477 DNA Barcoding of from the Galápagos: searching native and introduced Vanderheyden A.1,2, Sonet G.2, Herrera H.W. 2,3,8, Delsinne T.4, Donoso D.5, Lattke J.6, De Busschere C. 2, Wittocx H. 1,2, Hendrickx F. 2,7, Leponce M. 2 , Pauly A 2, Backeljau T.1,2 and Dekoninck W. 2* Abstract Until now 50 species have been recorded from the Galápagos Archipelago. Yet, for 26 of them it is still unclear if they are native to the Galápagos. This uncertainty is due to the fact that the ant fauna of mainland is too poorly known to unequivocally infer the status of species in the Galápagos. Here we explore the possibility to use amounts of COI sequence variation (a DNA barcoding fragment) as an indicator to distinguish between introduced and native species. For the presumed endemic species C. planus, Dorymyrmex pyramicus albemarlensis and N. steinheili, large intraspecific nucleotide diversity was observed that was structured according to island geography. We observed a similar pattern of variation for two species with an unknown status i.e. Pheiodole HH1 and Nylanderia sp., which suggests that these are probably native species. Camponotus conspicuous zonatus, Pheidole megacephala, Hypoponera opacior, Hypoponera opacipes, Monomorium floricola, Cardiocondyla emery and louisianae, showed no COI variation, which might indicate that they are recently introduced species.

Significance: Our results stress that future studies should include a sufficient number of distinct populations from the archipelago and from areas where the species are native to increase confidence in the status of a species. If these conditions are met, our initial results showed that COI may serve as an indicative tool to distinguish native from introduced species, even if mainland relatives are unknown. Nevertheless lack of variation within COI might also be caused by other factors than recent introduction and this will be discussed and illustrated. Results Distances within subpopulations Discussion Solenopsis globularia d 0.0033 99 E, I, S, Sc, So, Ba, M, F, Sf, Sb Based on variation of COI:

