Anthony K. Campbell, Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK.
Summary
1. Biodiversity involves diversity of species, genetics and habitats. But there is a fourth source of biodiversity – molecular biodiversity – without which evolution cannot occur, either in the origin of a new species, its survival and development, or its eventual extinction.
2. Molecular biodiversity is distinct from genetic diversity, though both ultimately depend on inheritable DNA. It occurs within one individual, between individuals of the same species, between related species, within and between phyla and ecosystems, and throughout evolution. There is also a crucial evolutionary role for ‘hidden’ molecular biodiversity in ‘bad’ genes. These highlight what Darwin and Wallace missed, the origin of a biological process, or a species.
3. Genetic engineering of bioluminescence, coupled with molecular imaging, has given us a wonderful technology for lighting up the molecular biodiversity of individual living cells, and even whole organisms. This has highlighted a major challenge – when is a biological process analogue or digital?
4.Synthesis and applications. The care of our planet, and ecology in the 21st century, depend on a new thinking based on molecular biodiversity. My go-home message is that ecologists must grasp the opportunities presented by advances in molecular and cellular biology. But, although molecules are at the centre of modern biology and medicine, science begins and ends with a curiosity about the whole – the cell, the organ or the individual organism, within a particular ecosystem.
The classic Rubicon question: when the population response is 50% of maximum, have all of the cells reached 50% of their maximum, or have only 50% of the cells been switched on?
Science begins with curiosity. So what relevance to ecology in the 21st century has a molecular scientist, who works on genetic engineering, wants to challenge Darwin and Gaia, and is sceptical about the global warming argument? I aim to persuade you that molecules are now the key to ecology. But only when we see Nature through Darwin's eyes can we take natural history into natural science, and back again. Beauty is in the eye of the beholder. The three-dimensional structure of a bioluminescent protein can be just as elegant and aesthetically exciting as a chough on a Pembrokeshire cliff top.
Imagine you are in the audience listening to my presentation. I ask everyone with one head to stand up. Then I ask people with blue eyes to sit down, followed by those with brown hair, those with hairy legs, those who know their blood cholesterol, those who don’t like milk, those who have a cold, and those who like chocolate. By the time we get to those who have a cold, there are just a handful of people still standing. This simple demonstration highlights the key to evolution – molecular biodiversity within a species. For each of these differences has its basis in molecules within each of us. Molecular individuality is the key to what Darwin and Wallace and natural selection never really addressed, the origin, as opposed to, the development of a species, and its eventual extinction. Analysis of molecules can provide ecologists with information crucial to whether an ecosystem will survive. So what do I mean by molecular biodiversity, and how is it different from genetic diversity?
What is molecular biodiversity?
Biodiversity is embodied in the richness of species (Hubbell 2001; Novacek 2001). Conventionally this is seen in three ways: diversity of species, diversity of genetics, and diversity of habitats. But there is a fourth: molecular biodiversity.
Molecular biodiversity is the richness of molecules found in life. Of the 90 naturally occurring elements, 27 are used by living systems (Campbell 1994). There are some interesting omissions, e.g. Al. The elements selected during evolution exist as molecular ions, e.g. Na+, Ca2+, Cl−, or as covalent complexes of two or more elements, e.g. . The molecules forming the structures and metabolism of life are: amino acids forming proteins; sugars forming the backbone of nucleic acids and energy stores such as starch; inorganic complexes such as phosphate and sulphate; lipids such as fatty acids forming triglycerides and phospholipids; and a diverse group of essential dietary organic molecules, the vitamins and intermediary metabolites. There are several molecular universalities in these, throughout the whole of life – prokaryote, eukaryote and archaebacteria. Without any one of them, life would not exist. Yet each has at least one exception that proves the rule.
ATP is the internal energy currency of all life's processes, the temporary energy store in muscle and many other eukaryotic cells being creatine phosphate. Yet fish use arginine phosphate. Most cells have high K+ and low Na+, but some cells can have the opposite. Nature has selected Na+ as the ion for excitability, yet some cells use Ca2+, and others Cl−. Sunlight is trapped by chlorophyll for photosynthesis, but linear tetrapyrroles can also trap light. All biological reactions are catalysed by proteins – enzymes. But RNA can be an ‘enzyme’. All inheritance is through DNA. Yet there are many RNA viruses, e.g. HIV. The genetic code is universal. DNA for all proteins starts with ATG coding for methionine, and ends in a stop codon, TAA, TAG, or TGA. Yet in mitochondria there are exceptions. Methionine in the mitochondrion is coded for by TAT, and TGA is for tryptophan, instead of stop. AGA and AGG are stops, instead of arginine. And then there is chirality. All proteins use left-handed amino acids, and all DNA uses right-handed sugars. Yet, when we run our muscles produce l-lactate from d-glucose, but our gut bacteria produce d-lactate, a biochemical diversity that is being exploited in the clinical diagnosis of gut infections. And bacteria use d-amino acids as a defence against attacking proteases that have evolved to attack proteins made of l-amino acids. Is there a mirror image of life somewhere out there? If there was it would have to be a complete mirror image, since proteins and nucleic acids could not have a consistent 3d-structure if each individual molecule was made of a random mixture of d- and l-sugar or amino-acid units. But these exceptions are not what I mean by molecular biodiversity.
