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Neotropical Entomology

Print version ISSN 1519-566X

Neotrop. entomol. vol.40 no.5 Londrina Sept./Oct. 2011 



Molecules, wing pattern and distribution: an approach to species delimitation in the "loxurina group" (Lepidoptera: Lycaenidae: Penaincisalia)



C PrietoI, II; E MicóI; E GalanteI

ICentro Iberoamericano de la Biodiversidad (CIBIO), Univ de Alicante, Alicante, Spain
IIGrupo de Estudios en Bioindicación y Conservación, Corporación Universitaria Autónoma Del Cauca, Popayán, Colombia





The wide range of morphological variations in the "loxurina group" makes taxa identification difficult, and despite several reviews, serious taxonomical confusion remains. We make use of DNA data in conjunction with morphological appearance and available information on species distribution to delimit the boundaries of the "loxurina" group species previously established based on morphology. A fragment of 635 base pairs within the mtDNA gene cytochrome oxidase I (COI) was analysed for seven species of the "loxurina group". Phylogenetic relationships among the included taxa were inferred using maximum parsimony and maximum likelihood methods. Penaincisalia sigsiga (Bálint et al), P. cillutincarae (Draudt), P. atymna (Hewitson) and P. loxurina (C. Felder & R. Felder) were easily delimited as the morphological, geographic and molecular data were congruent. Penaincisalia ludovica (Bálint & Wojtusiak) and P. loxurina astillero (Johnson) represent the same entity and constitute a sub-species of P. loxurina. However, incongruence among morphological, genetic, and geographic data is shown in P. chachapoya (Bálint & Wojtusiak) and P. tegulina (Bálint et al). Our results highlight that an integrative approach is needed to clarify the taxonomy of these neotropical taxa, but more genetic and geographical studies are still required.

Keywords: COI, Eumaeini, Thecloxurina, Andes, butterfly




Interspecific and intraspecific variation in DNA sequences has been used for assessing morphological variability between closely related species in several studies of invertebrate taxa (e.g. Falniowski & Wilke 2001, Mengual et al 2006, Iguchi et al 2007). The utility of short Cytochrome c oxidase subunit I (COI) regions has been offered as a tool for the discovery of cryptic butterfly and Diptera species (Hebert et al 2004a, Smith et al 2006, van Velzen et al 2007), in the understanding of the species boundaries of taxa (e.g. Micó et al 2003, StÅhls & Savolainen 2008) and to accurately classify species in a number of studies (e. g. Hebert et al 2004b, Kerr et al 2007).

The genus Penaincisalia (Eumaeine) was established by Johnson (1990) for a small group of high Andean butterflies related with Austral biomes. More recently, Robbins (2004) synonymized other four related genera (Thecloxurina, Pons, Abloxurina, Candora) with Penaincisalia forming a genus with highly variable wing shapes. Although the latter taxonomy of the Penaincisalia genus is relatively well accepted, additional morphological and molecular characters need to be explored to improve our knowledge on the relationships among the species of various Eumaeine genera and species groups.

Prieto (2008) considered six preliminary species groups within Penaincisalia, including the "loxurina group" (Thecloxurina, sensu Johnson 1992), this group is characterized for a hind wing vein CuA2 terminus extended as a rigid tail in both sexes. The "loxurina group" is restricted to the tropical Andean habitats where some species are abundant, particularly in the northernmost Andes. Only minor differences in wing pattern characters differ between taxa within the "loxurina group", and it is difficult to determine species boundaries, especially considering that the proposed diagnostic characters in the dorsal and ventral surfaces are frequently variable.

Several studies have been conducted on the species related with Penaincisalia loxurina (Felder & Felder) and despite several proposed classifications, checklists and nomenclatural descriptions of new species (Table 1), serious taxonomical confusion remains. Although Prieto (2008) recognized eight species in the "loxurina group" including several recently described taxa (Table 1), this is the first taxonomic study of this species group.

In some cases wing pattern of the species of the "loxurina group" is so variable that parapatric or allopatric populations have often been considered different species. This creates an undesirable over-abundance of redundant species names. On the other hand, synonymy may also occur by "lumping" together species into a single entity even though several species do exist. Most questions in evolutionary biology, ecology, conservation priorities or biogeography depend on our knowledge of species (Dayrat 2005, Bickford et al 2007), so there is a need for rigorously delimit species boundaries for producing accurate species inventories.

In spite of the morphological variability of the group, no genetic studies on Penaincisalia species have been reported as yet. In this study we use mtDNA COI sequences to clarify the taxonomy of Penaincisalia, particularly for the taxa where morphological and taxonomical confusion has been most apparent, under the concept of "integrative taxonomy", the use of DNA data in conjunction with morphological characters and available distribution information to define biological species for comparison with previously established species boundaries based on morphology (Dayrat 2005, Mengual et al 2006).Therefore, we aim to delineate the species boundaries of the "loxurina group" based on three criteria: a) sympatry/allopatry as an indication of interbreeding, b) wing pattern differentiation and intermediate forms as possible indicator of interbreeding, and c) genetic distance.


