VOLUME 66, N UMBER 4 185
J OURNAL OF T HE L EPIDOPTERISTS ’ S OCIETY
Volume 66 2012 Number 4
Journal of the Lepidopterists’ Society 66(4), 2012, 185 –198
DNA BARCODES AND INSIGHTS INTO THE RELATIONSHIPS AND SYSTEMATICS OF BUCKEYE BUTTERFLIES (NYMPHALIDAE: NYMPHALINAE: JUNONIA ) FROM THE AMERICAS
EDWARD PFEILER , Centro de Investigación en Alimentación y Desarrollo, A.C., Unidad Guaymas, Apartado Postal 284, Guaymas, Sonora C.P. 85480, México; email: [email protected]
SARAH JOHNSON , Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093
AND
THERESE A. M ARKOW Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093
ABSTRACT. Nucleotide sequence data from a segment of the mitochondrial cytochrome c oxidase subunit I (COI) gene, known as the barcode segment, were used to examine phylogenetic relationships and systematics of buckeye butterflies (Nymphalidae: Nymphalinae: Junonia ) from the New World, with emphasis on taxa from western North America . Three nominal species have been recognized for North America, J. evarete (Cramer), J. genoveva (Cramer), and J. coenia Hübner, with additional species recently pro - posed for the West Indies and northern South America . The distinctive Andean buckeye, J. vestina C. Felder & R. Felder, along with J. evarete and J. genoveva , are also components of the South American fauna . With the exception of J. vestina , butterflies comprising the New World Junonia have had a confused taxonomic history, and species assignments are often problematic . Our results show that the barcode segment resolves the two major clades of New World Junonia , referred to here as clades A and B, with similar high sup - port seen in an earlier phylogenetic study using both mitochondrial and nuclear genes . Within clade A, J. vestina resolved in a basal position to J. evarete from South America and the Caribbean . The data further suggest that species assignments in some populations of New World Junonia clustering in clade B ( J. coenia + J. genoveva ) need to be reevaluated . DNA barcodes, although failing to re - solve all recognized species and subspecies level taxa of New World Junonia , probably owing to relatively recent divergences, can pro - vide valuable tools for identifying the two major lineages, and when used in conjunction with morphological, ecological, behavioral and life history information can provide insights into the taxonomy and evolution of this difficult group. Additional key words: cytochrome c oxidase subunit I, dispersal, genetic distance, population structure, speciation.
Butterflies commonly known as buckeyes synonym of J. evarete (Comstock 1942)] . The genus (Nymphalidae: Nymphalinae: Junonia ) are widely Precis also has been used for the New World buckeyes, distributed in the Americas, being found from southern but butterflies belonging to this genus are now known to Canada to South America . In an early treatment of the be restricted to Africa (Wahlberg et al. 2005) . Recently, genus, Forbes (1928) recognized two species of Junonia Pelham (2008) recognized three nominal species of in the New World, J. vestina C. Felder & R. Felder, a Junonia belonging to the J. evarete complex as defined high altitude form found throughout the Andes of South here: J. evarete (Cramer), J. genoveva (Cramer) and J. America (Fig. 1), and J. lavinia (Cramer) [= J. evarete coenia Hübner, as well as three subspecies: J. evarete (Cramer)] in which he grouped all others forms that nigrosuffusa W. Barnes & McDunnough, J. evarete were morphologically similar and distinct from J. zonalis C. Felder & R. Felder and J. coenia grisea vestina. In the present paper we refer to buckeyes Austin & J. Emmel . In addition, ongoing taxonomic included in J. lavinia as the J. evarete complex [ Junonia studies on Junonia from the West Indies and northern lavinia is now recognized as a permanently invalid South America suggest that additional species level taxa 186 186 JOURNAL OF THE LEPIDOPTERISTS ’ S OCIETY
