DNA Barcodes Reveal Two Distinct Butterflies Are Male & Female Of Same Species

DNA barcodes reveal that a recently-described butterfly species is actually the female of another species known to science for more than a century

by GrrlScientist for Forbes | @GrrlScientist

Male Sunburst Cerulean-Satyr butterfly (Caeruleuptychia helios; Weymer, 1911) attracted to bait of fermented fish and urine. Villa Carmen Biological Station, Pilcopata, Cuzco, Peru, April 2016.
(Credit: Andrew Neild /
Florida Museum of Natural History.)

Using DNA barcodes, an international team of scientists recently discovered that a distinct butterfly species is actually the female of a different species known to science for more than a century. This species represents a case of extreme sexual dimorphism, a trait that is unusual amongst butterflies. Additionally, this study suggests that DNA barcodes can be an effective tool for identifying butterfly species.

Although taxonomy is an exacting science, it certainly is not an exact science, thanks to a variety of evolutionary challenges. One case in point: sexual dimorphism. This is a situation where males and females of the same species look different from each other. These differences can extend to all sorts of visible traits, from differences in color or color patterns to variations in physical structure or size. These differences arise due to a variety of influences, one of which is female mate choice (more here and here) and which can be accentuated by differing selection pressures on the sexes (more here). Sometimes, these sex-based differences are so dramatic that males and females end up being classified as different species (one example).

How can these taxonomic challenges be resolved?

This is where DNA barcoding enters the picture. Initially developed in 2003 by Paul Hebert at the University of Guelph, this molecular innovation is becoming more widely accepted as the primary tool for identifying and distinguishing species, although its applicability remains controversial for poorly understood and sparsely sampled groups, such as butterflies (ref and ref).

The principle of DNA barcoding is simple enough: it relies on sequencing and analyzing a short and highly variable stretch of DNA from a particular mitochondrial gene. Sequences in this stretch of DNA feature specific changes that are probably unique to each taxon, so analysis of this region can be used to identify species (ref). However, the effectiveness of this technique is dependent upon adequate sampling of closely-related species.

Over the years, DNA barcoding has proven to be a rapid and highly useful method for identifying species from a wide variety of animal groups, and several public reference libraries of DNA barcodes have been set up so unknown specimens can be assigned to known species.

The euptychiine butterflies (Euptychiina) comprise a diverse group of mostly brown tropical butterflies from the New World (the Neotropics). These butterflies are so poorly known and so difficult to classify that more than one butterfly expert has simply thrown his hands up in the air and collectively referred to them all as “Euptychia”. For this reason, the taxonomy of these little brown butterflies is — to put it bluntly — “still chaotic” (ref).

But this taxonomic turmoil means that these enigmatic butterflies represent a longstanding mystery in need of resolution; that they can serve as an opportunity to add sequence data to DNA barcode libraries and to test the applicability of DNA barcodes for classifying these butterflies.

To begin clearing up the taxonomic confusion surrounding euptychiine species is a challenge that inspired and motivated butterfly researcher Shinichi Nakahara, a research associate at the Florida Museum of Natural History. He organised and led an expedition of international scientists to South America to collect specimens for research.

Dr. Nakahara and his research team recently reported their findings for seven members in one of the larger groups of euptychiine butterflies, the genus Caeruleuptychia. This genus currently contains 15 species — although it is suspected to include more. This genus is exceptional amongst the euptychiine butterflies because a number of Caeruleuptychiaspecies have brilliant blue wings, which certainly sets them apart from their lackluster brethren.

A researcher walks down Trail 2 at Villa Carmen Biological Station, Pilcopata, Cuzco, Peru. This trail features secondary regrowth habitat where numerous species of Caeruleuptychia butterflies have been recorded. (doi:10.1163/1876312X-00002167)

Caeruleuptychia butterflies occur only in South America, entirely east of the Andes. Their ranges vary in elevation from sea level up to about 1600 metres (5250 feet), and they reach their highest diversity in low-elevation tropical rainforests of the western Amazon. Even though our knowledge of these colorful butterflies’ habits is hazy, previous work indicates that they tend to either fly at dusk or lurk in the shade, where their iridescent blue wings gleam and flash conspicuously in the shadows, attracting the attention of butterfly collectors. Perhaps this is why butterfly taxonomists are relatively familiar with the males of Caeruleuptychia species, whereas the females remained unknown.

The team described the morphology and analyzed DNA barcodes obtained from their newly-collected butterfly specimens, and from older specimens maintained in major collections throughout the Americas and Europe.

Their most unexpected discovery was a female brown-winged butterfly, Magneuptychia keltoumae, which was so rare and distinct that it had only been described in 2012, had an identical DNA barcode to the brilliant blue-winged sunburst cerulean-satyr butterfly, which had been described in 1911 (figure 1).

