DNA Sequencing and Analysis

Application in Taxonomic and Systematics Studies

Scientific research reports and papers can be difficult to understand: in part this is because of the unfamiliar terms used, which often have quite precise meaning, and in part because little is known about the methods and techniques which are employed. Below, I attempt to clarify some of these issues so far as taxonomic and systematics studies are concerned. Unfamiliar terms are used as little as possible but, since they are certain to be encountered in other reports and discussions on this type of research, I have appended a glossary of phylogenetic terms to this article.

Much research attention in recent years has been focussed on DNA sequencing, and most noticeable to the public eye has been the immense international effort which has gone into the human genome project; the mapping of every gene on every one of the twenty-three chromosomes we possess. An enterprise which is now getting near to its closing stages.

However besides this chromosomal DNA, often referred to as nuclear DNA, there is another source of DNA in the cells. This is mitochondrial DNA (mtDNA) which is carried in the mitochondria present in the cytoplasm of most cells and, in particular, the female germ cells or ova. Few mitochondria are present in the much smaller male germ cells which have very little cytoplasm. Consequently they are inherited almost exclusively through the female or maternal line. Although some cross-contamination, delightfully known as ‘paternal oozing’ may occur, this is not found to be significant.

Mitochondrial DNA, unlike the more familiar long strands of DNA forming the chromosomes in the nucleus of the cell, is a relatively very small segment which forms into a closed loop. Nevertheless it goes through a similar process of copying at each cell division and mistakes or errors in this process occur in the same way.

Genes on the mitochondria usually have little effect on the organism in whose cells they lie (although instances are known) but do have a major role to play within those individual cells. Mitochondrial DNA is particularly well suited to taxonomic and systematics studies for many reasons including ease of isolation, the possibility of using small sections, and a high mutation rate. This rate varies between different regions allowing the choice of region to be influenced by the time scale under consideration. One region that has been much studied in birds is that containing the cytochrome-b gene.

The basis for the genetic information carried by DNA is the genetic code provided by the sequence of nucleotide bases along the DNA strand. This is a linear triplet code using combinations of three bases from a choice of four labelled A, C, G, and T. Each three letter sequence is known as a codon and may be thought of as a ‘word’. Carrying this analogy forward; a number of successive ‘words’ comprise a genetic ‘sentence’, and this we know as a gene. (See “Speaking the Language of Recombinant DNA” at Access Excellence.)

Genes only occupy a small proportion of the total length of DNA and the intervening spaces are occupied by redundant or 'junk' code. Coding errors, mistakes in the copying process, in these sections are the most frequent to survive since they are unlikely to affect viability.

All this bears some comparison with computer code, with which programmers can do so much, although in this case the code is much more simple in having just two states; 1 and 0, or ‘on’ and ‘off’. Computer code is also often ‘bloated’ by redundancy produced by easy to speak (!) but inefficient program language.

Mistakes or coding errors in the copying process within DNA may involve just one base (letter) or whole strings of bases and involve replacement of one with another, or of deletions and insertions of fragments of varying size. The factor which gives real meaning to all this is that mutation has been found to occur at a constant characteristic frequency over time. This biological ‘time’ has been compared and correlated with geological time.

Scientists are now able to extract DNA, mitochondrial or nuclear, from cells and using biochemical ‘scissors’ cut out matching sections from many individuals for later comparison and analysis. In order to achieve this, techniques which have now become routine and largely automated, are employed to sequence or ‘read’ large numbers of matching sections.

The next stage is the comparison and analysis of the sequenced data both within a species or sub-species and related species or genera, etc. (And no doubt computer programs have also been developed to ease this process.) Knowing the number of errors which have occurred, and the characteristic rate at which these occur over time, a measure of biological and thus geological time can be inferred. Additionally, the degree of relatedness between individuals can be established and compared with morphological, fossil, and other evidence.

Using the information gathered it is now possible to develop a simple ‘tree’ diagram illustrating how from a common ancestor an Order, Family, Genus, or Species, has developed and diverged over perhaps many millions of years under the influences of environmental and biological pressures.

Clive Hesford, April 2000 (minor revisions 30th December 2000)

A Glossary of Phylogenetic Terms

[Reproduced from an excellent Introduction to Cladistics at the University of California]

adaptation: Change in a organism resulting from natural selection; a structure which is the result of such selection.

anagensis: Evolutionary change along an unbranching lineage; change without speciation.

ancestor: Any organism, population, or species from which some other organism, population, or species is descended by reproduction.

basal group: The smaller of two sister groups; often used as the outgroup for a study of the larger clade.

character: Heritable trait possessed by an organism; characters may be described in terms of their states, for example: “hair present” vs. “hair absent”, where “hair” is the character, and “present” and “absent” are its states.

clade: A monophyletic taxon; a group of organisms which includes the most recent common ancestor of all of its members and all of the descendants of that most recent common ancestor. From the Greek word “klados”, meaning branch or twig.

cladogenesis: The development of a new clade; the splitting of a single lineage into two distinct lineages; speciation.

cladogram: A diagram, resulting from a cladistic analysis, which depicts a hypothetical branching sequence of lineages leading to the taxa under consideration. The points of branching within a cladogram are called nodes. All taxa occur at the endpoints of the cladogram.

