Volume 31, Issue 4, Winter 2013

Five ‘Cryptic’ Bats for West Africa

Molecular biology reveals unexpected diversity in Senegal

By Darina Koubínová

The first molecular analysis of bats in Senegal revealed an exciting and unexpected diversity. A Czech-UK team identified a remarkable five cryptic species – groups of animals that appear, based on their external anatomy, to be a single species, but which are revealed through their genetics to be two or more distinct species that cannot interbreed. Our study indicates that these new species diverged from their African cousins about three to four million years ago, during periods of changing climate.

This story of discovery begins in 2004, when Professor Petr Koubek of the Institute of Vertebrate Biology in Brno, Czech Republic, led the first of seven expeditions to Senegal on Africa’s western tip. The team from several Czech institutions surveyed the varied fauna of the Niokolo-Koba National Park in southeastern Senegal.

The park’s 3,525 square miles (9,130 square kilometers) encompass woodland savannas, dry forests, gallery forests, bamboos and grasslands, plus vast wetlands. It is the largest and oldest national park in Senegal. The park is listed as an Endangered World Heritage Site because of poaching and plans to dam the river that flows alongside it.

As a zoology student at Charles University in Prague, I was invited by Professor Jan Zima to analyze the samples of small mammals collected from this project. Later, as a Ph.D. student, I focused specifically on bats. Biologist Nancy Irwin of the University of York was an invaluable teacher and mentor throughout this study conducted on vesper bats (Vespertilionidae), the largest and most diverse bat family, with more than 400 species.

Many vesper bats are very similar in appearance, especially in tropical regions, with few obvious external features to aid species identification in the field. So I used two lab-based genetic techniques to determine species. The first was cytogenetics, in which the number and shape of chromosomes are characterized. The second technique was molecular phylogenetics, where DNA is sequenced and the results compared to other individuals and species. This was the first time that the bat fauna in Senegal had been examined at a molecular and chromosomal level.

I recorded the number and appearance of the chromosomes, known as the karyotype, which can reveal relationships among groups of organisms and their evolutionary history. Individuals of a single species usually share the same karyotype characteristics, which can be unique and readily distinguishable. It is thought that animals with very different chromosomes cannot produce normal and/or fertile young.

While some bats differed in their karyotype from the same species in other areas, this was not always conclusive. We therefore sequenced DNA for up to eight genes from each of the samples. The power of such DNA analysis is dramatically enhanced by a freely accessible database of sequencing data from scientists worldwide (GenBank, maintained by the U.S. National Center of Biotechnology Information). This resource allows anyone to directly compare their DNA results to what has gone before. I was amazed at what comparisons revealed about these bats of Senegal.

Ten species were confirmed in our samples. And five of them were so different from their closest genetic relatives (their apparent species) in other parts of Africa that they should be considered distinct species.

The five species are provisionally identified as Schlieffen’s bat (Nycticeinops schlieffenii), dark-winged lesser house bat (Scotoecus hirundo), African/dusky pipistrelle (Pipistrellus hesperidus), banana pipistrelle (Neoromicia nana) and Somali serotine (N. somalica).

These bats clearly differ from others of their putative species in their DNA sequences, and also in the numbers or shapes of their chromosomes. Additional taxonomic study and revision will be required to describe these species formally, since there are often several subspecies that must be traced and investigated. There may, in fact, be more than one cryptic species to describe in some of these new taxonomic complexes. It could also be that the species from outside Senegal is the new (cryptic) one and the bats in Senegal will get to keep their original name.

The molecular data allowed me to consider when the Senegalese species diverged from their cousins elsewhere in Africa, splitting off from their original species to evolve into new ones. I estimate this divergence occurred about three to four million years ago, which happens to coincide with the start of a major episode of climate change in West Africa. The region previously was covered with rainforest, but pollen studies show that it began to dry out some three million years ago, transforming the landscape into grassy savannas with a massive decline in trees. Other studies suggest that the West African forests retreated into a small patch, which retained some of its original fauna.

Our data support the related notion of a “refugium” that sheltered some forest species in Senegal. Refugia are landscape fragments that largely escape periods of changing climate and retain previous environmental conditions, thus becoming a haven for remnants of species that would otherwise rapidly evolve or become extinct. This would help explain at least some of the unexpected diversity we found in this region – and perhaps provide a little insight on today’s changing climate.

DARINA KOUBÍNOVÁ completed her Ph.D. at the Charles University in Prague, Czech Republic, where she focused on molecular phylogeny and comparative cytogenetics of small mammals, mainly bats, from Senegal, West Africa.