FIG . 1. Map of North and South America showing collection localities and phenotypic variability of Junonia spp. at selected lo - calities where dorsal images were available. Red and green dots correspond to the two main clades (A and B, respectively) of New World Junonia (see Fig. 2). The shaded area represents the approximate geographic distribution of the Andean buckeye, J. vestina . Voucher codes for each species are given below (see Table 1 for details). Scientific names in parentheses are suggested changes in assignment based on data presented here (see Discussion regarding the assignment of J. nigrosuffusa ) or unpublished data (C. Brévi - gnon, pers. com.). 1, J. coenia grisea (CIAD 10–B03); 2, J. coenia coenia (NW38–18); 3, J. coenia coenia (female) (NW85–13); 4, J. coenia coenia (no image) (Bio175–17); 5, J. coenia coenia (no image) (DNA–ATBI–0802 and –0816); 6, J. coenia coenia (no im - age) (TDWG–0126); 7, J. evarete (= J. genoveva ; CIAD 10–B19; Estero del Soldado); 8, J. evarete nigrosuffusa (= J. nigrosuffusa ; CIAD 10–B24); 9, J. evarete (no image) (= J. genoveva ; JM6–10); 10, J. evarete (no image) (= J. genoveva ; MAL–02877); 11, J. evarete (= J. genoveva ; 05–SRNP–58293); 12, J. evarete (no image) (= J. genoveva ; YB–BCI12765); 13, J. genoveva (no image) (= J. neildi Brévignon [C. Brévignon, pers com.]; NW136–16); 14, Junonia sp. (no image) (= J. evarete ; NW153–12); 15, J. evarete (no image) (NW136–17); 16, Junonia spp. (no images) (UK4–14, –15, –16); 17, J. evarete (no image) (NW151–3); 18, J. evarete (NW126–20); 19, J. evarete (NW84–15); 20, J. genoveva ? (= J. evarete ; NW155–2); 21, J. vestina (no image) (NN07); 22, J. vestina (Las Culebrillas, Cañar, Ecuador; DNA not extracted). Photograph credits: 2, 3, 18–20, Nymphalidae Systematics Group (2009); 1, 7, 8, Wain Evans; 11, Janzen and Hallwachs (2009); 22, Jean-Claude Petit. VOLUME 66, N UMBER 4 187 are also present (Brévignon 2008, 2009). (633 bp) of the 658 bp barcode region was sequenced Although the buckeyes are a well-known and much by Kodandaramaiah and Wahlberg (2007), we were studied group of nymphalid butterflies, especially with particularly interested in determining if the barcode respect to genetic factors involved in evolution and segment alone could provide informative characters for development of eyespots and color patterns (Nijhout inferring phylogenetic relationships and addressing 1980; Reed et al. 2007; Monteiro 2008; taxonomic uncertainties in Junonia from the Americas . Kodandaramaiah 2009; Monteiro & Prudic 2010), and DNA barcodes, although sometimes of limited the chemical ecology and evolution of hostplant usefulness (Elias et al. 2007; Yassin et al. 2010), have preferences (Bowers 1984; Bowers & Puttick 1989; been shown to be highly reliable at species-level Bowers & Stamp 1997), the systematics of the J. evarete identifications within the Lepidoptera in the eastern complex has been plagued with uncertainty, with USA and northwestern Costa Rica, with a success rate species assignments often questionable and unreliable. of >97% for ~2000 morphologically-defined taxa The confusion can be traced, at least in part, to the (Hebert et al. 2003, 2010; Janzen et al. 2005; Hajibabaei pronounced phenotypic variability in wing maculation et al. 2006) . In the present study, we analyzed both new and coloration within the genus Junonia (Tilden 1971; and previously published COI sequences from a total of Hafernik 1982). The apparent loss of type specimens, 85 individuals of New World Junonia . vague or unknown type localities, and non-standardized use of common names have also added to the taxonomic MATERIALS AND METHODS confusion . Molecular phylogenetic evidence suggests Sampling. The new taxa of Junonia treated here that the ancestor of the J. evarete complex probably include (1) J. coenia grisea from far western USA colonized the New World from Africa or Asia relatively (California and southern Oregon) and the Baja recently, ~2–4 million years ago (Ma) (Kodandaramaiah California Peninsula, Mexico (type locality: South & Wahlberg 2007), implying that subsequent speciation Pasadena, Los Angeles County, California) (Austin & in this group also is relatively recent. Thus, the Emmel 1998); specimens for the present study were possibility for incomplete lineage sorting among collected at a residential development site in Santa diversifying taxa may be high. The many observations of Barbara, California, USA, (2) a population from hybridization among phenotypic variants of Junonia northwestern Mexico that feeds on black mangrove (Rutkowski 1971; Hafernik 1982) are consistent with Avicennia germinans (L.) L. (Acanthaceae) (Pfeiler this possibility. 