Hybridisation in Colias butterflies (Lepidoptera, Pieridae): identification of natural hybrids using unlinked molecular markers
DOI: 10.5281/zenodo.8353128

Hybridisation in Colias butterflies (Lepidoptera, Pieridae): identification of natural hybrids using unlinked molecular markers

Department of Karyosystematics, Zoological Institute, Russian Academy of Sciences, Universitetskaya nab. 1, 199034 Saint-Petersburg, Russia
Department of Entomology, Biological Faculty, Lomonosov Moscow State University, Leninskie Gory, GSP-1, korp. 12, 119991 Moscow, Russia; Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninsky Prospect 33, 119071 Moscow, Russia
Department of Ecology, Altai State University, Lenina Pr. 61, 656049 Barnaul, Russia; Institute of Biology, Tomsk State University, Lenina Pr. 36, 634050 Tomsk, Russia
Centro de Investigaciones en Optica, Lomas del Bosque 115, 37150 Leon, Mexico
Department of Ecology, Altai State University, Lenina Pr. 61, 656049 Barnaul, Russia; Institute of Biology, Tomsk State University, Lenina Pr. 36, 634050 Tomsk, Russia
Central Asia DNA barcodes interspecific hybridisation molecular markers


In this report, we analyze a wild-caught Colias specimen from Kyrgyzstan with unusual wing pattern, which was provisionally identified as a natural hybrid between Colias christophi helialaica Schulte, 1988 and Colias cocandica cocandica Erschoff, 1874 due to its intermediate phenotype. Using one mitochondrial (COI barcode) and three nuclear (Ca-ATPase, H3 and CAD gene fragments) markers, we confirm its hybrid origin and demonstrate that this specimen is a hybrid between a female of Colias christophi helialaica and a male of Colias cocandica cocandica. Our study shows that an analysis of mitochondrial and nuclear molecular markers can successfully discriminate natural hybrids in such a taxonomically challenging group of butterflies as the genus Colias.

Acta Biologica Sibirica 9: 605–616 (2023) doi: 10.5281/zenodo.8353128

Corresponding authors: Galina N. Shapoval (galinakuftina@mail.ru); Nazar A. Shapoval (nazaret@bk.ru)

Academic editor: A. Matsyura | Received 6 October 2022 | Accepted 15 November 2022 | Published 20 September 2023


Citation: Shapoval NA, Krupitsky AV, Yakovlev RV, Kir'yanov AV, Shapoval GN (2023) Hybridisation in Colias butterflies (Lepidoptera, Pieridae): identification of natural hybrids using unlinked molecular markers. Acta Biologica Sibirica 9: 605–616. https://doi.org/10.5281/zenodo.8353128


Central Asia, DNA barcodes, interspecific hybridisation, molecular markers


The genus Colias (Pieridae: Coliadinae) is a large taxonomically challenging group of butterflies with approximately 90 described species distributed throughout most of the world (Verhulst 2000; Greishuber and Lamas 2007; Greishuber et al. 2012; Greishuber 2014). In contrast to many other groups of Lepidoptera, species of the genus are highly conservative with respect to the male genitalia structures, which often lack reliable diagnostic characters (Greishuber 2014). The differences in the wing pattern between Colias species are also often very subtle or even vanishing (Hammond and McCorkle 2003). Furthermore, many taxa exhibit significant intraspecific variability in the wing colouration and pattern (Ellers and Boggs 2002; Hammond and McCorkle 2008). The wide colour polymorphism and the limited applicability of external and internal diagnostic features make reliable identification and species-level boundaries definition very challenging by morphological grounds. Although natural hybridisation is thought to be somewhat uncommon in animals, individuals with exceptional phenotypical traits, morphological characteristics at the intersection of different species, or of “intermediate” phenotypes regularly occur in nature (Uthicke et al. 2005; Lutterschmidt et al. 2007; Galindo-Ruiz et al. 2019; Eshenroder et al. 2021; Liberman et al. 2021; Gaudiano et al. 2022). Such individuals are often considered as putative hybrids. In entomological literature, numerous publications are regarded to possible cases of interspecific hybridisation between different species of Lepidoptera (Sperling 1990; Warren and Robbins 1993; ten Hagen 2003; Gil-T 2007; Mullen 2008; Jasso-Martínez et al. 2018; Shapoval et al. 2021), including the genus Colias (Ae 1959; Jahner et al. 2012; Dwyer et al. 2015; Dzurinka et al. 2021, 2022).

