Neotrop Entomol DOI 10.1007/s13744-016-0481-z SYSTEMATICS, MORPHOLOGY AND PHYSIOLOGY

DNA Barcoding of an Assembly of Montane Andean (): Geographical Scale and Identification Performance

1,2 2 2 2,3 2 4 5 MA MARÍN ,ICCADAVID ,LVALDÉS ,CFÁLVAREZ ,SIURIBE ,RVILA ,TWPYRCZ

1Depto de Biologia , Instituto de Biologia, Univ Estadual de Campinas – UNICAMP, Campinas, SP , Brazil 2Univ Nacional de Colombia, Sede Medellín, Grupo de Investigación en Sistemática Molecular, Medellín, Colombia 3Corporación Universitaria Lasallista, Antioquia, Colombia 4Instituto de Biología Evolutiva (CSIC-UPF), Barcelona, Spain 5Zoological Museum, Jagiellonian Univ, Kraków, Poland

Keywords Abstract , , , DNA barcoding is a technique used primarily for the documentation and mitochondrial DNA, molecular , identification of biological diversity based on mitochondrial DNA se- andean cloud forest, community ecology quences. Butterflies have received particular attention in DNA barcoding Correspondence studies, although varied performance may be obtained due to different MA Marín, Depto de Biologia Animal, Instituto de Biologia, Univ Estadual de scales of geographic sampling and speciation processes in various groups. Campinas – UNICAMP, Rua Monteiro Lobato, The montane Andean Satyrinae constitutes a challenging study group for 255 - Cidade Universitária Zeferino Vaz - Barão taxonomy. The group displays high richness, with more of 550 , and Geraldo, CEP, Campinas, SP 13083-970, Brazil; [email protected] remarkable morphological similarity among taxa, which renders their iden- tification difficult. In the present study, we evaluated the effectiveness of Edited by Alberto S Corrêa – ESALQ/USP DNA barcodes in the identification of montane Andean satyrines and the Received 15 June 2016 and accepted 28 effect of increased geographical scale of sampling on identification perfor- December 2016 mance. Mitochondrial sequences were obtained from 104 specimens of 39 species and 16 genera, collected in a forest remnant in the northwest * Sociedade Entomológica do Brasil 2017 Andes. DNA barcoding has proved to be a useful tool for the identification of the specimens, with a well-defined gap and producing clusters with unambiguous identifications for all the morphospecies in the study area. The expansion of the geographical scale with published data increased genetic distances within species and reduced those among species, but did not generally reduce the success of specimen identification. Only in Forsterinaria rustica (Butler, 1868), a taxon with high intraspecific varia- tion, the barcode gap was lost and low support for monophyly was ob- tained. Likewise, expanded sampling resulted in a substantial increase in the intraspecific distance in Morpho sulkowskyi (Kollar, 1850); Panyapedaliodes drymaea (Hewitson, 1858); obsoleta (Westwood, 1851); and Lymanopoda labda Hewitson, 1861; but for these species, the barcode gap was maintained. These divergent lineages are nonetheless worth a detailed study of external and genitalic morphology variation, as well as ecological features, in order to determine the poten- tial existence of cryptic species. Even including these cases, DNA barcoding performance in specimen identification was 100% successful based on monophyly, an unexpected result in such a taxonomically complicated group. Marín et al

Introduction 2011,Ashfaqet al 2013,Wilsonet al 2013,Dincă et al 2011, 2015, Yang et al 2015), and a total of 148,306 DNA barcodes Andean Satyrinae are available for named butterflies specimens () in the Barcode of Life Database (BOLD Systems v3, accessed In the Neotropics, the subfamily Satyrinae accounts for more on October 6, 2016). However, some studies have shown than 1200 known species in five tribes (Morphini, , that DNA barcodes are not completely accurate when iden- , , and ). Two Satyrini subtribes en- tifying specific taxonomical groups. Elias et al (2007), report- compass most of the species: , with more than 400 ed in Ithomiini (Danainae) that only 77% of species can be species, is a group of mostly lowland or premontane butterflies precisely identified using the