DASMAHAPATRA ET AL. barcoding

Title: Mitochondrial DNA barcoding detects “” that are not real

Kanchon K. Dasmahapatra 1 [email protected]

Marianne Elias 2 [email protected]

Ryan I Hill 3 [email protected]

Joseph I Hoffman 4 [email protected]

James Mallet 1,5 [email protected]

1 Department of Genetics, Evolution and Environment, University College London, 4 Stephenson Way, London NW1 2HE, U.K.

2 Institute of Evolutionary Biology, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, U.K.

3 Department of Integrative Biology, University of California, Berkeley, CA 94720, U.S.A.

4 Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, U.K.

5 Wissenschaftskolleg zu Berlin, Wallotstraße 19, 14193 Berlin, Germany

Author for correspondence: Kanchon K. Dasmahapatra Tel: +44 (0)207 6795100 Fax: +44 (0)207 6795052

1 DASMAHAPATRA ET AL. Mechanitis barcoding

SUMMARY

Mimicry and extensive geographic subspecies polymorphism combine to make species in the ithomiine genus Mechanitis (; ) difficult to determine. We use mitochondrial (mtDNA) barcoding, nuclear sequences and amplified fragment length polymorphism (AFLP) genotyping to investigate species limits in this genus. Although earlier biosystematics studies based on morphology described only four species, mitochondrial barcoding revealed eight welldifferentiated haplogroups, indicating the presence of four new putative “cryptic species”. However, using AFLP markers we find support for only one of the four putative new “cryptic species” as biologically meaningful. We demonstrate that in this genus deep genetic divisions expected on the basis of mtDNA are not always reflected in the nuclear genome, and advocate the use of AFLP markers as a check when mtDNA barcoding gives unexpected results.

Keywords: DNA barcoding, AFLP, Mechanitis, cryptic species, ithomiine

2 DASMAHAPATRA ET AL. Mechanitis barcoding

1. INTRODUCTION

DNA barcoding has recently been suggested as a quick method useful for rapid species discovery and biodiversity assessment (Borisenko et al. 2008; Hajibabaei et al. 2006; Stoeckle

& Hebert 2008). For taxa the majority of these studies have used a short section of mitochondrial DNA (mtDNA), namely the first ~650 bp of the 5’ end of the cytochrome oxidase I gene (CoI) (EliasGutierrez et al. 2008; Hebert et al. 2003; Rock et al. 2008). DNA barcoding has been argued to revolutionise allowing rapid species identification without need for detailed taxonomic expertise with increasing economy (Hajibabaei et al.

2007; Stoeckle & Hebert 2008). But the practice of mtDNA barcoding has received much criticism on methodological (Will & Rubinoff 2004), theoretical (Hickerson et al. 2006) and empirical grounds (Elias et al. 2007; Hurst & Jiggins 2005; Meyer & Paulay 2005; Wiemers

& Fiedler 2007). Despite the problems, undoubted successes for mtDNA barcoding have been the discovery of cryptic species overlooked by more traditional taxonomic methods (Burns et al. 2008; Smith et al. 2006).

Several studies have sought to overcome some of the problems inherent in mtDNA taxonomy by supplementing mtDNA sequences with nuclear sequences (Elias et al. 2007; Monaghan et al. 2005). However, the use of nuclear sequences for DNA taxonomy has been limited, largely due to the difficulty in finding and sequencing fastevolving nuclear loci (Dasmahapatra &

Mallet 2006). A possible alternative to nuclear sequence data is analysis based on amplified fragment length polymorphisms (AFLPs), which are typically fastevolving anonymous nuclear markers, readily amplifiable in any organism (Mueller & Wolfenbarger 1999; Vos et al. 1995). In this paper we first use mtDNA sequences to test species affiliations in the difficult ithomiine butterfly genus Mechanitis (Lepidoptera; Nymphalidae), and then further investigate these results utilising both nuclear gene sequences and AFLP markers.

3 DASMAHAPATRA ET AL. Mechanitis barcoding

Four Mechanitis species have been described and all are both abundant locally and widely distributed in the neotropics (Brown 1977). Like most other ithomiines, all of the Mechanitis species show multiple geographic subspecies variation and typically have orange, brown, yellow and black wing colouration (Brown 1979; Lamas 2004). The many geographic forms of Mechanitis, involved in Müllerian mimicry with various members of the ~360 species of

Ithomiinae as well as Heliconius species, had led to an extremely problematic specieslevel taxonomy. However, careful biosystematic studies in the 1970s gave rise to a reasonably stable classification, based on morphology, distribution, hybrid zones, and ecology (Brown

1977), that is still generally accepted (Lamas 2004). Here, we investigate the four putative species comprising this genus.

2. MATERIAL AND METHODS

All four species of Mechanitis were used in this study: M. mazaeus (M. m. deceptus, M. m. cf. phasianita, M. m. messenoides, M. m. pannifera, M. m. mazaeus), M. polymnia (M. p. proceriformis, M. p. casabranca, M. p. bolivarensis, M. p. eurydice), M. lysimnia (M. l. roqueensis, M. l. nesaea, M. l. solaria) and M. menapis (M. m. mantineus). Our samples reflect the wide distribution of the first three species: specimens were collected from north and central (eastern San Martin and southern Loreto), , northern and the Atlantic coast of (Table 1). M. menapis has a narrower distribution, replacing M. mazaeus west of the Andes (Brown 1979) and in Central America; our M. menapis specimens were obtained from western Ecuador. We sampled 121 specimens, mainly from Ecuador and

Peru, together with five from each of Venezuela and eastern Brazil for comparison (Table 1).

Details of sampling locations are provided in the Supplementary Materials (Table S1).

4 DASMAHAPATRA ET AL. Mechanitis barcoding

DNA was extracted from legs and thoraces using QIAamp DNA Micro and DNeasy Blood and Tissue Kits (QIAGEN). Approximately 640 bp of mtDNA comprising the 5’end of CoI, the “barcoding region”, was amplified and sequenced in all specimens. To examine the effect of using more mtDNA sequence, a further ~1500 bp, comprising the remaining 3’ portion of

CoI, the tRAleu gene, and the 5’ end of CoII, was also sequenced from 71 specimens representing all four species. To examine whether the patterns revealed by mtDNA were reflected in the nuclear genome, sequences were also obtained from three nuclear loci: Tektin

(58 sequences), Rpl5 (68 sequences) and Tpi (73 sequences) (Mallarino et al. 2005; Whinnett et al. 2005a). For Rpl5 and Tpi these sequences included representatives of all four species, but for Tektin no sequences were obtained for M. menapis. PCR primers and reaction conditions have been reported previously (Dasmahapatra et al. 2007; Elias et al. 2007;

Whinnett et al. 2005a); a detailed description is also provided in Table S2. Cycle sequencing was carried out using the Big Dye Terminator 3.1 Cycle Sequencing Kit (Applied

Biosystems). All sequences obtained for this study have been deposited in Genbank (awaiting

Accession numbers XXXXXXXXXXXX, EU068843EU068856, EU068966, EU068993

EU068900, EU069070EU069075).