2011) . This population is listed as J. evarete by Brown et In the only comprehensive (worldwide) molecular al. (1992) (an assignment initially followed here) and phylogenetic study conducted to date on Junonia , based referred to as an intermediate between J. evarete zonalis on 3090 base pairs (bp) from both mitochondrial and J. coenia by Hafernik (1982); our samples were (cytochrome c oxidase subunit I; COI) and nuclear collected at a mangrove estuary (Estero del Soldado) genes ( wingless and elongation factor-1 α), the three near San Carlos, Sonora, Mexico, (3) the taxon currently nominal species of the J. evarete complex partitioned recognized as J. evarete nigrosuffusa (Luna-Reyes et al. into two well-supported clades, one comprised of J. 2008; Pelham 2008), a large, dark subspecies inhabiting evarete (Brazil and Guadeloupe) and the other southwestern USA and Mexico, generally inland from consisting of J. coenia coenia (Utah and Tennessee, the immediate coast (type locality: southeastern USA) + J. genoveva (Martinique) (Kodandaramaiah & Arizona); our specimens were collected in the coastal Wahlberg 2007) . Because total sample size from the two foothills of the Sierra El Aguaje at San Carlos, Sonora, New World clades was low ( N = 8), and did not include Mexico . GenBank sequences were available for a any populations from western North America (with the population of Junonia from the Area de Conservación exception of a single individual from Utah), the Guanacaste (ACG), Guanacaste Province in relationships of these previously studied taxa to western northwestern Costa Rica assigned to J. evarete , whose populations of Junonia remain unclear. We also wished foodplants include Dyschoriste valeriana Leonard to assess whether molecular data from western (Acanthaceae) and Stachytarpheta jamaicensis (L.) Vahl populations would provide any additional insights into (Verbenaceae) (DeVries 1987; D.H. Janzen & M. the results of the hybridization studies of Hafernik Hajibabaei unpublished). Additional GenBank (1982) who found high genetic similarity among western sequences were obtained for specimens collected in taxa. southern Mexico, Panama, Brazil, Peru, French Guiana, Given the increase in available COI sequence data for central and eastern USA, and the Caribbean . Details on Junonia from the DNA barcode initiative taxa analyzed, collection data and GenBank accession (Ratnasingham & Hebert 2007), and the fact that most numbers for the complete data set are given in Table 1. 188 188 JOURNAL OF THE LEPIDOPTERISTS ’ S OCIETY a
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Molecular protocol and data analysis. Total 100 random additions of sequences . Clade support for genomic DNA was extracted from two legs of each Bayesian trees was estimated utilizing a Markov chain butterfly using the DNeasy TM (QIAGEN Inc., Valencia, Monte Carlo (MCMC) algorithm and expressed as CA) protocol . The polymerase chain reaction (PCR) was posterior probabilities; relative support for MP tree used to amplify the barcode segment of the COI gene topology was obtained by bootstrapping using 500 with primers LCO1490f and HCO2198r using standard pseudoreplicates. PCR conditions (Folmer et al. 1994) . Sequencing reactions were performed on an Applied Biosystems RESULTS (Foster City, CA) ABI 3730XL DNA sequencer at the Sequence data and genetic diversity. A complete DNA Sequencing Facility, University of Arizona, Tucson barcode segment (658 bp) was available for 68 of the 85 using the amplifying primers. Sequences were proofread individuals of Junonia shown in Table 1 . No stop codons and aligned in ClustalX 1.81 (Thompson et al. 1997) or indels were found in any of the sequences . There followed by manual editing. were 53 variable sites . Nucleotide composition was Calculations of Kimura (1980) 2–parameter (K2P) nearly identical in the 68 sequences (mean values: genetic distances ( d) among sequences were carried out 38.7% T, 14.6% C, 31.4% A and 15.2% G) . There was a in MEGA version 4.0 (Tamura et al. 2007) . Calculations strong bias against G at the third codon position (mean of genetic diversity indices and Tajima’s (1989) D were G content 1.4%; range 0.5–2.7%) . Inspection of the 658 performed in DnaSP version 5.00.04 (Librado & Rozas bp segment in the 68 samples revealed that of the 53 2009) . Relative rate tests (Tajima 1993) of sequence variable sites, none were present in the first 25 bases evolution were carried out in MEGA using J. orithya as that were deleted for phylogenetic analyses. the outgroup . Analysis of molecular variance (AMOVA, Genetic diversity indices for Junonia are shown in Excoffier et al. 1992) performed in ARLEQUIN version Table 2 . Two different patterns were observed . 