Sandra Damm - Invertebrates Plenary

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Character-based DNA Barcoding allows for integration of geography, ecology and morphology: The discovery of a cryptic species complex in dragonflies using Caos.

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  • Thank you very much! Good morning everybody! Ist a great pleasure for me to be here and to have the possibility to tell you something about my work on character-based barcoding on odonates and the discovery of the first cryptic dragonfly species.
  • Today we have around 2 million described species on earth, but the real number of species is unknown. There might be around 10-20 million species or even much more. Together with that we have an estimated extinction rate of around 0.25% per year. So many species werent even discovered before they get extinct. So for conserving biodiversity we need a fast tool to identify species and here DNA barcoding is a perfect tool to realize that. But there are also some problems of DNA barcoding - especially when new species are discovered. This is because genetic distances highly vary between species groups and therefore a general threshold to diagnose new species is not possible. Before I come to my case study, I want to show you a short example of two known dragonfly species to demonstrate the problems of the tradititonal distance based barcoding and highlight the advantages of character-based barcoding.
  • Here you can see the two dragonfly species, A.i. the Blue Emperor and A.p. the lesser Emperor. The tree shows the phylogenetic relationship between different Anax species based on CO1 and you can see that some individuals of Ai and Ap are closer related to each other than the population of Ai from Europe. Also the genetic distance between them is only 1.8%. The genetic distances between all other Anax species varied from 1.8% to 13%. So in comparision to other Anax species the distance of Ai and Ap is very low. Concerning these results and the proposed 3% rule to disciminate species, the two Anax species are not seperate species.
  • I want to show you now the same example when character-based barcoding is used. But before: What is chracter-based barcoding? This method was first described by Neil Sakar and collueges. It uses diagnostic characters in the sequence to identify species instead of genetic distances. Here you can see two sequences, and these characters (perhaps a A or C) is specific or diagnostic for species A. And so on. The advantages are that the characters matrix could be complemented with additional markers. When barcoding Odonates we find out that the combination of these two markers, but also including a different region of CO1 brings the best reulution to distinguish between species. and Characters of other disciplines could be incorporated e.g. morphology, ecology or geography.
  • If we now have a look at our example of the two Anax species we see first, the DNA based characters. Here the specific characters of CO1 and here, complemented with characters of ND1. Now we have 10 distinct DNA characters instead of four. Second we compared morphological characters of the two species And third we included the species distribution (Ai is widespread from southern Africa to northern europe, while Ap is more restricted) and a very specific ecological trait, the different behaviour of oviposition. If we sum up all characters we could come to the conclusion that the Ai and Ap are two separate species, distinguished by unambigious characters of not only DNA but also characters of other different disciplines. Now I will come to a real discovery of two new african dragonfly species.
  • During a biodiversity study on African Odonates we did population genetic analyses of Trithemis stictica. A widespread species in Souhern Africa and quite common at permanent water ponds with vegetation around. We included samples from South Africa up to Ethiopia to cover a main part of the species distribution. But the results were very surprising.
  • We first analysed the ND1 region and the more than 100 analysed individual were split in three genetic distinct groups with high genetic distances between them. The groups have no shared haplotypes and F statistic parameters like the Fst values indictated that there is not gene flow between the groups anymore. So the question arised:
  • Are the three genetic clades already separate species?
  • We included other Trithemis species to evaluate the genetic distances within the genus and we analysed also additional genetic markers, like the barcoding region of CO1, but also 16S and the nuclear ITS I to II. We can see that one genetic group was found on this branch with two other close relatives and genetic distance of e.g. 3% in CO1 And the other two are on a separate branch but with even higher genetic distances between them with e.g. 5.7% in CO1. So the genetic distances between all three groups are clearly at the species level and our results can be confirmed by four different genetic markers.
  • If we now analyse the CO1 and the ND1 region with the CAOS program, we find a high number of pure diagnostic characters in a pairwise comparison distinguishing the analysed species. Pure diagnostic characters are characters which are 100% identical in all analysed individual of one species and 100% different in a second species. In our case…..