100 Solenopsis gnoma 0.0015 Native : Ec, Sc Wasmannia auropunctata 0 • Pheidole HH01, Nylanderia sp, Nylanderia steinheili, Dorymyrmex 100 I, S, Sc, M, Ec 100 pyramicus albemarlensis & Camponotus planus Solenopsis sp1 Sc, S 0 100 Possible Native (not enough samples): 100 Solenopsis geminata 0.0378 I, M, Sc, Sb, Ec • 96 Cyphomyrmex rimosus & Cylindromyrmex whymperi 100 100 Cyphomyrmex HH04 Sc, S, Col 0.0014 New Records for Galapagos: 98 100 Cyphomyrmex rimosus Sc 0.0153 100 • Solenopsis sp1, Solenopsis sp2, Cardiocondyla mauritanica, 100 99 Strumigenys louisianae 100 I, Col, Sc, Ec 0.0471 S Cardiocondyla obscurior, Pheidole bilimeki & Paratrechina sp. 100 Strumigenys membranifera Trichomyrmex destructor S, B, IC 0 100 Monomorium pharaonis I, Sc, B, Sb 0.0057 100 Monomorium floricola Sc, Ec, S 0 100 Cardiocondyla minutior Ec, I 0.0014 100 Sufficient variation in COI can be an indication the species is native. If 100 Cardiocondyla mauritanica F, UF 0.0022 100 Cardiocondyla obscurior Sc, Se, Sc, Ma 0.0126 sequences of COI display a large distances within subpopulations (d>0.01) 98 99 82 Cardiocondyla emeryi 0.0504 and if this variation is island related. 100 Ec, M, I, Sc, M, I 90 Example: Pheidole HH01 certain sites (volcano 82 Pheidole HH01 0.0337 99 92 Id, Ia, Sc, Sb Alcedo vs volcano Darwin) or islands (Isabela, 97 100 Pheidole flavens Ec 100 Santa Cruz and San Cristobal) cluster separately. Pheidole megacephala IC, Sb 0 94 This is caused by natural distribution of species throughout the Pheidole williamsi 100 B3, F, Id, Ec, Ba, Ia, S 0.0076 Galapagos archipelago and random genetic drift of COI-gene. This in 100 Pheidole bilimeki Ec, Sc 0.0045 contrast to exotic species which were relatively recent introductions 100 Solenopsis sp2 Ec, Sb 0.0067 who display little to no variation of COI (d ~ 0) between specimens 97 collected on different islands. 83 Tetramorium lanuginosum 0.0088 100 80 Ba, M, B3, M, B2, So, S, M, I, I Example: Tetramorium bicarinatum sequences Tetramorium bicarinatum 0 100 S, Sc, I, Ec, F (Santiago, Santa Cruz, Isabela, Fernandina and 100 Tetramorium simillimum I, S 0.0089 mainland Ecuador) are identical. 99 Nylanderia sp This species is a cosmopolitan species and a recent introduction to the 82 S, Ia, Sc, Sb, Id, S, F, M, S 0.0230 Galapagos. 80 82 99 However! Multiple introductions of specimens from different source Nylanderia steinheili areas can cause confusion as different haplotypes can be introduced. A 100 0.0163 82 100 Sb, Sc, Col, Sc, Sc 99 large divergence in COI (d>0.01) can be mimicked by introduction from 100 Paratrechina longicornis Sc, IC, Sc 0.0152 Paratrechina sp. Sc multiple source areas. 100 Brachymyrmex heeri Ec, Sc, Ec 99 Example: bauri sequences do not cluster by island (Isabela sequences have two 100 100 Camponotus planus 0.0757 separate clusters). 100 F, Ia, I, Iw, Fl, P, Sc, Sb, Sc, Sb, Sb, M, S 100 100 Though variation occurs random, sequences do not cluster by island as is the case for variation of species from endemic populations. This 100 Camponotus conspicuus zonatus 0 99 P, S, Sf, Sb, Sc, Ec makes the distinction between exotic and native species and in return Camponotus macilentus 0.0034 suggestions for conservation difficult. 100 Id, M, Ia, Ia, F, S 100 Cylindromyrmex whymperi S, I 0.0135 0.0527 Based on this analysis 6 new species were recorded for Galapagos. 100 98 Odontomachus bauri 99 Ec, Sb, Sc, I, Ec, I Bringing the estimated total amount of recorded species to 56 ! 100 99 Hypoponera opaciceps Sc, Fl, I 0 100 I, Sc 100 Hypoponera opacior 0 85 100 Dorymyrmex pyramicus albemarlensis 100 0.0576 100 99 M, S, F, Ec 100 Tapinoma melanocephalum Sc, E, Ec Conclusion : 100 0 Outgroup Exotic species are arriving at Galapagos at an alarming speed, in 2009 alone 9 new records were made. Hence there are 15 new species ML Tree of ants collected from Galápagos and Ecuador, and some other countries introductions in less than 10 years! Status of species cannot always be Galápagos Islands: Ba = Bartholomew, B2 = Beagle 2, B3 = Beagle 3, S = Santiago, E = Espanola, F = correctly identified by use of COI-variation. These results stress the Fernandina, Fl = Floreana, I = Isabela (a:Alcedo-, d:Darwin-, w:Wolf- volcano), M = Marchena, P = Pinzon, Sb = San Cristobal, Sc = Santa Cruz, Sf = Santa Fe. BLACK/BOLD and underlined= Extracted need for continued monitoring and correct species identification in from specimen collected in mainland Ecuador (Ec = Ecuador) or collected elsewhere or Galapagos. This way potentially harmful species can be discovered and downloaded from BOLD (Col = Columbia, IC = Ivory Coast, Se = Seychelles, UF = USA Florida and possibly eradicated. Ma = Madagascar,)

Affiliations Funding Sources/Acknowledgements: 1 Evolutionary Ecology Group, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, This research was done in the context of the FWO research community GO9.9.11N and was funded Belgium. 2 Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussels, Belgium. 3 by FWO grant G0D2915N. We would like to thank the Charles Darwin Research Station (CDRS) and Charles Darwin Research Station, Puerto Ayora, Santa Cruz – Galápagos, Casilla 17-01-3891. 4 the Galápagos National Park for providing permits and field support, the Belgian Worktable for Société d'Histoire Naturelle Alcide-d'Orbigny, 57 rue de Gergovie, 63170 Aubière, France. 5 financial and logistic support at the CDRS, the Léopold III Founding and the Entomology Instituto de Ciencias Biológicas, Escuela Politécnica Nacional, Av. Ladrón de Guevara E11-253, Department of RBINS for their financial supports. This project is part of the project ‘Control of Apdo 17-01-2759, Quito, Ecuador 6 Departamento de Zoologia, Universidade Federal do Paraná, invasive invertebrates’ of the Charles Darwin foundation. This research also benefited from the Caixa Postal 19020, CEP 81531-980, Curitiba, . 7 Terrestrial Ecology Unit (TEREC),Biology financial support from the Belgian Directorate-General for Development Cooperation (DGD), Department, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium. 8 Facultad de partim Global Initiative, within the framework of the CEBioS programme. Recursos Naturales, Departamento de Entomología, Escuela Superior Politécnica de Chimborazo, Panamericana Sur km 1 ½, Riobamba, Ecuador. * Corresponding author. E-mail: [email protected]