Genetic divisions revealed by mitochondrial CoI may sometimes not be captured at autosomal nuclear loci. This could be due to lack of coalescence within species given the ~4× effective population size (E) of autosomal loci compared to cytoplasmic mtDNA, and also because exons typically sequenced readily across genera tend to evolve at slower rates than CoI. The

Rpl5 and Tpi loci we have used both contain fastevolving intronic regions. In addition, Tpi is a sexlinked locus having only ~3× E compared to mitochondrial loci. To obtain even higher resolution nuclear data, we genotyped 84 specimens representing all four species and all eight

5 DASMAHAPATRA ET AL. Mechanitis barcoding mtDNA haplogroups (Figure 1a) using AFLP markers. The samples were genotyped using four AFLP primer combinations: TaqICGA + EcoRIACA; TaqICAG + EcoRIAGC; TaqI

CAG + EcoRIATG; TaqICCA + EcoRIACA. AFLP primers and protocols used are described in Madden et al. (2004). AFLP profiles were visualised by autoradiography.

Samples with aberrant AFLP profiles were discarded (Bonin et al. 2004), and to ensure reliability, the remaining AFLP genotypes were scored by eye; 108 putative loci were polymorphic and could be scored reliably.

Neighbourjoining (NJ) trees for each of the four sequenced loci were constructed using

MEGA 4 (Tamura et al. 2007). The optimal number of genotypic clusters indicated by the

AFLP genotypes was established with the Bayesian programme STRUCTURE 2.2 (Pritchard et al. 2000), using standardized inference criteria (Evanno et al. 2005). Following a 100,000 step burnin period, data were collected over 100,000 MCMC repetitions. STRUCTURE analysis was carried out on the dataset increasing K from 1 to 10. At each value of K the analysis was carried out three times to check betweenrun consistency.

3. RESULTS

Topologies of the mtDNA NJ trees based on the 636bp CoI “barcoding” region and the full

2000bp are similar and show the same major mtDNA haplogroups. In Figure 1a we show the former, since sequences come from a larger number of specimens. The tree shows eight major clusters or mtDNA haplogroups: three each within M. mazaeus (mthaplogroups A, F and H) and M. polymnia (mthaplogroups B, C and D), and one each corresponding to M. menapis

(mthaplogroup G) and M. lysimnia (mthaplogroup E). Not included in these groups are the two specimens of M. lysimnia solaria from Venezuela that appear sister to the M. mazaeus

6 DASMAHAPATRA ET AL. Mechanitis barcoding mthaplogroup H, rather than lying within main M. lysimnia clade; this exception is described further in the Discussion. The raw average pairwise distance between mthaplogroups is

2.7%, the largest being among M. mazaeus mthaplogroups (≥ 3.5%) and the smallest between M. polymnia mthaplogroups C and D (1.6%), with all other distances ≥ 2.0%. The average intrahaplogroup variation is, in contrast, only 0.3%. Pairwise mtDNA distances between the eight main haplogroups are shown in Table S3.

Hardly any genetic variation was present among Tektin sequences. The only clear pattern observable is the single shared Tektin haplotype in M. lysimnia and M. polymnia, suggesting a sister relationship (Figure 1b). In contrast, high levels of polymorphism are present at both

Tpi and Rpl5 (Figures 1c, 1d). Neither gene shows distinctions among M. polymnia mt haplogroups B, C and D. In the Rpl5 genealogy, there is some support for separate evolution of M. mazaeus mthaplogroups H from A and F, although mthaplogroup H appears paraphyletic. Similarly, a monophyletic M. mazaeus clade consisting of mthaplogroups A and F, which is distinct from M. mazaeus mthaplogroup H, is also supported in the Tpi genealogy, as is a separate clade corresponding to M. menapis. Overall, the nuclear gene genealogies suggest widespread paraphyly of recognized species and mthaplogroups.

Betweenrun consistency was high in the STRUCTURE analysis of AFLP genotypes: replicate runs at each K value yielded virtually identical likelihoods. Figure 2a shows the average likelihood from these replicates. The optimal number of groups was four (Figures 2a,

2b): two within M. mazaeus, and one each matching M. lysimnia and M. polymnia. Of the two

M. mazaeus AFLP clusters, one corresponds exactly to mthaplogroup H (M. mazaeus mazaeus); while the other mazaeus cluster is a mixture of mthaplogroups A and F (M. mazaeus deceptus, M. mazaeus cf. phasianita, and M. mazaeus messenoides), with no

7 DASMAHAPATRA ET AL. Mechanitis barcoding evidence of subdivision between them. The M. lysimnia and M. polymnia AFLP clusters correspond perfectly with morphologybased species designations except that one M. lysimnia specimen shows genotypic evidence of being a hybrid between the two; and there is no evidence for subdivision of M. polymnia along mthaplogroup lines. There is no single AFLP genotypic cluster associated with M. menapis; instead it appears to share genotypes largely with M. mazaeus mthaplogroups A + F, and to a lesser extent with M. mazaeus mt haplogroup H and M. lysimnia. Increasing the number of clusters to eight, the total number of mthaplogroups, did not alter the pattern and Figure 2b (K = 4) is virtually identical to Figure

2c (K = 8) with the four additional clusters making negligible contributions. This is further evidence for the lack of nuclear divisions corresponding to mthaplogroups within M. polymnia and M. mazaeus (mthaplogroups A and F).

4. DISCUSSION

Mitochondrial DNA barcoding of the four species in the genus Mechanitis revealed deep genetic divisions corresponding to eight mthaplogroups (Figure 1a). One mthaplogroup each corresponded to M. lysimnia and M. menapis. However three mthaplogroups were present within each of M. mazaeus and M. polymnia suggesting the existence of four new putative cryptic species in addition to the four species already recognized in morphological and biosystematic work. In contrast, while our nuclear sequence data give little resolution, our

AFLP data strongly indicate the existence of only four genetic clusters: two within M. mazaeus and one each corresponding to M. lysimnia and M. polymnia (Figure 2b). Thus, we obtain disparate results using mitochondrial and nuclear markers.

