Diptera: Chironomidae: Orthocladiinae

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Bulletin of Entomological Research (2014) 104, 65–78 © Cambridge University Press 2013

doi:10.1017/S0007485313000515

Integrating DNA barcodes and morphology for species delimitation in the Corynoneura group (Diptera: Chironomidae: Orthocladiinae) F.L. Silva1* and S. Wiedenbrug2 1

Laboratory of Aquatic Entomology, Department of Hydrobiology, Federal University of São Carlos, P.O. Box 676, 13565-905, São Carlos, SP, Brazil: 2 Museum of Zoology, University of São Paulo, Av. Nazaré, 481, 04263-000, São Paulo, SP, Brazil Abstract

In this study, we use DNA barcodes for species delimitation to solve taxonomic conflicts in 86 specimens of 14 species belonging to the Corynoneura group (Diptera: Chironomidae: Orthocladiinae), from the Atlantic Forest, Brazil. Molecular analysis of cytochrome c-oxidase subunit I (COI) gene sequences supported 14 cohesive species groups, of which two similar groups were subsequently associated with morphological variation at the pupal stage. Eleven species previously described based on morphological criteria were linked to DNA markers. Furthermore, there is the possibility that there may be cryptic species within the Corynoneura group, since one group of species presented internal grouping, although no morphological divergence was observed. Our results support DNA-barcoding as an excellent tool for species delimitation in groups where taxonomy by means of morphology is difficult or even impossible. Keywords: DNA-barcoding, DNA markers, Orthocladiinae, Neotropical, Brazil (Accepted 17 September 2013; First published online 10 October 2013)

Introduction The Corynoneura group (Chironomidae) is a large and ecologically diverse taxon containing over 180 species of nonbiting midges worldwide and at least 42 in the Neotropical region. These minute (0.5–1.5 mm) dipterans are found in a broad range of aquatic habitats and are especially diverse in mountain streams in the Atlantic Forest (Wiedenbrug & Trivinho-Strixino, 2011). The larvae seem to be particularly abundant on litter and stones of streams, and they are associated with macrophytes from lentic habitats (Sanseverino & Nessimian, 2001; Henriques-Oliveira et al., 2003). The Atlantic Forest has been identified as a biodiversity hotspot of global significance due to its high concentration of endemic taxa and vulnerability to processes that threaten

*Author for correspondence E-mail: [email protected]

its unique biodiversity (Myers et al., 2000). The enormous biodiversity of this biome results in part from the wide range of latitude it covers, its variations in altitude, its diverse climatic regimes as well as the geological and climatic history of the whole region (Galindo-Leal & Câmara, 2003). Currently, the coverage of the Atlantic Forest in Southeast Brazil also has been significantly reduced due to a long process of exploitation and degradation (Zaher et al., 2011). The Corynoneura group comprises nine valid genera: Corynoneura Winnertz, Corynoneurella Brundin, Ichthyocladius Fittkau, Onconeura Andersen et Sæther, Notocladius Harrison, Physoneura Ferrington et Sæther, Tempisquitoneura Epler, Thienemanniella Kieffer and Ubatubaneura Wiedenbrug et Trivinho-Strixino. The adult identification of this group is based mostly on genitalic structures, as for many other Chironomidae. Their morphology-based identification is often difficult, especially for non-experts, and often demands time-consuming genitalic dissections. The identification of adults of the Corynoneura group is further complicated

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by a lack of diagnostic morphological features to distinguish several species. Moreover, identification of some species can be achieved only by individual rearing of larvae and collecting larval and pupal skins to establish the associations between life stages. Despite these drawbacks, the group has recently been studied taxonomically in East Asia and in the Afrotropical, Nearctic and Neotropical regions (e.g., Fu et al., 2009, 2010a, b; Wiedenbrug & Trivinho-Strixino, 2009, 2011; Wiedenbrug et al., 2009, 2012, 2013; Fu & Sæther, 2012). Consequently, species-level keys as well as extensive reference collections are available for most of the genera, making this an ideal group of Chironomidae for investigating molecular/ morphological correspondence. The family Chironomidae has been increasingly studied from the perspective of DNA barcoding. Most published studies involving the cytochrome c-oxidase subunit I (COI) gene in chironomids have used sequence data to perform phylogenetic analyses (e.g., Allegrucci et al., 2006; Martin et al., 2007), delimit species (e.g., Carew et al., 2011; Silva et al., 2013), document species diversity (e.g., Ekrem et al., 2010a), biomonitor environmental changes (Brodin et al., 2012) and associate different life stages based on genetic similarity (e.g., Stur & Ekrem, 2011). DNA barcoding employs a short gene fragment to identify species. It represents a shift from the near-exclusive reliance on morphological features for the identification and delimitation of species to an approach that includes molecular characters in species discrimination (Ekrem et al., 2010a). Orthocladiinae is the second most diverse chironomid subfamily after Chironominae (Ashe & Cranston, 1990). It was established by Edwards (1929) derived from Kieffer’s Orthocladiariae group (Cranston, 1995) and its monophyly is supported, with Prodiamesinae as its sister-group. Within the Orthocladiinae, the tribe Corynoneurini is also well supported with the studied genera Thienemanniella, Corynoneura and Notocladius (Cranston et al., 2011). In this study, we use DNA barcodes for species delimitation in order to solve taxonomic conflicts in the Corynoneura group (Orthocladiinae) in the Atlantic Forest. We use sequences from mitochondrial COI to determine genetic groups in the Corynoneura group and examine sequence variation between and within these groups. Our study focuses on the genera that occur in the benthic fauna of streams in the Atlantic Forest; these are Corynoneura, Onconeura, Thienemanniella and Ubatubaneura, which were previously revised by the second author.

