Short Communication
Short Communication
Mitochondrial chromosome as a marker of animal migratory routes: DNA barcoding revealed Asian (non-African) origin of a tropical migrant butterfly Junonia orithya in south Israel
expand article infoVladimir A. Lukhtanov, Elena A. Pazhenkova, Asya V. Novikova§
‡ Zoological Institute of Russian Academy of Science and St. Petersburg State University, St. Petersburg, Russia
§ Hebrew University of Jerusalem, Jerusalem, Israel
Open Access


The blue pansy Junonia orithya Linnaeus, 1758 (Lepidoptera, Nymphalidae) is widely distributed along the tropical areas of Africa, Asia and Australia. It is also known as a migrant species in the Levant. Here we record J. orithya in south Israel and provide a DNA-barcode-based evidence for its Asian (non-African) origin.


barcode libraries, COI , Iran, Jordan, migration, Nymphalidae


Despite its small size, the mitochondrial chromosome is a functionally important portion of the eukaryotic DNA (Taanman 1999). It is also an extremely useful genetic marker broadly used in genetic, phylogenetic, phylogeographic, biogeographic and taxonomic studies (Avise 2004, Talavera and Vila 2011, Lukhtanov et al. 2016). A relatively fast mutation rate and rapid sorting of mtDNA gene lineages, as well as absence (or at least rarity) of recombination usually result in high divergence of mitochondrial genomes among species and a comparatively small variance within species. For this reason, mtDNA-based species identification (so called DNA barcoding) has become a popular tool for identifying unknown specimens in terms of pre-existing classifications (Hebert et al. 2003, Chambers and Hebert 2016). This prompted DNA barcode databases ( and DNA reference barcode libraries (e.g. Dincă et al. 2011, 2015, Wilson et al. 2013).

Here we demonstrate how DNA barcode libraries result in an opportunity to study migration routes. Migration is a common phenomenon in animals (Dingle 2014, Chapman et al. 2015), but it is poorly studied in some groups, especially in insects. Within butterflies, with the exception of the relatively well studied monarch Danaus plexippus Linnaeus, 1758 (Brower 1995, Oberhauser et al. 2015) and the painted lady Vanessa cardui Linnaeus, 1758 (Talavera and Vila 2016), little is known about other species’ migratory routes.

The blue pansy Junonia orithya Linnaeus, 1758 belongs to a group of butterflies able to perform long-range migrations (Larsen 1984, Benyamini 2010). This species is widely distributed within the tropical areas of the Old World: in sub-Saharan Africa, Arabia, South and South-East Asia and Australia. It is known to be adapted to tropical environments and, according to Larsen (1984), could not normally survive winter even in the hottest spots of the Palaearctic region, such as the Jordan Valley in the Middle East. In south Iraq and south Iran, butterflies can be regularly observed in most months of the year, but in the central and northern parts of these countries they are less regular (Wiltshire 1957, Tshikolovets et al. 2014). Migrant individuals occur in Jordan (Larsen 1984, Katbeh-Bader et al. 2003, Benyamini 2010) and have been recently recorded in East Turkey (Biricik 2011). In Israel, records of J. orithya are known from the Jordan River valley in the north-east (Benyamini 2010). This distributional pattern fits well the hypothesis that specimens from Israel (as well as from the entire Middle East) might be connected to South Asia (and not to Africa, despite its geographical proximity), moving from south-east to north-west and finally reaching the most eastern parts of Israel.

On April 28, 2016 we collected a male of J. orithya in the southern tip of Israel in a small cultivated green patch near kibbutz Neot Smadar (30°02'40"N, 35°01'01"E, 409 m above sea level), enclosed by the Negev desert. Israel forms a biogeographic land bridge between Asia, Africa and Europe (Chipman et al. 2013); therefore the discovery of the blue pansy in the Negev, close to the African continent, raises a possibility of an African origin of J. orithya in Israel.

