The Bat with the Identity Crisis

or

“Will the real Myotis nattereri please stand up?”

By Andrew Harrington.

There is a misfortunate bat species that is going through a bit of an identity crisis at the moment. This poor creature is Natterer’s bat, or Myotis nattereri to give it its formal name (named after the distinguished 19th Century Austrian naturalist Johann Natterer, not because it makes a lot of noise, as I have sometimes been asked). Natterer’s bat is distributed across Europe and north-west Africa, and is mainly considered to be a woodland species- in other words, it has specialised in hunting for insects in areas of woodland and scrub. It tends to hunt in the canopy of woodlands, flying slowly along woodland paths and rides, or through the branches and leaves where it often picks insects and other invertebrates right off their hiding places on the leaves of trees- apparently it is even manoeuvrable enough to be capable of picking spiders right off their webs! In Ireland it is widespread but generally regarded as fairly uncommon- quite understandably, as woodland covers quite a low percentage of the land here compared with most other European countries.



However, it seems that Natterer’s bat has been hiding a split personality. What we recognised within Europe until a few years ago as a single species has recently been found to consist of not just two, but probably at least four separate bat species. This phenomenon is known as “cryptic species” and seems to occur quite commonly among bat species. In recent decades, several species have been found “in hiding” as they were thought to form part of another species and were not distinguished as their own separate entity.

This might sound a bit incredible at first, that bat experts couldn’t tell two (or more) genetically quite distinct mammal species apart, several times over. But it becomes more understandable when you consider several unusual features of bats. As they are small, active at night, fly around rapidly and make calls which were inaudible to humans until bat detectors were developed which could make sense of their ultrasonic vocalisations, they are very difficult to casually observe. Although bat detectors now make it easier to identify or at least narrow down the bat species which is flying past you in the dark, some species have very similar calls (bats of the Myotis genus in particular) and you have to use cues such as the habitat they are feeding in to point you towards the right species, and even that isn’t always totally certain- it depends on the context and your level of experience.

So, how do we spot cryptic species?

To be really sure of what bat species you are looking at, you need to hold it in your hand and take a good close look, but this requires special equipment, training and a government licence before you can trap the bat and find out what species it is. But even when you have a bat up close to identify it, there are pitfalls. Bats are evolutionarily limited in how much they can vary their bodily features due to the tight constraints of the need to be able to fly effectively, which is why to the untrained eye they look so similar to each other. As well as that, they fly at night so there is no real purpose in having bright, variable body colouration like birds do.

Therefore, we have to depend on often quite subtle body features to tell bat species apart. These include variations in fur colour, body size, forearm length, ear shape and length, and facial colour. In some cases, it is even necessary to look at the shape and length of the tragus (a lobe of skin over the earhole) or the shape and length of particular teeth- try that when the bat in your hand is trying to bite your finger to get away! This detailed examination of the bat is usually enough to identify which species it is, and until only fifteen years ago or so, was the essentially the sole basis on which bat species were described scientifically.

Up until the mid-1990s, a few cryptic species had been discovered sporadically over the decades when someone recognised that some bat species actually consisted of two distinct subsets which had subtle but consistent differences in some body features. For example, the grey long-eared bat (Plecotus austriacus) was recognised as a separate species in the early 1960s, as members of this species had previously been assumed to be brown long-eared bats (Plecotus auritus), but both are now known to be clearly distinct species. However, in the hand the difference can be relatively hard to spot, hinging on a slight difference in body colour and measurements.

Another example of a pair of cryptic species are the whiskered bat (Myotis mystacinus) and Brandt’s bat (Myotis brandtii), which were separated in 1970. These are truly difficult to tell apart as they are almost identical, both in their habits, echolocation calls and even in the hand. They are so hard to tell apart that it takes an expert eye to distinguish the tiny difference in the length of a single tooth, and a few other very slight differences which identify the species. These are cryptic species par excellence.

The DNA revolution

Since the 1990s, an explosion in the use of DNA technology allowed further, more detailed, study of these cryptic species. One of the first major discoveries was that the common pipistrelle (Pipistrellus pipistrellus) had been hiding a cryptic species, which was named the soprano pipistrelle (P. pygmaeus). Although this situation had been suspected for some years, as it had been noticed that there were two distinct subsets within the common pipistrelle with different echolocation frequencies and body features, a genetic study by Barratt et al. (1997) confirmed that the two subsets formed definitely distinct, if closely related, species.