3.5.1.3 (Excoffier & Lischer 2010) was used to test for Haplotype diversity ( h) and nucleotide diversity ( π) structure among selected populations of Junonia. The were relatively high ( h ≥ 0.900; π > 0.003) in J. evarete calculation of significance ( α = 0.05 ) of the fixation from Costa Rica and South America (including the index ΦST was based on 10,000 permutations of the data Caribbean) and in J. coenia coenia from the USA, but matrix . Estimates of the number of migrants per were lower ( h < 0.700; π < 0.002 ) in J. evarete and J. generation ( Nm) among populations were also calculated evarete nigrosuffusa from Sonora, Mexico and in J. in ARLEQUIN. coenia grisea from southern California, USA . The Phylogenetic analyses. For phylogenetic analyses differences in h and π seen in J. evarete from Sonora, all COI sequences were trimmed to 633 bp to Mexico and Costa Rica are notable given that sample correspond to the barcode region reported in sizes from the two localities were similar . Tajima's D was Kodandaramaiah and Wahlberg (2007) . Relationships not significant in any of the taxa . None of the relative among haplotypes were assessed with the neighbor- rates tests (Tajima 1993) were significant, indicating that joining (NJ) algorithm of Saitou and Nei (1987) carried a molecular clock could not be rejected for Junonia. The out in MEGA using a matrix of K2P distances . We used AMOVA revealed significant structure among two African species of Junonia as outgroups, J. orithya populations of J. evarete from Costa Rica ( N = 22) and
(GenBank EU053315) and J. westermanni (GenBank Estero del Soldado, Mexico ( N = 19) ( ΦST = 0.398; P < EU053319) . Both African species show a close 0.0001) . The estimated number of individuals migrating relationship with the New World Junonia between the two regions per generation ( Nm) was 0.756 . (Kodandaramaiah & Wahlberg 2007) . Junonia orithya , The AMOVA also showed significant structure between in particular, shares similarities in both wing pattern and the subspecies J. coenia coenia (N = 6) from eastern morphology of male genitalia with New World Junonia USA and J. coenia grisea (N = 7) from California ( ΦST = (Corbet 1948; Tilden 1971) . Statistical support for nodes 0.787; P < 0.001; Nm = 0.135). was obtained by bootstrap analyses using 1000 Phylogenetic relationships . Preliminary phylo- pseudoreplicates (Felsenstein 1985) . Confirmation of genetic analyses of the three New World taxa ( J. evarete , clades identified from NJ analysis was obtained by J. genoveva and J. coenia coenia ) from Kodandaramaiah constructing phylogenetic trees with (a) Bayesian and Wahlberg (2007), using only the 633 bp COI inference implemented in MrBayes version 3.1 barcode segment and J. orithya and J. westermanni as (Huelsenbeck & Ronquist 2001), sampling 4000 trees outgroups, resolved the J. evarete and ( J. coenia + J. and using both HKY and GTR nucleotide substitution genoveva ) clades (referred to below as clades A and B, models, and (b) maximum parsimony (MP) carried out respectively) in NJ, MP and Bayesian trees (not shown) in MEGA using the CNI heuristic search option and with similar (MP) or identical (Bayesian) clade support 192 192 JOURNAL OF THE LEPIDOPTERISTS ’ S OCIETY values reported by those workers from the combined coenia grisea was d = 1.0% . Mean values in all pairwise mitochondrial and nuclear data set of 3090 bp. comparisons between clades A and B, including The NJ tree of New World Junonia based on comparisons with individuals assigned to J. evarete barcodes, and representing both new and previously which appear in both clades, ranged from d = published data, is shown in Fig. 2 . The NJ tree again 4.0–4.5% . These values are higher than the genetic resolved clades A and B with high statistical support . In distances found between the two species from Africa addition to the single J. genoveva and two J. coenia used as outgroups, J. orithya and J. westermanni (d = coenia from Kodandaramaiah and Wahlberg (2007), all 