THERE are two ways in which the mystery behind the evolution of a species can be worked out: by examining fossil records, and by evaluating the rate of change in genes. Recently, the Indo-French Centre for the Promotion of Advanced Research funded a study which aimed at genetically breeding new strains of mice for laboratories. For the purpose, researchers had to define these mice at the genetic level and in the process, they stumbled onto startling evidence which led them to hypothesise that the first squeaks were possibly heard in India; they discovered that the Indian house mouse could be the ancestor of all its brethren the world over.

This hypothesis of the rodent's origins has been scientifically established by the collaborative efforts of researchers at the National Institute of Immunology (NII), New Delhi, the Unite d'Immunochemie Analytique Institut Pasteur, Paris, and the Universite Montpellier II, Montpellier, France. According to Rajesh Anand, in-charge of the Special Animal Facility at the NII, this discovery fills in a number of gaps which have persisted in the theory governing the evolution of the house mouse. The Indian house mouse exhibits not only the characteristics of the four well defined sub-species, but also demonstrates some features which are totally new to the scientific community. Genesis of a genus The mouse, being the most important of all laboratory animals, has contributed in no small measure to biological sciences and medicine. Its small Size, rapid rate breeding, easy reed in adaptability to varied environs and the fact that many of the ailments afflicting it are common to humans well, make it an invaluable aid in biomedical research, as well as in areas like genetics, oncology, pharmocology, toxicology, developmental biology and immunology.

Before this Indo-French collaborative study, whatever evidence we had of the evolutionary history of the house, mouse came from fossil finds. No genetic studies had been conducted to support palaeontological theories which said that mice originated in the Indian subcontinent.

Rodents are members of the taxonomic family Muridae. Fossil records of Anteinio, the most primitive murid - going back 14 million years to the Middle Miocene period - were recovered from the Indo-Pakistan region. Palaeontological evidence also indicates that the Mus auctor, which represents the first Mus genus, originated in the Indian subcontinent as well - about 5.5 million years ago, in the Late Miocene period. After studying the dental structure and morphology of the M auctor, scientists have concluded that it could very well be the ancestor of the entire genus, and not of the sub-genus Mus alone.

Once adjusted to a particular geographic location, a living organism undergoes genetic changes, and these changes are passed on from one generation to the next. This change in genetic information, called mutational change, takes place at random and is influenced by climatic, geographic or environmental factors. Such random mutations are not directional and indicate an active evolutionary process. By investigating the genetic relatedness of the house mouse Mus musculus with other Mus species, researchers found that it had separated only recently (one million years ago) from its nearest relations - M spretus, M spicilegus and M macedonicus. This separation, followed by the reproductive isolation of a small population, led to the establishment of the musculus species about half a million year ago. Its small size, rapid breeding rate and resilient behaviour guaranteed its ecological success in regions to which it was a stranger.

The M musculus can adapt to wide-ranging climatic conditions - from the mild temperate or the extreme cold of the sub-Antarctic islands, to the debilitating heat of near desert landscapes. In the case of the house mouse, evidence suggests that geographic migration of the original population has led to differentiation into new species and sub-species. Sub-species come into being when a species has already evolved and there is very little variation of individuals from the ancestral strain. The change that occurs, follows a particular direction and is not random.

Sub-species prove to be good biological markers for evolutionary studies. The M musculus species has four main sub-species (see map). Eastern Europe and northern Asia are homes to the Mus mus musculus, which is distinctive for its short tail and whitish belly. The domain of this sub-species extends over Vladivostok in the erstwhile USSR to Scandinavia and central Europe, where it intermingles with another subspecies, the M m domesticus, in a 30-40 km wide zone - called the 'zone of hybridisation'.

The M m dome5tiCUS is found in western Europe, northern Africa and in West Asia. With a long tail and variable coat colours from sandy yellow to dark brown, this house mouse sub-species is the primary source of laboratory mice strains. Southeast Asia - beginning from the eastern part of the Indian subcontinent - is the home of the M m casteneus. This sub-species is known to interbreed with M m musculus in China to form a hybrid population in Japan, which is sometimes referred to as the M m molossinus. These hybrid mice are brownish in colour, with long tails; they survive amidst human dwellings.

The domestic mice from Afghanistan belong to the M m bactrianus sub-species, which is thought to range southwards to the Indian subcontinent. But according to Anand, that was before the Indian mice were studied for genetic classification. "Our study reveals that these mice do not belong to any of the known sub-species, and therefore cannot be categorised into one till further research," he says.

The human factor
Another theory which could form the basis for explaining the interesting trends in worldwide colonisation by the house mouse is commensalism. This theory holds that passive transport by humans has led to the colonisation by the species, which has always been ecologically dependent on humans.

Commensalism of the house mouse in relation to humans is turhing out to be a major feature in the life history of the animal. Though the whole species is essentially commensal, there is a different level of commensalism within the subspecies. The M m castaneus is totally dependent on human dwelling, while the M m musculus survives in a large variety of human-related as well as wild environments. The other subspecies are intermediates.

A pertinent question that arises here is, when did man and animal begin living in close contact with each other. The scientific study under discussion in this article concludes that the commensalism of the M musculus started around 0.9 million years ago in the Indian subcontinent. Could it then be possible to theorise that the dwellings of the early humans, the Homo erectus, were where the Mw and the human first made contact with each other?

