1. DNA barcoding and evolutionary relationships in Accipiter Brisson,1760 (Aves, Falconiformes: Accipitridae) with a focus on African and Eurasian representatives

2. Nomenclature A. gentilis: Nl; havik, Eng; gosshawk, Fr; Autour des palombes A. nisus: Nl; sperwer, Eng; sparrowhawk, Fr; epervier d’Europe Buteo buteo: Nl; buizerd, Eng; Common buzzard Fr; Buse variable Buteo albicaudatus; Nl; witstaart buizerd, Eng; white tailed hawk, Fr; Buse à queue blanche

4. Content DNA barcoding a short introduction –General DNA barcoding –Mini barcodes –10X threshold, best compromise threshold –BCM and BM criteria –Character based identification Coverage –Sample origins –The dataset Methods –Character based method (CB) –The 10 X intraspecific distance threshold (10 X) –The Best compromise threshold (BCTh) –Best match and best close match explained Results for DNA barcoding –Character based identification –Different thresholds Phylogeny reconstruction –291 bp dataset –647 bp dartaset Discussion Conclusion

5. DNA barcoding Identification of species with an agreed upon (part of a) gene (COI) 5’-3’ part of Cytochrome C Oxidase subunit I ~650bp in length Polymerase chain reactions and Sanger sequencing

6. Mini barcodes Genetic material from museums is often degraded Shorter fragments are needed E.g. sequences of 250-300bp or, in some cases even shorter, also work for identification purposes Sonet et al. in press

7. Sample coverage and origins African and European taxa are well represented The new world partially represented Australia and S-E Asia poorly represented Most samples from RMCA/RBINS (n=83/9) Supplemented with available genbank sequences (n=28)

8. The resulting dataset 25 European and African Accipiter species with a total of 140 specimens sequenced for a 291bp (mini) and 647bp (standard) BC fragment of the COI gene three datasets analysed: –dataset A: 25 species, 291bp –dataset B: 19 species, 647bp –dataset C: 19 species, 291bp

9. Character based indentification –Easy it is about detecting unique differences –May be unreliable with insufficient sampling –No threshold whatsoever is used

10. 10 X intraspecific distances and the barcoding gap Distances Arbitrary Does not always hold (Tephritidae) However, works well in birds

11. The Best compromise threshold (BCTh) Based on the intercept between cumulative intra and interspecific distances Based on the dataset under study rather than a predetermined threshold Results from one dataset not applicable to another Lefébure et al. 2006

12. BM and Best Close match Best Match (BM), assignes the species name of its best- matching sequence regardless of how similar the query and reference sequences were. Identification is considered correct when both sequences were from the same species, incorrect if the query species differed from the closest reference species or ambiguous if multiple species were the BM of the query species Best close match (BCM) also considers the threshold

13. BM vs. BCM example 4 taxa 3 species: A. francesiae (n=2) A. badius (n=1) & A. toussenelii (n=1) BM would assign A. badius and A. toussenelii to the “ambiguous” or “incorrect” (dependent on the threshold) categories BCM would classify them as impossible when the difference is larger than the threshold

14. Results character based identification

15. Results BCTh vs. 10 X distance threshold

16. Different thresholds Partial overlap Caused by –A. nisus/rufiventris –A. virgatus/gularis –A. cooperii/gundlachi At or just below the BC treshold are –A. badius subsp –A. gentilis subsp Results BCTh vs. 10 X distance threshold A. rufiventrisA. nisus

17. Phylogeny reconstruction Hypothesis of evolution Clades are units of supported taxa (related by direct descent) This dataset is only suited for low taxonomic level inferences One gene  gene tree rather than species tree Comparison with the current taxonomy based on morphology and morphometrics

18. Phylogeny based on 291bp 10 supported clades –[tachiro] A. tachiro subclades –A. minullus –A. francesiae/ soloensis –[cooperii] –A. badius/ brevipes –A. nisus/ striatus/ erythronemius –A. ovampensis/ madagascariensis –[virgatus] including A. fasciatus

19. Phylogeny based on 291bp a closer look

20. Phylogeny based on 647bp 19 supported clades –[tachiro] A. tachiro A. toussenelii & subsp –A. soloensis & A. francesiae subsp –[gentilis] –[cooperii] –[nisus] (without A. ovampensis/ madagascariensis –[minullus] (without A. erythropus) –[virgatus] including A. fasciatus

21. Phylogeny based on 647bp a closer look

22. Discussion Thresholds –The 10 X threshold would have considered A. brevipes and A. badius, as well as A. gentilis and A. melanoleucus as belonging to the same species and Species concepts –Morphological –Distances based –Phylogenetic Support values –Models and interpretation remain issues of discussion Sampling –Incomplete or lacking in some cases

23. Conclusions Molecular techniques provide a powerfull method for reviewing taxonomy Museum material works! The content of the dataset matters COI can identify 19 out of the 25 species included COI carries a phylogenetic signal suitable for molecular taxonomy on a low taxonomic level Parts of genus Accipiter may need revision

24. Thank you for your attention