3.4%; not shown in Table 3), and also are higher than sequences of Junonia from the USA, Mexico, Costa Rica the value found between J. orithya and clade A 2 (d = and Panama clustered in clade B, including those from 3.9%); the values are slightly lower than d = 5.0 % taxa currently assigned to J. evarete and J. evarete found between J. orithya and clade B . Within clade A, nigrosuffusa. A short COI sequence (290 bp) assigned the genetic distance between the distinctive J. vestina to J. evarete from Quintana Roo, Mexico (Prado et al. (clade A 1) and J. evarete (clade A 2) was d = 2.1 % . The 2011) also clustered in clade B (not shown) . Within mean value between J. evarete from Costa Rica and clade B, a weakly-supported subclade consisting of J. Sonora, Mexico, localities separated by ~3250 km, was coenia grisea from southern California was found . All d = 0.5% . Within population d values for J. evarete other populations within clade B were unresolved . The were 0.0–0.5% (mean d = 0.1%) for Sonora and same topology, with similar support values, was 0.0–1.4% (mean d = 0.5%) for Costa Rica . One obtained on a representative subset of sequences from individual of J. evarete from Sonora shared the same all taxa using MP and Bayesian analyses (not shown). haplotype with an individual from Costa Rica (see Fig. All populations of Junonia from South America and 3). the Caribbean, with the exception of a single J. genoveva (= J. neildi Brévignon) from Martinique DISCUSSION (NW136–16), clustered in Clade A, including We have shown that phylogenetic analysis of a 633 bp individuals identified as J. evarete and J. genoveva. The segment of the mitochondrial COI gene, comprising resolution of J. vestina in a basal position in clade A most of the barcode segment, resolves the two main
(referred to here as clade A 1) was highly supported . The clades of New World Junonia reported previously using remaining clade A individuals were all closely related a larger data set of both mitochondrial and nuclear
(see below) and are grouped into clade A 2. genes (Kodandaramaiah & Wahlberg 2007) . Barcodes Pairwise comparisons of K2P genetic distances ( d) thus provide an informative and relatively inexpensive among New World Junonia are shown in Table 3 . Mean tool for phylogenetic studies of this group . Assigning genetic distances were low ( d ≤ 1.1%) for all individuals of the J. evarete complex to their respective comparisons between taxa within clade B . Genetic clade using morphological characters alone is unreliable distance between the subspecies J. coenia coenia and J. and has probably contributed much to the taxonomic
Table 2. Summary of genetic diversity indices and results of neutrality tests (Tajima’s D) in theCOI gene segment in Junonia .
Species N L k K h (± SD) π (± SD ) Tajima’s D
J. evarete (clade A 2)* 12 633 20 10 0.970 ± 0.044 0.00824 ± 0.00105 –0.97
J. evarete (Costa Rica) 22 658 18 14 0.926 ± 0.039 0.00455 ± 0.00075 –1.45
J. evarete (Sonora, Mexico) 19 658 3 5 0.696 ± 0.077 0.00133 ± 0.00023 0.06
J. e. nigrosuffusa (Mexico) 9 658 3 3 0.639 ± 0.126 0.00186 ± 0.00042 0.41
J. coenia coenia (USA) 5 633 4 4 0.900 ± 0.161 0.00253 ± 0.00076 –1.09
J. coenia grisea (USA) 7 658 2 3 0.667 ± 0.160 0.00116 ± 0.00035 –0.27 N, number of sequences; L = sequence length (number of bases); k = number of variable sites; K, number of haplotypes; h, haplo - type diversity; π, nucleotide diversity. Values for Tajima's D were not significant for any species at the 0.05 level. *All species from
South America and the Caribbean clustering in clade A 2, including those originally assigned to J. evarete, J. genoveva or Junonia sp. ≤ (see Fig. 2), were combined under J. evarete in Tables 2 and 3. Five shorter sequences ( 600 bp) from J. evarete (clade A 2) (NW36–2, UK4–15, UK4–18), J. e. nigrosuffusa (CIAD 10–B25) and J. coenia coenia (NW38–18) were omitted from Tables 2 and 3. VOLUME 66, N UMBER 4 193
TABLE 3. Mean K2P genetic distances ( d) among taxa and geographic populations of New World Junonia based on the COI gene
(633 bp). Values for d within taxa are shown along the diagonal. Shaded area shows taxa included in clade A, J. vestina (clade A 1) and J. evarete (clade A 2) from South America and the Caribbean (see footnote to Table 2). The remaining taxa all cluster in clade B from North America and the Caribbean.