8 DASMAHAPATRA ET AL. Mechanitis barcoding

Mitochondrial DNA barcoding relies on intraspecific genetic variation being much less than interspecific genetic variation. When this condition is met, a “barcoding gap” may exist

(Meyer & Paulay 2005), and clusters corresponding to genetically more homogenous entities can be discerned. Based on such cases, some proponents of mtDNA barcoding have advocated the use of a threshold of sequence divergence above which genetic clusters may be considered species (Hebert et al. 2004b). Subsequent careful studies have demonstrated the lack of a barcoding gap in a number of taxa when sampling of species, populations, and individuals within the study group is thorough (Elias et al. 2007; Meyer & Paulay 2005;

Wiemers & Fiedler 2007). In the case of Mechanitis, we have sampled all four described species in the genus. While we have not included all of the many geographic subspecies of these species, we have used specimens collected across a wide geographic area representing opposite ends of the species’ geographic distribution in S. America. With our current sampling, mitochondrial data show eight major genetic clusters or haplogroups, clearly separated from one another by a large average genetic distance of 2.7 %. We found no overlap between intra and intercluster genetic variation.

Only four of the eight mtDNA divisions are reflected in the nuclear genome. Evidence from nuclear sequence information is weak, very likely a result of incomplete lineage sorting due to recency of origin coupled with large effective population sizes within these very widespread and common species. However, even multilocus AFLP genotyping fails to detect groups corresponding to three of the four novel mthaplogroups. The M. menapis samples do not form their own AFLP genotypic cluster, instead appearing to share most alleles with the two allopatric M. mazaeus clusters, and as such these samples are associated with a particular genetic pattern (Figure 2b). However, no such patterns are associated with any of the three M. polymnia mthaplogroups or the M. mazaeus mthaplogroups A and F.

9 DASMAHAPATRA ET AL. Mechanitis barcoding

Two possibilities might explain the mismatch between mthaplogroups and AFLP genotypic clusters. First, that some of the mthaplogroups are not “real species,” which we take to mean taxa that can maintain multilocus genetic, ecological, behavioural, and/or morphological differences in sympatry. Second, the AFLP markers may not be sensitive enough to detect small differences between real but closely related species detected by mtDNA barcoding.

Several lines of evidence strongly indicate that the former is more likely. Detailed examination of the wing patterns of the specimens in the three M. polymnia mthaplogroups as well as M. mazaeus mthaplogroups A and F failed to reveal any correlation between wing patterning and mthaplogroup. In contrast, every AFLP genetic cluster detected by

STRUCTURE is correlated with wing phenotype (discussed below). This suggests that while the AFLP clusters are biologically relevant, some of the mitochondrial divisions are not.

AFLP markers provide a nuclear, multilocus, genomewide picture of genetic divergence and as they sample noncoding variation, they have relatively rapid rates of evolution. This sensitivity is routinely exploited to reveal population genetic patterns within single species

(Baus et al. 2005; ChaputBardy et al. 2008; Takami et al. 2004). The sensitivity of AFLP markers for detecting small genetic differences relative to mtDNA is also demonstrated by results from a parallel study on butterfly species within the ithomiine genus Melinaea. In this genus there are at least six clearly distinguishable morphological species (Melinaea satevis,

M. menophilus, M. marsaeus, M. idae, M. mneme, M. isocomma) which cannot be detected using mtDNA barcoding (Elias et al. 2007; Whinnett et al. 2005b), Dasmahapatra et al. in prep.). Yet where mtDNA barcoding failed to detect distinct groups, the same AFLP primer combinations used in this paper were able to confirm morphologybased species divisions

(Dasmahapatra et al. in prep.). In contrast to the genomewide picture obtained from AFLP markers, mtDNA reflects evolution only of the maternally inherited mitochondrial genome,

10 DASMAHAPATRA ET AL. Mechanitis barcoding which can be affected by factors such as interspecific hybridisation, and selection, such as via maternally transmitted endosymbionts like Wolbachia (Hurst & Jiggins 2005). As such, genomewide genetic clustering revealed by AFLP markers is likely to be more generally useful for discovery of “real species” than mtDNA barcoding.

Previous detailed morphological and biosystematic work (Brown 1977; Lamas 2004) recognised four species within Mechanitis (M. lysimnia, M. polymnia, M. mazaeus and M. menapis) each with multiple geographic subspecies. The nuclear data reported here strongly support monophyletic M. lysimnia and M. polymnia clades. However, paraphyly of M. lysimnia is suggested by two mtDNA sequences of Venezuelan M. lysimnia solaria (Figure

1a). Unfortunately, it was not possible to genotype these specimens using AFLPs as the DNA came from old dried specimens and was degraded. This split within M. lysimnia may be correlated with chromosome numbers (Brown et al. 2004), and a group of M. lysimnia subspecies including M. l. solaria might represent a separate species from M. lysimnia sensu stricto. Future investigation of phylogenetic relationships within Mechanitis should focus on this apparent division within M. lysimnia.

M. mazaeus is also paraphyletic, and two genotypic clusters are supported by nuclear data, one comprising mthaplogroup H and the other combining mthaplogroups A and F. M. mazaeus mthaplogroups A and F corresponds to melanic forms currently considered to represent subspecies messenoides ( south to Ecuador) and deceptus (Ecuador southwards), both of which are generally found at mid elevations on the eastern slopes of the Andes. They are mimetic of melanic sympatric subspecies and races of

Melinaea marsaeus, Melinaea isocomma, Heliconius numata, and other species (Brown 1977;

Brown 1979). Although there is some evidence for intermediate colour patterns (Brown 1977

11 DASMAHAPATRA ET AL. Mechanitis barcoding and Table 1), most specimens in M. mazaeus mthaplogroup H correspond to paler lowland

M. mazaeus (sensu stricto) involved in mimicking the generalized lowland ithomiine and heliconiine tiger patterns. We conclude that these “mazaeus” forms are best considered full species, as their distributions overlap extensively in the Eastern foothills of the Andes (Brown

1977) with little evidence of hybridization from AFLP loci. Therefore, M. mazaeus mt haplogroups A and F should probably now regain species level designation as M. messenoides

(including M. messenoides messenoides C. & R. Felder 1865, M. messenoides cf. phasianita and M. messenoides deceptus Butler 1873). The nuclear evidence points to the fourth species,

M. menapis, being a transAndean form close to M. mazaeus and M. messenoides, since it shares genotypes with both cisAndean M. mazaeus forms described above. M. menapis is not sympatric with M. mazaeus, and instead replaces it west of the Andes and in Central America.