The “African” hypothesis seems to be plausible because animals and plants of African origin comprise a large group in the ecosystems of Israel, especially in the south where African elements are predominant (Goren and Ortal 1999). For example, butterfly fauna of south Israel includes such species of African origin as Papilio saharae Oberthür, 1879, Euchloe aegyptiaca Verity, 1911, Euchloe falloui Allard, 1867, Epamera claucus Butler, 1885, Anthene amarah (Guérin, 1849), Azanus ubaldus (Cramer, 1782), Pseudophilotes abencerragus (Pierret, 1837) and Gomalia elma (Trimen, 1862) (Benyamini 2010).

Here we tested the Asian versus African hypotheses by analyzing the barcode COI region of the mitochondrial genome. First we inspected all COI barcodes available from GenBank ( and BOLD ( and revealed that J. orithya comprised two genetically differentiated clusters of individuals. One cluster was represented by butterflies from Africa (Kenya, South Africa, Zimbabwe), and another cluster was represented by butterflies from Asia and Australia. Despite huge territory occupied by each of these clusters and despite the substantial divergence between the clusters, each group was found to be very homogenous with respect to the COI gene.

Then we obtained DNA barcodes for the sample from Neot Smadar, Israel (BPALB098-16, GenBank accession number: KY118822) and in three additional samples from Iran (from the collection of the Zoological Institute, St. Petersburg: BPAL2941-15 KY118824, BPAL2942-15 KY118825, BPAL2943-15 KY118823). These samples were processed as previously described (Lukhtanov et al. 2014, 2015). We used Bayesian Inference (MrBayes 3.1.2) as described previously (Vershinina and Lukhtanov 2010) to reconstruct a phylogenetic tree (Fig. 1). We used the published sequences of J. neildi Brévignon, 2004 (Gemmell et al. 2014) to root the tree (Fig. 1). Uncorrected p-distances were calculated manually based on direct comparison of sequences. In addition to our own sequences, only published data (one sample from Zimbabwe, Africa, one sample from Australia and 31 samples from Asia) (Kodandaramaiah and Wahlberg 2007, Zhang et al. 2008, Gaikwad et al. 2012, Ashfaq et al. 2013, Wilson et al. 2013, Hebert et al. 2013, Srirama et al. 2014, Yamada et al. 2014) were used for the phylogenetic inference (Fig. 1) and p-distance comparison.

The analysis revealed that the collected sample from south Israel was identical with eight samples from South Asia: with three samples from Malaysia (KF226505.1, KF226504.1, KF226503.1) and with five samples from Pakistan (GU681856.1, HQ990373.1, HQ990374.1, HQ990375.1, KC755868.1) (p-distance = 0%). It was also very close to other samples from Oriental and Australian regions as well as to the samples from Iran (Fig. 1) (p-distance from 0.2 to 1.3%). At the same time it differed by 19 nucleotide substitutions from the Zimbabwean sample (p-distance = 2.9%).

Thus, the genetic connectivity identified in our study supports the hypothesis that J. orithya may colonize Israel and Iran from tropical regions in Asia. Given that the species seem not to overwinter in the north of the Middle East, its sporadic presence suggests that these are immigrants, but precise origins of the specimens studied here could not be traced because of lack of phylogeographic structure within the analysed Asian populations. Since the species is common in certain areas in Arabia (Wiltshire 1957), that might be one of the most likely origin for these specimens. An African origin is not supported for these specimens and whether the Asian/Arabian origins are annually recurrent should be studied in detail with temporal series of immigrants. A wider African sampling, particularly from the northern hemisphere and especially from the nearest areas to Israel is also required for further testing the African hypothesis.

Figure 1.

Bayesian tree of Junonia orithya samples based on analysis of the cytochrome oxidase subunit I (COI) gene (barcode region, 658 bp). Numbers at nodes indicate Bayesian posterior probability.


We thank Gerard Talavera for critical comments. The financial support for this study was provided by the grant N 14-14-00541 from the Russian Science Foundation to the Zoological Institute of the Russian Academy of Sciences.