Material and methods The taxa included in this study were selected to represent as many species of the Corynoneura group as possible. Fieldwork was conducted in streams of the Atlantic Forest, Southeast Brazil. The material was initially collected for taxonomic studies. Adults, pharate adults and pupal exuviae were collected with drift nets. Living larvae were collected with hand nets and isolated in small boxes (1 cm × 1 cm) halffilled with stream water. Neither substratum nor food was given, except for some detritus carried over with the water. Boxes were checked twice a day for emerged specimens. The larval and pupal exuviae and the adults were preserved in 96% ethanol. Tissue for DNA extraction was sampled under a stereo microscope and shipped to the Canadian Centre for DNABarcoding at the University of Guelph (CCDB,

www.dnabarcoding.ca) for sequence analysis. The remainder of most of the sampled specimens were macerated in KOH and slide-mounted in Euparal (BioQuip, Rancho Dominguez, CA, USA) for species identification with a compound microscope. Morphological identification of species of Onconeura and Ubatubaneura was based on the available literature (Wiedenbrug & Trivinho-Strixino, 2009, Wiedenbrug et al., 2009), while species of Corynoneura and Thienemanniella were described and included in part as type material (Wiedenbrug & Trivinho-Strixino, 2011; Wiedenbrug et al., 2013). Nomenclature and abbreviations follow Sæther (1980). DNA extraction, PCR and sequencing followed standard protocols and primers at the CCDB. Sequence information was entered in the Barcode of Life Database (BOLD, www. barcodinglife.org) along with an image and collateral information for each voucher specimen. The detailed specimen records and sequence information, including trace files, are available in BOLD under the project ‘DNA Barcoding Chironomidae Brazil’ (MPSW). All sequences have been submitted to GenBank (Appendix). A maximum likelihood (ML) tree was produced in MEGA 5.03 (Tamura et al., 2011) using the general time reversible model with invariable sites and gamma correction for rate heterogeneity (GTR + G + I), as the latter has been found as the most appropriate model of nucleotide substitution for our dataset by hierarchic likelihood ratio test in MEGA 5.03 ( lnL = 6051.349, BIC = 14061.461, AIC = 12461.841). Bootstrapanalysis used 1000 pseudoreplicates (Felsenstein, 1985). The analyses of neighbour-joining and intra- and interspecific genetic distances were based on the Kimura-2-parameter (K2-P) model (Kimura, 1980) for easier comparison with other barcode studies and were also calculated with MEGA 5.03. Additionally, we compared the former sequences to the full Corynoneura group dataset in BOLD in order to observe how our species groups would be defined when compared with other species of the Corynoneura group from other regions of the world. The sequences were selected using the BOLD identification engine. Only sequences published in GenBank were considered.

Results Partial COI gene sequences were obtained from 86 specimens of 14 species and three morphotypes belonging to the Corynoneura group (Appendix). Most of them were adults with associated pupal and larval exuviae. Corynoneura ferelobata, Corynoneura fortispicula, Corynoneura mediaspicula, Corynoneura sertaodaquina, Corynoneura unicapsulata, Onconeura oncovolsella, Onconeura cf. semifimbriata and Onconeura similispina composed well-supported monophyletic groups which were fully congruent with the morphological characteristics (fig. 1). Corynoneura mineira and Ubatubaneura atlantica were represented by single individuals. The specimens identified morphologically as Onconeura japi were divided into two separate barcode groups (fig. 1). Nucleotide sequences of these specimens differed by a minimum of 13.87% and in up to 102 nucleotide sites. The pupae of group 1 (fig. 2a, b) present abdominal tergite VI with oral shagreen grouped and L3 seta elongated similar to L2 seta, whereas the pupae of group 2 (fig. 2c, d) exhibit abdominal tergite VI with oral shagreen sparsely distributed and L3 seta short similar to L4. Similarly, the specimens identified as O. similispina also formed two distinct groups, but no morphological differences were observed. Nucleotide

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Fig. 1. For Figure legend see next page.

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Fig. 1. ML tree of the Corynoneura group, based on partial COI sequences (DNA barcodes) and the generalized time reversible substitution model. Numbers on branches are bootstrap values >80%.

