The complete sequence of the cytochrome c oxidase subunit I (COI) gene of Noctua
atlantica (Warren, 1905) has been determined and deposited in the NCBI GenBank under
the Accession number AY600452. Complete and partial COI sequences of other Lepidoptera have been collected and used to reconstruct a phylogeny with both the Neighbor-Joining and the Maximum Likelihood methods. A molecular clock calibrated for our models indicate a divergence time between Noctua atlantica and Noctua pronuba of 4.7-5.9 Million years, consistent with the geological age of the Azores and suggesting that the speciation process of N. atlantica occurred in this archipelago

L. Oliveira

N. Simões

Rafael Montiel

T. Martins

V. Vieira

Montiel, Rafael

Vieira, Virgílio

Martins, Tiago

Oliveira, Luísa

Repositório da Universidade dos Açores

 43The speciation of Noctua atlantica (Lepidoptera, Noctuidae) occurred in the Azores as supported by a molecular clock based on mitochondrial COI sequences RAFAEL MONTIEL, V. VIEIRA, T. MARTINS, N. SIMÕES & M.L. OLIVEIRA Montiel, R., V. Vieira, T. Martins, N. Simões & M.L. Oliveira 2008. The speciation of Noctua atlantica (Lepidoptera, Noctuidae) occurred in the Azores as supported by a molecular clock based on mitochondrial COI sequences. Arquipélago. Life and Marine Sciences 25: 43-48. The complete sequence of the cytochrome c oxidase subunit I (COI) gene of Noctua atlantica (Warren, 1905) has been determined and deposited in the NCBI GenBank under the Accession number AY600452. Complete and partial COI sequences of other Lepidoptera have been collected and used to reconstruct a phylogeny with both the Neighbor-Joining and the Maximum Likelihood methods. A molecular clock calibrated for our models indicate a divergence time between Noctua atlantica and Noctua pronuba of 4.7-5.9 Million years, consistent with the geological age of the Azores and suggesting that the speciation process of N. atlantica occurred in this archipelago. Key words: divergence, endemic, Neighbor-Joining tree, Noctua pronuba, sequences Rafael Montiel (e-mail: montiel@uac.pt), V. Vieira, T. Martins, N. Simões & L. Oliveira. Universidade dos Açores, Universidade dos Açores, Departamento de Biologia, CIRN, Rua da Mãe de Deus, PT- 9501-801 Ponta Delgada, Açores, Portugal. INTRODUCTION The Azorean archipelago consists of nine volcanic islands of variable geological ages ranging from 0.037 to 8.12 million years (Queiroz 1990). It is situated in the Atlantic Ocean at 1500 km from mainland Portugal. These characteristics make it very interesting model systems to study colonization, radiation and even speciation process of invertebrate and vertebrate taxa. In the Azores there have been identified 38 Lepidoptera endemic species, eight of which are Noctuidae (Karsholt & Vieira 2005).     Noctua atlantica (Warren, 1905) is an endemic species of Azores (Warren 1905; Meyer 1991; Vieira & Silva 1994; Hacker & Schmitz 1996; Vieira 1997) that inhabit all the Azores islands, excepting Santa Maria Island (Carvalho et al. 1999; Karsholt & Vieira 2005). It is found in natural ecosystems of “Laurisilva” (ancient native evergreen forest found in Macaronesia) (Oliveira et al. 2004) and has been catalogued as an endangered species, affected mainly by the specificity of its habitat. On the other hand,        N. pronuba (Linnaeus, 1758), of palaearctic origin and also found in the Azores, has a less specific habitat and a more diverse geographic distribution. Noctua atlantica is morphologically very similar to N. pronuba, having a smaller wingspan and wings with a less shining color (Carvalho et al. 1999). Thus, it is very interesting to analyze the genetic relationship of these two species in the context of lepidopteran genetic variability to address evolutionary hypotheses and to help in developing conservation planning.     