I define three types of molecular biodiversity. First, there is the biological use of the samemolecule in different (diverse) processes. Secondly, there is the multiple use of different(diverse) molecules in the same biological process, function, or phenomenon. Thirdly, there is the molecular biodiversity of cells, whereby the same molecule is expressed at a different level in individual cells. This results in each cell responding differently to particular levels of stimulus or pathogen. For example, one nerve cell body will require a specific number of dendritic processes to produce a summation of miniature end-plate potentials to generate an action potential, whereas a neighbouring cell may require a completely different number of dendritic processes to cross the Rubicon, and fire. It is this cellular individuality that particularly distinguishes molecular biodiversity from conventional genetic diversity. Both are ultimately dependent on inheritable DNA. But the consequences of molecular biodiversity for an individual or species are influenced in a major way by non-inheritable mechanisms. This is why molecular biodiversity is so important for ecology.
In each case the molecules fall under the same forces of natural selection. The differences may involve tiny or subtle differences, such as a single base change in the DNA, or a single amino acid residue in a protein. However, they may be two distinct organic molecules or protein sequences, or even two distinct biochemical pathways. An example of the latter involves the enzymes in the gut that hydrolyse lactose, the sugar in milk. In E. coli this is beta galactosidase, but our gut cells use a completely different protein, lactase. There is no sequence similarity between these two.
Molecular biodiversity can exist between two DNA sequences coding for the same protein, yet with ‘synonymous’ base changes between them. and it can be found between different DNA sequences, such as response elements controlling the synthesis of the same enzyme. Molecular biodiversity can be two enzymes that catalyse the same reaction. It can be pigments with differing structures that have the same colour. Even if a spectrophotometer can tell them apart, they will be susceptible to the same forces of natural selection. If two or more different molecules are to be considered partners in molecular biodiversity, three criteria must be satisfied. First, the molecules must have at least one structural difference between them. Secondly, the molecules must play the same role in a biological process. Thirdly, the molecules have to be susceptible to the same forces of natural selection when the organism is forced to adapt to environmental change.
Molecular biodiversity abounds throughout life, being found within one individual organism, between individuals of the same species, between related species, within and between phyla, within and between ecosystems, and throughout evolution. The molecular biodiversity may be overt, as it is in a coloured pigment, or it may be hidden from obvious sight, e.g. two different transcription factors capable of regulating the same DNA response element. Molecular biodiversity also shows us that even apparently ‘bad’ genes within a species are really necessary for the survival of a species over evolutionary time-scales. They have a selective advantage.
Molecular biodiversity holds the key to the appearance of a new species in the continued presence of the old, a problem for Darwin (1859/1868), and still a major one, as Lack (1947) struggled with in his famous ‘Darwin's finches’. The Galapagos have several species of finch with different beaks existing together on the same island, in the same habitat, and seeking the same food. A similar problem exists in the ‘Darwin finches’ of Pembrokeshire; for what else are the puffins, guillemots and razorbills? A mouse cannot mate with an elephant, not because of size, but because the DNA just won’t mix. This is the molecular definition of one species evolving into another. But it is not really the key to the origin of a new species, only a statement about two existing species. Molecular biodiversity of individuals within a species results in two or more populations separating into two distinct molecular clusters. Eventually a Rubicon is crossed – a sperm from one group cannot fertilize the egg of the other group. Alternatively, if it can, the maternal and paternal chromosomes have such different molecular biodiversity that the egg fails to develop, or produces an adult that is sterile. The converse is the cause of extinction. A species crosses the Rubicon towards extinction when the molecular biodiversity within that species fails to allow it to adapt to a changing environment (Campbell 1994).
Rubicon
What do an orgasm and a falling leaf have in common? Why should we consider both the flash of a firefly and the demise of the dinosaurs 65 million years ago? All of these are biological events, and they are digital. Darwin argued ‘Natura non facit saltus’ (Darwin 1859/1868; pp. 193, 326); Nature takes no leaps. Yet all biological phenomena are a combination of digital and analogue processes (Campbell 1994). Our body exists in states, involving thresholds, e.g. hunger, thirst, tiredness, fever and illness, headache, stationary, moving, sexy, awake, and asleep. Similarly our organs cross thresholds, as do the cells within them. Heart cells contract in synchrony to enable our heart to beat. The strength of the contraction of an arm muscle depends on the number of fibres that have fired, not on the strength within each individual fibre. A cell, an organ, an individual organism, or an ecosystem are taken to the banks of a Rubicon. A switch then operates, and once the living unit has crossed a biological event has occurred. The crossing may take a few milliseconds in the case of nerve firing or a firefly flashing, or it may take a million years in the case of the extinction of a dinosaur species. During evolution we see the ultimate Rubicon – the origin of a new process such as bioluminescence, a new cell type, or a new species.
At a cellular level the Rubicon principle is even more apparent. A time course or dose–response experiment of an agonist, drug or pathogen on a cellular process, generates a smooth curve (Fig. 1); Rubicon asks the question – at 50% of the population maximum have all of the cells been activated to half their individual maxima, or have only 50% of the cells been switched on? Using a bioluminescence reporter gene, such as firefly luciferase, the light output from a population of cells is measured. Gene activation increases or decreases light emission in a graded manner. But when we use molecular imaging to watch individual cells, particular cells are seen switching on or off at different times, and at different concentrations of agonist. Only when the switch has operated in a particular cell does the analogue component come into play, resulting in a different level of gene activation in each cell.