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Table 1. Variable and informative sites and average nucleotide composition in the analysed COI gene sequences. Nucleotide position 1st 2nd 3rd All

% Variable sites

% Informative sites

% Adenine

% Cytosine

% Guanine

% Thymine

18.5 3.1 78.4 46.5

17.0 1.2 81.8 93.1

27.3 14.5 45.2 29.0

15.9 25.7 7.5 16.3

28.2 16.1 3.9 16.1

28.6 43.7 43.4 38.6

Fig. 2. (a–d) Onconeura japi. (a–b) O. japi (group 1). (a) Tergite VI showing chaetotaxy and shagreen grouped distributed. (b) Sternite IV showing shagreen distribution and elongated lateral setae 3 (L3). (c–d) O. japi (group 2). (c) Tergite VI showing chaetotaxy and shagreen sparsely distributed. (d) Sternite IV showing shagreen distribution and short lateral setae 3 (L3).

sequences of these specimens diverged by a minimum of 11.61% and up to 93 nucleotide sites. DNA barcode sequences also indicated that two groups represented related species. Thienemanniella sp. group 1 (fig. 1) resembled Thienemanniella sp. group 2 (fig. 1) in all morphological diagnostic characters. Based on the molecular analysis some morphological differences in pupal characters could be observed. The differences are in the shape of the posterior spines of the male abdominal sternite VIII, and the displacement of Dc1 to ventral. Nucleotide sequences of these specimens differed by a minimum of 17.5% and in up to 106 nucleotide sites. Additionally, Ubatubaneura sp., a female without associated immatures, previously identified as U. atlantica, was also recognized as a possible distinct species; however, we could not link it to any morphological variation. Nucleotide sequences of U. atlantica and U. sp. differed by a minimum of 11.6% and in up to 66 nucleotide sites. Furthermore, the morphotypes Corynoneura sp. sexadentata and Corynoneura sp. septadentata gr. were represented by two groups, although no morphological variation was observed. Nucleotide sequences of these specimens differed by a minimum of 14.9% and up to 87 nucleotide sites. In this study, the aligned sequences were 659 bp long with 260 variable sites (46.5%), of which 242 (93.1%) were parsimony-informative. Most variable sites occurred in the third codon-position (Table 1). The sequences were heavily AT-biased, specifically in the third position which showed a combined average AT-composition of 88.6% (Table 1). The

pairwise distances for the analysed specimens, produced by the K2-P model, showed distinctly larger interspecific than intraspecific divergences (Table 2). All species were distinguishable by genetic distances. Average intra- and interspecific K2-P distances for all analysed species were 2.29% and 18.28%, respectively. Maximum intraspecific divergence was observed in O. japi (14.84%), followed by Corynoneura similispina (12.18%) and C. fortispicula (6.70%) (Table 2). Lowest interspecific distances were found between U. atlantica and U. sp. (average 11.6%), followed by C. ferelobata and U. sp. (average 12.2%). A ML tree (fig. 1) demonstrated that a substantial barcode divergence exists between all the analysed species of the Corynoneura group. For comparison, a neighbour-joining tree was also produced and yielded almost identical trees, except for C. mineira, which groups with C. sp. sexadentata + C. sp. septadentata gr. + O. oncovolsella + Onconeura cf. semifimbriata as its closest cluster (data not shown). Bootstrap support exhibited minor variation among all analyses, but were always 96% or higher for all species groups. Molecular analysis of COI gene sequences from 151 specimens of the full Corynoneura group dataset in BOLD supported 33 cohesive groups (fig. 3). A ML tree showed a significant barcode divergence between all the analysed groups (fig. 3). Bootstrap support was always 99% or higher for all groups. In short, groups obtained in the original data set were maintained, and the previous species already in BOLD also composed monophyletic groups.

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Table 2. Summary of intra- and interspecific Kimura 2-parameter distances between morphological species of the Corynoneura group. N/A indicates species with only one specimen examined. Species

Mean intraspecific K2-P distance

Maximum intraspecific K2-P distance

K2-P distance to nearest neighbour

Mean interspecific K2-P distance

5.2 3.8 1.9 N/A N/A 0.0 1.5 0.0 7.1 1.5 1.2 3.1 2.4 0.6 N/A N/A

5.2 6.7 3.5 N/A N/A 0.0 1.5 0.0 14.8 2.5 1.5 12.2 3.6 1.1 N/A N/A

11.3 12.6 13.3 13.5 12.6 13.3 13.6 12.7 13.7 12.7 12.7 12.9 12.4 13.5 10.8 10.8

18.9 18.2 18.8 17.9 16.8 17.6 18.5 17.7 19.2 17.8 18.3 18.6 17.3 17.6 19.7 17.9

Corynoneura ferelobata Corynoneura fortispicula Corynoneura mediaspicula Corynoneura mineira Corynoneura sp. septadentata gr. Corynoneura sertaodaquina Corynoneura sp. sexadentata Corynoneura unicapsulata Onconeura japi Onconeura oncovolsella Onconeura cf. semifimbriata Onconeura similispina Thienemanniella gr. 1 Thienemanniella gr. 2 Ubatubaneura atlantica Ubatubaneura sp.