barcode data at local scale (Viloria 2003), particularly diversified in the basin of the Amazon, (21 km2), while Wiemers & Fiedler (2007) reported 84% of Atlantic Forest, and the foothills of the Andes, and Pronophilina, accurately identified Polyommatinae (Lycaenidae) at a conti- more diverse in the highlands, is the richest group of montane nental scale. butterflies with over 550 described species (Lamas et al 2004), Dasmahapatra et al (2010) demonstrated that deep ge- and presents the greatest diversity in the tropical Andes. netic divergences detected with mtDNA barcoding in the Montane satyrines display high morphological similarity Mechanitis Fabricius, 1807 (Ithomiini), were not always among congeneric species (Forster 1964). For example, in reflected in the nuclear genome and suggested the use of Euptychiina, the genus Forsterinaria Gray, 1973 the only nuclear genes as a check step when mtDNA barcoding gives strictly montane within the subtribe, presents a demanding unexpected results. However, other studies supported the taxonomy (Zubek et al 2014). Its species show little differ- effectiveness of DNA barcoding for attribution of specimens ence in wing pattern and even the genitalia provide few to species, with an accuracy between 90–100% (Lukhtanov diagnostic characters for the immediate segregation of some et al 2009,Pradoet al 2011,Ashfaqet al 2013,Wilsonet al allopatric or sympatric species (Zubek & Pyrcz 2011). In some 2013,Dincă et al 2011, 2015), and its usefulness in the study Pronophilina, the situation is similar, particularly notable in of species complexes (Hebert et al 2004, Cong & Grishin diverse genera as Butler, 1867; Thieme, 2014, Seraphim et al 2014). The variable performance of 1905; and Manerebia Staudinger, 1897. These exhibit a re- DNA barcoding has been attributed to factors such as sam- markable external similarity among many species, which can pling, geographic scale, time divergence among species, mi- only be identified with certainty through dissection, and this tochondrial introgression, and dominant mode of speciation fact has led to much confusion in the literature and resulted (Moritz & Cicero 2004,Eliaset al 2007, Wiemers & Fiedler in a significant underestimation of their taxonomic diversity 2007,Bergstenet al 2012, Talavera et al 2013). Interestingly, (Pyrcz et al 2006). DNA barcode libraries based on regional sampling may still Additionally, montane satyrines present an intricate alti- be effective at much wider geographic scales (Huemer et al tudinal distribution. Pronophilina butterflies occur predomi- 2014). Without a cause that fully explains the variability in nantly in high elevation cloud forests near or within the the performance of DNA barcoding, it is necessary to test its forest-paramo ecotone, but they are distributed in narrow utility for different taxa, and under different ecological and bands of altitude (Adams 1985, Pyrcz & Wojtusiak 2002, sampling conditions. Pyrcz et al 2009, Casner & Pyrcz 2010, Viloria et al 2010, Neotropical Satyrinae constitute an interesting model Pyrcz & Garlacz 2012,Marínet al 2015) and demonstrate a group for testing the performance of DNA barcoding because large percentage of endemic species, frequently confined to of high diversity and notable morphological stasis. However, single massifs (Pyrcz & Rodríguez 2007, Pyrcz et al 2013, and in contrast with other butterflies groups, montane 2016). Within each part of the Andes, lower-elevation species Andean satyrines have received little attention in molecular with broad geographic ranges are replaced by higher-eleva- studies (e.g., Casner & Pyrcz 2010). Accordingly, in the pres- tion, narrowly distributed congeners. The result is a stair-step ent study, we evaluated the effectiveness of DNA barcodes distribution in mountain sides with unique species composi- on the identification of montane Andean satyrines and the tion in adjacent regions of an extended mountain chain or in effect of increased geographical sampling scale on the per- nearby cordilleras (Adams 1985,Pyrczet al 1999, Casner & formance of this methodology. Pyrcz 2010).