This I believe is the fundamental problem in contemporary biology. We still have no real mechanism or mathematics for these Rubicons. Many of the components responsible have been identified, but we don’t know precisely how the switch operates. Mathematical models exist for biochemical and physical process in cells – the Michaelis Menton equation for enzymes; the Goldman-Hodgkin-Katz equation for action potentials; the Mitchell equation for the proton motive force for ATP synthesis in a mitochondrion; receptor binding at the cell surface; models for Ca2+ signals in live cells (Baker et al. 2002); and the Hill equation for sigmoid curves. Yet these are all analogue equations. They treat the living unit as a single, giant thermodynamic system. There are no true digital mathematical models for biological processes that involve thresholds, switches, and rubicons. And ecosystems also have thresholds within them. The leaves falling in autumn at different times in a particular species of tree, and the first leaves in spring are obvious ones. The beech trees in my garden are always the last to come into leaf. If they were not, the bluebells in the wood would be smothered by the canopy of green before they could flower. This then is my problem with Gaia, elegant an idea as it is. The Earth is not an analogue system. If we are to understand global events such as warming, atmospheric change and the sea, there must be a digital component at the heart of the model.
I will now illustrate my argument with some examples selected from three areas that I love, and of which I have a little knowledge: bioluminescence, cell signalling and medical biochemistry.
Bioluminescence and cell signalling
Bioluminescence is the emission of light from living organisms (Campbell 1988). It is a major communication system in the biggest ecosystem on this planet – the deep sea. All bioluminescence is cold light, and it is ‘burning without fire’. All the energy from the chemical reaction goes into making light, instead of heat. Luminescence is the emission of light from atoms or molecules in electronically excited states. In chemiluminescence, and thus bioluminescence, the energy for exciting the electron comes from a chemical reaction, as opposed to the absorption of light in photoluminescence (fluorescence). A minimum of three components are required for bioluminescence – a small organic molecule (the luciferin), which is oxidized by oxygen or one of its derivatives, and a protein catalyst, the luciferase. Luciferin and luciferase are generic terms. There are five recognized chemical families of luciferin (Fig. 2): aldehydes, imidazolopyrazines, benzothiazole, linear tetrapyrrolles, and flavins. But many chemistries await discovery. The luciferase from each species has a unique protein sequence, but for each chemistry there is structural similarity between them. Thus, the luminous beetles all use a benzothiazole luciferin, but the luciferases are 50–80% similar between the thousand or so luminous species. The molecular biodiversity in bioluminescence is a real puzzle. It is as if the main groups of organisms are listed on a blackboard, and a handful of sand is thrown. The luminous species are those where the sand sticks (Harvey 1952). There is no obvious phylogenetic pattern to bioluminescence. and major groups are completely missing. There are no luminous spiders or crabs, except those infected with glowing bacteria. There are no higher plants that produce visible light, except those that have been genetically engineered. And there are no visibly luminous humans.
Five chemical families of luciferin in bioluminescence
Bioluminescence is a beautiful example of molecular biodiversity in cell signalling. All cells have evolved with specialized functions. These can be switched on or off. A nerve fires, a muscle twitches, an insulin cell secretes, a foetal cell differentiates, a lymphocyte produces antibody, a fertilized egg divides, a luminous cell flashes or glows, and a cell connecting a leaf to its branch dies by apoptosis, so that it falls in autumn. All of these cellular events have to be signalled. An external agent, such as an electrical impulse, touch, a neurotransmitter, a hormone, a pathogen, or internal programming, acts on the outer membrane of the cell, triggering a chemical process within. This requires an intracellular signalling system involving second messengers. Ca2+ is a universal intracellular signal in all animal, plant and some microbial cells (Campbell 1983, 1994). Cyclic AMP, cyclic GMP and nitric oxide are three others. Ca2+ acts as a switch, whereas cyclic AMP appears often to be an analogue regulator. All these intracellular signals act by binding to a receptor protein, e.g. a kinase to put phosphate onto serine, threonine, tyrosine, or histidine, which initiates a signalling cascade ending in the event. Their importance is exemplified by the incredible estimate that over 5% of all enzymes are kinases.
Molecular biodiversity in action
within an individual
Genetically, at the same locus, there can be hundreds of molecular differences – polymorphisms. Many are expressed differently between individuals, but some are found within the same individual. Plasma enzyme measurement is a key diagnostic test for damage to a particular tissue. Several are based on iso-enzymes with subtly different biochemical properties, e.g. substrate Km, occurring in different amounts in particular tissues, e.g. troponin T and I, or creatine kinase MB for cardiac muscle after a heart attack; alkaline phosphatase for bone or liver; and lactate dehydrogenase in liver or muscle. The pump that concentrates Ca2+ within the endoplasmic reticulum of every cell in our body has three isoforms, SERCA I, II and III. Excitable tissues such as muscle have mostly I, whereas non-excitable ones such as liver have mainly II. In bioluminescence, too, we find unexpected molecular biodiversity, even within one cell type in an individual. Aequorin, the photoprotein that generates light in the luminous jellyfish Aequorea, has at least six isoforms with just a few amino acids different between each gene product. These have subtle differences in spectrum and Ca2+ sensitivity, and can all be isolated from just one individual. We found a similar diversity in the green fluorescent protein (GFP), that changes the colour of the light emitted in several coelenterates, including Aequorea and its British relative Obelia.