convergence: Similarities which have arisen independently in two or more organisms that are not closely related. Contrast with homology.

crown group: All the taxa descended from a major cladogenesis event, recognized by possessing the clade’s synapomorphy. See: stem group.

derived: Describes a character state that is present in one or more subclades, but not all, of a clade under consideration. A derived character state is inferred to be a modified version of the primitive condition of that character, and to have arisen later in the evolution of the clade. For example, “presence of hair” is a primitive character state for all mammals, whereas the “hairlessness” of whales is a derived state for one subclade within the Mammalia.

diversity: Term used to describe numbers of taxa, or variation in morphology.

endosymbiosis: When one organism takes up permanent residence within another, such that the two become a single functional organism. Mitochondria and plastids are believed to have resulted from endosymbiosis.

evolution: Darwin’s definition: descent with modification. The term has been variously used and abused since Darwin to include everything from the origin of man to the origin of life.

evolutionary tree: A diagram which depicts the hypothetical phylogeny of the taxa under consideration. The points at which lineages split represent ancestor taxa to the descendant taxa appearing at the terminal points of the cladogram.

extinction: When all the members of a clade or taxon die, the group is said to be extinct.

gradualism: A model of evolution that assumes slow, steady rates of change. Charles Darwin’s original concept of evolution by natural selection assumed gradualism. Contrast with punctuated equilibrium.

homology: Two structures are considered homologous when they are inherited from a common ancestor which possessed the structure. This may be difficult to determine when the structure has been modified through descent.

hypothesis: A concept or idea that can be falsified by various scientific methods.

ingroup: In a cladistic analysis, the set of taxa which are hypothesized to be more closely related to each other than any are to the outgroup.

lineage: Any continuous line of descent; any series of organisms connected by reproduction by parent of offspring.

monophyletic: Term applied to a group of organisms which includes the most recent common ancestor of all of its members and all of the descendants of that most recent common ancestor. A monophyletic group is called a clade.

outgroup: In a cladistic analysis, any taxon used to help resolve the polarity of characters, and which is hypothesized to be less closely related to each of the taxa under consideration than any are to each other.

paraphyletic: Term applied to a group of organisms which includes the most recent common ancestor of all of its members, but not all of the descendants of that most recent common ancestor.

parsimony: Refers to a rule used to choose among possible cladograms, which states that the cladogram implying the least number of changes in character states is the best.

phylogenetics: Field of biology that deals with the relationships between organisms. It includes the discovery of these relationships, and the study of the causes behind this pattern.

phylogeny: The evolutionary relationships among organisms; the patterns of lineage branching produced by the true evolutionary history of the organisms being considered.

plesiomorphy: A primitive character state for the taxa under consideration.

polarity of characters: The states of characters used in a cladistic analysis, either original or derived. Original characters are those acquired by an ancestor deeper in the phylogeny than the most recent common ancestor of the taxa under consideration. Derived characters are those acquired by the most recent common ancestor of the taxa under consideration.

polyphyletic: Term applied to a group of organisms which does not include the most recent common ancestor of those organisms; the ancestor does not possess the character shared by members of the group.

primitive: Describes a character state that is present in the common ancestor of a clade. A primitive character state is inferred to be the original condition of that character within the clade under consideration. For example, “presence of hair” is a primitive character state for all mammals, whereas the “hairlessness” of whales is a derived state for one subclade within the Mammalia.

pseudoextinction: The apparent disappearance of a taxon. In cases of pseudoextinction, this disappearance is not due to the death of all members, but the evolution of novel features in one or more lineages, so that the new clades are not recognized as belonging to the paraphyletic ancestral group, whose members have ceased to exist. The Dinosauria, if defined so as to exclude the birds, is an example of a group that has undergone pseudoextinction.

punctuated equilibrium: A model of evolution in which change occurs in relatively rapid bursts, followed by longer periods of stasis.

radiation: Event of rapid cladogenesis, believed to occur under conditions where a new feature permits a lineage to move into a new niche or new habitat, and is then called an adaptive radiation.

rank: In traditional taxonomy, taxa are ranked according to their level of inclusiveness. Thus a genus contains one or more species, a family includes one or more genera, and so on.

relatedness: Two clades are more closely related when they share a more recent common ancestor between them than they do with any other clade.

reticulation: Joining of separate lineages on a phylogenetic tree, generally through hybridization or through lateral gene transfer. Fairly common in certain land plant clades; reticulation is thought to be rare among metazoans.

selection: Process which favors one feature of organisms in a population over another feature found in the population. This occurs through differential reproduction – those with the favoured feature produce more offspring than those with the other feature, such that they become a greater percentage of the population in the next generation.

sister group: The two clades resulting from the splitting of a single lineage.

stasis: A period of little or no discernible change in a lineage.

stem group: All the taxa in a clade preceding a major cladogenesis event. They are often difficult to recognize because they may not possess synapomorpies found in the crown group.

synapomorphy: A character which is derived, and because it is shared by the taxa under consideration, is used to infer common ancestry.

systematics: Field of biology that deals with the diversity of life. Systematics is usually divided into the two areas of phylogenetics and taxonomy.

taxon: Any named group of organisms, not necessarily a clade.

taxonomy: The science of naming and classifying organisms.

vicariance: Speciation which occurs as a result of the separation and subsequent isolation of portions of an orginal population.