Sequences from the cytochrome c oxidase subunit I (COI) have been used to address phylogenetic problems at a wide range of hierarchical levels, from species to orders (Caterino & Sperling 1999). Hebert et al. (2003a)  44proposed that a DNA barcoding system for animal life could be based upon sequence diversity in COI and have applied this method to lepidopterians successfully (Hebert et al. 2003a; 2003b; although see also Will & Rubinoff 2004). Furthermore, Gaunt & Miles (2002) found that the COI is better suited to conduct studies based on the molecular clock assumption than other gene sequences, such as 16S, 18S, cytochrome b (cob) or elongation factor 1α (EF-1α). The molecular clock hypothesis is the basis of the modern molecular phylogenetic approach for dating and resolving evolutionary divergence (Gaunt & Miles 2002).     This is the first report on a DNA sequence of N. atlantica. We present its complete COI gene sequence that is compared with COI sequences of other lepidoptereans, including N. pronuba. Also, by calibrating a molecular clock, we determine an approximate time of divergence for these species and its relationship with the geological history of the archipelago. This represent a first step to develop studies aimed to understand how does genetic variation in N. atlantica compare with levels observed in other non-endemic Azorean lepidoptera. MATERIAL AND METHODS Noctua atlantica specimens were collected at Monte Escuro (800 m altitude) in São Miguel Island. Specimens were reproduced in laboratory and a 3rd-instar larva (from first generation) was selected for analysis.     For DNA extraction, larva was grinded under liquid nitrogen, re-suspended in 500 µl of digestion buffer (100 mM Tris HCl pH 8.0, 80 mM EDTA, 1% SDS, 160 mM sucrose) and digested overnight at 37 ºC with 25 µg proteinase K. After centrifugation, the supernatant was extracted once with phenol:chloroform:isoamyl alcohol (25:24:1) and once with chloroform:isoamyl alcohol (24:1). DNA was ethanol precipitated and re-suspended in 100 µl of H20.      Conserved primers TY-J-1460 and TL2-N-3014 (Simon et al. 1994) were used to PCR amplify a segment between genes for tRNA-Tyr and tRNA-Leu encompassing the complete gene for the mitochondrial cytochrome oxidase I (COI). The amplified fragment was cloned into a TOPO-TA vector (Invitrogen) and three clones were selected for direct sequencing. Universal primers T3 and T7 were used for ends sequencing, and primers NaCOXI-F104 (5'-ATTTTTGGAATTTGAGCTGG-3') and NaCOXI-R777 (5'-ATATAAACTTCTGGATGACC-3') were used for internal sequencing.     Fifteen COI sequences from species representing 4 Lepidopterean families, taken from the NCBI GenBank, were used for comparison (Table 1). For some species (as N. pronuba) the complete COI sequence was not available. For these reason our alignment was limited to 615 pb of the 5’ end. Ten out of these 15 sequences were from Noctuidae (seven Noctuinae). Sequences were aligned with Clustal W (Thompson et al. 1994) and used to construct Maximum Likelihood (ML) trees with Treepuzzle (Schmidt et al., 2002) and Neighbor-Joining trees using the Tamura-Nei gamma distance with Mega2 (Kumar et al. 2001). Treepuzzle was used to estimate the gamma parameter alpha. The standard errors for the Tamura-Nei distances were estimated analytically, and for the ML distances they were derived from the standard errors of the ML branch lengths. The robustness of the resulting trees was assessed by bootstrapping (1000 resamplings) and by quartet puzzling (10,000 puzzling steps) for the Neighbor-Joining and ML trees, respectively. The divergence time between Tegeticula synthetica and T. yuccasella (32.4 My), estimated