Morphologically based biosystematic work (Brown 1977) has shown itself to be more useful in this genus than mtDNA barcoding. Brown (1977) and Lamas (2004) accepted four species on the basis of morphology, a result largely upheld by our multilocus nuclear analysis. The only major discrepancy is that intermediate colour patterns in Ecuador (Table 1) apparently indicating hybridization between upland M. mazaeus deceptus/messenoides, and lowland mazaeus (sensu stricto) led Brown (1977) to lump the highland melanic forms incorrectly as subspecies of mazaeus. In comparison, mtDNA barcoding reveals eight clear monophyletic mthaplogroups within Mechanitis, three of which are not detected in our nuclear analysis, suggesting that they do not correspond to “real species.” Mitochondrial DNA barcoding provides a very sensitive technique to find new taxa, but a downside is that such taxa may have no basis in biological reality.

12 DASMAHAPATRA ET AL. Mechanitis barcoding

Focussing on the butterfly genus Mechanitis we have used nuclear sequences and sensitive

AFLP genotyping to demonstrate how deep genetic divisions in mtDNA are not always reflected by corresponding divisions in the nuclear genome. Such “cryptic species” clusters may instead represent locally divergent populations that have undergone a bottleneck, rather than having speciated in the sense of producing divergent populations that can coexist.

Putative cryptic species detected by mtDNA barcoding merit closer investigation via analysis of nuclear genetic data, or more indepth examination of ecology and taxonomy e.g. (Hebert et al. 2004a; Smith et al. 2006). Owing to their ease of amplification across taxa, sensitivity to small genetic differences and genomewide coverage, we advocate the use of AFLP markers in cases where mtDNA barcoding reveals unexpected results, such as a failure to recover known taxonomic divisions or the presence of additional “cryptic” taxa. Traditional mtDNA sequencing was always a useful way of investigating possible cryptic biological groupings long before “DNA barcoding” was first promoted (Hebert et al. 2003). The simple argument that mtDNA barcoding may be used in accurate species discovery, or even in identification of known species, ignores the crucial differences between mtDNA and supermarket barcodes. In a supermarket, each product type has a unique barcode, shared by all other examples of that type. In organisms, there is no such unique barcode, and a onetoone correspondence between mtDNA sequence and species does not exist.

Acknowledgements

We are grateful to Gerardo Lamas of the Museo de Historia Natural, UNMSM, Lima, Peru, and Keith Willmott from the Florida Museum of Natural History, Gainesville, USA, for tireless lepidopteran expertise. Fraser Simpson assisted with many aspects of this project.

Collecting permits and support were provided by INRENA (Peru) and the Ministerio del

Ambiente and the Museo Ecuatoriano de Ciencias Naturales (Ecuador). Logistic support in

13 DASMAHAPATRA ET AL. Mechanitis barcoding

Ecuador was given by the Napo Wildlife Centre and La Selva Jungle Lodge. We thank

NERC, DEFRA–Darwin, the Leverhulme Trust and the Margaret C. Walker fund for teaching and research in systematic entomology for funding different aspects of this project.

14 DASMAHAPATRA ET AL. Mechanitis barcoding

M. mazaeus M. lysimnia M. polymnia M. menapis

subspecies n subspecies n subspecies n subspecies n

Peru deceptus 8 roqueensis 8 proceriformis 12

cf. phasianita 7 eurydice 1

mazaeus 11

Ecuador deceptus 13 roqueensis 7 cf. proceriformis 8 mantineus 3

messenoides 16

mazaeus 12

messenoides × mazaeus 4

deceptus × mazaeus 1

Venezuela pannifera 2 solaria 2 bolivarensis 1

Brazil nesaea 2 casabranca 3

Table 1 Subspecies and numbers of specimens collected in different countries.

15 DASMAHAPATRA ET AL. Mechanitis barcoding

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Whinnett, A., Brower, A. V. Z., Lee, M. M., Willmott, K. R., & Mallet, J. 2005a Phylogenetic

utility of Tektin, a novel region for inferring systematic relationships amongst

Lepidoptera. Ann. Entomol. Soc. Amer. 98, 873886.

Whinnett, A., Zimmermann, M., Willmott, K. R., Herrera, N., Mallarino, R., Simpson, F.,

Joron, M., Lamas, G., & Mallet, J. 2005b Strikingly variable divergence times inferred

across an Amazonian butterfly 'suture zone'. Proc. Roy. Soc. B 272, 25252533.

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butterflies (Lepidoptera: Lycaenidae). Frontiers in Zoology 4.

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replace morphology for identification and classification. Cladistics 20, 4755.

20 DASMAHAPATRA ET AL. Mechanitis barcoding

FIGURE CAPTIONS

Figure 1

Neighbourjoining trees constructed from (a) mitochondrial sequences and (b, c and d) nuclear sequences, showing relationships between the eight Mechanitis mitochondrial haplogroups. Nodes marked with filled and open symbols have > 90 % and > 50 % (and < 90

%) bootstrap support respectively. Scale bars represent raw percentage sequence divergence.

(dec. = deceptus; maz. = mazaeus; mess. = messenoides)

Figure 2

STRUCTURE analysis of AFLP genotypes. a) Log likelihood of data as a function of K, the number of clusters. Likelihoods from each of the three replicate runs at each K are indistinguishable and the average likelihood is shown. Highest likelihood is achieved with four clusters, even though eight mthaplogroups are present. STRUCTURE results for b) the optimal number of genotypic clusters, K = 4, and c) the number of mthaplogroups, K = 8.

Each of the 85 individuals is represented by a vertical bar broken into K coloured segments.

The length of each colour in the bar indicates the posterior mean proportion of ancestry from each genetic clusters. b) and c) are virtually identical, indicating the absence of any significant additional genetic structure for more than four clusters.