  • Ashfaq M, Akhtar S, Khan AM, Adamowicz SJ, Hebert PDN (2013) DNA barcode analysis of butterfly species from Pakistan points towards regional endemism. Molecular Ecology Resources 13: 832–843. doi: 10.1111/1755-0998.12131
  • Avise JC (2004) Molecular Markers, Natural History and Evolution. Sinauer Associates, Sunderland, 541 pp.
  • Benyamini D (2010) A Field Guide to the Butterflies of Israel. Keter Publishing House, Jerusalem, 248 pp.
  • Biricik M (2011) First record of Junonia orithya (Linnaeus, 1758) (Lepidoptera: Nymphalidae) in Turkey. Zoology in the Middle East 53(1): 130–132. doi: 10.1080/09397140.2011.10648871
  • Brower LP (1995) Understanding and misunderstanding the migration of the monarch butterfly (Nymphalidae) in North America: 1857–1995. Journal of the Lepidopterists’ Society 49: 304–385.
  • Chambers EA, Hebert PDN (2016) Assessing DNA barcodes for species identification in North American reptiles and amphibians in natural history collections. PLoS ONE 11(4): e0154363. doi: 10.1371/journal.pone.0154363
  • Chapman JW, Reynolds DR, Wilson K (2015) Long-range seasonal migration in insects: mechanisms, evolutionary drivers and ecological consequences. Ecology Letters 18: 287–302. doi: 10.1111/ele.12407
  • Chipman AD, Dor N, Bonato L (2013) Diversity and biogeography of Israeli geophilomorph centipedes (Chilopoda: Geophilomorpha). Zootaxa 3652(2): 232–248. doi: 10.11646/zootaxa.3652.2.2
  • Dincă V, Zakharov EV, Hebert PDN, Vila R (2011) Complete DNA barcode reference library for a country’s butterfly fauna reveals high performance for temperate Europe. Proceedings of the Royal Society B: Biological Sciences 278: 347–355. doi: 10.1098/rspb.2010.1089
  • Dincă V, Montagud S, Talavera G, Hernández-Roldán H, Munguira ML, García-Barros E, Hebert PDN, Vila R (2015) DNA barcode reference library for Iberian butterflies enables a continental-scale preview of potential cryptic diversity. Scientific Reports 5: 12395. doi: 10.1038/srep12395
  • Gaikwad SS, Ghate HV, Ghaskadbi SS, Patole MS, Shouche YS (2012) DNA barcoding of nymphalid butterflies (Nymphalidae: Lepidoptera) from Western Ghats of India. Molecular Biology Reports 39(3): 2375–2383. doi: 10.1007/s11033-011-0988-7
  • Gemmell AP, Borchers TE, Marcus JM (2014) Molecular population structure of Junonia butterflies from French Guiana, Guadeloupe, and Martinique. Psyche 2014: 897596. doi: 10.1155/2014/897596
  • Goren M, Ortal R (1999) Biogeography, diversity and conservation of the inland water fish communities in Israel. Biological Conservation 89(1): 1–9. doi: 10.1016/S0006-3207(98)00127-X
  • Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences 270: 313–321. doi: 10.1098/rspb.2002.2218
  • Hebert PDN, deWaard JR, Zakharov EV, Prosser SWJ, Sones JE, McKeown JTA, Mantle B, La Salle J (2013) A DNA ‘barcode blitz’: rapid digitization and sequencing of a natural history collection. PLoS ONE 8(7): e68535. doi: 10.1371/journal.pone.0068535
  • Katbeh-Bader A, Amr ZS, Isma’el S (2003) The butterflies of Jordan. Journal of Research on the Lepidoptera 37: 11–26.
  • Kodandaramaiah U, Wahlberg N (2007) Out-of-Africa origin and dispersal-mediated diversification of the butterfly genus Junonia (Nymphalidae: Nymphalinae). Journal of Evolutionary Biology 20(6): 2181–2191. doi: 10.1111/j.1420-9101.2007.01425.x
  • Larsen T (1984) A tropical migrant butterfly new to the eastern Mediterranean Junonia orithya here LANG (Lepidoptera, Nymphalidae). Atalanta 15(1/2): 101–102.