Discussion DNA barcodes have proven useful for identifying a variety of chironomid species (Carew et al., 2005, 2007; Stur & Ekrem, 2011; Silva et al., 2013). We found COI useful for summarizing the sequence diversity and detecting taxonomically challenging species and species groups within the Corynoneura group from the Atlantic Forest. The most common species previously described by means of morphology were linked to molecular markers. Similarly, Ekrem et al. (2007) were able to associate species of Micropsectra (Chironominae) previously described based on morphological criteria to genetic markers. A ML tree based on analysis of pairwise COI distances of the Brazilian species of the Corynoneura group indicated that most species showed substantial barcode divergence, but there was some evidence for cryptic diversity. There is strong evidence for at least three groups indicating new species in the Corynoneura group in southeast Brazil. Specimens of the two observed groups of Thienemanniella were collected from two ecosystems in São Paulo State, one in São Carlos municipality and one in Ubatuba municipality (Appendix). The two groups exhibited exclusive distributions, indicating some geographical structure in the grouping. These species were recently described by Wiedenbrug et al. (2013) as Thienemanniella sancticaroli (group 1) and Thienemanniella ubatuba (group 2), based on the results of the present study. Likewise, Carew et al. (2011) morphologically delimited species of Procladius based on groups previously defined by DNA barcodes. According to our results O. japi consists of two groups. The analysis of specimens belonging to these groups also indicated some morphological differences only noticed at the pupal stage. The diverging specimens were collected from four different sites in Southeast Brazil, three in Ubatuba municipality and one in Paraisópolis municipality (Appendix). All specimens from both the groups were collected at the two municipalities; therefore, there was no geographical structure in the grouping. Despite these minor differences, in the absence of additional evidence we hesitate to consider species distinctions among these specimens. In contrast, no morphological differences could be determined between Ubatubaneura species; only two specimens of this genus were examined,

of which one was an unassociated female, thus more material is necessary to understand the variation within this genus. Cryptic species may be present within the Corynoneura group. The taxa currently defined as O. similispina showed internal grouping of some individuals in the pairwise analyses, and no morphological variation was observed. Similarly Silva et al. (2013) obtained two separate clusters of Labrundinia tenata specimens (Tanypodinae), although they were unable to determine any distinct morphological difference distinguishing the clusters. On the other hand, Sinclair & Gresens (2008) found one cluster represented by two species of Cricotopus (Orthocladiinae), with minor genetic variation and consistent morphological differences. These results indicate that a more intensive investigation of these taxa would be needed to positively assign new species. This should include a higher number of specimens collected throughout the species range, different life stages and multiple DNA markers, particularly those from a nuclear origin. According to Wiedenbrug & Trivinho-Strixino (2011) the species C. septadentata, C. sertaodaquina and the morphotypes C. sp. sexadentata and C. sp. circulimentum, the last two described as female and immatures, are very similar species with a few differences in the immature stages. In our study, it was possible to verify a clear distinction between C. sertaodaquina and C. sp. sexadentata. On the other hand C. sp. septadentata gr. was not clearly identified because of the lack of larvae, but its very fine shagreen at pupal sternite II indicates that it might be C. sp. circulimentum. In this case, more material is needed, not only to understand the group genetically, but also in order to obtain diagnostic morphological characters of the males that are still missing for both morphotypes. The addition of other species of the Corynoneura group into the barcoding analysis was useful to confirm the species groups obtained in the original data set. Species groups with similar morphology, O. japi group 1 and 2 and Thienemanniella group 1 and 2, were sustained. Moreover, the internal grouping previously observed in O. similispina was maintained, not composing a distinct monophyletic group. The use of DNA barcodes is demonstrated to be problematic on higher geographical scales (Bergsten et al., 2012), since in this case the intraspecific variation increases

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Fig. 3. For Figure legend see next page.

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Fig. 3. For Figure legend see next page.

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Fig. 3. ML tree of the full Corynoneura group data set in BOLD (only sequences in GenBank were analysed), based on partial COI sequences (DNA barcodes) and the generalized time reversible substitution model. Numbers on branches are bootstrap values >90%.

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while the interspecific decreases. However, in our study, although the specimens included in the analysis of the full Corynoneura group data set were originated from different regions of the world, the species groups remained distinct and well supported. DNA barcodes have been argued to be unreliable for consistent species identifications by many authors due to a number of drawbacks (DeSalle et al., 2005; Will et al., 2005; Rubinoff et al., 2006; Ebach, 2011). Recent speciation, incomplete lineage sorting, interspecific hybridization and infection by endosymbiotic bacteria such as Wolbachia (Funk & Omland, 2003; Whitworth et al., 2007; Foottit & Adler, 2009) may all interfere with the performance of DNA barcoding in insects (Virgilio et al., 2010). In Chironomidae, Martin et al. (2002) verified incongruence in phylogenetic analyses of Chironomus (Chironominae) within a dataset composed of specimens from Nearctic and Palearctic populations and attributed this issue to mtDNA gene flow resulting from hybridization. However, numerous studies have been successful in species delimitation of Chironomidae (Carew et al., 2005, 2007; Ekrem et al., 2007, 2010b; Sinclair & Gresens, 2008; Silva et al., 2012, 2013 and this study). Our analysis of DNA barcode sequences using ML analyses supported 14 cohesive species groups belonging to the Corynoneura group, of which two similar groups were subsequently associated with morphological variation at the pupal stage. Eleven species previously described, based on morphological criteria, were linked to DNA markers. Moreover, there is the possibility that there may be cryptic species within the Corynoneura group, since one group of species presented internal grouping, although no morphological divergence was observed. Finally, regardless of the promising results, the incorporation of nuclear genes is valuable for species delimitation and might strengthen results, as they are independent of the maternal inherited mitochondrial genes.