within a species
Individual differences in eye, hair and skin colour, the amount of body hair, likes and dislikes, are all caused by molecular differences within our species. The four molecules mainly responsible for such biodiversity between individuals are: protein, DNA, small organic molecules, and ions. Thus, clinical tests for cancer utilize foetal antigens, e.g. prostate specific antigen (PSA), foetal haemoglobin, colonic, ovarian, and oat cell carcinoma antigens. All of these differ in protein sequence between foetus and adult. Molecular biodiversity lies at the heart of selective advantages between sexes of the same species. In the British glow-worm Lampyris noctiluca the female is wingless (Fig. 3). During July, she glows green for several hours, with two bars and two dots. The selective advantage enables the male to find her, and mate. Her lantern then goes out, and she lays her eggs. These will hatch in about 4–5 weeks. Both the eggs, and the resulting tiny larvae and pupa glow. Larvae, like the males, have two small dots in their last segment, smaller than those on the last segment of the female. These flash within a duration of seconds, in contrast to their faster firefly relatives. But even in the male the light is not used during mating. I have seen a male dancing around a female, but no light was produced. Wallace gives us the clue to the selective advantage. Flashing occurs when the larvae or males are disturbed. As Darwin discovered, some beetles taste very unpleasant. Many are brightly coloured and are thus very noticeable. They produce bitter substances that make then unpalatable.
The European glow-worm, Lampyris noctiluca: (a) male, female and eggs; (b) female by its own light.
between related species
There are only four families of beetles that produce visible light (Campbell 1988). The US firefly, Photinus pyralis, is in the same family as the British glow-worm – the Lampyridae. Yet one produces yellow light, the other green. One flashes the other glows. We have cloned and sequenced the DNA coding for the luciferase in the British Lampyris. It is 80% identical in sequence with Photinus luciferase (Sala-Newby et al. 1996). Both start with M = methionine. Both end with SKL, which targets the protein to the peroxisome. But the Lampyris sequence is three amino acids shorter than Photinus, and in the middle of the protein there are just a handful of amino acids that change the solvent cage surrounding the oxyluciferin, so that the light emission spectra are different. Yet these biochemical differences do not explain how a firefly flashes and glow-worm glows. The molecular biodiversity of this lies in the signalling system which controls access of the bioluminescence system to oxygen. Nitric oxide inhibits oxygen uptake in a small band of mitochondria close to the plasma membrane of the photocytes, thereby allowing access of oxygen to the luciferase in the peroxisomes further inside the cell. To our eyes beetles can produce green, green-yellow, yellow, orange and rarely, as in the South American railroad worm, red light. The latter has the fantastic ability to produce two colours from different cells in the same larva. It has 11 green-yellow light organs on its body, and two red headlights on its head. But can the firefly or glow-worm tell the difference between them? In a firefly meadow the molecular biodiversity in the control mechanisms make different species flash at different rates, and with different special patterns. The males recognize the females. Except for one femme fatale that flashes the same way as that of another species, and then eats the unsuspecting male! Each summer the children associated with our Darwin Centre project carry out my daughter's experiment – what is a glow-worm's favourite colour? Using coloured light sticks or badges we observe which colour attracts the flying males. Only green and red seem to attract them. None land on the blue ones.
within a phylum
Bioluminescence gives us a beautiful example of molecular biodiversity in the same phenomenon between genera and families in the same phyla. In coelenterates the luciferin is an imidazolopyrazine known as coelenterazine (Fig. 2). In jellyfish such as Aequorea and Obelia the protein catalyst binds the luciferin and oxygen so tightly that the whole complex can be isolated as one. Just add Ca2+ and it flashes. In contrast, the closely related Anthozoans, such as Renilla, do not have a photoprotein. They have a luciferase, which is very different in sequence from the photoproteins. Here the light flash is controlled differently. Ca2+ in this case releases the luciferin from a binding protein, rather than triggering the luciferase itself. And even when we examine the green fluorescent proteins (GFP) in hydrozoans and anthozoans we find very different proteins sequences. Yet both produce a green fluor, with an almost identical spectrum, and the same change in colour emitted from each species.
Even more dramatic molecular biodiversity is found in luminous arthropods. There are at least six distinct chemistries within the arthropods. Coelenterazine is used by decapod shrimp and copepods; another imidazolopyrazine by ostracods; a linear tetrapyrrole by euphausiid shrimp; a benzothiazole by the luminous beetles; luminous bacteria, found in some amphipods and nematodes, use a hydroxyflavin luciferin; and in the dipteran New Zealand glow-worm the luciferin has yet to be identified. Similarly, in luminous molluscs we find molecular biodiversity, with different luciferins within the same phylum: coelenterazine or luminous bacteria in squid; and an as yet unidentified luciferin in the bivalve Pholas (Dunstan et al. 2000).