SHORT TITLE: Mechanitis barcoding

21 E258 M. mazaeus deceptus: Ecuador 20846 M. mazaeus deceptus: Ecuador E257 M. mazaeus deceptus: Ecuador a) mtDNA E370 M. mazaeus deceptus: Ecuador b) Tektin c) Tpi d) Rpl5 E255 M. mazaeus deceptus: Ecuador 20648 M. mazaeus messenoides: Ecuador E256 M. mazaeus deceptus: Ecuador 20289 M. mazaeus messenoides: Ecuador 20820 M. mazaeus messenoides: Ecuador 4-274 M. mazaeus cf phasianita: Peru M. mazaeus 4-272 M. mazaeus cf phasianita: Peru 4-273 M. mazaeus cf phasianita: Peru mt-haplogroup H 20208 M. mazaeus mess. x maz.: Ecuador LS03-81 M. mazaeus messenoides: Ecuador 5-821 M. mazaeus deceptus: Peru M. mazaeus 4-306 M. mazaeus cf phasianita: Peru LS03-150 M. mazaeus messenoides: Ecuador mt-haplogroup A 5-822 M. mazaeus deceptus: Peru LS02-220 M. mazaeus deceptus: Ecuador LS05-02 M. mazaeus messenoides: Ecuador 4-276 M. mazaeus cf phasianita: Peru 2-348 M. mazaeus deceptus: Peru 20705 M. mazaeus messenoides: Ecuador LS02-219 M. mazaeus messenoides: Ecuador 20345 M. mazaeus deceptus: Ecuador 20281 M. mazaeus messenoides: Ecuador 20853 M. mazaeus deceptus: Ecuador 20809 M. mazaeus deceptus: Ecuador 20315 M. mazaeus messenoides: Ecuador LS02-49 M. mazaeus deceptus: Ecuador 20265 M. mazaeus deceptus: Ecuador 4-555 M. polymnia proceriformis: Peru 4-89 M. polymnia proceriformis: Peru 2-246 M. polymnia proceriformis: Peru M. polymnia 4-436 M. polymnia proceriformis: Peru 4-435 M. polymnia proceriformis: Peru mt-haplogroup B 4-2 M. polymnia proceriformis: Peru 3-51 M. polymnia casabranca: Eastern Brazil 3-52 M. polymnia casabranca: Eastern Brazil 3-53 M. polymnia casabranca: Eastern Brazil M. polymnia 4-438 M. polymnia proceriformis: Peru JM84-12 M. polymnia bolivarensis: Venezuela mt-haplogroup C 4-142 M. polymnia proceriformis: Peru 2-3131 M. polymnia eurydice: Peru 20494 M. polymnia cf proceriformis: Ecuador 4-141 M. polymnia proceriformis: Peru 4-140 M. polymnia proceriformis: Peru 2-245 M. polymnia proceriformis: Peru M. polymnia 20297 M. polymnia cf proceriformis: Ecuador 20339 M. polymnia cf proceriformis: Ecuador mt-haplogroup D LS02-176 M. polymnia cf proceriformis: Ecuador 20298 M. polymnia cf proceriformis: Ecuador 20340 M. polymnia cf proceriformis: Ecuador RH00-62 M. polymnia cf proceriformis: Ecuador 20728 M. polymnia cf proceriformis: Ecuador 3-139 M. lysimnia nesaea: Eastern Brazil 4-279 M. lysimnia roqueensis: Peru 4-1 M. lysimnia roqueensis: Peru 2-485 M. lysimnia roqueensis: Peru 3-50 M. lysimnia nesaea: Eastern Brazil 4-152 M. lysimnia roqueensis: Peru 4-552 M. lysimnia roqueensis: Peru 4-278 M. lysimnia roqueensis: Peru 2-1222 M. lysimnia roqueensis: Ecuador M. lysimnia LS04-13 M. lysimnia roqueensis: Ecuador 20338 M. lysimnia roqueensis: Ecuador mt-haplogroup E 20646 M. lysimnia roqueensis: Ecuador E358 M. lysimnia roqueensis: Ecuador 20273 M. lysimnia roqueensis: Ecuador 20586 M. lysimnia roqueensis: Ecuador LS01-67 M. lysimnia roqueensis: Ecuador E497 M. mazaeus messenoides: Ecuador 20765 M. mazaeus deceptus: Ecuador 20810 M. mazaeus messenoides: Ecuador 20223 M. mazaeus mess. x maz.: Ecuador 20514 M. mazaeus messenoides: Ecuador 20783 M. mazaeus messenoides: Ecuador 20782 M. mazaeus mess. x maz.: Ecuador 20512 M. mazaeus messenoides: Ecuador M. mazaeus 20819 M. mazaeus messenoides: Ecuador 20316 M. mazaeus mess. x maz.: Ecuador mt-haplogroup F 2-1743 M. mazaeus deceptus: Peru 4-406 M. mazaeus cf phasianita: Peru 4-85 M. mazaeus deceptus: Peru MEPeru25 M. mazaeus deceptus: Peru MEPeru65 M. mazaeus deceptus: Peru 5-381 M. mazaeus cf phasianita: Peru 2-1895 M. mazaeus deceptus: Peru M. menapis E544 M. menapis mantineus: West Ecuador E589 M. menapis mantineus: West Ecuador mt-haplogroup G E60 M. menapis mantineus: West Ecuador JM84-7 M. lysimnia solaria: Venezuela M. mazaeus JM84-8 M. lysimnia solaria: Venezuela LS02-177 M. mazaeus mazaeus: Ecuador M. mazaeus 20282 M. mazaeus mazaeus: Ecuador mt-haplogroups 20505 M. mazaeus mazaeus: Ecuador mt-haplogroup H 20701 M. mazaeus mazaeus: Ecuador LS02-96 M. mazaeus mazaeus: Ecuador A + F LS01-127 M. mazaeus mazaeus: Ecuador JM84-6 M. mazaeus pannifera: Venezuela 0.1 % 5-751 M. mazaeus mazaeus: Peru 5-56 M. mazaeus mazaeus: Peru 4-269 M. mazaeus mazaeus: Peru 4-270 M. mazaeus mazaeus: Peru 4-136 M. mazaeus mazaeus: Peru M. mazaeus 5-382 M. mazaeus mazaeus: Peru 20256 M. mazaeus mazaeus: Ecuador mt-haplogroup H 4-267 M. mazaeus mazaeus: Peru LS02-164 M. maz. x dec.: Ecuador 20506 M. mazaeus mazaeus: Ecuador 20784 M. mazaeus mazaeus: Ecuador 0.2 % 20322 M. mazaeus mazaeus: Ecuador 20781 M. mazaeus mazaeus: Ecuador 4-271 M. mazaeus mazaeus: Peru 0.6 % 1 % 2-241 M. mazaeus mazaeus: Peru E359 M. mazaeus mazaeus: Ecuador 4-275 M. mazaeus mazaeus: Peru JM84-4 M. mazaeus pannifera: Venezuela