Material and Methods

Specimens and molecular techniques

We analysed partial nucleotide sequences of mtDNA COI of 17 specimens belonging to seven species and one subspecies of the "loxurina group" from several populations occurring along the tropical Andes (Fig 1). Two additional Penaincisalia species, Penaincisalia browni (Johnson) and Penaincisalia magnifica (Johnson), belonging to sister species group (Prieto 2008) were sequenced as outgroups.



Thorax and legs were used for DNA extraction from single individuals of either dry, pinned or ethanol preserved specimens. DNA was extracted from these parts using the QIAGEN DNeasy Tissue extraction kit. We used the forward primer C1-J-1751 (5'-GGATCACCTGATATAGCATTCCC-3') and the reverse primer TL2-N-3014 (5'-TCCAATGCACTAATCTGCCATATTA-3') (Simon et al 1994) in PCR amplifications in 25 µl reactions containing 3 µl DNA extract, 1 µl of each primer (primers at 10 pmol/ µl), 0.25 µl of AmpliTaq DNA polymerase (250 units, 5 U/µl), 3 µl 25 mM MgCl2, 2.5 µl Buffer (Perquin-Elmer®), 4 µl 10 mM dNTP (Perquin-Elmer®) and ultra-pure water. Thermocycler conditions were 96ºC for 1 min (1x), followed by 29 cycles at 96ºC for 30s, 50ºC for 15s, and 60ºC for 4 min. PCR products were purified using the QIAquick® PCR Purification kit QIAGEN. Amplified PCR samples were sequenced including a second forward primer, C1-J-21835 (5'-CAACATTTATTTTGATTTTTTGG -3') (Simon et al 1994) with an ABI PRISM 310 (Applied Biosystems) sequencer.

Data analysis

The sequences were inspected, edited for base-calling errors and submitted to GenBank (accession numbers are presented in Table 2).

We used the program PAUP* version 4.0b10 (Swofford 2002) for a parsimony analysis (MP) using the heuristic search procedure. Gaps were treated as missing data. We also inferred the phylogenetic relationships among all species using maximum-likelihood (ML). Bootstrap support values (Felsenstein 1985) were calculated using 200 replicates for the MP and ML analyses, using HKY85 model (ln = -1691.639).

Results and Discussion

The final aligned sequences yielded 635 nucleotides that also include 60% of the "Folmer fragment" which is the fragment proposed by the DNA Barcoding Council. Of the obtained 635 nucleotides, 98 were variable and 72 sites were parsimony-informative. Parsimony analysis produced seven equally parsimonious trees, with a consistency index (CI) of 0.74, a retention index (RI) of 0.79. Results of the ML analysis are shown in Fig 2, one of the seven MP trees is shown in Fig 3. Monophyly of the "loxurina group" was not rejected and was supported with very high bootstrap values for maximum-likelihood and parsimony analyses (Figs 2, 3).





Uncorrected pairwise divergences between ingroup taxa ranged from 2.52% to 6.15%, and among individuals within each clade from 0.0 % to 0.78% (Table 3, Fig 2). Divergences between outgroup and ingroup taxa ranged from 6.29% to 7.57% (Table 3, Fig 2). A total of ten different haplotypes were detected in the "loxurina" species-group. Most clades were associated with distinct morphologies, especially in wing upper surface appearance. In addition, within the clade I, the dorsal surface for each individual is very distinct (Fig 3).

Congruence among data

Several species were easily defined, as the morphological, geographic and molecular data were congruent. Penaincisalia sigsiga (Bálint et al) was considered as a reproductively isolated taxon due to its relatively high genetic divergence when compared with all species of the "loxurina group" (4.7% to 6.15%) (Table 3), external morphology and confirmed sympatry with the most similar species P. atymna (Hewitson). Although sympatry was not confirmed for P. cillutincare (Draudt) with any other "loxurina group" species, its relatively high genetic divergence when compared with other "loxurina group" species (3.5% to 4.7%) (Table 3) and morphological differentiation suggest that this is a reproductively isolated taxon following current usage (Fig 3).

Clade IV (P. atymna) exhibits the highest intraspecific divergence (0.0% to 0.78%) with the specimen from the Quindío population as the most distinctive (Fig 3). Although clade III [P. loxurina (C. Felder & R. Felder) from Colombia] and P. loxurina from Peru presented higher genetic divergences than the average within clades, these entities are geographically isolated and differ morphologically by the lighter dorsal blue surface and the larger size of P. loxurina from Peru. Both morphological characters are very variable in the group, allowing us to consider that both are slight geographical forms of the same species.

