Character-based DNA barcoding for identifying conservation units in Odonates

1. Character-based DNA barcoding for identifying conservation units in Odonates J. Rach1, R. DeSalle2, I.N. Sarkar2, B. Schierwater1,2 & H. Hadrys1, 3 1ITZ- Ecology & Evolution, TiHo Hannover, Germany 2Division of Invertebrate Zoology, American Museum of Natural History, New York, USA 3Dept. Ecology & Evolutionary Biology, Yale University, New Haven, USA

2. Thank you to: • DAWB (CBOL)/DIMACS • Sandra Giere • Antonia Wargel • Janne Timm • Linn Groeneveld • Nadine Habekost • Kai Kamm • DFG & BMBF

3. Character-based DNA barcoding: A rapid and reliable method for the identification of conservation units in dragonflies

4. Contents • Introduction: • - Why barcoding dragonflies? • - Why character-based DNA barcoding? • - Which genetic marker is appropriate? • Methods • - Character-based DNA barcoding • Case studies • I. Species identification • II. Discrimination of conservation units • 4. Conclusions & Future prospects

5. 1. Introduction: Why barcoding dragonflies? Odonata (demonstrator system): - Small insect order - Model organisms for ecology and evolution - Wide range of habitat specificity (generalists / specialists) - Fast respond to environmental changes

6. 1. Introduction: Why barcoding dragonflies? - Prime indicators for all types of fresh water ecosystems Terrestrial Aquatic Increasing importance for conservation management

7. 1. Introduction: Why barcoding dragonflies? Identification through phenotypic traits is difficult: - Wing veneation: requires a lot of experience - Colours: Bright colours of males fade quickly after death; females of same genus inconspiciuous - Ecological and behavioural patterns: difficult and time-consuming - Larvae: discrimination often impossible ♀ ♀

8. 1. Introduction: Why barcoding dragonflies? Rapid and reliable identification of dragonflies valuable for conservation management: If phenotypic traits do not serve  Need of genetic approaches! How to get DNA non-invasive: Exuvia Middle leg (Hadrys et al. 1992)

9. 1. Introduction: Why character-based DNA barcoding? Distance approaches can be misleading: - High intraspecific genetic variability (e.g. geographical clusters) can hinder assignment of unknown samples to their species - Distances between species often lower than within species - Thresholds cannot be defined (might lead to overestimated biodiversity)

10. 1. Introduction: Why character-based DNA barcoding? Diagnostic characters useful for DNA barcoding: Identification at any taxonomic level Character-based DNA barcodes for species and single populations

11. 1. Introduction: Which genetic marker is appropriate? CO1 (cytochrome c oxidase 1) supposed to be appropriate for DNA barcoding of most animal groups: Has not been applied for Odonates before: • Search for conserved primer sequences • Optimization of PCR conditions • Test for suitability

12. 1. Introduction: Which genetic marker is appropriate? ND1 (NADH dehydrogenase subunit 1) is a suitable marker: - Sequences easy to obtain and analyse - Detection of geographical patterns - Identification of conservation units Cryptic speciation in Trithemis stictica

13. 2. Methods: Character-based DNA barcoding 1. Standard Methods - PCR with gene specific primers - Sequencing (MegaBACE 500) - Alignment (MUSCLE) - NJ tree based on Kimura-2-parameter (K2P) distances (PAUP)

14. 2. Methods: Character-based DNA barcoding 2. Establishment of character-based DNA barcodes: - Search for diagnostic characters by application of CAOS algorithm - Development of perl scripts to assist further analyses - Selection of nucleotide positions for final DNA barcodes by eye

15. 2. Methods: Character-based DNA barcoding 2. Establishment of character-based DNA barcodes: 0 1 1 0 1 0 II. Search for characteristic attributes with CAOS algorithm I. Phylogenetic Tree III. Find unique combinations of character states

16. 2. Methods: Character-based DNA barcoding Types of characteristic attributes (CAs): - Pure (Pu): Exist in all elements of a group but not in alternate group - Private (Pr): Only present in some members of a group but absent from alternate group - Simple (s): At a single nucleotide position - Compound (c): combination of states  sPu and sPr CAs shared by at least 80% of members of a group were used (Filtered by diagViewer)