A number of Colias specimens, which are thought to represent natural hybrids, are known from private and museum collections (Schurian and Rose 1978; Schulte 1988). However, given the limited number of reliable diagnostic characters coupled with broad intraspecific variability common for many taxa of the genus, a caution is required in the interpretation of such individuals as hybrids. Recent reports have shown that the assumptions on the hybrid origin of phenotypic intermediates based on the analysis of external morphology alone can be misleading (Qin et al. 2023). In such cases, combined analysis of morphological and molecular data can serve as a powerful approach for both species identification and delimitation, and for natural hybrids discrimination (Shapoval et al. 2021).

At the beginning of July 2022, a putative hybrid specimen sharing the external characters of Colias christophi helialaica Schulte, 1988 and Colias cocandica cocandica Erschoff, 1874 was collected by Alexander Baryshev (Penza, Russia) in the Alay Mountains (Kyrgyzstan). In this study, we employ the combined approach (morphological and multigene data analysis) to confirm the hybrid origin of this specimen, ascertain parental species and identify the direction of the hybridisation.

Materials and methods

The putative hybrid and specimens of the putative parental species (Fig. 1) were collected in the Alay Mountains (Kyrgyzstan) by Alexander Baryshev (Penza, Russia) during the field studies in 2022. One mitochondrial (cytochrome oxidase subunit I (COI)) and three nuclear (Ca-ATPase (sarco/endoplasmic reticulum calcium ATPase), H3 (Histone h3) and CAD (Carbamoyl-Phosphate Synthetase 2, Aspartate Transcarbamylase, and Dihydroorotase)) genes were used as molecular markers. One or two legs from each specimen were taken for DNA extraction using the CTAB-based method (Murray and Thompson 1980) with some modifications (Lukhtanov and Shapoval 2008; Lukhtanov et al. 2008). Mitochondrial DNA barcode (a 658 bp fragment of the COI gene) was amplified using LCO1490/HC2198 primer pair (Folmer et al. 1994). A 445 bp fragment of the Ca-ATPase, a 328 bp fragment of the H3 and a 847 bp fragment of the CAD genes were amplified using Ca-ATPase_F/Ca-ATPase_R (Wahlberg et al. 2016), H3aF/H3aR (Colgan 1998), CAD743nF/CADmidR CADmidF/CAD1028R (Wahlberg and Wheat 2008) primer pairs, respectively. The PCR amplifications were performed in a 25 µL reaction volume per sample. Each reaction contained 1 µL template DNA (ca. 10–50 ng genomic DNA), 1.3 µL of both forward and reverse primers aliquoted to a standard concentration of 10 µM, 5 µL of 5× ScreenMix (Evrogen, Moscow, Russia) and 16.4 µL of ddH₂O. The temperature profiles for COI, Ca-ATPase, H3 and CAD genes were as follows: initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50 °C (COI)/55°C (Ca-ATPase, CAD)/60°C (H3) for 30 s and extension at 72°C for 1 min 30 s, with a final extension at 72°C for 10 min. Amplified fragments were purified using QIAquick PCR Purfication Kit (Qiagen, USA). Purification was carried out according to the manufacturer’s protocol. Purified PCR products were used for direct sequencing or subsequent cloning. We cloned the nuclear CAD gene fragments for the putative hybrid specimen for which standard sequencing revealed intra-individual heterogeneities in the form of single nucleotide polymorphism. The cloning procedure was performed following previously described protocols (Lukhtanov et al. 2015; Shapoval and Lukhtanov 2015). 20 clones for each CAD fragment were sequenced. Sequencing of the doublestranded product was carried out at the Research Resource Center for Molecular and Cell Technologies (St. Petersburg State University, St. Petersburg, Russia) using ABI 3500xL analyzer (Applied Biosystems, Waltham, MA, USA).