DNA Barcoding in Butterflies Material and Methods

Butterflies have been widely used in DNA barcoding studies Specimens sampling (Hebert et al 2004, Elias et al 2007,Janzenet al 2009, Linares & Soto-Calderón 2009,Lukhtanovet al 2009,Silva- Butterflies were collected in a forest remnant of 51.71 km2 Brandão et al 2009, Dasmahapatra et al 2010,Pradoet al close to Medellín city in the Colombian Central Cordillera DNA Barcoding of Andean Satyrinae of the Andes (6°05′–6°13′N/75°39′–75°43′W), from March 2011 Analysis of genetic variability to February 2012 at 2500–2890 m. In this area, maximum annual rainfall is approximately 3000 mm (Corantioquia Pairwise distances were calculated in the software MEGA 5.2 2004, Hermelin 2007). According to Holdridge (1967), this life (Tamura et al 2011) using the uncorrected p distance model zone corresponds to montane rain forest and lower montane according to Collins et al (2012) and Srivathsana and Meiera wet forest. (2012), remaining analyses were conducted in the R package External morphology was used to examine and iden- Spider (Brown et al 2012). For analytical purposes, a distance tify collected specimens, in addition for Pedaliodes spe- matrix of combined dataset (specimens local study area plus cies, internal genital morphology was examined at the GenBank) was generated with pairwise deletion of missing Laboratorio de Biología y Sistemática de Insectos sites. For each species, mean intraspecific divergence, maxi- (Insectario) of the Universidad Nacional de Colombia, mum intraspecific divergence, and minimum interspecific Sede Medellín and the Zoological Museum of the distances were computed. Jagiellonian University (Kraków, Poland). Each specimen studied was labeled, assigned a code number, and de- Evaluation of DNA barcoding utility for species identification posited in the Museo Entomológico Francisco Luis Gallego (MEFLG, Medellín, Colombia). All sequences The existence of a “barcode gap” was checked, by plotting generated in this study were deposited in GenBank maximum intraspecific divergences against minimum inter- under the following accession numbers: KU359838– specific distances, with a 1:1 slope representing the point at KU359939, and KU588156–KU588157 with images of which the difference between them is zero and there is “no voucher specimens in Barcode of Life Data barcode gap.” Two datasets were used to test for the exis- Systems—BOLD (http://boldsystems.org/). To test the tence of a barcode gap: one dataset with only specimens of effect of expanded geographic sampling on barcode the local study area and the other with a combined dataset performance, sequences for an additional 157 at wider geographical scale. Barcode efficacy was tested specimens of 83 species and 18 genera, sampled from using distance-based methods, enabling to make specimen Peru to Venezuela were obtained from GenBank and identifications even in some instances in the presence of included in the analysis (Table S1). The identification paraphyly (Collins & Cruickshank 2013). Based in the concept of these specimens was verified, with the photos de- that the distances between three sequences do not have to posited in the NSG Voucher Specimen Database be equilateral and a fixed threshold value cannot be main- http://www.nymphalidae.net/all_species.php (accessed tained (Meier et al 2006), this defines how similar (threshold on September 14, 2016) (Peña & Malm 2012). value) a set of barcodes needs to be before it can be identi- fied to be the same species (Zhang et al 2012). The threshold value can be estimated for a given data set by obtaining a DNA extraction and PCR amplification frequency distribution of all intraspecific pairwise distances and determining the threshold distance below 95% of all Two legs were removed from each specimen with ster- intraspecific found distances (Meier et al 2006,Zhanget al ile forceps and transferred to microtubes of 1.5 μland 2012). We used the threshID function because it considers all preserved at −20°C. Total genomic DNA was extracted sequences within the given threshold and makes an analysis using the grind buffer method described by Rosero et al similar to the “Identify Specimen” tool provided by the BOLD (2010). The PCR reaction had a final volume of 50 μl (Brown et