  • Lukhtanov VA, Shapoval NA, Dantchenko AV (2014) Taxonomic position of several enigmatic Polyommatus (Agrodiaetus) species (Lepidoptera, Lycaenidae) from Central and Eastern Iran: insights from molecular and chromosomal data. Comparative Cytogenetics 8(4): 313–322. doi: 10.3897/CompCytogen.v8i4.8939
  • Lukhtanov VA, Sourakov A, Zakharov EV (2016) DNA barcodes as a tool in biodiversity research: testing pre-existing taxonomic hypotheses in Delphic Apollo butterflies (Lepidoptera, Papilionidae). Systematics and Biodiversity 14(6): 599–613. doi: 10.1080/14772000.2016.1203371
  • Lukhtanov VA, Dantchenko AV, Vishnevskaya MS, Saifitdinova AF (2015) Detecting cryptic species in sympatry and allopatry: analysis of hidden diversity in Polyommatus (Agrodiaetus) butterflies (Lepidoptera: Lycaenidae). Biological Journal of the Linnean Society 116(2): 468‒485. doi: 10.1111/bij.12596
  • Oberhauser KS, Nail KR, Altizer S (Eds) (2015) Monarchs in a Changing World: Biology and Conservation of an Iconic Butterfly. Cornell University Press, 321 pp.
  • Srirama R, Gurumurthy BR, Senthilkumar U, Ravikanth G, Shaanker RU, Shivanna MB (2014) Are mini DNA-barcodes sufficiently informative to resolve species identities? An in silico analysis using Phyllanthus. Journal of Genetics 93(3): 823–829. doi: 10.1007/s12041-014-0432-6
  • Taanman J-W (1999) The mitochondrial genome: structure, transcription, translation and replication. Biochimica et Biophysica Acta 1410: 103–123. doi: 10.1016/S0005-2728(98)00161-3
  • Talavera G, Vila R (2011) What is the phylogenetic signal limit from mitogenomes? The reconciliation between mitochondrial and nuclear data in the Insecta class phylogeny. BMC Evolutionary Biology 11: 315. doi: 10.1186/1471-2148-11-315
  • Talavera G, Vila R (2016) Discovery of mass migration and breeding of the painted lady butterfly Vanessa cardui in the Sub-Sahara: the Europe–Africa migration revisited. Biological Journal of the Linnean Society. doi: 10.1111/bij.12873
  • Tshikolovets V, Naderi A, Eckweiler W (2014) The Butterflies of Iran and Iraq. Tshikolovets Publications, 366 pp.
  • Vershinina AO, Lukhtanov VA (2010) Geographical distribution of the cryptic species Agrodiaetus alcestis alcestis, A. alcestis karacetinae and A. demavendi (Lepidoptera, Lycaenidae) revealed by cytogenetic analysis. Comparative Cytogenetics 4(1): 1‒11. doi: 10.3897/compcytogen.v4i1.21
  • Wilson JJ, Sing KW, Sofian-Azirun M (2013) Building a DNA barcode reference library for the true butterflies (Lepidoptera) of Peninsula Malaysia: What about the subspecies? PLoS ONE 8(11): e79969. doi: 10.1371/journal.pone.0079969
  • Wiltshire EP (1957) The Lepidoptera of Iraq. Revised Edition. Nicholas Kaye, Bagdad, 162 pp.
  • Yamada K, Kawanishi Y, Yamada A, Tokuda G, Gurung RD, Sasaki T, Nakajima Y, Maekawa H (2014) A novel cluster of mariner-like elements belonging to mellifera subfamily from spiders and insects: implications of recent horizontal transfer on the South-West Islands of Japan. Genetica 142(2): 149–160. doi: 10.1007/s10709-014-9762-9
  • Zhang M, Zhong Y, Cao T, Geng Y, Zhang Y, Jin K, Ren Z, Zhang R, Guo Y, Ma E (2008) Phylogenetic relationship and morphological evolution in the subfamily Limenitidinae (Lepidoptera: Nymphalidae). Progress in Natural Science 18(11): 1357–1364. doi: 10.1016/j.pnsc.2008.03.025
login to comment