The sequences were checked, edited and aligned using CHROMAS 2.6.6 (http://www.technelysium.com.au/), Geneious v.8.1.6 (Kearse et al. 2012) and BioEdit v.7.0.3 (Hall 2011) software. Primer sequences were cropped. Three nuclear genes were concatenated resulting in the final dataset comprising a total of 1620 bp (445 bp of the Ca-ATPase, 328 bp of the H3 and 847 bp of the CAD). The mitochondrial COI gene was analysed independently. Since Colias croceus (Geoffroy, 1785) was earlier inferred as one of the basal clades within the genus Colias, we used previously obtained mitochondrial and nuclear sequences of this species as outgroup to root the mitochondrial and nuclear phylograms. The COI dataset of the phylogenetic analysis included in total sequences of 11 specimens: an outgroup, a putative hybrid, three C. cocandica cocandica samples and six C. christophi helialaica sequences (five of them were sequenced during this study and one more sequence was obtained from BOLD). The list of the specimens used for the molecular analysis with the full collection data and GenBank/BOLD Accession Numbers are given in the Table 1. The best-fitting substitution models for the COI dataset and for the concatenated Ca-ATPase+H3+CAD nuclear genes fragments were estimated based on the Bayesian information criterion (BIC) using jModelTest v.2.1.10 (Darriba et al. 2012). The analyses were performed using the MrBayes v3.2.7a software (Ronquist et al. 2012) with the nucleotide substitution model HKY + I for COI dataset and F81 for the concatenated nuclear fragments as suggested by jModelTest v2.1.10. Two independent MCMC runs of 10 million generations, with four simultaneous chains (one cold and three heated) for each analysis, were performed. The sampling of trees and parameters were set to every 1000 generations. TRACER, v1.7.1 was used for summarising the results of the Bayesian phylogenetic analysis (http://beast.bio.ed.ac.uk/Tracer). The first 10% of trees were discarded as burn-in prior to computing a consensus phylogeny and posterior probabilities. The consensus of the obtained trees was visualised using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). Genetic distances among COIbarcodes were calculated using MEGA v.7.0.14 (Kumar et al. 2016).

Figure 1.Putative hybrid specimen (center) and its putative parental species, female of C. christophi helialaica (left) and male of Colias cocandica cocandica (right) [Kyrgyzstan, Alay Range, Tengiz-Bay Pass, h = 3680 m, 39°40'17.2" N; 72°08'06.9" E, 08–10.VII.2022, A. Baryshev leg.].

Taxon Sample ID GenBank/BOLD Accession Number Locality
C. cocandica × C. christophi BR01 OR364041 OR539248 OR539249 OR539259 OR539260 OR539271 OR539272 Kyrgyzstan*
C. christophi BR02 OR364042 OR539252 OR539264 OR539276 Kyrgyzstan*
C. christophi BR03 OR364043 OR539253 OR539265 OR539277 Kyrgyzstan*
C. christophi BR04 OR364044 OR539254 OR539266 OR539278 Kyrgyzstan*
C. christophi BR05 OR364045 OR539255 OR539267 OR539279 Kyrgyzstan*
C. christophi BR06 OR364046 OR539256 OR539268 OR539280 Kyrgyzstan*
C. christophi SACOL191-18 Kyrgyzstan*
C. cocandica BR08 OR364047 OR539250 OR539261 OR539273 Kyrgyzstan*
C. cocandica BR09 OR364048 OR539251 OR539262 OR539274 Kyrgyzstan*
C. cocandica BRX10 OR364049 OR539257 OR539263 OR539275 Kyrgyzstan*
C. croceus CL151 OR178497 OR539252 OR539269 OR539270 Russia, Kaliningrad**
Table 1.List of studied specimens.* – Kyrgyzstan, Alay Range, Tengiz-Bay Pass, h = 3680 m, 39°40'17.2'' N; 72°08'06.9'' E, 08–10. VII.2022, A. Baryshev leg.; ** – Russia, Kaliningrad Oblast, Zelenogradsky District, Courish Spit, “Fringilla” field station 55°05'17" N; 20°44'04" E, 28.VIII.2016, A. Shapoval leg.