Figure 1 a) K clusters 0 2 4 6 8 10

-1900

-2300

Ln L(K) -2700

-3100

M. menapis b) K = 4

M. lysimnia M. polymnia M. mazaeus

1.0

0.8

0.6

0.4

0.2

0.0 E G B C D H F A

mitochondrial haplogroups

M. menapis c) K = 8

M. lysimnia M. polymnia M. mazaeus

1.0

0.8

0.6

0.4

0.2

0.0 E G B C D H F A

mitochondrial haplogroups

Figure 2 DASMAHAPATRA ET AL. Mechanitis barcoding

SUPPLEMENTARY MATERIAL

Species Subspecies mthaplogroup AFLP genotypic cluster Specimen Collection locality Latitude Longitude (by wing pattern) ID M. mazaeus messenoides × A ‘messenoides/deceptus’ 20208 Napo Wildlife Centre, Ecuador 0° 31' 45 S 76° 25' 01 W mazaeus M. mazaeus deceptus A ‘messenoides/deceptus’ 20265 Napo Wildlife Centre, Ecuador 0° 30' 33 S 76° 26’ 12 W M. mazaeus messenoides A ‘messenoides/deceptus’ 20281 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus messenoides A ‘messenoides/deceptus’ 20289 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus messenoides A ‘messenoides/deceptus’ 20315 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus deceptus A ‘messenoides/deceptus’ 20345 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus messenoides A ‘messenoides/deceptus’ 20648 Napo Wildlife Centre, Ecuador 0° 30’ 33 S 76° 26’ 12 W M. mazaeus messenoides A ‘messenoides/deceptus’ 20705 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus deceptus A ‘messenoides/deceptus’ 20809 Napo Wildlife Centre, Ecuador 0° 29’ 37 S 76° 26’ 48 W M. mazaeus messenoides A ‘messenoides/deceptus’ 20820 Napo Wildlife Centre, Ecuador 0° 30’ 33 S 76° 26’ 12 W M. mazaeus deceptus A ‘messenoides/deceptus’ 20846 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus deceptus A ‘messenoides/deceptus’ 20853 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus deceptus A ‘messenoides/deceptus’ 2348 Pongo de Caynarachi, Peru 6° 20' 7 S 76° 15' 15 W M. mazaeus cf. phasianita A ‘messenoides/deceptus’ 4272 Bosque von Humboldt, INIA, Peru 8° 49' 48 S 75° 03' 28 W M. mazaeus cf. phasianita A ‘messenoides/deceptus’ 4273 Bosque von Humboldt, INIA, Peru 8° 49' 48 S 75° 03' 28 W M. mazaeus cf. phasianita A ‘messenoides/deceptus’ 4274 Bosque von Humboldt, INIA, Peru 8° 49' 48 S 75° 03' 28 W M. mazaeus cf. phasianita A ‘messenoides/deceptus’ 4276 Bosque von Humboldt, INIA, Peru 8° 49' 48 S 75° 03' 28 W M. mazaeus cf. phasianita A ‘messenoides/deceptus’ 4306 Km 7.2, Pongo – Barranquita, Peru 6° 17' 20 S 76° 13' 41 W M. mazaeus deceptus A - 5821 Quebrada Yanayacu, Peru 6° 44' 26 S 75° 58' 54 W M. mazaeus deceptus A - 5822 Quebrada Yanayacu, Peru 6° 44' 26 S 75° 58' 54 W M. mazaeus deceptus A ‘messenoides/deceptus’ E255 Podocarpus National Reserve, Ecuador 4° 06' 80 S 78° 57' 90 W M. mazaeus deceptus A - E256 Podocarpus National Reserve, Ecuador 4° 06' 80 S 78° 57' 90 W M. mazaeus deceptus A ‘messenoides/deceptus’ E257 Podocarpus National Reserve, Ecuador 4° 06' 80 S 78° 57' 90 W M. mazaeus deceptus A ‘messenoides/deceptus’ E258 Podocarpus National Reserve, Ecuador 4° 06' 80 S 78° 57' 90 W M. mazaeus deceptus A - E370 Sucua, Ecuador 2° 27' 35 S 78° 10' 11 W M. mazaeus deceptus A - LS0249 Garza Cocha, Sucumbios, Ecuador 0° 29’ 47 S 76° 22’ 25 W M. mazaeus messenoides A - LS02219 Garza Cocha, Sucumbios, Ecuador 0° 29’ 47 S 76° 22’ 25 W M. mazaeus deceptus A - LS02220 Garza Cocha, Sucumbios, Ecuador 0° 29’ 47 S 76° 22’ 25 W M. mazaeus messenoides A - LS0381 Garza Cocha, Sucumbios, Ecuador 0° 29’ 47 S 76° 22’ 25 W M. mazaeus messenoides A - LS03150 Garza Cocha, Sucumbios, Ecuador 0° 29’ 47 S 76° 22’ 25 W M. mazaeus messenoides A - LS0502 Sani Isla, Ecuador 0° 30’ 41 S 76° 21’ 04 W M. mazaeus messenoides F ‘messenoides/deceptus’ 20223 Napo Wildlife Centre, Ecuador 0° 30’ 33 S 76° 26’ 12 W × mazaeus