  • In our case….. we find e.g. 43 pure diagnostic characters in CO1 between T. stictica and the other two new genetic clades. Also between the two new clades we found 28 diagnostic characters. Using the CAOS barcoding algorith, we also generated a character matrix to distinguish between all seven here analysed Trithemis species (including the two new clades). And we can see that with this matrix we are able to identify the seven species with only 15 selected nucleotide positions. This includes also our two potentionally new species. So, no doubt, with genetics alone we were nearly sure that we have discovered two new dragonfly species. But to really diagnose and describe the new species we included also other disciplines. I will start with the Morphology:
  • In the field the three species were identified as T. stictica with a normal morphological identification key. But after a closer look back in the lab we found
  • But after a closer look back in the lab we found…… Differences in the colouration of the eyes – T. stictica has unicoloured eyes, while the other two have bicoloured eyes.
  • In addition, T. stictica has a clear wingbase, while in the other two the wingbase is yellow coloured.
  • The only difference we found between the other two is a significant difference in size. But, size is not a good criteria to disibguish between odonate species, because it is could be highly variable btween individuals of the same species, dependent on e.g. food availability in the larval stage. Because Odonates have this special reproductive system with the lock and key mechanism of the genitals, we also did SEM analyses of the secondary genitalia of the males.
  • Here you can see the outer part of the secondary genitalia, and here inside lays the penis like structure with the distal segment at the end. Here again we found a difference between T stictica and the other two, but also here no difference between the two new one. The shape of the distal segment is, by the way, the reason why we identified the one genetic group as T. stictica, because the origial description of the species shows exactly this shape.
  • The mapping of the population sites to the genetic groups revealed a widespread distribution of T. stictica, here illustrated as red dots, from Namibia, South Africa, Kenya Tanzania and Ethiopia.
  • The other two species have a regionally restricted distribution. They, so far as we know today, occur only at the region of the Zambezi and the Okavango floodplains. Here they share one population site, where they occur sympatrically.
  • The only real difference beside the genetics between the two new species, we found in their ecology. T. Stictica occured often in shaded habitats, in highlands and at natural sources in mountain regions.
  • One the two new species occured at rivers with a galery forest and most important, at fast running waters, often with rapids.
  • And the third species occured at open habitats, most often swamps and standing waters.
  • To sum up all the results of the different disciplines we can say that we have successfully discovered two new (and cryptic) dragonfly species using an integrative approach for species discovery which is not only distance based. But in our case study the two new species would have also been discovered using genetic distances alone But these results are confirmed by a high number of diagnostic characters using CAOS DNA barcoding, with characters of morphology (here it meens the discrimination against T. stictica), Reproductive isolation by morphology and indirect by disruption of gene flow despite of the same geographical distribution, Ecology and Geography.
  • To conclude Character-based barcoding may be a promising alternative or a perfect complement to the distance based aapproach because it allows for a unambigous identification of known species and a reliable discovery of new species In addition the building of a comprehensive database which integrates characters of multiple disciplines can serve as a perfect backbone to evaluate biodiversity on the one hand, but provides also useful informations for conservation management like in our case study the species specific habitats. And at the end, we were able to discover two new species in group of insects which was not supposed to evolve cryptic species until now and there might be much more waiting to be described.
  • I want to thank all those people for helping us during our study, with analyses and discussion especially Bernd Schierwater…. And for providing or helping to get samples from different parts of Africa…… And the BMBF for finacial support!
  • And I thank you for your attention!