Figure 1: Sunburst cerulean-satyr butterfly (Caeruleuptychia helios). Male (top row: upperside and underside of the wings) and female (bottom row: upperside and underside of the wings). (Shinichi Nakahara et al., 2017, doi:10.1163/1876312X-00002167)

A combination of morphology and DNA barcode data also identified a new species, dubbed Trembath’s cerulean-satyr, Caeruleuptychia trembathi. This species, which is found in high elevation cloud forests in eastern Ecuador, is also dramatically sexually dimorphic, with blue-winged males and brown-winged females (figure 2).

Figure 2. Trembath’s cerulean-satyr butterfly (Caeruleuptychia trembathi). Male (top row; upper and underside of the wings) and female (bottom row; upper and underside of the wings). (Shinichi Nakahara et al., 2017, doi:10.1163/1876312X-00002167)

The researchers were quite surprised by these findings.

“None of us thought about this possibility before, and we were all surprised by this outcome of our DNA analysis,” Dr. Nakahara said. “Given that males and females of most euptychiine butterflies look more or less the same, I guess no one thought that the female would be so different compared to the male.”

This study not only increases the known number of sexually dimorphic butterfly species, but it also establishes Caeruleuptychia as one of the most sexually dimorphic genera of butterflies yet identified, although not all species placed in this genus are sexually dimorphic.

“Although many have blue wings, there are some Caeruleuptychia species that have brownish wings (typical of Euptychiina),” Dr. Nakamura elaborated in email. “[B]ut they may be classified as a different genus in the future.”

This research also raises some interesting questions: although only seven Caeruleuptychia species were included in this study, might future investigations discover that all Caeruleuptychia species are sexually dimorphic? Why do males and females look so different? What are the roles of wing color patterns in signalling between males and females of these species?

“Our taxonomic study of Caeruleuptychia represent just the proverbial tip of the iceberg, with the ongoing study of the remaining species of the genus (by myself and collaborators) suggesting that our understanding of the species diversity of this genus is far from complete,” Dr Nakamura said in email.

Since the team has obtained diagnostic DNA barcodes for more than 300 euptychiine species, it would appear there will be plenty of new discoveries to look forward to.

“[I]n other words, more work is needed to further understand these elements of Neotropical biodiversity.”

Shinichi Nakahara, Thamara Zacca, Blanca Huertas, Andrew F. E. Neild, Jason P. W. Hall, Gerardo Lamas, Lauren A. Holian, Marianne Espeland, and Keith R. Willmott (2017). Remarkable sexual dimorphism, rarity and cryptic species: a revision of the ‘aegrota species group’ of the Neotropical butterfly genus CaeruleuptychiaForster, 1964 with the description of three new species (Lepidoptera, Nymphalidae, Satyrinae), Insect Systematics & Evolution, published online on 27 July 2017 ahead of print | doi:10.1163/1876312X-00002167

Also mentioned:

Paul D. N. Hebert, Alina Cywinska, Shelley L. Ball, and Jeremy R. deWaard (2003). Biological identifications through DNA barcodes, Proceedings of the Royal Society of London B: Biological Sciences 270:313–321 | doi:10.1098/rspb.2002.2218

Martin Wiemers and Konrad Fiedler (2007). Does the DNA barcoding gap exist? — a case study in blue butterflies (Lepidoptera: Lycaenidae), Frontiers in Zoology 4(1):8–24 | doi:10.1186/1742–9994–4–8

Christopher P Meyer and Gustav Paulay (2005). DNA Barcoding: Error Rates Based on Comprehensive Sampling, PLoS Biology 3(12):e422 | doi:10.1371/journal.pbio.0030422

Read more butterfly science:

GrrlScientist. “Fragmented Habitats Accelerate Butterfly Evolution — And Extinction”, Forbes, 25 February 2016. (Medium link.)

GrrlScientist. “Birth of the blue morphos”, The Guardian, 13 April 2015. (Medium link.)

GrrlScientist. “Why does this butterfly hiss?”, The Guardian, 3 February 2014. (Medium link.)

GrrlScientist. “Watch: A century of butterflies and moths”, The Guardian, 14 August 2012. (Medium link.)

GrrlScientist. “Watch: Weird and wonderful butterflies in the Cal Academy Collection”, The Guardian, 26 April 2012. (Medium link.)

GrrlScientist. “Watch: How to fix a broken butterfly wing”, The Guardian, 30 November 2011. (Medium link.)

GrrlScientist. “Watch: Supergene controls butterfly mimicry”, The Guardian, 18 August 2011. (Medium link.)

GrrlScientist. “The hot and cold of butterfly dancing”, The Guardian, 1 February 2011. (Medium link.)

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Originally published at Forbes on 4 August 2017.

PhD evolutionary ecology/ornithology. Psittacophile. scicomm Forbes, previously Guardian. always Ravenclaw. discarded scientist & writer, now an angry house elf