1 2 3 4 5 6 7 8
1 J. coenia coenia (USA) ( N = 5) 0.003
2 J. coenia grisea (USA) ( N = 7) 0.010 0.001
3 J. e. nigrosuffusa (MX) (N = 9) 0.003 0.010 0.002
4 J. genoveva (Martinique) ( N = 1) 0.004 0.009 0.003 -----
5 J. evarete (Sonora, MX) ( N = 19) 0.004 0.008 0.003 0.003 0.001
6 J. evarete (Costa Rica) ( N = 22 ) 0.007 0.011 0.006 0.006 0.005 0.005
7 J. evarete (S. Amer.) ( N = 12) 0.043 0.043 0.042 0.045 0.044 0.042 0.008
8 J. vestina (Peru) ( N = 1) 0.040 0.044 0.040 0.043 0.041 0.042 0.021 ----- confusion . Because of evidence for relatively recent fossil evidence provide estimated dates which are in divergences in the New World Junonia , however, relatively close agreement, implying that clades A and barcodes alone may be of limited usefulness for B began to diverge shortly after colonization of the inferring intra-clade relationships and species New World . Because we found no evidence that identifications, especially within clade B . All new nucleotide substitution rate in the COI gene in Junonia barcode sequences from populations from western is different from that typically seen in many insects, the North America, comprising several recognized taxa, low genetic divergences within clade B likely indicate a clustered in clade B and most showed low genetic relatively recent (late Pleistocene or Holocene) divergences ( d < 1%) . The western J. coenia grisea , radiation and speciation within this group . The low however, resolved as a weakly-supported subclade genetic divergences also could result from incomplete within clade B, supporting its designation as a lineage sorting and extensive hybridization among subspecies of J. coenia (Austin & Emmel 1998) . The diversifying taxa, possibly suggesting just a single, AMOVA showed significant population structure polytypic species . There is evidence, however, apart among J. coenia grisea and J. coenia coenia , also from the pronounced intra-clade phenotypic variability consistent with subspecies status . Additionally, our (Fig. 3), to support recognizing distinct species level analyses revealed that none of the North American taxa within clade B that barcodes are unable to detect. Junonia from Mexico and Central America currently The low genetic divergences and presumed recent recognized as J. evarete , including J. evarete speciation among recognized taxa of Junonia nigrosuffusa from Mexico, clustered with J. evarete comprising clade B are consistent with the conclusions from South America and the Caribbean (clade A 2). of laboratory hybridization studies showing a high These results suggest that either the taxon currently degree of genetic similarity among North and Central recognized as J. evarete is paraphyletic, or taxonomic American Junonia (Hafernik 1982) . The taxa used in assignments of the western populations need to be the hybridization experiments and phenetic analyses of reconsidered (see below). Hafernik (1982) included J. coenia (populations from Assuming a molecular clock rate of ~2% pairwise both Texas and California representing what are now sequence divergence per million years for insect COI recognized as subspecies J. coenia coenia and J. coenia (Brower 1994; Craft et al. 2010; Pfeiler et al. 2010) we grisea , respectively), J. evarete nigrosuffusa (southern estimate that clades A and B began to diverge ~2.2 Ma . Texas and southeastern Arizona; treated as a full species Based on fossil evidence, Junonia is thought to have by Hafernik) and J. evarete zonalis (southern colonized the New World about 2–4 Ma Guatemala and northwestern Costa Rica) . Caribbean (Kodandaramaiah & Wahlberg 2007) . Mean genetic populations, including J. genoveva , were excluded from distances between clade A and the outgroup taxa from the study [ J. genoveva is currently listed for southern Africa were 3.9 and 4.3% for J. orithya and J. Texas (Opler et al. 2011; Warren et al. 2011)] . Several westermanni , respectively, suggesting the ancestor of lines of circumstantial evidence, however, suggest that the clade A lineage began to diverge from the African the reference populations of Junonia from Central taxa ~2 Ma . Thus, molecular clock considerations and America used by Hafernik (1982) may have been from 194 194 JOURNAL OF THE LEPIDOPTERISTS ’ S OCIETY
FIG . 2. Neighbor-joining (NJ) tree showing relationships among New World Junonia based on COI barcode sequences. Voucher codes are listed for each of the ingroup species (see Table 1 for details). GenBank accession numbers are shown for the outgroups, J. orithya and J. westermanni from Africa. Red and green bars represent the two main clades (A and B, respectively) of New World
