Efficiency of the DNA-barcode expertise and its relationships with the practice of alpha-taxonomy illustrated by the stu

1. Efficiency of the DNA-barcode expertise and its relationships with the practice of alpha-taxonomy illustrated by the study of the genus Eumunida (Decapoda). Sarah Samadi (IRD) UMR 7138, Dept Syst & Evol, MNHN, Paris. Nicolas Puillandre, Enrique Macpherson, Josie Lambourdière, Marie-Catherine Boisselier

2. The context Given the estimated amount of biological species that needs to be described  Offering an efficient expertise tool is an important challenge for the taxonomy of the 21st century. Our positions > The taxonomic expertise directly lies on the taxonomic knowledge which continually evolves with the new data provided by the taxonomic research. > New expertise tools must be directly linked to taxonomic research. > The constitution of DNA barcode dataset on a given set of organisms must follow the requirements of specimens sampling of the taxonomic research.

3. TAXONOMIC EXPERTISE =  to link a specimen to a species name TAXONOMIC RESEARCH =  To propose hypotheses of species delimitations > What is a species ? > How in practice do we delimit species ? > How names are linked to these hypotheses ?

4. Extinction Speciation A species A species Speciation Speciation Delimiting species =  recovering the structure of the genealogical network A species is a permanently diverging lineage De Quieroz, 1998 Samadi & Barberousse, 2006 A species is a piece of genealogical network that is definitively divergent from others part of the network

5. (i) alpha-Taxonomy (morphology or molecules) (phenetic criteria) (i) Similarity in heritable characters (ii) Populations genetics and biology (biological criteria) (ii) Interfecondity, gene flows (iii) Phylogenetic reconstruction (phylogenetic criteria) (iii) Share a common history using heritable characters Delimiting species =  resolving the structure of the genealogical network How in practice do we resolve the structure of the network ? Samadi & Barberousse, 2006  a species is a reproductive community  Divergence with others such species is definitive

6. DNA offers many polymorphic and heritable characters on which the different criteria can be applied … But with some sampling requirements > to measure similarity or to estimate gene flows sampling must includeseveral specimens > Sampling must cover the known geographic distribution > Phylogenetically close species must be included (the evolutionary context) > In addition : The analysis of type-specimens permits to link the DNA-barcode to the names

7. COI is DNA fragmentthat offers many polymorphic and heritable characters But … other data should be integrated to test the primary hypotheses > unlinked molecular characters (nuclear versus mitochondrial) > Morphological characters > Life histroy traits > Ecological data (biotic and abiotic environnement …)

8. The genus Eumunida Well studied by « classical » taxonomy Collections preserved in ethanol Many specimens available for each species (MUSORSTOM cruises) http://www.mnhn.fr/musorstom/ Many type specimens available in the collections of the MNHN (Paris) Among the 26 described species we were able to extract and sequence DNA from specimens of the 17 species available in the collections of the MNHN, that cover the known distribution area Type specimens were available for 13 species 9 holotypes & 24 paratypes

9. The genus Eumunida > 229 specimens > 17 species / 26 described > 9 holotypes et 24 paratypes (13 species) > COI (« barcode fragment», 658pb) > 28S (C1’-D2, 867pb) Distribution of pairwise genetic distances for COI In black, the distribution of the distances between pairs of type specimens

10. The genus Eumunida > 229 specimens > 17 species / 26 described > 9 holotypes et 24 paratypes (13 species) > COI (« barcode fragment», 658pb) > 28S (C1’-D2, 867pb) 28S fragment is less variable than COI but the genetic clusters are the same COI data are more easily obtained For some old specimens we did not obtain the 28S sequence COI was obtained for all except one specimens

11. E. sternomaculata E. similor E. spinosa E. keiji E. treguieri E. multineata E. annulosa (holotype) E. laevimana E. marginata E. minor E. capilata E. bispinata E. picta E. squamifera E. funambulus The genus Eumunida > 229 specimens > 17 species / 26 described > 9 holotypes et 24 paratypes (13 species) > COI (« barcode fragment», 658pb) > 28S (C1’-D2, 867pb) When no type specimens is included in a genetic cluster the morphological determination key is used The type specimens are used to give names to the genetic clusters

12. E. sternomaculata E. similor E. spinosa E. keiji E. treguieri E. multineata One cluster correspond to three names (including the types of two of them) E. annulosa (holotype) E. laevimana E. marginata E. minor E. capilata E. bispinata E. parva + E. karubar + E. smithii E. picta E. squamifera The genus Eumunida > 229 specimens > 17 species / 26 described > 9 holotypes et 24 paratypes (13 species) > COI (« barcode fragment», 658pb) > 28S (C1’-D2, 867pb) The morphological diagnostic for one cluster correspond to a name already attributed, using the position of the holotype, to another cluster E. annulosa Overall, the species recognized using the traditional approach are recovered … but E. annulosa (holotype) E. funambulus

13. Spines on the carpus of the chelipeds The genus Eumunida One cryptic species is detected E. annulosa E. annulosa (holotype) This character is diagnostic only for adult specimens  Such characters are often used in determination keys In such a case the DNA-barcode expertise is more efficient than the traditional morphological approach Moreover it works for all the stages of the life cycle

14. The genus Eumunida Type specimens and localities are mixed for the three species Three species correspond to a unique genetic cluster S P K S K H K P K P K P K P P K P P P K P Ph P P K E. smithii E. karubar E. parva P P The hypothesis of synonymy is supported by the 28S data

15. The genus Eumunida > 229 specimens > 17 species / 26 described > 9 holotypes et 24 paratypes (13 species) This sampling and the associated molecular data set allow us to : > COI (« barcode fragment», 658pb) > 28S (C1’-D2, 867pb) (i) Support most of the primary species hypotheses based on morphology using the phenetic criterion on molecular data … but also to bring up new hypotheses (ii) For some of the species to show that gene flows occurs among populations over the geographic range of the species (Biological criterion) (… more details for two of them in Samadi et al, Mar Biol 2006) (iii) Using a close out-group outside the genus, to reveal that each of the proposed species has it own evolutionary history (Phylogenetical criterion)

16. When the taxonomy is well established The DNA-barcode can be used as a determination key But sampling must cover the intra and inter specific variability, the geographic distribution and when possible type specimens When sampling is adequate such data should enhance taxonomy and bring up new hypotheses But only if we go back to specimens and morphology Thus, DNA-barcode is an efficient expertise tool only if sampling is adequate and directly linked to taxonomical research.

17. acknowledgments The crew of the R/V Alis The staff of the ‘Service de systématique moléculaire’ at the ‘MNHN, Paris’ Régis Cleva (curator of crustacean collections) The ‘Consortium national de recherche en génomique’, Genoscope