synapomorphic intronpositions with other holometabolicorders

•Apis has plesiomorphic intron positions

•Resulting phylogenetically informative intron distributions areinvestigated in representative species of other holometabolic insect orders

expansion of the genome-scaled studies of holometabolic insects

2/19

INTRODUCTION



•Additional to Hymenoptera and Coleoptera other groups are involved inthe basal evolutionary splitting event of Holometabola:

Neuropteroidea, Strepsiptera, Siphonaptera and Mecoptera

Büning (2005)

?

?

?

?

3/19

INTRODUCTION



•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

INTRODUCTION



•Side result of our analysis: Tribolium-specific introns

Determination of the relative age of the intron positionInvestigation: hypothetical sister group relationship Coleoptera+Neuropterida

Alternatively: group consisting of Hymenoptera, Neuropterida,Mecoptera and Siphonaptera assister group to all other Holometabola(Whiting 2002)

Sister group relationship Coleoptera + Neuropteridarelatively 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

INTRODUCTION







(Büning 2005)


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

METHODS



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

METHODS



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

133-1 146-2

Part of alignment(MacVector)

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

7/19

METHODS



•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 3882-2NIP

2088-3 2088-1 2088-4 2088-2 2088-5NIP

NIP

NIP

NIP

3024-3 3024-1 3024-2 3024-4

4441-3 4441-1 4441-4 4441-2

6763-2 6763-36763-1

Degenerate primers for each gene fragment

NIP

8/19

RESULTS



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

ColeopteraNeuro-pterida

Meco-pteroidea

support Holometabola (inclusive

of Neuropterida)exclusive Hymenoptera

Intron 295-0= synapomorphic

character

?? ?? ? ? ? ?? ?

?= no sequence available x= no intron exist

xxx

9/19

RESULTS




Intron positions: Tribolium: 160-1 (synapomorphic) / Apis: 159-1 (plesiomorphic)


Insertion of 160-1

300-320 Mya


Meco-pteroidea

Intron 160-1 might have originated by

sliding of the intron 159-1

support Coleoptera +

Mecopteroidea

? ? ? ? ? ? ? ?? ?? ? ?


10/19

RESULTS



Results: gene fragment 66563882 (TFIIH)

Intron positions: Tribolium: 110-0 / Apis: 119-2 (plesiomorphic)


Insertion of 110-0

290 Mya


Meco-pteroidea

support the sister group relationship Coleoptera+

Neuropterida

Insertion of 110-0 = occur after splitting up

(Coleoptera + Neuropterida)

from Mecopteroidea

?? ? ?? ? ? ? ? xxxx


11/19

RESULTS






Insertion of 165-1

300-320 Mya


Meco-pteroidea

Confirmation: Intron 165-1

= synapomorphic character

support Coleoptera + Mecopteroidea

??? ? ? ? ???x x x x x


12/19

RESULTS



Results: gene fragment 66553024


Insertion of 146-2

? -290 Mya



Meco-pteroidea

Intron 146-2 seems very young

probably long space of time between

intron loss and gain


(Coleoptera + Neuropterida)

from Mecopteroidea

? ? ? ? ? ? ? ? ?x x x x x x x


13/19

RESULTS






Insertion of 590-1

210 -285 Mya


Meco-pteroidea

Case of intron migration?


Coleoptera from Neuropterida

?? ? ? ?? ? ??x x x x


= Intron 591-1

14/19

RESULTS




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

RESULTS





Insertion of 490-0

220 -255 Mya



Meco-pteroidea

Intron position 490-0 = Intron of

the Polyphaga

?? ? ??? x


16/19

DISCUSSION



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

DISCUSSION



Summary

18/19

• Results support: Büning (2005)

DISCUSSION



Further investigations

•Expansion of the genome-scaled studies of holometabolic insects

Inclusion of Strepsiptera and Siphonaptera

•Bioinformatics Group (Prof. Stadler): development of tools for automaticgene 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

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 !

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

Comparison of genomic and cDNA Intron positions for phylogenetic analysis

INTRODUCTION



•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

INTRODUCTION







(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

RESULTS




Intron positions: 295-0 (synapomorphic) / Apis:289-0 (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 -? = no sequence available - = no intron exist

•Intron 295-0= synapomorphic character

•support Holometabola (inclusive

Neuropteroidea)exclusive Hymenoptera

RESULTS





Order Genus Intron

Coleoptera: Tenebrio 160-1

Leptinotarsa 160-1

Harmonia ?

Clytus 160-1

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 haveoriginated by sliding of the 159-1 intron

RESULTS





Order Genus Intron


Agelastica -

Coccinella 110-1

Rhagonycha ?

Phyllopertha ?

Phosphuga ?

Hydaticus ?

Amara, Harpalus -

Neuroptera: Chrysoperla 110-1



•support the sister group relationship

Coleoptera+Neuropteroidea

•Insertion of the intron 110-0 = occur after splitting up

(Coleoptera + Neuropteroidea)from Mecopteroidea

RESULTS





Order Genus Intron


Agelastica -

Coccinella 165-1

Rhagonycha ?

Phyllopertha ?

Phosphuga ?

Hydaticus ?

Amara, Harpalus -

Neuroptera: Chrysoperla -

Megaloptera: Sialis -


•Intron 165-1 = support Coleoptera + Mecopteroidea

•Confirmation: Intron 165-1= synapomorphic character

RESULTS





Order Genus Intron

Coleoptera: Tenebrio -

Leptinotarsa ?

Harmonia -

Rhagonycha -

Phyllopertha -

Phosphuga ?

Hydaticus ?

Amara -


Megaloptera: Sialis ?

Mecoptera: Panorpa -? = no sequence available - = no intron exist



•Intron seems very young

probably long space of timebetween intron loss and gain

RESULTS





Order Genus Intron


Leptinotarsa 67-1

Harmonia ?

Rhagonycha ?

Phyllopertha 67-1

Phosphuga ?

Hydaticus -

Amara -


Megaloptera: Sialis -




RESULTS





Order Genus Intron


Leptinotarsa 591-1

Harmonia ?

Rhagonycha -

Phyllopertha ?

Phosphuga -

Hydaticus ?

Amara -

Neuroptera: Chrysoperla -




Coleoptera from Neuropteroidea

•Case of intron migration?

RESULTS





Order Genus Intron


Leptinotarsa 490-0

Harmonia ?

Rhagonycha 490-0

Phyllopertha 490-0

Phosphuga -

Hydaticus 505-2

Amara 505-2



Mecoptera: Panorpa 505-2? = no sequence available - = no intron exist

•Intron position 490-0 = Intron of

the Polyphaga

INTRODUCTION



•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)

INTRODUCTION




Investigation: hypothetical sister group relationship Coleoptera+Neuropterida



(Büning 2005)

by Tribolium-specific introns Evaluation of these hypotheses

INTRODUCTION







(Büning 2005)


Evidence for the Coleoptera + Neuropterida group:

Neuropterida show apomorphic intron positions of Tribolium

RESULTS






Insertion of 67-1

220 -290 Mya


Meco-pteroidea


Coleoptera from Mecopteroidea

?? ? ? ? ? ? ? ?x x x x


„ nips: analysis of unsequenced holometabolic groups “ bled, slovenia, feb 18, 2008 carina...

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