They showed this by examining the bats’ genetic sequence, in particular focusing on the mitochondrial DNA (mtDNA), a subset of the cell’s genetic code which in animals is only passed on in the female line (i.e. from a mother to her offspring). Mitochondrial DNA has the advantage that although there may be slight variations in the code between members of the same species, the level of variation is usually quite low. In contrast, the level of mtDNA variation between even closely related species is usually high enough for this to be a useful method to tell species apart. In general, a difference of more than 5% between the mtDNA sequences of two individuals notionally from the same species indicates that there may be a cryptic species present, and 10% or more sequence divergence is a strong indicator of the presence of separate species (Baker and Bradley, 2006). In this case, it was found that the mtDNA sequence divergence between P. pipistrellus and P. pygmaeus was about 11%, whereas the sequence divergence within each species was less than 1%. However, while mtDNA can be highly indicative of a split into two species, caution is needed as it does not tell the whole story. To confirm that two possible cryptic species are truly separate, we need to examine their nuclear DNA (nDNA), which unlike mtDNA is inherited from both parents. nDNA can indicate if interbreeding is occurring between two potential cryptic species which we have identified from differences in their mtDNA; if interbreeding is indeed occurring, then they actually constitute a single species and the cryptic species hypothesis has to be thrown out. To be fully sure that you have discovered a new cryptic species, you have to examine both types of DNA. In the case of the two pipistrelle species it was confirmed in 2007 that nDNA genetic markers showed that no interbreeding was occurring between them, confirming their status as separate species (Racey et al., 2007).

Barratt et al.’s landmark 1997 study was the beginning of extensive investigation into the genetics and evolutionary history of European bat species, which is still ongoing and far from complete. Since 2000 it seems that new, previously unrecognised cryptic species have been regularly coming out of the woodwork. In this short period, newly identified bat species in Europe include the Alcathoe bat (Myotis alcathoe), steppe whiskered bat (M. aurascens), Alpine long-eared bat (Plecotus macrobullaris), Sardinian long-eared bat (P. sardus) and Balkan long-eared bat (P. kolombatovici), among others, all of which have been identified in large part on the evidence from genetic studies.

Natterer’s bat springs a surprise… or four

In this context, it came as no surprise that Natterer’s bat too was hiding a cryptic species up its sleeve. Some years ago, Ibañez et al.’s (2006) study on the genetic diversity of bat species in the Iberian peninsula found that Natterer’s bat had the highest level of mtDNA sequence divergence within what was assumed to be a single species, which at an incredible 16% exceeded by far what would be considered usual. Apart from the “typical” M. nattereri which they found only in northern Europe, they found not one, but two other highly divergent lineages of “M. nattereri” limited to Iberia. One of these, present mainly in southern Iberia, was found to have distinctive habitat and roost preferences as well as a mtDNA sequence which diverged from the true M. nattereri by 16%. This lineage of bats was so distinctive from M. nattereri that Ibañez et al. immediately felt that it merited elevation to new species status, naming it Myotis escalerai (or Escalera’s bat). Another highly divergent lineage was discovered in northern Iberia, with a mtDNA sequence divergence of 10% from M. nattereri, but as the authors could not identify characteristics of its morphology or ecology that could distinguish it easily from M. nattereri, they refrained from immediately describing it as a new species at the time. However, they had also looked at the nDNA of both M. escalerai and this unnamed cryptic species (later dubbed “Myotis species A”), which confirmed that they were not interbreeding with M. nattereri and therefore most likely were new species in their own right. In a single study, Natterer’s bat had spawned two new cryptic species!

Since then, a stream of more recent studies has shed further light on the odd situation of “the bat formerly known as Natterer’s” (Mayer et al., 2007; Garcia-Mudarra et al., 2009; Salicini et al., 2011; Galimberti et al., 2012). Most recently, Salicini et al. (2013) looked at the genetics of this group of species by taking samples from 136 bats from 12 countries, giving the clearest picture yet of the situation. Firstly, the range of the recently-identified Escalera’s bat (M. escalerai) has been extended all across Iberia, the Balearic Islands and far south-western France, on the north side of the Pyrenees. A new species of bat also previously thought to be part of M. nattereri, named for now “Myotis species B” (first discovered by Garcia-Mudarra et al., 2009), has been shown by Salicini et al. to be the nearest relative of M. escalerai, and is present in Morocco. However, although closely related, they have been shown not to be interbreeding, so they are distinct species.

