15 May 2014
By Magdalena Zarowiecki
Many biologists dream about finding a new species, but how do you know when you have found one? The theory of evolution teaches us that - just like us - species are born, live, procreate (speciate) and die (become extinct). So at any one time a species might be dividing into two or more species; a so-called species complex. In these cases, it is not easy to know how many species you have.
In order to try to understand how many species you have, it is a good idea to try to reconstruct the species’ evolutionary history. We can use genetic signatures to reconstruct what happened to that species in the past; where it came from, if it was abundant, and how it has spread across a geographical area, diversified and evolved.
My target species complex was the malaria-transmitting mosquito Anopheles sundaicus. For Anopheles sundaicus there were many reasons to believe it was a species complex; it is spread across a huge area – from India to the Philippines. People have shown that the larvae can live both in fresh-water and in very salty sea-water, and even more importantly – in some places it is transmitting malaria very efficiently, and in other places not at all.
The Anopheles sundaicus mosquito larvae are found in lagoons and rock pools on beaches in Southeast Asia, so I travelled to Thailand and Myanmar to collect some mosquitoes. It might sound like a glamorous beach-holiday, but it was not; we had to spend our mornings wading around in mud and scorching sun trying to find mosquito larvae. In the afternoons we borrowed a local cow and put it into a tent, capturing the mosquitoes coming to bite the cow. Early in the morning we had to collect the mosquitoes in the tent, while trying not to offend the cow, who was understandably a little bit grumpy before breakfast. Luckily, we had a lot of help from local malaria control teams.
Back in the UK, we sequenced the DNA from hundreds of mosquitoes for three genes; two genes from the cell nucleus, (internal transcribed spacer 2, ITS2 and mannose-phosphate isomerase, Mpi), and one mitochondrial gene (cytochrome oxidase 1, CO1) (mitochondria are organelles inside cells that create energy, and they have their own small genomes). From these DNA sequences we used statistical methods to reconstruct the population history of these mosquitoes for the last million years.
We were also able to tell whether a unique DNA sequence (allele) is only present in one population, or shared between several populations. If we had DNA sequences from two different species we would expect them not to share many alleles, and for their alleles to be quite different. The opposite could also happen; that there are two populations which are closely related, and in that case you expect many alleles to be similar, and many to be shared. Population genetic methods can be used to calculate which scenario is most likely.
Trying to reconstruct the evolutionary history of Anopheles sundaicus was made all the more difficult by Southeast Asia itself. During the last million years, the sea-level in Southeast Asia has changed many times; sometimes islands were scattered like they are today, and sometimes the sea level was more than 100 metres lower than today – joining Borneo, Thailand, Malaysia and many Indonesian islands into one massive peninsula (see figure). Such massive landmass reformation would undoubtedly have had a very large effect on the distribution and migration of the mosquitoes (and humans too). There were even times where they theoretically could tiptoe on dry land from Northern Borneo to Vietnam, and our population genetic analysis shows that it is quite likely that they actually did that.
So how many species are there of Anopheles sundaicus then? We cannot know for sure, but we can see that populations across their entire geographical distribution are surprisingly similar, and that there are alleles shared across such large distances as between Indonesia and Burma, Vietnam and Borneo. So although these populations are separate now, they were probably united as recently as some 100,000 years ago. If they stay isolated they might become separate species, but future climate change might also prevent speciation from happening, by generating new and unexpected mosquito encounters.
Dr Magdalena Zarowiecki is a Postdoctoral Fellow studying tropical diseases in Matthew Berriman’s Parasite genomics research group at the Wellcome Trust Sanger Institute. The research was conducted as part of her Ph.D. at Natural History Museum, London, with the help of collaborators in Cambodia, Indonesia, Myanmar, The Philippines, Thailand, Vietnam, the U.S. and the U.K., and many Southeast Asian mosquitoes.
Zarowiecki M et al. Repeated landmass reformation limits diversification in the widespread littoral zone mosquito Anopheles sundaicus sensu lato in the Indo-Oriental Region. Molecular Ecology 2014; 23: 2573–8.