al 2012). The efficiency in specimen identification and included: 2 mM MgCl2,1×Buffer,0.2mMdNTPs, with the combined dataset was analyzed using an optimum 0.2 μM of each primer, 0.04 U/μlofTaqpolymerase threshold value. The identification status of each specimen (Fermentas, St. Leon, Germany), and 4 μLofDNA.The was coded as: “correct” if the species within the given primers used were LCO1490 5′ GGTCAACAAATCAT threshold were the same, “incorrect” if the species within AAAGATATTGG 3′ and HCO2198 5′ TAAACTTCAGGGTG the given threshold were different, “ambiguous” if there ACCAAAAAATCA 3′ (Bogdanowicz et al 1993,Folmer were more than one species within the given threshold, et al 1994). Amplification was verified in gel electro- and “no identification” if no species were within the thresh- phoresis with 1.2% agarose, TBE and 80 V, and visual- old distance. The cumulative error based on false positives ized using Gel Star™ (1: 100). The PCR products were and false negatives for each threshold value among 0 and purified on Millipore filtration plates and sequenced 0.04 (8% of genetic distance) was estimated to determinate using the service Macrogen Inc., Korea. The amplicons an optimum threshold value (Meyer & Paulay 2005). were sequenced in both directions. Sequence quality was Additionally, in order to determine if species were recov- evaluated with the program FinchTV 1.3.1 (Geospiza Inc.), ered as monophyletic, a monophyly test was conducted. This and aligned using the software MUSCLE (Edgar 2004). incorporates a bootstrap resampling (1000 replicates) to Marín et al determine the support for the monophyly. Species with a sequence for each of the 39 species and 16 genera studied bootstrap support lower than “thresh” (70%) were coded (Table S2). For the local study area, the minimum interspe- as “with weak support”. Two trees were obtained, a cific divergence was 3.46% between orcus Neighbor Joining NJ tree with the software MEGA 5.2 (Latreille 1813) and Pronophila epidipnis (Thieme, 1907), and (Tamura et al 2011) using uncorrected p distances, and a the maximum intraspecific distance was 1.2% in Lymanopoda maximum likelihood (ML) tree, with 200 heuristic searches labda (Hewitson, 1861), resulting in a well-defined barcode of 20,000 generations, using the model GTR + G in the pro- gap (Fig 1a). With increased geographical sampling, the spe- gram GARLI 2.0 (Zwickl 2006), both with bootstrap resam- cies of the local study area exhibited a minimum interspecific pling of 1000 replicates. divergence of 2.7% between Forsterinaria rustica (Butler, 1868) and Forsterinaria guaniloi (C. Peña & Lamas, 2005), and the maximum intraspecific distances of 4.0% in Results Morpho sulkowskyi (Kollar, 1850) and 4.5% in Panyapedaliodes drymaea (Hewitson, 1858) (Table S3). The DNA barcode sequences greater than 500 base pairs were intraspecific distance in F. rustica was 3.5%, which is greater recovered from 104 specimens, representing at least one than its minimum interspecific distance, thus no barcode gap

Fig 1 Evaluation of barcode gap and cumulative error with uncorrected p distance. a Dot plot for each individual of the study site, plotted the distance of the furthest conspecific against the distance to the nearest nonconspecific, above the 1:1 line indicate the presence of a “barcode gap.” b Dot plot for each individual with the increased geographic dimension of sampling; black dots are specimens of the study site and white dots are specimens of others localities. c Box plots comparing the genetic variation between minimum interspecific distance and maximum intraspecific distance using only specimens of the study site and with the inclusion of specimens of the geographical extension. d Cumulative error based on false positives plus false negatives for each threshold value until 0.04. DNA Barcoding of Andean Satyrinae was detected in this case. Likewise, Lymanopoda labda and L. which only five species had been DNA barcoded previously obsoleta (Westwood, 1851) presented large intraspecific dis- (October 6, 2016). tances of 3.5 and 3.0%, and minimum interspecific distances Identifications of congeneric specimens in the most diverse of 6.0 and 3.8%, respectively; although in these cases, the genera (Pedaliodes, Lymanopoda,andManerebia)weresuc- barcode