The phylogenetic analysis based on the mitochondrial COI marker recovered C. christophi helialaica and C. cocandica cocanidca as two strongly supported clades (Fig. 2) separated by at least 17 fixed nucleotide substitutions (mean uncorrected p-distance is 2.66%). The analysed specimens of C. cocandica cocandica formed a single haplotype, while C. christophi helialaica specimens constituted two haplotypes, differing in one nucleotide substitution (A => G) in the position 508. The putative hybrid specimen shared the mitochondrial DNA barcode with C. christophi helialaica (specimens SACOL191-18, BR02 (OR364042), BR03 (OR364043), BR05 (OR364044)).

With respect to the nuclear Ca-ATPase and H3 genes, all the studied specimens displayed no intra-individual polymorphism (usually represented by dual peaks of similar height in the electropherograms), and no fixed differences between C. christophi helialaica and C. cocandica cocandica were found. The analysis of the nuclear CAD gene fragments revealed a relatively low level of intra-individual heterozygosity for the species analysed. Aside from heterozygous sites, the CAD sequences possessed several fixed nucleotide substitutions separating C. christophi helialaica and C. cocandica cocandica (namely, T=>A in the position 298, A=>T in the position 579 and T=>C in the position 730). The CAD fragments of the putative hybrid specimen were heterozygous in these positions, bearing nucleotides of both C. christophi helialaica and C. cocandica cocandica (Fig. 3).

Figure 2.The Bayesian consensus trees of the analysed specimens of C. christophi helialaica, C. cocandica cocandica and the putative hybrid, inferred from COI sequences (left) and concatenated alignment of three nuclear markers (Ca-ATPase, H3, CAD) (right). Numbers at nodes indicate Bayesian posterior probabilities. Scale bar = 0.06 and 0.0006 substitutions per position for mtDNA tree and nucDNA tree, respectively.

Figure 3.Electropherograms of two CAD nuclear gene fragments of parental species (a,b) and putative hybrid (c). Fixed interspecific nucleotide differences between C. christophi helialaica and C. cocandica cocandica are boxed. Arrows indicate mixed signals in putative hybrid.

To elucidate the visible heterogeneities, CAD fragments of the putative hybrid specimen were cloned and 20 clones per fragment were sequenced. Since the all cloned sequences formed two distinct variants, the identical clones were merged and included in the concatenated molecular matrix as two independent samples, “clone group #1” and “clone group #2”. Bayesian analysis based on the concatenated alignment of the nuclear markers recovered two highly supported clades, corresponding to C. christophi helialaica and C. cocandica cocandica. “Clone group #1” and “clone group #2” clustered within the C. cocandica cocandica and C. christophi helialaica clades, respectively, confirmed our suggestion that the heterozygosity of the studied specimens revealed by the direct sequencing of the nuclear genes is a result of shared combination of the both lineages, C. christophi helialaica and C. cocandica cocandica.