  • Sandra Damm - Invertebrates Plenary

    1. 1. University of Veterinary Medicine Hannover Foundation Biodiversity Transect Analysis in Africa (BMBF) Character-based DNA barcoding allows for integration of geography, ecology and morphology Discovery of a cryptic species complex in dragonflies using CAOS Sandra Damm & Heike Hadrys University of Veterinary Medicine Hannover ITZ, Ecology & Evolution
    2. 2. DNA barcoding <ul><li>2 million described species </li></ul><ul><li>Estimated number of species: 10-20 million or higher </li></ul><ul><li>Estimated extinction rate: 0.25% per year </li></ul><ul><li>DNA Barcoding: perfect tool for fast identification </li></ul><ul><li>of known species </li></ul><ul><li>Discovery of new species – Some problems </li></ul><ul><li>Genetic distances variable between species groups </li></ul><ul><li>No general threshold possible to diagnose new species </li></ul>
    3. 3. Distance-based barcoding Anax imperator Anax parthenope Genetic distance between the two species: 1.8% Genetic distances between species within this genus: 1.8 – 13% Regarding the 3% rule  No separate species Distance-based DNA barcoding (COI)
    4. 4. Anax imperator Anax parthenope Character-based barcoding CAOS barcoding <ul><li>Definition of diagnostic characters of CO1 using CAOS ( C haracter A ttribute O rganization S ystem) (Sakar et al. 2002) </li></ul><ul><li>Advantage: </li></ul><ul><li>Character matrix could be complemented with additional markers (for Odonates ND1 is highly suitable) </li></ul><ul><li>and </li></ul><ul><li>with characters of morphology, ecology, geography or others </li></ul>Species A Species B
    5. 5. Anax imperator Anax parthenope Morphology DNA CAOS barcoding  Two different species: supported by unambiguous characters of different disciplines Character-based barcoding A B C D E F G H I J K L M A. imperator 0 1 1 0 1 1 1 0 0 0 1 1 1 A. parthenope 1 0 0 1 0 0 0 1 1 1 0 0 0 COI ND1 101 132 135 152 10 28 120 287 346 326 A. imperator G A G A T A C A T A A. parthenope C G A G G T T T C G Geography Ecology A. imperator Africa, Europe, Asia Oviposition without guarding male A. parthenope North Africa, Southern Europe, Asia Oviposition with guarding male
    6. 6. Population genetic analyses in Trithemis stictica <ul><li>widespread species in southern Africa </li></ul><ul><li>permanent water pond with vegetation </li></ul>Case study – Population genetics
    7. 7. Maximum Parsimony tree of ND1 sequences of 108 analysed „ T. stictica “ individuals Damm et al. (2010) Molecular Ecology <ul><li>Three genetic distinct groups </li></ul><ul><li>High genetic distances </li></ul><ul><li>No shared haplotypes </li></ul><ul><li>Complete genetic isolation </li></ul><ul><li>(F st -values ≥ 0.89) </li></ul>Case study – Population genetics T. stictica T. spec. nov T. spec. nov
    8. 8. Are the three genetic clades already separate species?
    9. 9. ND1: 2.2% CO1: 3.3% 16S: 1.2% ITS: 1.0% ND1: 5.0% CO1: 5.7% 16S: 1.1% ITS: 1.0% Bayesian analyses based on 16S, ND1, CO1 and ITS sequences <ul><li>Genetic distances at the </li></ul><ul><li>species level </li></ul><ul><li>Confirmed by four different </li></ul><ul><li>sequence markers </li></ul>Damm et al. (2010) Molecular Ecology Case study – Phylogenetic analyses
    10. 10. Case study – Character-based barcoding Damm et al. (2010) Molecular Ecology Pure diagnostic characters Pairwise comparison ND1 COI T. stictica (C1) / T. spec. nov. (C2) 26 43 T. stictica (C1) / T. spec. nov. (C3) 27 43 T. stictica (C1) / T. nuptialis 4 19 T. stictica (C1) / T. grouti 21 20 T. spec. nov. (C2)/ T. spec. nov. (C3) 13 28 T. spec. nov. (C2) / T. nuptialis 32 51 T. spec. nov. (C2) / T. grouti 30 49 T. spec. nov. (C3) / T. nuptialis 28 52 T. spec. nov. (C3) / T. grouti 30 50 A A B B
    11. 11. Case study – Character-based barcoding High number of pure diagnostic characters in a pairwise comparison 15 selected nucleotide positions of CO1 to distinguish between seven different Trithemis species Damm et al. (2010) Molecular Ecology Combination of species specific characters Pairwise comparison ND1 COI T. stictica (C1) / T. spec. nov. (C2) 26 43 T. stictica (C1) / T. spec. nov. (C3) 27 43 T. stictica (C1) / T. nuptialis 4 19 T. stictica (C1) / T. grouti 21 20 T. spec. nov. (C2)/ T. spec. nov. (C3) 13 28 T. spec. nov. (C2) / T. nuptialis 32 51 T. spec. nov. (C2) / T. grouti 30 49 T. spec. nov. (C3) / T. nuptialis 28 52 T. spec. nov. (C3) / T. grouti 30 50 COI Nucleotide positions   Species 45 144 162 180 279 288 294 297 330 333 360 393 396 454 459 T. stictica (C1) C C A C T A A T T G T A A A T T. spec. nov. (C2) C G A A A A T T T T C A A C T T. spec. nov. (C3) A G A A G G C T T G T G A C T T. grouti A G G C T A A T T G T A A T C T. nuptialis C G G C T A A C C G T A A A T T. annulata A T T C A A A T T A T A C A T T. furva A A A T T A A A T T A T T T T