This research was originally reported in Frontiers in Zoology, an open journal that can be accessed online without charge, as “Hidden Diversity in Senegalese Bats and Associated Findings in the Systematics of the Family Vespertilionidae” (2013); Darina Koubínová, Nancy Irwin, Pavel Hulva, Petr Koubek and Jan Zima.

Of Molecules, Cryptic Bats and Refugia

by Nancy Irwin

Darina Koubínová’s impressive study demonstrates just how international bat science has become: bats collected in Senegal were studied in Prague, Brno and Studenec in the Czech Republic and York in the United Kingdom; the results were published in a German journal in English. It was remarkable for a Ph.D. student to find 5 new cryptic species in one study, and the conclusion – that this cryptic diversity is evidence of a refugium – has global significance.

The Czech Republic has long been at the forefront of bat ecological research in Europe. In 1968, the country hosted the first international bat conference in the town of Hluboká nad Vltavou, only to be interrupted by the unexpected arrival of Soviet tanks. I have been fortunate to work recently with a growing corps of talented scientists who come from this long tradition of bat research.

In 2009, Darina spent a month at the University of York, where technician Michaela Nelson and I helped her design the gene-sequencing part of her project and get the process working well before she returned to the Institute of Vertebrate Zoology in the Czech Republic. Over the next four years, Dr. Pavel Hulva of Charles University and I helped her define the nature and origins of the biodiversity she discovered.

Traditionally, bats were classified on easily identified external characteristics, such as measurements of the forearm, leg and ear. But with the advent of genetic analysis in the 1990s, it became increasingly common to find that what had always been considered a single bat species was, based on DNA differences, actually two or more.

A famous example of this was the most-studied bat in Europe, the common pipistrelle (Pipistrellus pipistrellus). Originally considered the only bat species in Europe to echolocate at two peak frequencies, it was shown in 1997 to include two completely different species – each using a different peak frequency. I was fascinated to learn that scientists had for so long been looking at bats from a human perspective, while the bats actually see, hear and share different cues about each that are very different from anything we had been thinking about. Thus began my long-standing interest in bats that are difficult to identify.

Since the work on the common pipistrelle, we’ve seen a massive increase in reports of “new” cryptic species of bats. This partly reflects the development of much more sophisticated tools for identifying bats and much cheaper access to molecular studies, as well as increasing research in rarely studied areas such as Africa.

The impact of molecular research is huge. In 1994, Walker’s Mammals of the World recognized 925 bat species. By 2005, that number had reached 1,116 – roughly 21 new species a year. The latest reported total is approximately 1,300 bat species. And these changes go well beyond just describing new species. The research explores how those species are related to each other, with occasional changes in genera, tribes and subfamilies.

It is fundamental to know what species you’re dealing with before effective conservation plans can be made, unless entire geographic areas are being protected. But how do you choose which areas to protect if you don’t know what’s there? With cryptic species, their conservation needs will, almost by definition, be hidden. If three cryptic species are mistakenly considered a single species, for example, then that species likely would be assumed to have a large range and a large population size with no severe threats to the population as a whole. The reality, however, might well be that at least one of the cryptic species could be limited in numbers and area and face critical local threats.

A history of refugia can go a long way towards explaining unexpected diversity in West Africa. A refugium is a limited geographical area that retains original environmental conditions during a period of climate change. Multiple species are isolated together in a refugium, so scientists often find that their evolutionary isolation from their nearest relatives dates to about that time. You might think of a refugium as an ark where many species find sanctuary from surrounding ecosystems in which they could no longer survive. Other members of the same species might travel to a different part of the continent and find refuge in a different refugium, a different ark.

When they come back together, these species have been isolated from each other for so long that they have changed so much that they can no longer mate with each other. So while they remain close relatives, they are no longer part of the same species.

The change involved in Darina’s research is the well-known drying of West Africa three to four million years ago, when it changed from wet forest to dry savannah. Multiple studies are documenting forest-adapted species in West Africa that seem to have high genetic diversity within a species, as well as species that are found nowhere else. This suggests that an isolated forest fragment remained during the drying period where animals and plants managed to survive. Then, when the climate became wetter again, these fauna and flora dispersed into the landscape and recolonized their former range. Many species in Africa are thought to have very widespread distributions, but new studies are suggesting this may not always be accurate. West African biodiversity may be greater than previously thought.

What is even more striking is that these results are only a small part of the accumulating findings that will emerge from the expeditions and collections of the Czech science teams.

NANCY IRWIN was funded for this work by NERC as a Daphne Jackson Research Fellow at the University of York in the United Kingdom. She is currently studying urban monitoring programs and New Guinean bats and has developed a research interest in emerging diseases in bats.

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