Acknowledgments The authors thank Paul Hebert and the staff at the Biodiversity Institute of Ontario, University of Guelph, Canada for sequencing the specimens used in this study. Sequencing in Guelph was supported from Genome Canada through a grant by the Ontario Genomics Institute to Paul Hebert. The authors are indebted to Mateus Pepinelli for his assistance. The authors also thank Nathan Viets for language improvements and Torbjørn Ekrem and an anonymous reviewer for comments and suggestions on the manuscript.

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DOI: http://dx.doi.org/10.4039/tce.2013.44, Published online: 5 September 2013. Sinclair, C.S. & Gresens, S.E. (2008) Discrimination of Cricotopus sp. (Diptera: Chironomidae) with mitochondrial gene cytochrome oxidase I sequences. Bulletin of Entomological Research 98, 555–563. Stur, E. & Ekrem, T. (2011) Exploring unknown life stages of Arctic Tanytarsini (Diptera: Chironomidae) with DNA barcoding. Zootaxa 2743, 27–39. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28, 2731–2739. Virgilio, M., Backeljau, T., Nevado, B. & Meyer, M.D. (2010) Comparative performances of DNA barcoding across insect orders. BMC Bioinformatics 11, 206. Whitworth, T.L., Dawson, R.D., Magalon, H. & Baudry, E. (2007) DNA barcoding cannot reliably identify species of the blowfly genus Protocalliphora (Diptera: Calliphoridae). Proceedings of the Royal Society B: Biological Sciences 274, 1731–1739. Wiedenbrug, S. & Trivinho-Strixino, S. (2009) Ubatubaneura, a new genus of the Corynoneura group (Diptera: Chironomidae: Orthocladiinae) from the Brazilian Atlantic Forest. Zootaxa 1993, 41–52. Wiedenbrug, S. & Trivinho-Strixino, S. (2011) New species of the genus Corynoneura Winnertz (Diptera, Chironomidae) from Brazil. Zootaxa 2822, 1–40. Wiedenbrug, S., Mendes, H.F., Pepinelli, M. & TrivinhoStrixino, S. (2009) Review of the genus Onconeura Andersen et Sæther (Diptera: Chironomidae), with the description of four new species from Brazil. Zootaxa 2265, 1–26. Wiedenbrug, S., Lamas, C.J.E. & Trivinho-Strixino, S. (2012) A review of the genus Corynoneura Winnertz (Diptera: Chironomidae) from the neotropical region. Zootaxa 3574, 1–61. Wiedenbrug, S., Lamas, C.J.E. & Trivinho-Strixino, S. (2013) A review of neotropical species in Thienemanniella Kieffer (Diptera, Chironomidae). Zootaxa 3670, 215–237. Will, K.P., Mishler, P.D. & Wheeler, Q.D. (2005) The perils of DNA barcoding and the need for integrative taxonomy. Systematic Biology 54, 844–851. Zaher, H., Barbo, F.E., Martínez, P.S., Nogueira, C., Rodrigues, M.T. & Sawaya, R.J. (2011) Répteis do Estado de São Paulo: Conhecimento Atual e Perspectivas. Biota Neotropica 11, 1–15.

Taxon

Locality

Stage

Voucher number

Accession number

Sweden, Skane, Havaeng, Baltic Sea Coast, 10.viii.2010 Sweden, Uppland, Graeddoe Asken, Baltic Sea Coast, 28.vii.2010 Sweden, Uppland, Graeddoe Asken, Baltic Sea Coast, 28.vii.2010 Sweden, Uppland, Graeddoe Asken, Baltic Sea Coast, 21.viii.2010 Norway, Sor-Trondelag, Roeros kommune, spring C3, 07.vi.2006 Norway, Sor-Trondelag, Roeros kommune, spring C3, 07.vi.2006 Norway, Sor-Trondelag, Roeros kommune, spring C3, 22.vi.2006 Norway, Sor-Trondelag, Roeros kommune, spring C3, 20.vii.2006 Norway, Sor-Trondelag, Roeros kommune, spring C3, 20.vii.2006 Norway, Sor-Trondelag, Roeros kommune, spring C3, 20.vii.2006 Norway, Sor-Trondelag, Roeros kommune, spring A1, 29.viii.2005 Norway, Sor-Trondelag, Roeros kommune, spring C3, 13.vi.2007 Norway, Sor-Trondelag, Roeros kommune, spring C3, 02.vi.2007 Norway, Sor-Trondelag, Roeros kommune, spring B1, 31.viii.2006 Norway, Sor-Trondelag, Roeros kommune, spring A2, 03.viii.2006 Norway, Sor-Trondelag, Roeros kommune, spring C3, 03.viii.2006 Norway, Sor-Trondelag, Roeros kommune, spring C3, 11.vi.2006 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 05.1.2010 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 05.1.2010 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 05.1.2010 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 05.1.2010 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 05.1.2010