Junonia . Clade A 1 is comprised of J. vestina ; clades A 2 and B contain the members of the J. evarete complex. Bootstrap support val - ues are shown on branches; values <60% were omitted. Scale bar indicates sequence divergence. VOLUME 66, N UMBER 4 195 the clade B lineage, most probably from the taxon J. values seen for species level taxa in Lepidoptera based genoveva. Specimens from these reference populations on barcodes (Hajibabaei et al. 2006; Hebert et al. were taken at Escuintla, Guatemala and Cañas, Costa 2010) . Finding high genetic identity in hybridization
Rica, both from the Pacific slope and ~700 and ~75 km, studies between individuals of clades A 2 and B would respectively, from the Area de Conservación not be expected in two distinct taxa with relatively high Guanacaste (ACG) . Although multiple species of genetic divergences . For example, in Jamaica where J. Junonia occur in certain regions, no COI genotypes evarete and J. genoveva both occur, no evidence was similar to those found in South American and found for natural hybridization among the two taxa Caribbean populations of J. evarete have thus far been (Turner & Parnell 1985) . However, the conclusions of detected in the 45 barcode sequences obtained for Hafernik (1982) are consistent with our findings if the Junonia from the ACG (D.H. Janzen & M. Hajibabaei, Central America taxon used in that study was from the unpublished) . Junonia evarete genotypes also were not J. genoveva lineage and not a subspecies of J. evarete. present in the two samples from Morelos, Mexico, or in Our argument assumes that J. evarete was correctly the samples from Quintana Roo, Mexico (Prado et al. identified in the earlier molecular study of 2011) and Panama (Fig. 1) . Because of the genetic Kodandaramaiah and Wahlberg (2007) . Photographs of similarities and lack of reproductive isolation, Hafernik J. evarete studied by those authors (see Fig. 1) match (1982) concluded that J. evarete nigrosuffusa and J. closely the phenotype of the recently assigned neotype evarete zonalis represented a cline from Central of J. e. evarete from Suriname, South America (Neild America to southern Texas and should be considered 2008), suggesting that the identification was correct. conspecific . We have shown, however, that J. evarete Based on the above arguments we propose that the nigrosuffusa from Mexico and J. evarete from South population of Junonia from Mexico that utilizes black America and the Caribbean show a mean genetic mangrove ( Avicennia germinans ) as a larval host, as divergence ( d = 4.2%; Table 3) well within the range of well as the specimens shown in Table 1 from Morelos
FIG . 3. Comparison of adult females of Junonia from western North America showing phenotypic variability. (A) J. evarete ni - grosuffusa (= J. nigrosuffusa ; see Discussion) (San Carlos, Sonora, Mexico; CIAD 10–B32); (B) J. evarete (= J. genoveva ) (Estero del Soldado, near San Carlos, Sonora, Mexico; CIAD 10–B11); (C) J. evarete (= J. genoveva ) (Area de Conservación Guanacaste, Gua - nacaste Province, Costa Rica; 05–SRNP–58220); (D) J. coenia grisea (Santa Barbara, California, USA; CIAD 10–B04). Haplotypes for COI were identical for specimens A, B and C; specimen D differs by 5 nucleotide substitutions. Specimens A, B and D are wild- caught; specimen C was reared. Specimen B from Estero del Soldado is a worn individual; ground color of recently eclosed speci - mens is deep brown (Pfeiler 2011). Scientific names in parentheses are suggested changes in assignment based on data presented here. Photograph credits: (A), (B) and (D), Wain Evans; (C), Janzen and Hallwachs (2009). 196 196 JOURNAL OF THE LEPIDOPTERISTS ’ S OCIETY
(JM6–10 and NW162–7) and Quintana Roo, Mexico & Brock 1991; Brown et al. 1992; Brown 2004) . In (MAL–02877), Panama (YB-BCI12765), and the northwestern Mexico, J. genoveva and J. nigrosuffusa are population from Costa Rica that utilizes Dyschoriste generally ecologically isolated and morphologically valeriana and Stachytarpheta jamaicensis , be removed distinct (Fig. 3), with larvae of the two species utilizing from J. evarete and provisionally reassigned to J. different host plants (Tilden 1971; Hafernik 1982; genoveva. These new assignments agree with an earlier Bailowitz & Brock 1991; Brown et al. 1992; Vargas et al. observation that a possible subspecies of J. genoveva 1996; Warren et al. 1998; Pfeiler 2011) . Our field occurs in coastal regions of western Mexico (Vargas et al. observations in the San Carlos region of Sonora have 1996) . Ongoing research on Junonia from the revealed no evidence for hybridization, although adults Caribbean, however, suggests