gap was maintained (Fig 1b). In summary, the intra- cessful. Similar studies with butterflies had difficulties in identi- and interspecific genetic variation that was documented for fication of congeneric species (Elias et al 2007,Wiemers& the study area did not show significant differences with Fiedler 2007, Dasmahapatra et al 2010, Prado et al 2011, those obtained when sequences from the extended geo- Kreuzinger et al 2015,Dincă et al 2011, 2015). For instance, graphical area were included (Fig 1c). However, there were Elias et al (2007) studied Ithomiini butterflies at a similar geo- some outliers, namely M. sulkowskyi, F. rustica, L. labda, graphical scale (21 km2) and found that the efficiency of speci- L. obsoleta,andP. drymaea, with intraspecific distances men identification was much lower among closely related spe- greater than 2%, that overlapped with interspecific variation. cies whose biogeographic and evolutionary histories affected Optimization threshold analysis using the whole dataset the barcode performance. It has also been proposed that sym- returned an optimum value of 0.016 (3.2% distances) where patric species likely share more barcode sequences than allo- the cumulative error was minimized at 15%. Errors were kept patric species (Lukhtanov et al 2009). Discrete mtDNA clusters below 20% when using threshold values between 0.008 provide strong evidence for independently evolving populations (1.6%) and 0.026 (5.2%) (Fig 1d), although the cumulative or species, although apparently their evolution is suppressed error indicates the possible presence of misidentified taxa, even under very low levels of dispersal (Papadopoulou et al the concordance with independent morphological identifica- 2008). In this study, the identification success of congeneric tion indicates that it is unlikely to be misidentifications. Using specimens could be explained by the particular evolutionary the lowest threshold of 1.6%, all species were correctly iden- history of montane Andean Satyrinae, with independently tified. Ambiguity in identification started to appear at 2.8% evolving populations and reduced levels of dispersal, forming for F. rustica and, when increasing the threshold value to a speciation pattern predominantly allo- and parapatric, likely 4.0%, five more ambiguous identifications appeared: Eretris induced by Pleistocene glacial cycles (Adams 1985, 1986,Casner porphyria (C. Felder & R. Felder, 1867); Eretris apuleja (C. &Pyrcz2010). This scenario is evident in the high percentage of Felder & R. Felder, 1867); P. epidipnis; P. orcus; and species with narrow elevation ranges, which diversified by L. obsoleta. The NJ and ML tree (Fig 2,FigS1)showedall dispersal into adjacent elevation strata, followed by special- the specimens clustered according to morphological identifi- ization and reduced expansion across elevation gradients cation for both limited and extended datasets, although a (Casner & Pyrcz 2010). lower bootstrap support (<70%) was found for three species, In addition to being useful for specimen identification, DNA M. sulkowskyi, F. rustica,andP. drymaea, and a bootstrap barcodes revealed deep intraspecific differences in some spe- support below 50% was found for L. obsoleta. cies, which deserve to be studied in more detail. The most remarkable is F. rustica, which lost the barcode gap, had an ambiguous identification, and exhibited low bootstrap sup- Discussion port. DNA barcode data suggest that this taxon may, in fact, represent two separate species. However, it is necessary to Specimens identification carry out a detailed morphological analysis to test this hy- pothesis. Similarly, a substantial increase in the intraspecific The DNA barcode was found to be effective in the identifica- distance was observed for some other species that neverthe- tion of montane Andean Satyrinae, showing its usefulness in less retained the barcode gap; for example, M. sulkowskyi differentiating morphologically very similar species (Fig 3). A with high intraspecific distance between the subspecies (3.3 well-defined gap was observed and clusters with unambigu- to 4%) and a minimum interspecific divergence from its ous identifications for all 39 species from the study area sister-species Morpho lympharis (Butler,1873)of4.3%. obtained, invariably matching with morphospecies attribu- However, Cassildé et al (2012) and Nattier et al (2016)con- tion. Based on cluster analysis, the increase of the geograph- firmed the separate specific status of M. sulkowskyi and ical area studied, retained the success in specimen identifi- M. lympharis. As our data indicate that their genetic distance cation, despite the fact that the barcode gap was lost for one is similar to those among the subspecies of M. sulkowskyi, species (F. rustica). This study contributes to the construction which may suggest the presence of more species in the of a DNA barcode reference library for Neotropical montane group. Admittedly, the DNA barcoding alone is not enough butterflies, with the publication of 29 DNA barcodes for for- to define the limits between species. Therefore, we suggest a merly unavailable species. This is particularly relevant for detailed review of subspecies of M. sulkowskyi with a wider Pedaliodes, the most species-rich genus of Neotropical but- sampling, the inclusion of nuclear genes and detailed mor- terflies with over 240 known species (Lamas et al 2004), for phological and ecological data. Marín et al

Fig 2 Maximum likelihood (ML) tree based on the GTR + G model and obtained using the expanded geographic sampling matrix of 261 sequences from 112 species. The nodes with multiple specimens or multiple species were collapsed to a vertical line or triangle with the horizontal depth indicating the level of divergence. Bold taxa indicate study site specimens and simple text taxa are specimens or species out of study site. Nodes marked with a black dot are conflictive identifications in the DNA barcode analysis, the thresh indicate the threshold value where the identification status is valued as “ambiguous,” the boot <70 indicate monophyly test with a bootstrap support lower than 70%.

The intraspecific divergence of more than 3% between geo- P. drymaea contrasts with those frequently observed among graphically isolated specimens of L. obsoleta, L. labda,and lowland satyrines, which rarely exceed 2% (Seraphim et al DNA Barcoding of Andean Satyrinae

Fig 3 Morphologically similar species of Pedaliodes of the local study area, dorsal (left)and ventral (right): a Pedaliodes rodriguezi Pyrcz & Andrade, 2013. b Pedaliodes hebena Pyrcz & Viloria, 1999. c Pedaliodes transmontana Pyrcz & Viloria, 1999. d Pedaliodes phrasicla (Hewitson, 1847). e Pedaliodes simpla costipunctata Weymer, 1912. f Pedaliodes obstructa Pyrcz & Viloria, 1999. g Pedaliodes montagna Adams & Bernard, 1981. h Pedaliodes pisonia (Hewitson, 1862). i Pedaliodes obstructa Pyrcz & Viloria, 1999. j Pedaliodes praemontagna Pyrcz & Viloria 2007. k Pedaliodes praemontagna Pyrcz & Viloria 2007. l Pedaliodes pollonia Adams, 1986.

2014). Such deep divergences are frequently interpreted as an identify intraspecific taxonomical levels, with several species indication of cryptic species (Prado et al 2011, Cong & Grishin showing intraspecific clusters with large genetic distances 2014, Seraphim et al 2014,Kreuzingeret al 2015). In montane (Supporting information). These clusters recognize allopatric satyrines, greater intraspecific genetic distances coincide with subspecies for M. sulkowskyi, and evolutionary significant the expected isolation and low degree of dispersal of most units for F. rustica, Lymanopoda ionius,andL. obsoleta montane species, generally distributed in discrete populations, (Westwood, 1851). Thus, our results provide evidence for geographically structured and genetically divergent across the distinguish geographic units of , and identify ge- range of the morphologically coherent lineages. Considering netic lineages that reflect phylogeographic processes. this, it is necessary to analyze more specimens including a The presence of monophyletic intraspecific clusters in mid- detailed study of external morphology, genitalia, and ecologi- elevations species typical of open areas and secondary forest cal features to properly evaluate their taxonomic status. such as M. sulkowskyi, F. rustica,andL. obsoleta,indicatealocal and regional structuring of the genetic diversity even in widely DNA barcoding in the study and conservation of Andean distributed Andean satyrines, with lineages suggesting