Excitement as DNA barcoding technique leads to unmasking of new species tempered with fear that some are already at risk of extinction

• Counting the species: how DNA barcoding is rewriting the book of life
• Read more in our series Biodiversity: what happened next?

A growing number of “cryptic species” hiding in plain sight have been unmasked in the past year, driven in part by the rise of DNA barcoding, a technique that can identify and differentiate between animal and plant species using their genetic divergence.

The discovery of new species of aloe, African leaf-nosed bats and chameleons that appear similar to the human eye but are in fact many and separate have thrilled and worried conservationists. Scientists say our planet might be more biologically diverse than previously thought, and estimates for the total number of species could be far higher than the current best guess of 8.7 million. But cryptic discoveries often mean that species once considered common and widespread are actually several, some of which may be endangered and require immediate protection.

The Jonah’s mouse lemur was only unveiled to the world this summer but is already on the verge of extinction. The newly described Popa langur in Myanmar, previously confused with another species, numbers around 200 and is likely to be classified as critically endangered, threatened by habitat loss and deforestation.

The Jonah’s mouse lemur is on the verge of extinction, despite the fact that its existence was only announced this summer. Photograph: Marina Blanco/Handout

The discovery of these cryptic species has been driven in part by the rise of DNA barcoding, a technique that can identify and differentiate between animal and plant species using their genetic divergence. African elephants, Indian vine snakes and South American neotropical birds are among the growing number of unmaskings. Thousands more are expected in the coming years, from living creatures and museum samples.

“DNA barcoding is a tool that allows us to detect differences among species at a finer scale than before, like a microscope allows us to see fine details of surface structure that are invisible to the naked eye,” says Brian Brown, entomology curator at the LA Natural History Museum, who is using the technique for research on flies. “It gives us a way to delimit some of the previously suspected, but unexplored, diversity within what we call species. It is showing that the world is even more wonderfully biodiverse than we suspected.”

The first cryptic species discoveries made using DNA barcoding were in Guanacaste conservation area (ACG) in northwestern Costa Rica, now the most DNA barcoded place on Earth. In a paper entitled Ten Species in One, Canadian professor Paul Hebert, known as the “father of DNA barcoding”, revealed the true identities of the two-barred flasher butterfly in 2004, along with University of Pennsylvania professors Daniel Janzen and Winnie Hallwachs, who have dedicated their lives to the ACG.

It was an insect that had bothered Janzen for decades. Taxonomic consensus told the 81-year-old evolutionary ecologist that the caterpillar samples he collected in the ACG were those of a common, unremarkable tropical butterfly found from Texas to northern Argentina. But he did not believe it.

Janzen had always been puzzled by the diversity of two-barred flasher caterpillars – Astraptes fulgerator – and the variety of plants on which they feasted. So when in 2004 he had the opportunity to test a controversial new technique called DNA barcoding put forward by Hebert (then primarily known for his expertise on water fleas), he knew which insect samples he would send.

The diversity of two-barred flasher caterpillars was a clue to the eventual discovery that they were at least ten distinct species. Photograph: PNAS

The results were thrilling. In his study area alone, the barcoding analysis indicated the two-barred flasher butterfly was, in fact, at least 10 genetically distinct species. The revelation of the butterfly as a cryptic species could mean across the rest of Latin America there are thousands of unidentified insect species waiting to be described – along with many that have never been collected and examined.

The findings were highly controversial and provoked a backlash from taxonomists and biologists who questioned whether genetic information should be included in identifying a species. Others did not agree that a binary, genetic threshold should be imposed on the continual process of evolution. For centuries, humanity’s understanding of life on Earth was based on the physical form. Every organism in the library of life fits within a hierarchy of classifications based on appearance, according to the modern taxonomic system first developed by Swedish botanist Carl Linnaeus.

“God created, Linnaeus organised,” he immodestly told people.

Today, the technique is commonly used alongside traditional Linnaean-based methods, quickly separating samples before further genetic and morphological analysis. Among the sceptics nearly 20 years ago was Brown, who is now responsible for a large unmasking: species once lumped together as Megaselia sulphurizona, a type of humpback fly, also collected in the ACG.

DNA barcoding analysis of samples across Latin America revealed 16 separate species, according to his unpublished research with co-authors.

“I thought I could tell my species perfectly well by looking at genitalia,” says Brown, referring to the common practice of identifying insects by studying their reproductive organs. “I didn’t really care if I was going as quickly as possible. But when I started working on this group of small flies, I realised what I was calling one species was actually 16 and that I wasn’t able to identify them morphologically like I thought.”

I see my flies and there are maybe 100,000 … maybe a million undescribed species of them in the world. We don't know - Brian Brown

Another convert, Michael Sharkey, an entomologist and emeritus professor at the University of Kentucky, DNA barcoded the insects he had classified for his PhD, only to realise that most of the species concepts he had proposed after three years of hard work were incorrect.

“It would have been much better if I had never published. I am happy to have had that experience though; it has taught me that despite best efforts morphological evidence is not sufficient. Barcodes will have their drawbacks as well, but they are a vast improvement,” he wrote of the experience.

A display of beetles at the Montreal Insectarium, Quebec, Canada. Photograph: Kenneth Taylor/Alamy

Either way, the direction of travel is clear. “We’re not going to be looking at genital apertures in beetles in 50 years from now to tell which species were on a tree,” Hebert says.

Brown says that if, as he suspects, some species are much rarer than previously thought that only makes conservation efforts more urgent.

“I look at my flies and there are maybe 100,000, maybe a million, undescribed species of them in the world. We don’t really know. But if we don’t use methods that take into account genetic divergence, we’re never going to get close to the truth.”