There have been no reports till now to suggest how this ecological dependence of mice on humans started. Before they came in contact with humans, mice were feral creatures living in the wild and foraging for food. But ecological evidence related to the mice species tinder observation point to the origin of commensalism from a palaeo-ecological point of view. This theory is based on a study of M m domesticus and its wild cousin M macedonicus in and around Israel. In Israel, these two species live in harmony with each other - the M m domesticus in human dwellings and the M macedonicus in the wild,. But in the Jordan valley and Negev desert, which fall outside the Mediterranean zone, the M macedonicus disappears, and the M m domesticus dominates the permanent feral populations. The presence of the M macedonicus has resulted in the house mouse taking shelter in human habitats; wherever the M macedonicus is absent, it has survived in the non-human niche. Thus, the partition of the species probably occurred when there was fierce competition within the wild mice populations for food and shelter.

The appearence of the house mouse in the eastern Mediterranean region is synchronous with the establishment of the earliest villages and the first signs of human settlement in the area. The advent and later differentiation took place about 0.35 million years ago. A study of the spread of human society in the Fertile Crescent, west of the Zagros mountains in Asia Minor, found that this corresponded with the distribution of M macedonicus populations in the region. It is believed that this, in all probability, opened up a new ecological niche for the emergence of the M m domesticus, in which any conflict with its wild cousin was avoided through the human partition created between the two species.

Idea behind the project
The basic concept governing the Indo-French project lies in the science of heredity. Hereditary traits, which are conserved through generations, have a preeminent role to play in the present study. To understand the fundamentals of heredity and evolution, it is necessary to comprehend the nature and constitution of genes.

Genes are made Up Of DNA (Deoxyribonucleic acid) sequences. DNA is a heteropolymer of four nuclecticles, the sequencing of which is important as it determines the amino acid chain in a protein. A set of three nucleotides in a DNA sequence denotes one amino acid in a protein. Proteins are heteropolymers of 20 amino acids, which are the basic building blocks of any living organism. The amino acid sequence determines the structure and function of the protein.

These functional proteins or alleles (a possible form of a given gene) are passed from parent to offspring, and therefore also have a role to play in hereditary exchange of information. For the purposes of the present study, mice from various localities in Delhi, the Nilgiris, Lahore and Teheran were trapped. Polymorphism or variation in proteins and allelic combinations in the genes of chromosomes and mitochondria were studied before it was concluded that mice from India, and particularly those from north India, are the original house mouse strains.

Since genomes or DNA sequences are very large, and contain several million nucleotides, they need to be cut down to a size that is easy to study and analyse. This is done with the help of enzymes called restriction endonucleases which cut the long DNA sequences into smaller bits. The DNA fragments are then introduced into minute depressions on a gelled plate through which electric current is passed. Being electrically charged, the nucleotides start moving from the negatively charged side to the positively charged one. This leaves behind a pattern on the plate, called the restriction pattern.

The restriction pattern for a gene is constant for each population of a species, pointing to which allele is present in that particular gene. Differences in these patterns are indicative of variations between populations, and are used to interpret evolutionary relationships within a species or to understand the evolution of a sub-species. The degree of polymorphism increases with the number of differences in the pattern obtained.

Similar tests, carried out with nuclear genes which are present in the chromosomes in the cell nucleus, revealed that the north Indian mice species exhibit maximum variability, which is indicative of a high degree of polymorphism. The Indian mouse exhibited genetic characteristics which did not fit in with any one of the sub-species which had been extensively studied. Besides, it showed a large number of alleles which had not been reported earlier in any existing mice population.

A statistical analysis of the variability data was collected and then fed into a computer to mark out the genetic distance that exists between the Indian mice and other well defined sub-species. This programme has what is called the 'phylogenetic tree' of the Mus musculus. The data on the Indian mouse filled in the gap in the centre of the tree, with the Pakistan and Nilgiri mice occupying neighbouring sites on either side of the Mus musculus.

According to Anand, the migration pattern of the species shows that from a central point, the species radiated outwards in various directions. Therefore, it may be safety concluded that the Mus musculus species originated in this region and then gradually colonised the rest of the continent. The presence of hybrid species in Europe and eastern Asia suggests that they developed due to secondary contacts. This has led scientists to hypothesise that the house mouse may be a 'ring species'; or alternatively, that it is in a highly active state in the evolutionary process.

Apart from establishing that the house mouse had its origins in north India, the entire exercise has gone to highlight the importance of this polymorphic species as a tool in the hands of various biomedical researchers. The Institut Pasteur was interested in studying a particular immune cell receptor, called the v-beta 17 T-cell receptor, which produces protective mechanisms in the form of antigens when the body is attacked by a foreign particle. The results showed a high degree of variability in this factor, indicating that the Indian mice strains possibly have an immune system which is different from any of the existing sub-species. That is to say, they exhibit a different response in terms of T-cell activation, deletion, energy and tolerance.

Once this variable immune response is established, the entire biological and medical community would be interested in these mice as new genetic models for comparative analysis in various fields, be it oncology or pharmacology. The Nit has already been involved in the process of developing new strains of laboratory mice, and now the additional factor of polymorphism has raised high their hopes of attracting a large number of scientists from different fields who would be interested in studying this species. A new strain of well defined mice, bred under laboratory conditions, will be ready after the 20th generation is produced. The NII's mice are in their 14th generation right now, and it will take another two to three years before the mice would be ready for supply to laboratories.