17. 2. Methods: Character-based DNA barcoding Analyses were assisted by a set of perl scripts: 1. “BarcodeFilter”: sorts out non-relevant nodes Nodes within species cluster are not relevant for barcoding species

18. 2. Methods: Character-based DNA barcoding 2. “BarcodeMaker”: Convertion of “diagViewer- attributes file” into tab delimited file importable to Microsoft Excel:

19. 2. Methods: Character-based DNA barcoding 3. “BarcodeHistMaker”:Counting numbers of CAs at each nucleotide position (selection of sequence fragment with highest number of CAs:

20. Case studies (Study I) Case Study I: Species identification 842 ND1 sequences (65 species) - Suitability of ND1 for DNA barcoding - Applicability of the CAOS algorithm for character-based DNA barcoding

21. Overlap of species cluster Case studies (Study I) NJ tree based on K2P distances: Overview tree: ND1 sequence of one individual of each species

22. Case studies (Study I) Results: Character-based DNA barcodes Unique combinations of character states at 13 selected nucleotide position

23. Case studies (Study I) NJ tree based on K2P distances: Overview tree: ND1 sequence of one individual of each species Overlap of species cluster

24. Case studies (Study I) Results: Character-based DNA barcodes Family Aeshnidae: Combination of character states shared by two or more species  Additional analysis with CAOS algorithm

25. Case studies (Study I) Results: Character-based DNA barcodes for Aeshnids - Search for diagnostic characters within whole ND1 fragment  better resolution

26. Case studies (Study I) NJ tree based on K2P distances: Overview tree: ND1 sequence of one individual of each species Overlap of species cluster

27. Case studies (Study I) Results: Character-based DNA barcodes • - Combination of character states shared by several individuals of Calopteryx splendens (cs) and of Calopteryx virgo (cv) • No diagnostic characters found through additional analysis with CAOS algorithm • Hybridisation • Wrong identification • Recent radiation

28. Case studies (Study I) Summary: Case study I - 60 of 65 species distinguishable through unique combinations of character states within ND1 fragment - ND1 suitable - Diagnostic characters easily found by application of the CAOS algorithm

29. Case studies (Study II) Case Study II: Discrimination of conservation units Subset of Case study I; 122 ND1 sequences (9 species) + 101 CO1 sequences (same 9 species) - Suitability of CO1 for DNA barcoding - Ability of both markers to discrimininate conservation units

30. Case studies (Study II) NJ trees based on K2P distances CO1 ND1 ND1

31. Case studies (Study II) Results: Character-based DNA barcodes unique combinations of character states at 11 selected nucleotide positions of CO1 fragment  CO1 also suitable

32. Case studies (Study II) Results: Identification of populations CO1 ND1 X  Combination of CO1 and ND1 to improve identification success

33. Case studies (Study II) Results: Identification of conservation units CO1 ND1 Pb77 Pb113 Pb78 Pb79

34. Kwando Popa Falls Zebra River Case studies (Study II) Results: Identification of cryptic species CO1 ND1 * One individual of Tst119 shares a combination of character states with 6 individuals of Tst128 Tst128 Populations: Tst94: Tst118: Tst119: Pb128: Tst119 Tst118 Tst94 = Kenya

35. Case studies (Study II) Summary: Case study II • - All nine species distinguishable through unique combinations of character states within ND1 and CO1 fragments • - Both markers suitable • - Character-based DNA barcodes established for conservation units of several species

36. Conclusions Character-based approaches are: Rapid - Application of CAOS algorithm - Assignment of samples through a few nucleotide positions Reliable - Discrete characters • Combination of ND1 and CO1 increases success - DNA barcodes for several conservation units

37. Conclusions Character-based DNA barcoding: A rapid and reliable method for the identification of conservation units in dragonflies !

38. Future Prospects • Next steps: • - More species • - More individuals of some species • - Development of data base • Character-based DNA barcodes for genera • Application of character-based DNA barcodes •  Identification of adults, exuvia and larvae •  Long-time monitoring

39. Thank you !!