Mitochondrial DNA provides an insight into the mechanisms driving diversification in the ithomiine butterfly Hyposcada anchiala (Lepidoptera: Nymphalidae: Ithomiinae)

Geographic subspecies of several ithomiine butterflies on the lower east Andean slopes display a black and orange “melanic tiger” aposematic wing pattern that occurs from Colombia to Bolivia, while geographically adjacent lowland subspecies typically bear a coloured, “tiger” pattern. However, it is not clear whether subspecies with similar wing patterns in different regions have arisen through independent events of convergent adaptation, possibly through parapatric differentiation, or result from allopatric differentiation, as proposed by the refuge hypothesis. Here, we examine geographic patterns of divergence in the widespread and common ithomiine butterfly Hyposcada anchiala. We present phylogenetic hypotheses for 5 subspecies of H. anchiala, based on 1567 bp mitochondrial DNA. All topologies indicated that a single switch in mimetic pattern best explained the wing patterning of the H. anchiala studied here. This finding suggests that the subspecies of H. anchiala studied here result from at least two stages of differentiation, and is consistent with a single colonisation into a novel altitudinal zone coincident with a wing pattern switch, followed by subsequent divergence within, rather than across altitudinal zones. The subspecies divergences indicated diversifications were consistent with the Pleistocene. Furthermore, the lowland subspecies were more recently derived than the montane taxa, in contrast to predictions of the “Andean species pump” hypothesis. 633 * Corresponding author; e-mail: [email protected] kilometres (KRW pers. obs.), so the potential for colonisation of new mimetic environments is high. This debate still remains today partly because of the difficulties in testing between vicariance or ecological adaptation as causes of divergence. Here, we examine the possible roles of these two causes in explaining divergence in a single west Amazonian ithomiine species, Hyposcada anchiala. The Ithomiinae (Lepidoptera: Nymphalidae) is an exclusively Neotropical subfamily which includes ~355 species (Lamas, 2004), all of which are thought to be highly unpalatable. These butterflies form part of multiple, diverse mimicry rings in a single location (Beccaloni, 1997a,b; Joron & Mallet, 1998). Hyposcada anchiala occurs from eastern Panama to Bolivia and western Brazil, and contains 12 recognised subspecies (Lamas, 2004). These subspecies display wing patterns of two distinct mimicry complexes, the orange and black tiger (melanic tiger) complex and the tiger complex. These complexes are strongly altitudinally zoned, constrained to high altitude (submontane) and low altitude sites respectively. In this paper we focus on relationships between the submontane melanic tigers, H. a. mendax Fox from N. Peru and H. a. fallax (Staudinger) from S. Peru and Bolivia, and the geographically adjacent, lowland tiger-pattern subspecies, H. a. interrupta Tessmann in the north and H. a. richardsi Fox, in the south (Fig. 1.). If individuals cluster by subspecies, there are 26 possible rooted cladograms for H. a. mendax, H. a. fallax, H. a. interrupta and H. a. richardsi. Of those, 6 are not informative about the associated mimetic-altitudinal shifts, for example an unresolved quadchotomy, 7 support just one mimeticaltitudinal shift, and 13 support two mimetic-altitudinal shifts. An example of the 7 topologies which support just a single shift in mimicry and altitude is a bifurcating topology, with both montane tiger melanic subspecies in one clade, and both non-melanic tiger, lowland subspecies in the other clade (as depicted in Fig. 1), this can best be explained by a vicariant diversification of two previously widespread ancestral populations. The other 13 topologies support two shifts of mimicry and altitude, and thus ecological diversification (Benson, 1982; Endler, 1982), an example is a sister pairing of the 634 Fig. 1. Map showing the butterfly collection localities for H. a. mendax, H. a. fallax, H. a. interrupta, H. a. richardsi and H. a. ecuadorina. The lines connecting figured butterflies indicate sampling locations for the molecular samples. Inset shows two of the 26 possible rooted topologies for H. a. mendax, H. a. fallax, H. a. interrupta and H. a. richardsi. two subspecies from N. Peru (one montane, melanic and one lowland, non-melanic tiger), and a sister pairing of the two subspecies from S. Peru (one montane, melanic and one lowland, non-melanic