1 DASMAHAPATRA ET AL. Mechanitis barcoding

M. mazaeus messenoides. × F ‘messenoides/deceptus’ 20316 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W mazaeus M. mazaeus messenoides F - 20512 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus messenoides F - 20514 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus deceptus F - 20765 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus messenoides × F ‘messenoides/deceptus’ 20782 Napo Wildlife Centre, Ecuador 0° 29’ 37 S 76° 26’ 48 W mazaeus M. mazaeus messenoides F ‘messenoides/deceptus’ 20783 Napo Wildlife Centre, Ecuador 0° 29’ 37 S 76° 26’ 48 W M. mazaeus messenoides F ‘messenoides/deceptus’ 20810 Napo Wildlife Centre, Ecuador 0° 29’ 37 S 76° 26’ 48 W M. mazaeus messenoides F ‘messenoides/deceptus’ 20819 Napo Wildlife Centre, Ecuador 0° 30’ 33 S 76° 26’ 12 W M. mazaeus deceptus F ‘messenoides/deceptus’ 21743 Puente Aguas Verdes, Peru 5° 39' 50 S 77° 38' 58 W M. mazaeus deceptus F ‘messenoides/deceptus’ 21895 Km 30, Tarapoto – Yurimaguas, Peru 6° 24' 81 S 76° 18' 50 W M. mazaeus deceptus F ‘messenoides/deceptus’ 485 Río Previsto suspension bridge, Peru 9° 06' 13 S 75° 44' 28 W M. mazaeus cf. phasianita F ‘messenoides/deceptus’ 4406 Km 16, Tarapoto – Yurimaguas, Peru 6° 27' 18 S 76° 17' 54 W M. mazaeus cf. phasianita F ‘messenoides/deceptus’ 5381 Cerro de Mira Culo, Peru 7° 27' 12 S 75° 50' 16 W M. mazaeus messenoides F ‘messenoides/deceptus’ E497 Montalvo, Ecuador 2° 15' 60 S 80° 38' 60 W M. mazaeus deceptus F ‘messenoides/deceptus’ MEPeru25 Tarapoto, San Martin, Peru M. mazaeus deceptus F ‘messenoides/deceptus’ MEPeru65 Tarapoto, San Martin, Peru M. mazaeus mazaeus H ‘mazaeus’ 20256 Napo Wildlife Centre, Ecuador 0° 30’ 33 S 76° 26’ 12 W M. mazaeus mazaeus H ‘mazaeus’ 20282 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus mazaeus H ‘mazaeus’ 20322 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus mazaeus H - 20505 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus mazaeus H ‘mazaeus’ 20506 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus mazaeus H ‘mazaeus’ 20701 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. mazaeus mazaeus H ‘mazaeus’ 20781 Napo Wildlife Centre, Ecuador 0° 29’ 37 S 76° 26’ 48 W M. mazaeus mazaeus H ‘mazaeus’ 20784 Napo Wildlife Centre, Ecuador 0° 29’ 37 S 76° 26’ 48 W M. mazaeus mazaeus H ‘mazaeus’ 2241 Km 7.2, Pongo – Barranquita, Peru 6° 17' 20 S 76° 13' 41 W M. mazaeus mazaeus H ‘mazaeus’ 4136 El Pandisho, Lago Yarinacocha, Peru 8° 19' 02 S 74° 35' 28 W M. mazaeus mazaeus H* ‘mazaeus’ 4184 Caño Tushmo, Lago Yarinacocha, Peru 8° 20' 25 S 74° 35' 30 W M. mazaeus mazaeus H ‘mazaeus’ 4267 Bosque von Humboldt, INIA, Peru 8° 49' 48 S 75° 03' 28 W M. mazaeus mazaeus H ‘mazaeus’ 4269 Bosque von Humboldt, INIA, Peru 8° 49' 48 S 75° 03' 28 W M. mazaeus mazaeus H ‘mazaeus’ 4270 Bosque von Humboldt, INIA, Peru 8° 49' 48 S 75° 03' 28 W M. mazaeus mazaeus H ‘mazaeus’ 4271 Bosque von Humboldt, INIA, Peru 8° 49' 48 S 75° 03' 28 W M. mazaeus mazaeus H ‘mazaeus’ 4275 Bosque von Humboldt, INIA, Peru 8° 49' 48 S 75° 03' 28 W M. mazaeus mazaeus H ‘mazaeus’ 556 Río Pauya/Río Cushabatay confluence, Peru 7° 09' 18 S 75° 43' 24 W M. mazaeus mazaeus H ‘mazaeus’ 5382 Cerro de Mira Culo, Peru 7° 27' 12 S 75° 50' 16 W M. mazaeus mazaeus H - 5751 Quebrada Yanayacu, Peru 6° 44' 26 S 75° 58' 54 W M. mazaeus mazaeus H ‘mazaeus’ E359 Communidad Shuar Mirador, Ecuador M. mazaeus pannifera H - JM844 El Miano, 4 km E, Venezuela 10° 15' 46 N 66° 17' 50 W

2 DASMAHAPATRA ET AL. Mechanitis barcoding

M. mazaeus pannifera H - JM846 El Miano, 4 km E, Venezuela 10° 15' 46 N 66° 17' 50 W M. mazaeus mazaeus H - LS01127 Garza Cocha, Sucumbios, Ecuador 0° 29’ 47 S 76° 22’ 25 W M. mazaeus mazaeus H - LS0296 Sani Isla, Ecuador 0° 30’ 41 S 76° 21’ 04 W M. mazaeus mazaeus × H - LS02164 Garza Cocha, Sucumbios, Ecuador 0° 29’ 47 S 76° 22’ 25 W deceptus M. mazaeus mazaeus H - LS02177 Garza Cocha, Sucumbios, Ecuador 0° 29’ 47 S 76° 22’ 25 W M. lysimnia solaria (H) - JM847 Cumanacoa Cocokar, Km 17, Venezuela 10° 14' 20 N 63° 55' 15 W M. lysimnia solaria (H) - JM848 Cumanacoa Cocokar, Km 17, Venezuela 10° 14' 20 N 63° 55' 15 W M. polymnia proceriformis B ‘polymnia’ 2246 Km 7.2, Pongo – Barranquita, Peru 6° 17' 20 S 76° 13' 41 W M. polymnia proceriformis B ‘polymnia’ 42 Castillo Grande, Tingo Maria, Peru 9° 16' 11 S 76° 00' 29 W M. polymnia proceriformis B ‘polymnia’ 489 Río Previsto suspension bridge, Peru 9° 06' 13 S 75° 44' 28 W M. polymnia proceriformis B* ‘polymnia’ 4212 Km 59, Pucallpa Tingo Maria, Peru 8° 38' 28 S 74° 57' 13 W M. polymnia proceriformis B ‘polymnia’ 4435 Km 22, Nuevo Lima Selva Andina, Peru 7° 11' 36 S 76° 20' 06 W M. polymnia proceriformis B ‘polymnia’ 4436 Km 22, Nuevo Lima Selva Andina, Peru 7° 11' 36 S 76° 20' 06 W M. polymnia proceriformis B ‘polymnia’ 4555 Km 22.5, Nuevo Lima La Perla del 7° 10' 56 S 76° 10' 52 W Ponacillo, Peru M. polymnia casabranca C ‘polymnia’ 351 Campinas, Brazil 22° 49' 34 S 47° 4' 4 W M. polymnia casabranca C ‘polymnia’ 352 Campinas, Brazil 22° 49' 34 S 47° 4' 4 W M. polymnia casabranca C ‘polymnia’ 353 Campinas, Brazil 22° 49' 34 S 47° 4' 4 W M. polymnia proceriformis C ‘polymnia’ 4438 Km 22, Nuevo Lima Selva Andina, Peru 7° 11' 36 S 76° 20' 06 W M. polymnia bolivarensis C - JM8412 El Miano, 4 km E, Venezuela 10° 15' 46 N 66° 17' 50 W M. polymnia cf. proceriformis D ‘polymnia’ 20340 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. polymnia cf. proceriformis D ‘polymnia’ 20728 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. polymnia cf. proceriformis D - 20297 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. polymnia cf. proceriformis D - 20298 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. polymnia cf. proceriformis D - 20339 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. polymnia cf. proceriformis D - 20494 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. polymnia proceriformis D - 2245 Km 7.2, Pongo – Barranquita, Peru 6° 17' 20 S 76° 13' 41 W M. polymnia eurydice D ‘polymnia’ 23131 Palma Real, Peru 12° 37' 18 S 72° 41' 25 W M. polymnia proceriformis D ‘polymnia’ 4140 El Pandisho, Lago Yarinacocha, Peru 8° 19' 02 S 74° 35' 28 W M. polymnia proceriformis D ‘polymnia’ 4141 El Pandisho, Lago Yarinacocha, Peru 8° 19' 02 S 74° 35' 28 W M. polymnia proceriformis D ‘polymnia’ 4142 El Pandisho, Lago Yarinacocha, Peru 8° 19' 02 S 74° 35' 28 W M. polymnia cf. proceriformis D - LS02176 Garza Cocha, Sucumbios, Ecuador 0° 29’ 47 S 76° 22’ 25 W M. polymnia cf. proceriformis D - RH0062 Garza Cocha, Sucumbios, Ecuador 0° 29’ 47 S 76° 22’ 25 W M. lysimnia roqueensis E - 20273 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. lysimnia roqueensis E ‘lysimnia’ 20586 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. lysimnia roqueensis E - 20646 Napo Wildlife Centre, Ecuador 0° 30’ 33 S 76° 26’ 12 W M. lysimnia roqueensis E ‘lysimnia’ 20338 Anangu Boca del Rio, Ecuador 0° 31’ 43 S 76° 23’ 41 W M. lysimnia roqueensis E - 2485 Km 7.2, Pongo – Barranquita, Peru 6° 17' 20 S 76° 13' 41 W