within an ecosystem
Molecular biodiversity between species is essential for the survival and evolution of any ecosystem. This is well illustrated in the deep sea. More than half of the Earth's surface is covered by sea more than 1000 m deep. Here virtually everything is luminous. The small deep-sea fish Cyclothone is probably the most common vertebrate in the world, and it is luminous. Some years ago we showed that the most common chemistry responsible for bioluminescence in the sea is coelenterazine (Campbell & Herring 1990; Thomson et al. 1997). Coelenterazine occurs in luminous animals from at least seven phyla: Sarcomastigophora (e.g. Radiolarians); Cnidarians (e.g. hydroids and jelly fish); Ctenophores; Molluscs (e.g. squid); Arthropods (e.g. shrimp); Chaetognates (e.g. arrow worms); and Chordates (e.g. fish). Some have Ca2+ activated photoproteins, some use luciferases. Some squirt, whereas some produce the light intracellularly. Some flash, while others glow. Here, in one chemistry, we find massive molecular and cellular biodiversity within one ecosystem. But several of these organisms lose their luminescence when kept in captivity. Aequorea, Obelia and the midshipman fish Porichthys have to be fed the luciferin, or the animals lose the ability to emit light. Furthermore we found many non-luminous organisms, such as copepods, also contained significant amounts of this luciferin. Thus, molecular biodiversity of bioluminescence highlights a crucial food chain in the sea. But coelenterazine may be a misnomer. The only organism we have found so far that can synthesize this luciferin de novo is the decapod shrimp Systellaspis (Thompson et al. 1995).
The selective advantage of bioluminescence is sex, safety or sustenance. Glow-worms and fireflies, and the polychaete worm Odontosyllis, use luminescence to attract a mate. Hatchet fish and lantern fish flash dozens, even hundreds, of tiny photophores on their under surface as a camouflage against the dim ambient light in the deep sea, to hide their otherwise dark silhouette. Decapod shrimp, and some squid, squirt out a luminous cloud or trail that blinds the predator, or hides it. Copepods release small packets of luminescence during their mating display, which can act as a decoy for potential predators. The anglerfish hangs its luminous lure, containing glowing bacteria, in front of its mouth to attract food.
Many deep-sea shrimp viewed on the surface are red. This is a warning for us not to assume that a phenomenon we see is the same as that where the organism actually lives. I use this in my lectures to intrigue the audience. Why are these shrimp red? Have they been cooked? Have I painted them red? In fact, surprisingly, they are not red at all. Things are not always what they seem. In the deep sea the small amount of light in the twilight zone is blue, from bioluminescence, the red component of sunlight being scattered or absorbed above. Shine blue light on a ‘red’ shrimp and it is black. But the dragon fish Malacosteus has two pairs of light organs on its head, one emitting blue, the other red light. This fish can see the light from its own red torch, but the shrimp cannot. This then is molecular biodiversity generating an invisible torch to light up its, now red, prey.
hidden molecular biodiversity
And what of the shells shown in Fig. 4? There are several obvious differences. One is brown, the other white. One is bigger than the other. They are different species. One is alive today, the other is two million years old. But perhaps the most interesting difference is the hidden molecular biodiversity responsible for the shape of the shells. The contemporary one has a right-handed spiral, the fossil a left-handed spiral. The basis of this shape biodiversity lies in the molecular biodiversity of the homeobox genes that control shape.
Two shells that demonstrate hidden molecular biodiversity: (a) Buccinum undatum, a living whelk found in Anglesey, 2000; (b) Neptunca contrania, a fossil whelk found at Red Crag, Walton on Naze, Essex (Pleistocene, 2MYA).
Another process in life not often associated with molecules, and molecular biodiversity, is behaviour. Identical twins often have the same mannerisms, and talk with the same accent and inflections, as well as looking alike. Behaviour is molecular. This was beautifully illustrated recently with studies on a ‘knock-out’ mouse. The gene removed was a major factor responsible for transcribing DNA in the brain. Complete removal of the gene apparently had no effect on the mice. They looked and behaved normal, and developed into an apparently healthy adult. Yet, one day, an observant animal technician noticed something different about them. When a baby mouse escaped from the nest, normal mothers collect it in their mouths, and return it. However, the knock-out mice did not do this. The only obvious difference between the normal mice and the knock-outs was a behavioural one.
Another example of ‘hidden’ molecular biodiversity involves senses beyond our experience: bats sensing ultrasound; pigeons sensing magnetic fields; plants sensing gravity; insects sensing pheremones; and bees sensing UV light. Bees find nectar because plants have pigments in their petals that absorb UV light, appearing black to their eyes, but invisible to ours.