tiger) (as depicted in Fig. 1.). Under this scenario, local processes drove the diversification of an ancestral N. Peru H. anchiala into two northern subspecies, a process paralleled independently in S. Peru. Here, we use 1567 bp mitochondrial DNA (mtDNA) to reconstruct relationships within H. anchiala and examine whether resulting phylogeographic patterns are more consistent with expectations of geographic (vicariant) or ecological diversification. MATERIAL AND METHODS Genomic DNA was analysed from the four described H. anchiala subspecies, plus an additional non-melanic tiger pattern subspecies, H. a. ecuadorina Bryk. Outgroup taxa were chosen to represent two closely related species, H. virginiana (Hewitson) and H. zarepha (Hewitson), as determined by the sequencing of five Hyposcada species with two, independent nuclear regions, wingless and elongation factor 1-a (data not shown) as well as mtDNA data (Table 1). A specimen of H. virginiana in 20% dimethylsulphoxide, 0.25M EDTA and saturated NaCl solution (DMSO solution) was identified and provided by Chris Jiggins (University of Edinburgh) and a dried specimen of H. a. richardsi was provided by GL. All other specimens were collected by the authors, preserved in DMSO solution, and identified to subspecies by GL and KRW. DNA was extracted from one third of the abdomen (H. a. richardsi) or one third of the thorax (all other specimens) using the DNAeasy kit (Qiagen, West Sussex, UK), according to the manufacturer’s instructions, with an initial 3 h incubation at 55°C, and a final elution volume of 300 μl. Genomic DNA extracts were preserved at –20°C. Dried wings were retained as vouchers at University College London, except for the H. a. richardsi and H. virginiana which were retained by the donors. PCRs were performed using primers Jerry and Pat (COI), or George and Imelda (COII) (Simon et al., 1994), in a 25 μl volume, using 2 μl template DNA under the following conditions: 1 × PCR buffer, 0.6 mM dNTPs, 4 mM MgCl2, 0.5 μM each primer, 0.025 U/μl Taq, and ddH2O, with an amplification profile of 94°C for 2 min, followed by [COI: 4 cycles of (94°C for 45 s, 51°C for 45 s, 72°C for 60 s) then 29 cycles of (94°C for 45 s, 51°C for 45 s, 72°C for 90 s)] or [COII: 33 cycles of (94°C for 45 s, 55.5°C for 45 s, 72°C for 90 s)] and a final 5 min extension at 72°C. The H. a. richardsi sample proved difficult to amplify, probably due to its preservation with paradichlorobenzene (PDB), a chemical known to form covalent bonds to DNA. Weak H. a. richardsi PCR products were therefore excised from an agarose gel and incubated at 70°C for 10 min in 1.5 ml ddH2O. 2 μl of this suspension was used for reamplification following the above protocol. Final PCR products were purified using the QIAquick PCR purification kit (Qiagen, West Sussex, UK), according to the manufacturer’s protocol and sent to a commercial facility for cycle sequencing using the PCR primers, precipitation and sequencing. Sequences were edited using SeqEd v1.0.3 (Applied Biosystems, Inc., Foster City, USA). PAUP version 4.0b 10 (Swofford, 2000) was used to calculate the numbers of variable and parsimony informative sites, and absolute and Hasegawa-Kishino635 DQ078362 300–450 PERU: San Martín: Chumía. 06°36 ́57S, 76°11 ́10W 02-197 H. zarepha flexibilis DQ078476 900–1000 PANAMA: Cerro Campana. 08°68 ́74N 79°91 ́97 W 8283 H. virginiana DQ078475 270 PERU: Madre de Dios: Río Los Amigos. 12°35 ́S 70°5 ́W G1 H. anchiala richardsi DQ078474 450 ECUADOR: Napo: Jatun Sacha. 01°04 ́S, 77°36 ́W Ec 452 H. anchiala ecuadorina DQ078477 350 PERU: Madre de Dios: Mazuko. 13°06 ́S 70°22 ́W 02-3519 H. anchiala fallax DQ078361 800–1000 PERU: San Martín: La Antena. 06°27 ́18S, 76°17 ́54W 02-2141 H. anchiala mendax DQ078360 800–1000 PERU: San Martín: La Antena. 06°27 ́18S, 76°17 ́54W 02-1602 H. anchiala mendax DQ078359 1150 PERU: San Martín: Puente Serranoyacu. 05°40 ́S, 77°40 ́W 02-716 H. anchiala mendax DQ078358 1150 PERU: San Martín: Puente Serranoyacu. 05°40 ́S, 77°40 ́W 02-1645 H. anchiala mendax DQ078357 1150 PERU: San Martín: Puente Serranoyacu. 05°40 ́S, 77°40 ́W 02-1644 H. anchiala mendax DQ078312 150 PERU: San Martín: km 7.2 PongoBarranquita. 06°17 ́20S, 76°13 ́41W 02-1293 H. anchiala interrupta DQ078356 150 PERU: San Martín: km 7.2 PongoBarranquita. 06°17 ́20S, 76°13 ́41W 02-2105 H. anchiala interrupta DQ078355 150 PERU: San Martín: km 7.2 PongoBarranquita. 06°17 ́20S, 76°13 ́41W 02-512 H. anchiala interrupta Genbank accession Approximate collection altitude (m) Collection locality Voucher number Taxon TABLE 1. Specimen information.