    12. 12. Trithemis stictica T. spec. nov. T. spec. nov. Case study – Morphology
    13. 13. Trithemis stictica <ul><li>Colouration of the eyes </li></ul>T. spec. nov. T. spec. nov. Damm & Hadrys (2009) International Journal of Odonatology Case study – Morphology
    14. 14. Trithemis stictica T. spec. nov. T. spec. nov. <ul><li>Colouration of the eyes </li></ul><ul><li>Colour of the wing basis </li></ul>Damm & Hadrys (2009) International Journal of Odonatology Case study – Morphology
    15. 15. <ul><li>Significant size differences </li></ul>Trithemis stictica T. spec. nov. T. spec. nov. Damm & Hadrys (2009) International Journal of Odonatology Case study – Morphology
    16. 16. T. spec. nov. T. spec. nov. T. stictica Males secondary genitalia  Reproductive isolation distal segment Damm & Hadrys (2009) International Journal of Odonatology Case study – Morphology
    17. 17. Damm et al. (2010) Molecular Ecology Case study – Geography
    18. 18. Damm et al. (2010) Molecular Ecology Caprivi region Case study – Geography
    19. 19. <ul><li>shaded habitat </li></ul><ul><li>highlands and natural </li></ul><ul><li>sources in mountain </li></ul><ul><li>regions </li></ul>Trithemis stictica T. spec. nov T. spec. nov Damm et al. (2010) Molecular Ecology Case study – Ecology
    20. 20. <ul><li>shaded habitat </li></ul><ul><li>highlands and natural </li></ul><ul><li>sources in mountain </li></ul><ul><li>regions </li></ul><ul><li>rivers with galery forest </li></ul><ul><li>fast running waters </li></ul>Trithemis stictica T. spec. nov T. spec. nov Damm et al. (2010) Molecular Ecology Case study – Ecology
    21. 21. <ul><li>shaded habitat </li></ul><ul><li>highlands and natural </li></ul><ul><li>sources in mountain </li></ul><ul><li>regions </li></ul><ul><li>rivers with galery forest </li></ul><ul><li>fast running waters </li></ul><ul><li>open habitat </li></ul><ul><li>swamp-like habitat </li></ul>Trithemis stictica T. spec. nov T. spec. nov Damm et al. (2010) Molecular Ecology Case study – Ecology
    22. 22. Case study – Summary Integrative approach for species discovery Geography Distance-based DNA barcoding  Two new species Confirmed by Morphology Reproductive Isolation (directly and indirectly) Ecology High number of diagnostic characters Damm et al. (2010) Molecular Ecology DNA Morphology Reproductive Isolation Ecology Geography Seq. div. diagnostics Size parameters Cornuti shape F st ND1 CO1 ND1 CO1 Hw Bs Hw AbdL S4 differences ND1 COI T. stictica / Clade 2 9.0 7.9 26 43 * ** - - significant 0.960 0.984 T. stictica open habitat widespread T. stictica / Clade 3 8.5 8.3 27 43 *** *** *** *** significant 0.944 0.966 Clade 2 swamp-like habitats Caprivi region Clade 2 / Clade 3 5.0 5.7 13 28 - - ** ** weak 0.906 0.921 Clade 3 fast running water Caprivi region
    23. 23. Conclusions Trithemis stictica Trithemis morrisoni Trithemis palustris Character-based barcoding allows <ul><li>Identification of known species </li></ul><ul><li>Reliable discovery of new species </li></ul><ul><li>Comprehensive database by integrating characters of multiple disciplines </li></ul><ul><li>Many important information for conserving biodiversity </li></ul> Discovery of the first cryptic dragonfly complex in Odonates
    24. 24. <ul><li>Bernd Schierwater </li></ul><ul><li>Sabrina Simon </li></ul><ul><li>Jessica Rach </li></ul><ul><li>Eugene Marais (National Museum of </li></ul><ul><li>Namibia, Windhoek) </li></ul><ul><li>Michael Samways (University of </li></ul><ul><li>Stellenbosch, South Africa) </li></ul><ul><li>K.D. Dijkstra </li></ul><ul><li>Frank Suhling </li></ul><ul><li>Jens Kipping </li></ul><ul><li>Viola Clausnitzer </li></ul><ul><li>BMBF BIOTA South </li></ul>Acknowledgements
    25. 25. Thank you for your attention!

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