Molecular biodiversity also exists in the major cations used by living systems: Na+ and K+are involved in the osmotic balance of cells, and excitability in nerves and muscle; Mg2+ is used in all reactions involving ATP; and the ‘hard’ materials in Nature use compounds of Si and Ca2+. Silicates form the hard ‘shells’ in diatoms and foraminifera, but are perhaps too brittle in larger species. These use calcium. Ca2+ has functions that are both overt and hidden. Nature has selected various complexes of calcium: calcium phosphate in bones and teeth; calcium carbonate in the shells of molluscs; and calcium sulphate in the balance organs of jellyfish. Calcium oxalate precipitates occur in plants, and as kidney stones in humans. But Ca2+ also has a major hidden function – it is a universal signal switching cells on and off. And there is huge molecular biodiversity in this role within an individual, between species, and between phyla (Campbell 1983). An increase in free Ca2+ in the cytosol is the signal for all forms of muscle contraction; vesicular secretions, such as insulin and neurosecretion; intermediary metabolism, such as glycogen breakdown and in the mitochondria; egg fertilization; certain events in the cell cycle; cell defence; and death by apoptosis or necrosis. Thus, Ca2+ inside the cell is the signal for nerves to fire, and communicate with the next cell. It is the signal for each heart beat. It enables glucose to signal the secretion of insulin in the β cells of the pancreas. Ca2+signals a jellyfish to flash. And intracellular Ca2+ is involved in many of the body's defence systems. It signals a neutrophil to release toxic oxygen species to kill invading bacteria. It provokes antibody production in lymphocytes. and it enables some viruses to take over a cell without being killed by the body's immune system. This occurs through loss of Ca2+from the major intracellular compartment storing Ca2+, the endoplasmic reticulum. This structure has a signalling system within it that communicates with other parts of the cell – the nucleus, the plasma membrane, the mitochondrion, and the cytosol. When the necessary Rubicon is crossed the cell decides to fire, to divide, to defend itself against attack or to die. But the crucial thing is how the Ca2+ is controlled, and acts. In each of these processes these are molecularly different. This is the molecular biodiversity of Ca2+signalling. And in plants, and bacteria (Jones et al. 2002) too, free Ca2+ also plays a crucial role. In plants, wind and cold induce Ca2+ transients which activate genes that act to protect the plant against stress (Knight et al. 1991; Campbell et al. 1996). In bacteria Ca2+ can be involved in virulence, defence, gene expression, competence, movement by chemotaxis, and cell growth. The key to these diverse actions of Ca2+ is the molecular biodiversity of the Ca2+ signal itself – where in the cell it acts, together with the proteins and ligands it reacts with.
Essential to this story has been the measurement of free Ca2+ in live cells. The photoproteins aequorin and obelin, and then fluors such as fura2, have revolutionized cell biology by enabling free Ca2+ signals to be measured and imaged in living cells, whole organs, and even whole organisms (Campbell et al. 1988; Sala-Newby et al. 2000). By targeting these indicators, the free Ca2+ can be measured in defined compartments of live cells – the endoplasmic reticulum mitochondria, the nucleus, the plasma membrane, and other sites (Sala-Newby et al. 2000). Ca2+ signals can be simple transients, or they can spike or oscillate. The spatio-temporal nature of the Ca2+ signal varies between cell types, and even between individual cells from the same tissue. The Ca2+ signal can be a puff, a spark, a cloud, a wave, or a tide. We have found that even within a population of the same cells, there is major molecular biodiversity in the Ca2+ signals (Davies et al. 1991). Thus, the dose–response of each cell in a population is a Rubicon (Fig. 1). This molecular biodiversity within the same cell species is critical for the functioning of a multicellular tissue.
Evolution has selected Ca2+ for cell signalling because of its chemical properties: high affinity of anion binding; selection over Mg2+ at micromolar Ca2+ in the presence of millimolar Mg2+. A key feature of Ca2+ binding is its rapid release when Ca2+ is removed from the cell. If Zn2+ had been selected in evolution, the relaxation of muscle would take seconds or minutes, as opposed to milliseconds. A nerve would remain switched on for minutes, instead of milliseconds. Zn2+ however, is used in DNA transcription, a slow process. Cu2+ and Fe2+/Fe3+ are oxido–reduction cations in energy, and electron exchange, reactions. Even vanadium is used by some organisms. It is particularly high in tunicates. Here ‘tunichromes’ can oxidize to form pink pigments that are similar to an oxidation product of adrenalin, also a catechol, adrenochrome. These are the likely origin of the luciferin in luminous tunicates, not intracellular bacteria as has been claimed.
Hidden molecular biodiversity is also found in non-metallic ions. Cl−, , and are vital in all cells, but can work differently. Oxygen metabolites, e.g. , are involved in cell killing, but also in egg fertilization. But there is one obvious element missing: aluminium is the most common metallic element in the Earth's crust. It is highly reactive, yet there is no known use in any biochemical process. It is toxic, generating oxygen species which can damage proteins, DNA and membranes.
Imagine a fertilized egg dividing once a day. How big will the baby be in 9 months? By 1 week the embryo will be the size of a small seed. But by 27 weeks it will be the size of an asteroid. At delivery the baby will be the size of the Milky Way! This astronomical calculation highlights the importance of hidden molecular biodiversity in cell death. Without cell death we would have webbed fingers, we would not recover from a cold, nor would our brains have developed. Without cell death a tadpole can’t change into a frog, or a leaf cannot fall in autumn.
‘bad’ molecular biodiversity
We all carry at least three ‘bad’ genes, estimated from the prevalence of inherited disease. ‘Bad’ genes cause disease. The protein products of these ‘bad’ genes either do not form at all, or have mutations that cause pathological effects in people unfortunate enough to express the disease. The ‘bad’ genes may be dominant or recessive. The commonest inherited disease, cystic fibrosis (25% carry it in the human population), involves a chloride channel, CFTR. Familial hypocholesterolaemia causing heart attacks in the young involves abnormal lipoprotein genes. and muscular dystrophy involves a huge membrane protein, dystrophin. But there are two surprises, revealed now by molecular techniques. First, there is massive molecular biodiversity in the mutations. For example, in cystic fibrosis several hundred have now been identified in the CFTR from different individuals and families. Secondly, if these genes are ‘bad’, why are they still present in the human gene pool? There has been plenty of time for natural selection to select them out. But Darwin and Wallace have taught us that even these so-called ‘bad’ genes must have a selective advantage. Haemoglobin mutations cause sickle cell anaemia. But the malaria trypanosome has to go through the red cell. Thus, if you are heterozygous for these ‘bad’ haemoglobin genes you are less likely to die of malaria. Mutant CFTR may cause problems for Salmonella growth in the intestine. Molecular biodiversity in ‘bad’ genes is an essential part of an evolving gene pool.