from COI sequence data by Gaunt & Miles (2002), was used to calibrate the molecular clock for our data. RESULTS After remove the primer sequences, we obtained 1579 base pairs (bp) of good quality sequence (without ambiguities) from the three clones analyzed. Blast search (Altschul et al. 1997) at the NCBI GenBank allowed us to identify the complete gene for the cytochrome c oxidase subunit I (COI) of Noctua atlantica, from position 11 to 1555 and comprising 1545 bp. The 45Table 1. COI sequences used in this study. a NCBI GenBank. b Economically important pest according to Zhang (1994). c Present in the Azores archipelago.  Taxa Acession No. a Pest b Distribution  Family NOCTUIDAE      Subfamily Noctuinae       Agrotis ipsilon c AF549736 yes Cosmopolitan    Agrotis volubili AF549702 yes Nearctic    Anaplectoides prasina AF549765 yes Palaearctic    Feltia jaculifera    U60990 yes Nearctic    Feltia tricosa AF549766 no Nearctic    Noctua atlantica c AY600452 no Azores (endemic)    Noctua pronuba c AF549752 yes Palaearctic   Subfamily Plusiinae       Allagrapha aerea AF549755 yes Nearctic    Diachrysia chrysitis AJ420361 yes Palaearctic    Diachrysia tutti AJ420369 no Palaearctic  Family NYMPHALIDAE      Subfamily Nymphalinae       Nymphalis californica AY248789 yes Nearctic    Vanessa cardui c AY248782 yes Cosmopolitan    Vanessa virginiensis c AY248783 no Nearctic  Family HESPERIIDAE      Subfamily Pyrginae       Pyrgus communis AF170857 no Nearctic  Family PRODOXIDAE       Tegeticula sintetica AY327144 no Nearctic    Tegeticula yuccasella AY488835 no Nearctic  10 bp located upstream correspond to the 3’ end of the gene for tRNA-Tyr and the 24 bp downstream comprise the 5’ end of the tRNA-Leu gene. The COI start codon is ATA and the stop codon is TAA. As expected for insect mitochondrial genomes this gene has a high A+T content (73.07%). The N. atlantica sequence data has been deposited in the NCBI GenBank, under the Accession number AY600452.     We estimated Tamura-Nei and ML distances from the sequences used for comparison (Table 2, see the end of manuscript). Figure 1 shows the unrooted Neighbor-Joining tree obtained from the Tamura-Nei distances.  DISCUSSION We have shown that Noctua pronuba is the closest species to N. atlantica, showing little divergence between them (Fig. 1). Assuming that the genetic distances observed between T. synthetica and T. yuccasella, were produced in 32.4 My, the divergence time between Noctua atlantica and N. pronuba is 4.9 (± 2.1) My for the  46Table 2. Neighbor-Joining gamma distances (below diagonal) and Maximum Likelihood distances (above) estimated from COI sequences; a Standard error = 0.006;  b Standard error = 0.007.   A. ipsilon  A. volubilis  A. aerea  A. prasina  D. chrysitis  D. tutti  F. jaculifera  F. tricosa  N. atlantica  N. pronuba  N. californica  P. communis  T. synthetica  T. yuccasella  V. cardui  V. virginiensis Agrotis ipsilon  0.048 0.142 0.081 0.149 0.137 0.092 0.083 0.090 0.099 0.177 0.213 0.347 0.284 0.203 0.197 Agrotis volubilis 0.055  0.146 0.080 0.150 0.141 0.081 0.077 0.083 0.089 0.202 0.198 0.311 0.311 0.189 0.208 Allagrapha aerea 0.160 0.148  0.125 0.098 0.097 0.149 0.147 0.144 0.155 0.220 0.247 0.358 0.325 0.216 0.206 Anaplectoides prasina 0.116 0.091 0.141  0.134 0.123 0.092 0.093 0.056 0.064 0.204 0.206 0.358 0.296 0.195 0.201 Diachrysia chrysitis 0.160 0.147 0.104 0.138  0.008 0.167 0.161 0.148 0.153 0.212 0.249 0.319 0.300 0.231 0.204 Diachrysia tutti 0.151 0.141 0.107 0.130 0.009  0.156 0.148 0.140 0.145 0.202 0.241 0.299 0.277 0.218 0.193 Feltia jaculifera 0.100 0.083 0.151 0.098 0.167 0.155  0.031 0.124 0.134 0.198 0.234 0.365 0.331 0.204 0.221 Feltia tricosa 0.094 0.082 0.153 0.108 0.159 0.147 0.036  0.116 0.125 0.169 0.208 0.319 