M M M M M M M M M M M M M M M M F F, LEX, PEX PM F, LEX, PEX F, LEX, PEX P, LEX M, LEX, PEX M, LEX, PEX L P PM

BSCHI042 BSCHI059 BSCHI060 BSCHI107 CHSOE007 CHSOE008 CHSOE015 CHSOE022 CHSOE023 CHSOE024 MIDGE053 MIDGE564 MIDGE573 MIDGE612 MIDGE650 MIDGE660 MIDGE812 MPSW041 MPSW052 MPSW035 MPSW036 MPSW038 MPSW043 MPSW048 MPSW049 MPSW050 MPSW051

KC250769 KC250767 KC250768 KC250770 HM406102 HQ105041 HM406104 HQ105042 HQ105043 HM406106 HQ105039 HQ105034 HQ105035 HQ105036 HQ105037 HQ105038 HQ105040 KF489885 KF489822 KF489857 KF489814 KF489886 KF489856 KF489868 KF489887 KF489865 KF489875

C. fortispicula C. fortispicula C. fortispicula C. fortispicula C. fortispicula C. fortispicula C. fortispicula C. fortispicula C. lacustris C. lacustris C. lacustris C. lacustris Corynoneura lobata C. lobata Corynoneura mediaspicula C. mediaspicula C. mediaspicula C. mediaspicula C. mediaspicula Corynoneura mineira Corynoneura sp. septadentata Corynoneura sertaodaquina

Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 05.1.2010 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 05.1.2010 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 05.1.2010 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 05.1.2010 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 29.xii.2009 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 29.xii.2009 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 29.xii.2009 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 01.v.2009 Norway, Sor-Trondelag, Roeros kommune, spring C1, 04.vii.2005 Norway, Sor-Trondelag, Roeros kommune, spring C1, 10.vii.2005 Norway, Sor-Trondelag, Roeros kommune, spring C1, 10.vii.2005 Norway, Sor-Trondelag, Roeros kommune, spring C1, 10.vii.2005 Norway, Sor-Trondelag, Roeros kommune, spring B2, 31.viii.2006 Norway, Sor-Trondelag, Roeros kommune, spring B2, 22.vi.2006 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, MG, Gonçalves, Cachoeira das Andorinhas, 14.x.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 29.xii.2009 Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira da Renata, 15.x.2008

PF P, LEX PF PF, LEX PM, LEX F, LEX, PEX F, LEX, PEX F, LEX, PEX M M M M M F M, LEX, PEX F, LEX, PEX PM, LEX F, LEX, PEX F, LEX, PEX F, LEX, PEX PF F, LEX, PEX

MPSW054 MPSW055 MPSW056 MPSW058 MPSW059 MPSW060 MPSW063 MPSW094 MIDGE205 MIDGE206 MIDGE208 MIDGE805 MIDGE618 MIDGE847 MPSW037 MPSW040 MPSW045 MPSW047 MPSW103 MPSW044 MPSW053 MPSW025

KF489835 KF489845 KF489852 KF489830 KF489853 KF489862 KF489859 KF489889 HQ105044 HQ105045 HQ105046 HQ105047 HQ105048 HQ105049 KF489847 KF489833 KF489877 KF489832 KF489882 KF489825 KF489860 KF489840

* * * * * * * * * * * * * * * * * *

* * * * * * * *

F.L. Silva and S. Wiedenbrug

Corynoneura carriana C. carriana C. carriana C. carriana Corynoneura fittkaui C. fittkaui C. fittkaui C. fittkaui C. fittkaui C. fittkaui C. fittkaui C. fittkaui C. fittkaui C. fittkaui C. fittkaui C. fittkaui C. fittkaui Corynoneura ferelobata C. ferelobata Corynoneura fortispicula C. fortispicula C. fortispicula C. fortispicula C. fortispicula C. fortispicula C. fortispicula C. fortispicula

76

Appendix : List of analysed specimens with associated sample localities, voucher reference numbers and GenBank accessions. M = male, F = female, P = pupa, L = larva, PM = pharate male, PF = pharate female, PEX = pupal exuviae, LEX = larval exuviae. * indicates the former sequences sampled in the Atlantic Forest, Brazil.

Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira da Renata, 28.xii.2009 Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira da Renata, 29.xii.2009 Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira da Renata, 29.xii.2009 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 16.x.2008 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 29.xii.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009

PF, LEX P, LEX F, LEX, PEX F, PEX F, LEX, PEX P, LEX M, LEX, PEX F, LEX, PEX M, LEX, PEX

MPSW061 MPSW062 MPSW067 MPSW019 MPSW057 MPSW034 MPSW039 MPSW042 MPSW046

KF489871 KF489819 KF489869 HM379587 KF489858 KF489880 KF489850 KF489866 KF489842

* * * * * * * * *

C. unicapsulata C. unicapsulata C. unicapsulata Corynoneura cf. scutellata Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Corynoneura sp. Onconeura japi O. japi O. japi O. japi O. japi O. japi O. japi O. japi O. japi O. japi O. japi O. japi O. japi O. japi O. japi O. japi O. japi O. japi Onconeura oncovolsella

Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Norway, Sor-Trondelag, Roeros kommune, spring A2, 13.vi.2007 Norway, Sor-Trondelag, Roeros kommune, spring B2, 22.vi.2006 USA, California, Los Angeles, E. Fork San Gabriel, 17.vi.2010 USA, California, Los Angeles, E. Fork San Gabriel, 17.vi.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira do Engenho, 14.x.2008 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 16.x.2008 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 16.x.2008 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 16.x.2008 Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira do Engenho, 16.x.2008 Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira da Renata, 15.x.2008 Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira da Renata, 27.xii.2009 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 11.iv.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 13.x.2009 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 11.iv.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 01.v.2009 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 01.v.2009 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 11.iv.2009 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 01.v.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira da Renata, 27.xii.2009 Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira do Engenho, 14.x.2008

M, LEX, PEX F, LEX, PEX F, LEX, PEX M F L L L L L L L L L L L L L L L L L L L L L L M, LEX, PEX F, LEX, PEX F, LEX, PEX F, LEX, PEX M, LEX, PEX M, LEX, PEX M, PEX F, LEX, PEX M, LEX, PEX M, LEX, PEX F, LEX, PEX M, LEX, PEX M, LEX, PEX M, LEX, PEX M, PEX F, LEX, PEX M, PEX F, LEX, PEX F, LEX, PEX

MPSW099 MPSW101 MPSW102 MIDGE547 MIDGE843 CFWIB799 CFWIB800 SWRC035 SWRC101 SWRC130 SWRC142 SWRC187 SWRC192 SWRC193 SWRC249 SWRC316 SWRC351 SWRC425 SWRC430 SWRC446 SWRC455 SWRC456 SWRC489 SWRC544 SWRC610 SWRC671 SWRC700 MPSW007 MPSW009 MPSW011 MPSW022 MPSW023 MPSW024 MPSW090 MPSW091 MPSW092 MPSW105 MPSW107 MPSW108 MPSW109 MPSW110 MPSW111 MPSW112 MPSW113 MPSW115 MPSW002

KF489816 KF489823 KF489876 HQ105033 HQ105032 HQ939815 HQ939816 JF286776 JF286777 JF286778 JF286779 JF286780 JF286782 JF286781 JF286783 JF286784 JF286786 JF286788 JF286787 JF286790 JF286789 JF286792 JF286791 JF286794 JF286795 JF286785 JF286793 HM379582 HM379583 HM379585 KF489811 HM379588 KF489838 KF489844 KF489888 KF489824 KF489831 KF489843 KF489820 KF489874 KF489815 KF489812 KF489834 KF489881 KF489837 HM379579

* * *

* * * * * * * * * * * * * * * * * * *

Integrating DNA barcodes and morphology in the Corynoneura group

Corynoneura sertaodaquina C. sertaodaquina C. sertaodaquina Corynoneura sp. sexadentata C. sp. sexadentata Corynoneura unicapsulata Corynoneura unicapsulata C. unicapsulata C. unicapsulata

77

78

Appendix : (Cont.) Taxon

Stage

Voucher number

Accession number

Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira do Engenho, 14.x.2008 Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira da Renata, 15.x.2008 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 01.v.2009 Brazil, SP, São Carlos, UFSCar, Fazzari stream, 18.xii.2008 Brazil, SP, São Carlos, UFSCar, Fazzari stream, 18.xii.2008 Brazil, SP, São Carlos, UFSCar, Fazzari stream, 18.xii.2008 Brazil, SP, São Carlos, UFSCar, Fazzari stream, 18.xii.2008 Brazil, SP, São Paulo, Parque Estadual do Jaraguá, 11.ii.2008 Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira do Engenho, 14.x.2008 Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira do Engenho, 14.x.2008 Brazil, SP, Ubatuba, Sertão da Quina, Sítio Santa Cruz, Bridge, 29.xii.2010 Brazil, SP, Ubatuba, Sertão da Quina, Sítio Santa Cruz, Bridge, 6.i.2011 Brazil, SP, Ubatuba, Sertão da Quina, Sítio Santa Cruz, Bridge, 6.i.2011 Brazil, SP, Ubatuba, Sertão da Quina, Sítio Santa Cruz, Bridge, 6.i.2011 Brazil, SP, Ubatuba, Sertão da Quina, Sítio Santa Cruz, Bridge, 6.i.2011 Brazil, SP, Ubatuba, Sertão da Quina, Sítio Santa Cruz, Bridge, 6.i.2011 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Brazil, MG, Paraisópolis, Stream Rua Joaquim Costas n°1, 11.x.2009 Norway, Sor-Trondelag, Roeros kommune, spring C3, 11.vi.2006 Norway, Sor-Trondelag, Roeros kommune, spring B2, 22.vi.2006 Norway, Sor-Trondelag, Roeros kommune, spring C1, 10.vii.2005 Norway, Sor-Trondelag, Roeros kommune, spring B2, 6.vii.2006 Norway, Sor-Trondelag, Roeros kommune, spring B2, 20.vii.2006 Norway, Sor-Trondelag, Roeros kommune, spring B1, 18.viii.2006 Norway, Sor-Trondelag, Roeros kommune, spring B1, 18.viii.2006 Norway, Sor-Trondelag, Roeros kommune, spring B1, 18.viii.2006 Norway, Sor-Trondelag, Roeros kommune, spring B1, 3.viii.2006 Norway, Sor-Trondelag, Roeros kommune, spring B1, 20.vii.2006 Norway, Sor-Trondelag, Roeros kommune, spring B1, 20.vii.2006 Norway, Sor-Trondelag, Roeros kommune, spring A3, 11.vi.2006 Norway, Sor-Trondelag, Roeros kommune, spring A1, 22.vi.2006 USA, California, Ventura, Conejo Creek, 21.vi.2010 USA, California, Ventura, Conejo Creek, 21.vi.2010 USA, California, Ventura, Conejo Creek, 21.vi.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 USA, Pennsylvania, Chester, White Clay Creek, 10.iii.2010 Brazil, SP, São Carlos, UFSCar, Fazzari stream, 18.xii.2008 Brazil, SP, São Carlos, UFSCar, Fazzari stream, 18.xii.2008 Brazil, SP, São Carlos, UFSCar, Fazzari stream, 18.xii.2008 Brazil, SP, Ubatuba, Sertão da Quina, Sítio Santa Cruz, Bridge, 21.i.2011 Brazil, SP, Ubatuba, Sertão da Quina, Sítio Santa Cruz, Bridge, 21.i.2011 Brazil, SP, Ubatuba, Sertão da Quina, Sítio Santa Cruz, Bridge, 21.i.2011 Brazil, SP, Ubatuba, Sertão da Quina, Sítio Santa Cruz, Bridge, 21.i.2011 Brazil, SP, Ubatuba, Sertão da Quina, Sítio Santa Cruz, Bridge, 8.i.2011 Brazil, SP, Ubatuba, Sertão da Quina, Sítio Santa Cruz, Bridge, 24.i.2011 Brazil, SP, Ubatuba, Sertão da Quina, Cachoeira da Renata, 7.vii.2009 Brazil, SP, Ubatuba, Stream besides Ruínas da Lagoinha, 11.iv.2009