On another branch of this family tree, “Myotis species A”, which as you saw above had been originally discovered living in northern Iberia in 2006, has since then also been found to occur in central Iberia, southern France, on the slopes of the Alps in northern Italy and Austria, and in central and southern Italy. There is some disagreement about the status of the southern Italian population of this species though, as it very different in its mtDNA from the other populations (sequence divergence of about 5%). Salicini et al. suggest that this probably forms a sub-species of Myotis species A, but Galimberti et al. (2012) claim that these probably form a fifth separate species, which they have predictably named “Myotis species C”. The problem with this population of southern European bats is that no study has yet compared its nuclear DNA with samples from Myotis species A to check whether these two groups interbreed, so really the jury is still out on which of the “sub-species of Myotis A” or “new species Myotis C” hypotheses is the correct one.

Lastly, the poor “true” M. nattereri is now a shadow of its former self, as it seems it doesn’t occur in Iberia, North Africa or Italy at all! Bats definitely belonging to M. nattereri haven’t yet been found in any of these areas so it now seems that its range is somewhat less than what was once thought, but it is still known to occur across Europe from the Balkans north-west through central and northern Europe as far as the British Isles. Its nearest relative is now thought to be the mysterious Myotis species A, as they are known to be distinct species and do not interbreed.

An insight into the past of Natterer’s bat

Interestingly, Salicini et al. think that the two main branches of bat species described here are not even that closely related- M. escalerai and Myotis species B are more closely related to other bat species from the Middle East, and that their ancestors may have colonised Iberia via North Africa instead of Europe. On the other hand, the ancestors of the other branch probably colonised Europe via the Balkans, being subsequently pushed back by the Pleistocene ice ages to the refuges of the Balkan and Italian peninsulas, where they evolved into M. nattereri and Myotis species A, respectively. Another unusual feature of the way these four species is now distributed across Europe is that it appears that M. nattereri somehow managed to recolonise far more of Europe after the last ice age than either M. escalerai or Myotis species A. This is a pattern seen in other bat species such as the lesser horseshoe bat, Rhinolophus hipposideros, where the majority of post-glacial recolonisation of Europe has come from the Balkans. In contrast, the more common pattern seen in other mammals is of more equal recolonisation of Europe coming from the refuge areas of Iberia, Italy and the Balkans, such as in the European badger, west European hedgehog and brown bear. Why M. escalerai and Myotis species A have apparently never colonised much further north than the Pyrenees or the Alps is currently unclear.

There is still very little known about these three newly discovered cryptic species in terms of their preferred habitats, prey and roosting places, or about their population size, so little that no-one has yet gathered enough information about them to formally describe any of them as a new species. There obviously still remains an awful lot of work to do to clarify such questions about these newest additions to the list of European bat species, and it is important in particular for their conservation into the future as they appear to have such limited geographic ranges, which makes them vulnerable to threats such as climate change or habitat loss. For M. nattereri too it is important to gather more information on its distribution, as it seems to be absent from a large section of what was once assumed to be part of its range. However, the story of Natterer’s bat hasn’t been fully told yet, and there may still be more cryptic species to discover in other little-studied parts of its range. This is what makes the study of bats so exciting at present- the feeling that at any time, yet another important discovery is waiting to be made and that we have only really scratched the surface of what is there to be found.

References

Baker R.J. (2006). Speciation in mammals and the genetic species concept, Journal of Mammalogy, 87: 643-662. DOI: http://dx.doi.org/10.1644/06-mamm-f-038r2.1

Barratt, E., Deaville, R., Burland, T., Bruford, M., Jones, G., Racey, P., & Wayne, R. (1997). DNA answers the call of pipistrelle bat species Nature, 387 (6629), 138-139 DOI: 10.1038/387138b0

Galimberti, A, et al., 2012. Integrated Operational Taxonomic Units (IOTUs) in echolocating bats: a bridge between molecular and traditional taxonomy. PLoS ONE 7: e40122.

García-Mudarra, JL, et al., 2009. The Straits of Gibraltar: barrier or bridge to Ibero-Moroccan bat diversity? Biological Journal of the Linnaean Society 96: 434-450.

Ibáñez, C, et al., 2006. The Iberian contribution to cryptic diversity in European bats. Acta Chiropterologica 8: 277-297.

Mayer, F, et al., 2007. Molecular species identification boosts bat diversity. Frontiers in Zoology 4: 4.

Racey, PA, et al., 2007. Microsatellite DNA polymorphism confirms reproductive isolation and reveals differences in population genetic structure of cryptic pipistrelle bat species. Biological Society of the Linnaean Society 90: 539-550.

Salicini, I, et al., 2011. Multilocus phylogeny and species delimitation within the Natterer’s bat species complex in the Western Palearctic. Molecular Phylogenetics and Evolution 61: 888-898.