What Darwin missed
How could a new phenomenon in evolution arise by gradual change? How could a new species arise from a population by graded change, without separation by time, geography, or behaviour? This is the Darwin finch's problem. As Hoyle pointed out, gradual random change is not enough if a species is to be split into two. A Rubicon must be crossed. Yet Darwin wanted no leaps. Punctuated equilibrium is a nice idea, but still doesn’t resolve this central dilemma. It is an analogue concept.
In Chapter VI, p. 191 of The Origin of Species, Darwin wrote: ‘The electric organs of fishes offer another case of special difficulty, for it is impossible to conceive by what steps these wondrous organs have been produced. The luminous organs which occur in a few insects … offer… a difficulty almost exactly parallel with that of the electric organ’. Glow-worm luciferase is 547 amino acids long, and the DNA sequence coding for this is 1641 bases (Sala-Newby et al. 1996). Imagine a sphere based on the Milky Way, 70 million light years in diameter. Packed with hydrogen atoms, it would contain 2·13 × 1078 atoms. But the number of combinations for the glow-worm gene, with four possible bases at each location, is 41641 = 10987, literally an astronomical number. This is Hoyle's problem with Darwin. And there are many other similar problems – the origin of new phenomena, new organs, new cell types, new species, and extinction. The holocaust at the K–T boundary 65 million years ago wiped out some 80% of all life, including the dinosaurs. Even if catalysed by an asteroid or volcanic eruption these cannot explain why it took millions of years to lose so many species, and yet many survived. There are two causes of extinction – evolution into a new species, or evolutionary death. Gain or loss of molecular biodiversity is the solution.
Darwin missed something else, crucial to our own molecular biodiversity. In the UK our diet is dominated by the cow. We consume large amounts of beef and milk. In many restaurants it is difficult to find any course without added milk products. Cheese and white sauces, custard, and cream abound. But amazingly all mammals, apart from white Northern Europeans (Caucasians), and a few other ethnic groups such as the Bedouins and African dairying tribes such as the Fulani, lose some 80–90% of their ability to digest lactose after weaning. Lactose, galactose β 1,4 glucose, is the sugar in the milk of all mammals, apart from the Pinnepedia (seal-lions and walruses). Lactose is hydrolysed by the enzyme lactase, found only in the small intestine. The resulting monosaccharides are then absorbed into the blood. All non-Caucasians, including black Africans, Asians, Chinese, Japanese, Native American Indians, Jews and several other ethnic groups, are hypolactasic, involving some 5–10 million in the UK and 3000–4000 million people world-wide. Any adult in these groups has a threshold to milk products, resulting in lactose intolerance. This threshold can be just a few millilitres, or more than a glass of milk (200 mL, approximately 10 g lactose). We have discovered a new syndrome caused by lactose (Matthews & Campbell 2000; Campbell & Matthews 2001) – systemic lactose intolerance. We name it MATHS – Muscle and joint pain, Allergy, Tachycardia and Tiredness, Headache, nausea and vomiting caused by sugar overload in the intestine. Lactose is added to many foods without being on the label. We proposed that this syndrome is caused by toxins generated by bacteria in the large intestine. It provides a molecular link between the gut and diseases in systemic tissues – cancer, rheumatoid arthritis, and multiple sclerosis. This is the syndrome that plagued Darwin for 40 years (Colp 1977; A.K. Campbell & S.B. Matthews, unpublished). But what is the selective advantage of retaining lactase after weaning? Monkeys don’t keep cattle. Dairying is only 6000 years old. and how is the Rubicon crossed? In hypolactasia gut cells appear to have 5–10% of the cells with lactase found in a suckling infant, rather than 100% of the cells with 5% of the enzyme. Lactose intolerance has much to teach us about our own origins.
Bringing molecular biodiversity to life
In North Burgundy there is a tiny ‘cuvette’ valley, south of Auxerre, where three villages nestle – Irancy, St Bris, and Coulange. These, with Epeneuil, produce the most northerly red burgundy. Not the greatest, but still based on the pinot noir grape. It may take some 5 years to mature. Understanding the chemistry of wine has much to teach medicine. In the lab we study cancer cells for just a few days, or at the most weeks. Yet even the most rampant tumour takes months, even years, to develop to a lethal point. Likewise, the time scale of ecology is over months or years, even decades. As you drive into the valley, towards the village of Irancy, the observant natural historian will notice not only vines covering the hillsides, but also beautiful roses along the edge – red, white and yellow, adorning the inspiring French countryside. But these roses are not there simply for decoration. They are indicators! As with any true indicator they are elegant, have a range of colours, and do not disturb the habitat in which they have been planted. If the roses start to suffer, there is a virus or fungus about. The wine-grower must act quickly if he is to save his crop, or produce a good vintage when the grapes are harvested. Likewise in cell signalling, discoveries have been critically dependent on indicators from the inventiveness of the experimenter.