0.297 0.198 0.211 Noctua atlantica 0.106 0.087 0.157 0.064 0.148 0.142 0.129 0.126  0.013 b 0.196 0.187 0.304 0.280 0.207 0.219 Noctua pronuba 0.125 0.097 0.178 0.078 0.156 0.149 0.146 0.143 0.014 a  0.198 0.209 0.311 0.295 0.223 0.239 Nymphalis californica 0.196 0.204 0.251 0.230 0.220 0.213 0.207 0.171 0.203 0.207  0.239 0.309 0.289 0.135 0.137 Pyrgus communis 0.244 0.201 0.276 0.228 0.254 0.252 0.248 0.217 0.203 0.237 0.281  0.286 0.261 0.225 0.213 Tegeticula synthetica 0.398 0.325 0.392 0.440 0.341 0.326 0.405 0.340 0.320 0.335 0.314 0.317  0.076 0.375 0.366 Tegeticula yuccasella 0.305 0.315 0.361 0.328 0.324 0.301 0.342 0.303 0.288 0.316 0.300 0.274 0.093  0.341 0.343 Vanessa cardui 0.230 0.208 0.242 0.218 0.231 0.223 0.218 0.216 0.225 0.259 0.164 0.246 0.450 0.356  0.056 Vanessa virginiensis 0.208 0.216 0.225 0.221 0.203 0.196 0.229 0.221 0.233 0.274 0.160 0.231 0.425 0.357 0.065   Tamura-Nei distance, and 5.5 (± 3) My for the ML distance. In order to confirm this result we estimated, using the same methods, the distances between Pachliopta neptunus and Papilio garamas (data not shown), with an estimated divergence time of 89.1 My (Gaunt & Miles 2002). Using this data we obtained divergence times for N. atlantica and N. pronuba of 5.1 (± 2) My and 5.7 (± 3.2) My for the Tamura-Nei and ML distances, respectively.     These divergence times were obtained using the most conservative estimations reported by Gaunt & Miles (2002), and therefore can be considered to be near to the upper limit. Also it must be considered that the gene divergence usually predates species divergence (Nei 1987; Nichols 2001). Thus, our estimations are consistent with the Azorean origin of N. atlantica. The divergence coincides with the geological age of the Azorean archipelago either occurring just after formation of the first Island (Santa Maria) or subsequently, following the conformation of the archipelago due to volcanic phenomena. Even, the speciation could be related to the appearing of São Miguel, that occurred 4.01 My ago (Queiroz 1990).     An alternative hypothesis could be that N. atlantica occupied a broad geographical range in ancient times and ultimately become confined to the Azores due to ecological changes and niche reduction. This hypothesis could be supported if N. atlantica arose as species before the formation of the Azores archipelago, however, if these hypothetical climatic changes occurred more recently, this alternative hypothesis could also be consistent with a recent speciation of N. atlantica. In this study we analyzed the divergence between N.  atlantica  and  N.  pronuba,  however,  further  47 Fig. 1. Unrooted Neighbor-Joining tree constructed from Tamura-Nei gamma distances (alpha parameter = 0.23). The tree topology was identical for the ML tree. Numbers in branches are bootstrap (and quartet puzzling) support values.  insights into the age of these species could be obtained from the analysis of other Noctua species from Macaronesia, like N. carvalhoi (also an Azorean endemic species), N. teixeirai (endemic in Madeira), and N. noacki (endemic in Canary islands), and comparing their genetic distances relatively to N. pronuba, since this widely distributed species could be the ancestor of all the endemic Noctua species from Macaronesia. Also intra-specific variability in these species should be assessed. ACKNOWLEDGEMENTS Rafael Montiel received a postdoctoral fellowship from the Fundação para a Ciência e a Tecnologia, Portugal (SFRH/BPD/13256/2003).  48REFERENCES Altschul, S.F., T.L. Madden, A.A. Schäffer, J. Zhang, Z. Zhang, W. Miller & D.J. Lipman 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389-3402. Carvalho, J.P., V.F.F. Vieira. & M.U. Carvalho 1999. Borboletas nocturnas dos Açores. Ed. Amigos dos Açores. Ponta Delgada. 