restrict- Satyrines ed gene flow. This, added to the high proportion of endemic taxa (Pyrcz 2004,Pyrcz&Rodríguez2007,Pyrczet al 2016), and The results of this study show that DNA barcoding is a useful the existence of unique species assemblages in each mountain technique for the identification of montane Andean system (Viloria et al 2010,Marínet al 2015,Pyrczet al 2016), Satyrinae, especially that the increased geographical scale suggest that the assemblages of Andean satyrines may act as did not significantly reduce the success of specimen identifi- metacommunities, with defined groups of interacting species in cation. Additionally, the results are consistent with the defi- spatially isolated habitat patches. If this hypothesis is confirmed, nition of DNA barcoding sensu lato, with identification of each mountain system could be considered an independent specimens at different taxonomical level (Valentini et al valuable unit of biodiversity. Despite some limitations of 2009), that help to glimpse the genetic variation in species single-marker approaches, DNA barcoding emerges as a practi- and genera. For Andean satyrines, the barcodes allowed to cal tool for comparative phylogeography at the community and Marín et al metacommunity levels, providing information on the spatial Casner KL, Pyrcz TW (2010) Patterns and timing of diversification in a variation and historical features of landscapes through drift tropical montane genus, Lymanopoda (, Satyrinae). Ecography 33:251–259 and migration (Joly et al 2014). Cassildé C, Blandin P, Silvain J-F (2012) Phylogeny of the genus Morpho DNA barcoding is a technique that extends beyond speci- Fabricius 1807: insights from two mitochondrial genes (: mens identification. Applied to specific taxonomic groups it pro- Nymphalidae). Ann Soc Entomol Fr (N.S.) 48:173–188 vides information for understanding species boundaries and for Collins RA, Boykin LM, Cruickshank RH, Armstrong KF (2012) Barcoding’s next top model: an evaluation of nucleotide substitution models for the prioritization of biodiversity conservation (Kress et al 2015). specimen identification. Methods Ecol Evol 3:457–465 Intraspecific variation in DNA barcodes has a unexploited po- Collins RA, Cruickshank RH (2013) The seven deadly sins of DNA tential for ecological applications such as in spatial ecology, eco- barcoding. Mol Ecol Resour 13:969–975 evolutionary dynamics, and for investigating the genetic conse- Cong Q, Grishin NV (2014) A new (Lepidoptera, quences of environmental change (Joly et al 2014). Likewise, it is Nymphalidae, Satyrinae) is sympatric and synchronic with H. sosybius in southeast US coastal plains, while another new a useful tool for the study of community assembly processes Hermeuptychia species - not hermes - inhabits South Texas and and its phylogenetic structure (Kress et al 2009, Boyle & Northeast Mexico. ZooKeys 91:43–91 Adamowicz 2015). Large-scale DNA barcoding allows a simulta- Corantioquia (2004) Programa integral de educación y comunicación a neous assessment of variation in community composition las comunidades de Angelópolis, Caldas y La Estrella, para la conservación y el manejo sostenible de los recursos naturales de la across of multiple hierarchical levels and can be used to discern reserva forestal alto el romeral y la vía parque Angelópolis-Caldas. the effects of neutral and non-neutral macroecological process- Fundación con Vida, Medellín, p 204 es (Baselga et al 2015,Vodă et al 2016). Dasmahapatra KK, Elias M, Hill RI,HoffmanJI,MalletJ(2010) Mitochondrial DNA barcoding detects some species that are real, and some that are not. Mol Ecol Resour 10:264–273 ă Acknowledgements The authors would like to thank Noemy Dinc V, Montagud S, Talavera G, Hernández-Roldán J, Munguira ML, Seraphim, Leila Shirai, Rita Isabel Veléz, and the anonymous reviewers García-Barros E, Hebert PD, Vila R (2015) DNA barcode reference for critically reading the manuscript. This work was supported by the library for Iberian butterflies enables a continental-scale preview of Corporación Universitaria Lasallista under grant [FCAA-07 2010] and potential cryptic diversity. Sci Rep 5:12395. doi:10.1038/srep12395 Fundación BBVA (Proyecto MARIPOSA) [BIOCON08_021]. 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