I became curious about how jelly fish and other creatures make their own light, and can produce all the colours of a rainbow. I realized that using genetic engineering we could engineer sites into bioluminescent proteins that would change the colour or intensity of the light emitted, and thereby enable us to measure chemical reactions in live cells. These are Rainbow proteins, and there are three types. (Campbell 1989 & 1997). We have used this technology to light up the molecular biodiversity of proteases in cell suicide – apoptosis (Waud et al. 2001). Curiosity about the molecular biodiversity of bioluminescence has revolutionized biology and medicine. Twenty years ago I realized that a flash can be better than a glow. As a result we developed a way of replacing radioactivity in a crucial area of clinical diagnosis. It is now used in some 100 million tests per year world wide.
Molecular biodiversity can also light up the chemistry of the human mind? Public understanding of science and health is the issue of our time. Bioluminescence and molecular biodiversity have marvellous potential for inspiring young people about the invention–discovery cycle that lies at the heart of curiosity-driven science, which is also essential for entrepreneurship. We have established the Darwin Centre in Pembrokeshire (http://www.darwincentre.com) as a vehicle for this. Science is the driving force in our culture and economy. Yet the gulf between science and the arts seems as wide as it did when C.P. Snow gave his famous lecture on the two cultures some 40 years ago. Contrary to popular belief, Darwin was not the man who proved the Bible was wrong. The Darwin Centre aims to be a multicultural sabbatical centre exploiting molecular biodiversity to the full. School science projects linking molecules to ecology are presented at Science Aglow. Bioluminescence is a wonderful phenomenon, putting a spark back into school and University science.
Conclusions
I have tried to highlight three types of molecular biodiversity, and to address four main questions: What is molecular biodiversity? What is its importance? How can we study it? Why is it fundamental to the origin of a new species and its eventual extinction? Molecular biodiversity is crucial to the origin, survival and evolution of species, phyla and ecosystems. It exists overtly and hidden. It reveals hidden ecosystems and highlights the evolutionary role of ‘bad’ genes. It provides a new perspective on natural selection, and highlights the need for a new technology to light up molecular biodiversity. And most important, it brings science together as one.
As a species evolves, the molecular biodiversity increases. When it reaches the banks of a Rubicon, certain individuals within the population can only produce fertile offspring when their molecular biodiversity is compatible. For a sperm to fertilize an egg and then develop into a fertile offspring the two chromosomes have to replicate, and express their proteins at roughly the same rate. Differences in codon usage between maternal and paternal chromosomes, and biochemical differences in enzymes catalysing the same reaction from each chromosome, will result in divergence in the rates of metabolic processes. Then a molecular Rubicon will be crossed – the chromosomes from the mother are not compatible with those from the father. This couple is infertile. They become parts of two subpopulations, which can then diverge to form two distinct species, even in the same environment. Furthermore, at meiosis, cross over (recombination) can only occur if the non-coding regions are essentially compatible. In contrast, when the molecular biodiversity is reduced below a set number, the species crosses the Rubicon towards extinction. We now need a mathematical relationship that converts this molecular biodiversity principle into a law, analogous to the Hardy–Weinberg law of evolution. Steve Jones in his interesting book Almost like a Whale, argues that the changes in a virus such as HIV are living proof of natural selection and the origin of species. Yes the mutations in HIV, since it first appeared some 60–70 years ago, support natural selection. BUT, it is still HIV! No new viral species has evolved. The go-home message for molecular biodiversity is that life, evolution, and the care of our planet depend on it.
My ultimate message can be summed up by a quote from Frederick Gowland Hopkins, the founder of British biochemistry, who won the Nobel prize for discovering tryptophan in 1908, and who was inspired to enter molecular biodiversity as a schoolboy trying to extract the colour from butterfly wings and isolating the substance squirted out by bombardier beetles.
‘All true biologists deserve the coveted name of naturalist. The touchstone of the naturalist is his abiding interest in living Nature in all its aspects.’ (Presidential address to the London Natural History Society, 1936.)
Our world that has been brought together by the incredible inventions of the 20th century. Yet science has become increasingly polarized between curiosity and invention, from the arts, from spirituality and religion. We have lost the ability to communicate with ourselves. E-mail is not enough. We scientists need to retake control of the agenda by using our inspiration for science, our curiosity, our inventiveness, our scholarship, to inspire others. Once you are hooked on science, it is for life! Molecules are beautiful. Molecules are fun. Curiosity inspires, discovery reveals. Ecology must find a way to save those molecules!
. The molecules forming the structures and metabolism of life are: amino acids forming proteins; sugars forming the backbone of nucleic acids and energy stores such as starch; inorganic complexes such as phosphate and sulphate; lipids such as fatty acids forming triglycerides and phospholipids; and a diverse group of essential dietary organic molecules, the vitamins and intermediary metabolites. There are several molecular universalities in these, throughout the whole of life – prokaryote, eukaryote and archaebacteria. Without any one of them, life would not exist. Yet each has at least one exception that proves the rule.
, and 
are vital in all cells, but can work differently. Oxygen metabolites, e.g. 
, are involved in cell killing, but also in egg fertilization. But there is one obvious element missing: aluminium is the most common metallic element in the Earth's crust. It is highly reactive, yet there is no known use in any biochemical process. It is toxic, generating oxygen species which can damage proteins, DNA and membranes.
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