that the mangrove of both lineages are occasionally encountered feeding buckeye probably consists of more than one species, together (Pfeiler 2011) . There are reports, however, of including the recently-named J. litoralis Brévignon and intermediates between the coastal J. genoveva and J. J. neildi Brévignon (Brévignon 2009) . In addition, the nigrosuffusa in other regions of western Mexico (Vargas clade A 2 individual from São Paulo Brazil (Fig. 1, locality et al. 1996), as well as intermediates between J. coenia 20), was reared on Avicennia sp. indicating that and J. nigrosuffusa from southeastern Arizona (K. representatives of both clades A and B have adapted to Hansen pers. com.) . We suggest that, at least for feeding on black mangrove . A more thorough northwestern Mexico, J. nigrosuffusa and J. genoveva examination of relationships among taxa of Junonia in meet the two basic criteria consistent with ecological the Americas that utilize black mangrove and other host speciation, i.e. evidence for ecologically-based divergent plants may ultimately require revision of our provisional selection and assortative mating (Chamberlain et al. assignment. 2009) . Strong adult dispersal capability (Adler & Dudley Although significant structure was found between the 1994), together with the ability of larvae to adapt to a populations of Junonia from Estero del Soldado, Mexico variety of host plants from different families, are traits and Costa Rica, the low mean genetic distance between that would favor survival and potentially lead to the two populations ( d = 0.5%) agrees well with ecological speciation during the radiation of the New intraspecific divergences in Lepidoptera based on World Junonia . barcodes (Hajibabaei et al. 2006; Hebert et al. 2010) . In summary, we have shown that COI barcodes can Phenotypic differences of adults, however, together with distinguish J. vestina from members of the J. evarete the different host plants utilized by larvae, suggest that complex, and can resolve the two subspecies of J. coenia , these two populations may warrant recognition as but overall are of limited usefulness in species distinct subspecies . Also, the higher haplotype and identifications within the complex itself . Nonetheless, nucleotide diversities of the Costa Rica population barcodes are a valuable tool in taxonomic studies of this compared with the Sonora population (Table 2) suggest group for their ability to easily identify the two major that dispersal and colonization proceeded from a clades of the J. evarete complex found in the New World, southern source population northward along the Pacific which is difficult, if not impossible, by morphological slope of North America (Pfeiler et al. 2012) . Haplotype analysis alone . The ability to unambiguously identify and nucleotide diversities were also relatively low in J. clades A and B will contribute to our understanding of evarete nigrosuffusa and J. coenia grisea (Table 2), but the degree of phenotypic variability and larval host plant low sample sizes did not allow for unambiguous preferences within each lineage . More extensive interpretations of demographic patterns. sampling will be required to determine the complete The barcode data also suggest that J. evarete distribution of the two clades in the New World [e.g., nigrosuffusa be removed as a subspecies of the J. evarete records of J. evarete zonalis in southern Florida (Warren lineage, as it clearly nests within clade B rather than et al. 2011) suggest the presence of clade A in the USA, clade A 2 (Fig. 2) . Two possible alternative assignments, and clade B probably occurs South America], but given previously proposed by others, are consistent with the the widely separated geographic localities in the genetic data . These include recognizing nigrosuffusa as a Americas sampled to date (Fig. 1), it seems unlikely that subspecies of J. genoveva (Vargas et al. 1996; Warren et barcodes will demonstrate additional deep divergences al. 1998; Glassberg 2001), or as a subspecies of J. coenia within the J. evarete complex . Other molecular markers, [as originally described by Barnes and McDunnough however, such as amplified fragment length (1916)] . A third possibility, also previously proposed but polymorphisms (AFLPs), show promise of being able to supported only by morphological and ecological data, is reveal recent divergences that barcodes fail to detect to recognize the taxon as a full species (Tilden 1971; (Dasmahapatra et al. 2010). Emmel & Emmel 1973; Miller & Brown 1981; Bailowitz VOLUME 66, N UMBER 4 197
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