115 pp. Caterino, M.S. & F.A.H. Sperling 1999. Papilio phylogeny based on mitochondrial cytochrome oxidase I and II genes. Molecular Phylogenetics and Evolution 11: 122-137. Gaunt, M.W. & M.A. Miles 2002. An insect molecular clock dates the origin of the insects and accords with Palaeontological and Biogeographic landmarks. Molecular Biology and Evolution 19: 748-761. Hacker, H. & W. Schmitz 1996. Fauna und Biogeographie der Noctuidae des makaronesischen Archipels. Esperiana 4: 167-221. Hebert, P.D.N., S. Ratnasingham & J.R. de Waard 2003a. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceeding of the Royal Society of London (Series B Biological Sciences) 270 (Suppl. 1): 96-99. Hebert P.D., A. Cywinska, S. L. Ball & J. R deWaard 2003b. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London, Series Biological Sciences 270: 313-321.  Karsholt, O. & V. Vieira 2005. Lepidoptera. Pp.207-210 in: Borges, P.A.V., R. Cunha, R. Gabriel, A.F. Martins, L. Silva and V. Vieira (Eds.). A list of the terrestrial fauna (Mollusca and Arthropoda) and flora (Bryophyta, Pteridophyta and Spermatophyta) from the Azores.. Direcção Regional de Ambiente e do Mar dos Açores and Universidade dos Açores, Horta, Angra do Heroísmo and Ponta Delgada. 318 pp. Kumar, S., K. Tamura, I. Jakobsen & M. Nei 2001. MEGA2: Molecular Evolutionary Genetics Analysis software. Arizona State University, Tempe, Arizona, USA. Meyer, M. 1991: Les Lépidoptères de la region macaronésienne. II-Liste des Macro-Hétérocères observes en juillet-août 1990 aux Açores (Lepidoptera: Geometridae, Sphingidae, Noctuidae). Linneana Belgica 13: 117-134. [Lepidoptera from the Macaronesia region; in French]     Nei, M. 1987. Molecular Evolutionary Genetics. Columbia University Press, New York. 512 pp. Nichols, R. 2001. Gene trees and species trees are not the same. Trends in Ecology and Evolution 16: 358-364. Oliveira, M.L.M., V. Vieira & P.V. Garcia 2004. Effect of temperature on the biology of Noctua atlantica (Lepidoptera: Noctuidae), a species endemic to the Azores. European Journal of Entomology 101: 423-426. Queiroz, M.G.P.S. 1990. Aspectos vulcanológicos do Maciço das Sete Cidades. Centro de Vulcanologia do INIC, Universidade dos Açores, Ponta Delgada. 139 pp. Schmidt, H., K. Strimmer, M. Vingron & A. von Haeseler 2002. TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18: 502-504. Simon, C., F. Frati, A. Bechenbach, B. Crespi, H. Liu & P. Flook 1994. Evolution, weighting and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Annals of the Entomological Society of America 87: 651-701. Thompson, J.D., D.G. Higgins & T.J. Gibson 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673-4680. Vieira, V. & L. Silva 1994. Rhopalocera and Heterocera (Insecta, Lepidoptera) observed in four islands of the Azores. Boletim do Museu Municipal do Funchal 46: 85-94. Vieira, V. 1997. Lepidoptera of the Azores islands. Boletim do Museu Municipal do Funchal 49(273): 5-76. Warren, W. 1905. Lepidoptera collected by W.R. Ogilvie-Grant on the Azores and Madeira in 1903. Novitates zoologicae 12: 439-447. Will, K.W. & D. Rubinoff 2004. Myth of the molecule: DNA barcodes for species cannot replace morphology for identification and classification. Cladistics 20: 47–55. Zhang, B-Ch. 1994. Index of economically important Lepidoptera. CAB International. 599 pp.     Accepted 5 November 2008. 

The speciation of Noctua atlantica (Lepidoptera, Noctuidae) occurred in the Azores as supported by a molecular clock based on mitochondrial COI sequences

The speciation of Noctua atlantica (Lepidoptera, Noctuidae) occurred in the Azores as supported by a molecular clock based on mitochondrial COI sequences

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