3 DASMAHAPATRA ET AL. Mechanitis barcoding

M. lysimnia roqueensis E - 21222 Km 8, Tarapoto – Yurimaguas, Peru 6° 27' 48 S 76° 19' 20 W M. lysimnia nesaea E ‘lysimnia’ 350 Campinas, Brazil 22° 49' 34 S 47° 04' 04 W M. lysimnia nesaea E ‘lysimnia’ 3139 Serra Bonita, Brazil 15° 25' 18 S 39° 30' 12 W M. lysimnia roqueensis E ‘lysimnia’ 41 Castillo Grande, Tingo Maria, Peru 9° 16' 11 S 76° 00' 29 W M. lysimnia roqueensis E* ‘lysimnia’ 4128 Río Previsto suspension bridge, Peru 9° 06' 13 S 75° 44' 28 W M. lysimnia roqueensis E - 4152 Caño Tushmo, Lago Yarinacocha, Peru 8° 20' 25 S 74° 35' 30 W M. lysimnia roqueensis E ‘lysimnia’ 4278 Bosque von Humboldt, INIA, Peru 8° 49' 48 S 75° 03' 28 W M. lysimnia roqueensis E ‘lysimnia’ 4279 Bosque von Humboldt, INIA, Peru 8° 49' 48 S 75° 03' 28 W M. lysimnia roqueensis E ‘lysimnia’ 4552 Km 22.5, Nuevo Lima La Perla del 7° 10' 56 S 76° 10' 52 W Ponacillo, Peru M. lysimnia roqueensis E ‘lysimnia’ E358 Communidad Shuar Mirador, Ecuador M. lysimnia roqueensis E - LS0167 Garza Cocha, Sucumbios, Ecuador 0° 29’ 47 S 76° 22’ 25 W M. lysimnia roqueensis E - LS0413 Garza Cocha, Sucumbios, Ecuador 0° 29’ 47 S 76° 22’ 25 W M. menapis mantineus G mixed E60 Mindo, Ecuador 0° 1' 60 S 78° 47' 60 W M. menapis mantineus G mixed E544 Los Cedros, Ecuador 0° 19' 48N 78° 48' 36 W M. menapis mantineus G mixed E589 Los Cedros, Ecuador 0° 19' 48N 78° 48' 36 W

Table S1 Details of samples and sampling localities

* Only a shorter length of mtDNA sequence (~560 bp) could be obtained for these individuals. This was sufficient for mthaplogroup classification, but these specimens are not represented in the mtDNA NJ tree (Figure 1a).

4 DASMAHAPATRA ET AL. Mechanitis barcoding

T MgCl dNTPs Amplicon Primers Primer sequences (5’3’) a 2 / ºC (mM) (mM) mtDNA Lep3.1f a TCTACAAATCATAAAGATATTGGAACATT 55 2.3 0.2 barcode Lep3.1r a AAATTTTAATTCCTGTTGGTACAGC mtDNA Jerry b CAACATTTATTTTGATTTTTTGG 57 2.3 0.2 CoI Patlep a TCCATTACATATAATCTGCCATATTA mtDNA Georgeith * TAGGWTTAGCWGGAATACC 58 2.3 0.2 CoII Evaith * GAGACCAATACTTGCTTTCAGACATCT Jess * AGCAANCAGATWAATGAAATCRTYA Tpi 63 – 56 3.0 0.8 Nab * TAGCCAYTTACGNAGAGANGC tek3 CGCAGTTTYTGATRCTYT Tektin c 57 2.75 0.68 teka ACCAGTGGRGAYATYCTWGG Rpl5F44 TCCGACTTTCAAACAAGGATG Rpl5 d 55 2.3 0.2 Rpl5R441 CATATCCTGGGAATCTCTTGATG

Table S2. PCR conditions for the amplicons used in this study.

All reactions were carried out in 10µl volumes using 10×buffer (Sigma), 0.5µM each of forward and reverse primers and 0.25U Taq polymerase (Sigma). For Tpi 0.16 µg/ml BSA was also added. PCR cycling conditions were: initial denaturing at 94ºC for 2 minutes; followed by 35 cycles of 94ºC for 45s, annealing temperature (T a) for 45s, 72ºC for 60s; final extension at 72ºC for 5 minutes. For Tpi , PCR cycling conditions were the same except that a touchdown procedure was used with T a dropping from 63ºC to 56ºC over 14 cycles, followed by a further 23 cycles at 56ºC. * These primers were developed for this study. Tpi primers Jess and Nab, were developed by Alaine Whinnett. a (Dasmahapatra et al. 2007); b (Simon et al. 1994); c (Whinnett et al. 2005a); d (Mallarino et al. 2005)

5 DASMAHAPATRA ET AL. Mechanitis barcoding

Species M. lysimnia M. menapis M. polymnia M. mazaeus mt haplogroup E G B C D A F H M. lysimnia E 0.6 2.3 2.4 2.3 2.0 2.8 2.8 3.1 M. menapis G 0.1 2.5 2.5 2.1 2.9 2.1 2.6 M. polymnia B 0.2 2.3 2.2 2.8 3.0 3.3 C 0.2 1.6 2.7 3.3 3.3 D 0.2 2.7 2.9 3.2 M. mazaeus A 0.6 3.6 4.1 F 0.4 3.5 H 0.5

Table S3. Raw percentage pairwise distances between mtDNA haplogroups. Intrahaplogroup distances are italicised.

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