The hybrid specimen was found flying together with other Colias species, namely C. eogene C. Felder & R. Felder, 1865, C. alpherakii Staudinger, 1882, C. christophi helialaica and C. cocandica cocandica. Externally, the hybrid specimen resembles the two distantly related taxa, C. cocandica cocandica and C. christophi helialaica, combining the morphological characters of both species. Greenish colouration of the dorsal side of the wings and the hindwing with angled outer margin are the characters of C. cocandica cocandica; well-developed white submarginal band and rounded outer margin of the hindwing are the characters of C.christophi helialaica. Interestingly, the left and right sides of the hybrid specimen significantly differ in size and wing shape (Fig. 4). The left forewing and the hindwing to some extent resemble those of C. cocandica cocandica (smaller size and angled outer margin of the hindwing), while the right forewing and the hindwing similar to those of C. christophi helialaica (larger size and rounded outer margins).

Figure 4.Wireframe representation of the forewings and hindwings of the Colias cocandica cocandica x C. christophi helialaica hybrid (a). Left side (black) and mirrored right side (red), showing the differences in wing shape and wing size (b).

Ongoing interspecific hybridisation has been suggested for several Colias taxa. One of the examples is North American sister taxa C. eurytheme Boisduval, 1852 and C. eriphyle Edwards, 1876. The latter taxon has often been considered as a subspecies of C. philodice Godart, 1819, a close relative of C. eurytheme widely distributed throughout the eastern USA. The phylogenetic relationships among C. eurytheme, C.eriphyle and C. philodice remain poorly understood, and the taxonomic status of these taxa are a matter of debates (Layberry et al. 1998; Wheat and Whatt 2008). Notwithstanding, regularly occurring individuals of an intermediate phenotype observed in the areas of sympatry of C. eurytheme and C. eriphyle have been regarded in the literature as hybrids of the two taxa, although no molecular analysis confirming hybrid origin of such specimens has been performed (Gerould 1946; Taylor 1972; Jahner et al. 2012). Recent molecular studies confirmed continuous gene flow and introgression between C. eurytheme and C. eriphyle, however, the specimens of the intermediate phenotype were found to be genetically indistinguishable from C. eriphyle, being a “colour morphs” of C. eriphyle, not genetic hybrids (Dwyer 2015).

Another pair of taxa, C. croceus C. erate (Esper, 1805), have been postulated to occasionally hybridise (Dinca et al. 2011; Dzurinka et al. 2021, 2022), given that numerous specimens with intermediate morphological features (e.g., with yellow-orange wing colouration) occur in nature. However, the lack of reliable morphological diagnostic characters and wide colour variation coupled with mitochondrial barcodes and nuclear DNA sharing among these two taxa (Dzurinka et al. 2022) make DNA-based and morphology-based identifications of their putative hybrids unreliable.

Finally, hybridisation has been asserted, without evidence, as a cause for poorly understood and unclear morphological variations in other Colias species (Hovanitz 1963). Thus, we can conclude that many cases of hybridisation and identification of the reported phenotypic intermediates as true hybrids are not firmly supported. Such conclusions require confirmation by a comprehensive analysis, involving both detailed morphological analysis and targeted gene/genome sequences.

In this study, we show that the analysis of the mitochondrial and nuclear DNA genes coupled with the analysis of the external morphology can successfully discriminate natural hybrids in such a taxonomically complicated group of butterflies as the genus Colias. We analyse a Colias specimen with unusual wing pattern, which was provisionally identified as a natural hybrid. Molecular analysis has shown that this specimen bears the mitochondrial haplotype of C. christophi helialaica, while the nuclear fragments are heterozygous, sharing a combination of C. cocandica cocandica and C. christophi helialaica lineages. The molecular results are confirmed by the intermediate morphological characters, clearly seen in the hybrid individual: it combine both the traits of C. cocandica cocandica and C. christophi helialaica. Our findings empirically demonstrate the possibility of genetic introgression between distantly related and morphologically very different species of the genus Colias.


The authors are grateful to Alexander Baryshev (Penza, Russia) for providing material for DNA analysis. The study was supported by the grant of Russian Science Foundation grant No. 22-24-01086 (molecular analysis) and by the state research project No. 122031100272-3 (morphological analysis).


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