The species P. ludovica (Bálint & Wojtusiak) and the subspecies P. loxurina astillero (Johnson) had identical COI sequences (clade II) and could not be separated based on the sequences analysed (Figs 2, 3; Table 3). Moreover, it was not possible to distinguish these taxa based on morphological characters. The genetic distances among the ingroup clades were larger than those within clades with the exception of clades II and III. The average genetic distance among clades II and III was not significantly larger than within clades (P > 0.09). Moreover, in our samplings, sympatry was not confirmed between clades II (P. loxurina astillero + P. ludovica), III (P. loxurina) and P. loxurina from Peru (Figs 2, 3). Thus, clade II most likely constitutes a subspecies of T. loxurina restricted to the eastern slope of the Ucayali river on the eastern mountain range in "Madre de Dios" (Peru) (Fig 1).

Incongruence among data

Two strongly divergent phenotypes were grouped in clade I (Fig 3) [P. chachapoya (Bálint & Wojtusiak) + P. tegulina] and had very low genetic divergence ("P" = 0.011). The phenotype of P. tegulina would be considered as a wing pattern variant of P. cillutincarae. However, our results show that this phenotype (from Peru) is more closely related to P. chachapoya (from Peru).

The genetic divergence between P. chachapoya and P. tegulina was lower than the genetic divergence between clades. Although, this could indicate that these two phenotypes constitute the same species, it could be a case of "false negative", where little or no sequence variation in the COI fragment is found between different biospecies (Meyer & Paulay 2005, Wiemers & Fiedler 2007) easily distinguishable by their strong phenotypical differentiation. To decide whether these lineages belong to distinct species or to the same polymorphic species requires further study.

Penaincisalia chachapoya has been considered as a geographic form of P. loxurina (Robbins 2004). However, although P. chachapoya and P. loxurina has not been found exactly at the same locality, sympatry is not rejected due to the inexistence of geographic barriers between the very close localities where they have been collected and to the similarity of their ecosystems (Fig 1). This fact, together with their observed genetic distinctness, suggests P. chachapoya and P. loxurina are most likely reproductively isolated.

Results from morphological and molecular analyses suggest the following conclusions: P. ludovica constitutes the same entity as P. loxurina astillero and constitutes a subspecies of P. loxurina. Although we cannot decide whether P. chachapoya and P. tegulina are distinct species based on our results, we can confirm that the P. tegulina phenotype is not related to P. cillutincarae from Argentina.

Limits of non-integrative analysis of the taxonomy of Penaincisalia

In agreement with Dayrat (2005), our results show that traditional morphology-based taxonomy has limits. Cases such as P. chachapoya, P. loxurina and P. ludovica show the need to test observed "morphodiversity" via different approaches and with different kinds of data in order to delimit species boundaries. On the other hand, the genetic methods alone may present some problems, such as the fact that gene trees may differ from species trees (Pamilo & Nei 1988, Maddison 1997). Based on genetic data alone, P. chachapoya and P. tegulina could have been considered as the same species, but their strong wing pattern differentiation suggests otherwise. Therefore, as Valdecasas et al (2008) suggested, part of the problem of species delineation is the fact that molecular biology, cytogenetics, enzymology, ecology, among others approaches for species delimitation also have certain limits.

While the success rate of barcoding undoubtedly varies among groups, problems in insects and other invertebrates have been frequently observed, including mitochondrial introgression between taxa and interbreeding that obscures species identification and limits (e.g. Croucher et al 2004, Kaila & StÅhls 2006). Moreover, some groups in which recent speciation rates are high and effective population sizes large and stable, as in many tropical insects, are particularly likely to be subject to difficulties (Elias et al 2007). As the "loxurina group" seems to be a result of recent speciation processes (Prieto 2008), the use of molecular methods alone to understand species boundaries could be unreliable. Thus, a multidisciplinary approach to taxonomy of this group is necessary.

In the studied group, as well as many tropical insects, measuring intra- and interspecific sequence divergence is hampered by the fragmentary knowledge of most taxa. More information is necessary on the amount of intraspecific genetic and morphological variation among closely related species. Our study group needs to be rigorously tested with sequence data from samples that cover the geographic range more comprehensively. Further studies of COI profiles, correlated with sequence data from additional nuclear genes and informative morphological, geographical and ecological characters are necessary for the understanding of species boundaries of this group of butterflies.



We thank Ximo Mengual for his observations and recommendations throughout this study, Zsolt Bálint for useful comments and information, Pierre Boyer, Tomasz Pyrcz, Janus Wojtusiak for providing specimens, Cristobal R Malaver, Vladimir Sandoval, Monica Ramirez and Charles Muñoz for their help in the field. Keith Willmott for helpful comments and corrections. This work was partially supported by the AECI project (A/6788/06). Field work was partially supported by the "Tropical Andean Butterfly Diversity Project TABDP".



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Carlos Prieto
Grupo de Estudios en Bioindicación y Conservación
Corporación Universitaria Autónoma Del Cauca
Popayán, Colombia

Received 17 May 2010 and accepted 29 December 2010



Edited by Marcelo Duarte - MZ/USP

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