M, LEX, PEX M, LEX, PEX M, LEX, PEX F, LEX, PEX M, LEX, PEX M, LEX, PEX F, LEX, PEX M, LEX, PEX F, LEX, PEX M, LEX, PEX F, LEX, PEX M, LEX, PEX M, LEX, PEX F, LEX, PEX F, LEX, PEX F, LEX, PEX M, LEX, PEX M, LEX, PEX M M M M M M M M M M M F F L L L L L F, LEX, PEX M, LEX, PEX F, LEX, PEX F, PEX F, LEX, PEX F, LEX, PEX M, LEX, PEX F, LEX, PEX F, LEX, PEX F, LEX, PEX F, LEX, PEX

MPSW004 MPSW015 MPSW093 MPSW026 MPSW028 MPSW029 MPSW032 MPSW001 MPSW005 MPSW010 MPSW072 MPSW073 MPSW074 MPSW075 MPSW076 MPSW077 MPSW104 MPSW114 CHSOE005 CHSOE047 MIDGE209 CHSOE032 CHSOE040 MIDGE632 MIDGE635 MIDGE644 MIDGE656 MIDGE704 MIDGE707 MIDGE824 MIDGE873 CFWIH072 CFWIH462 CFWIH464 SWRC288 SWRC292 MPSW027 MPSW030 MPSW031 MPSW065 MPSW066 MPSW068 MPSW069 MPSW070 MPSW071 MPSW064 MPSW081

HM379580 HM379586 KF489827 KF489848 KF489826 KF489870 KF489855 KF489884 HM379581 HM379584 KF489872 KF489878 KF489817 KF489828 KF489846 KF489821 KF489849 KF489839 HM406101 HQ105378 HQ105377 HQ105376 HM406107 HQ105368 HQ105369 HQ105370 HQ105371 HQ105372 HQ105373 HQ105374 HQ105375 JF870590 JF870663 JF870664 JF288127 JF288128 KF489863 KF489818 KF489829 KF489879 KF489841 KF489873 KF489854 KF489867 KF489864 KF489883 KF489836

* * * * * * * * * * * * * * * * * *

* * * * * * * * * * *

F.L. Silva and S. Wiedenbrug

O. oncovolsella O. oncovolsella O. oncovolsella Onconeura cf. semifimbriata O. cf. semifimbriata O. cf. semifimbriata O. cf. semifimbriata Onconeura similispina O. similispina O. similispina O. similispina O. similispina O. similispina O. similispina O. similispina O. similispina O. similispina O. similispina Thienemanniella minuscula T. minuscula T. minuscula Thienemanniella cf. vittata T. cf. vittata T. cf. vittata T. cf. vittata T. cf. vittata T. cf. vittata T. cf. vittata T. cf. vittata T. cf. vittata T. cf. vittata Thienemanniella sp. Thienemanniella sp. Thienemanniella sp. Thienemanniella sp. Thienemanniella sp. Thienemanniella sp. Thienemanniella sp. Thienemanniella sp. Thienemanniella sp. Thienemanniella sp. Thienemanniella sp. Thienemanniella sp. Thienemanniella sp. Thienemanniella sp. Ubatubaneura atlantica Ubatubaneura sp.

Locality