1The idea in one paragraph

There are millions of species, and nobody can study them one at a time, so biologists sort them into groups. The traditional way was a ladder of ranks, from kingdom down to species, built mostly on how organisms look. The trouble is that looks mislead and ranks are arbitrary. Cladistics sorts organisms by ancestry instead: every group, a clade, is one ancestor and all of its descendants, worked out mostly from DNA and protein sequences. A cladogram is the branching diagram that records the result. The same sequence differences, if they build up at a steady rate, also act as a molecular clock for when groups split. Cladistics has shown some old groups to be false and added a whole new level to classification: the three domains.

2Why classify at all

There are about two million named species and many more unnamed. Without a system, each would be an isolated fact. Classification puts them into groups within groups, and that does three jobs.

• It makes diversity manageable. You can learn what a mammal is once, rather than for each of more than 6,000 mammal species.
• It gives every organism a place and a name, so that scientists anywhere know which organism is meant.
• It opens the way to further study. Once an organism is classified, its relatives are known, and research on one can be compared with, and guide, research on the others.

3The traditional hierarchy, and where it breaks

The traditional system, from Linnaeus, places every species in a nested series of taxa (singular taxon), each with a fixed rank. The lion, for example:

It is tidy and still used every day, but it does not always match the pattern of divergence that evolution produced, for three reasons.

Ranks are arbitrary. Evolution produces a continuous branching, with splits at every depth. The ranks pretend that there are exactly seven levels of branching, and that a "family" of beetles is the same kind of thing as a "family" of mammals. Nothing in biology makes those equal: they may differ hugely in age and in number of species.

Groups were built on appearance, and appearance can mislead. Organisms can look alike because they face the same way of life rather than because they are related. That is convergent evolution, and A4.1 covers it.

Some traditional groups leave out descendants. Figure 1 shows the famous case. Traditionally, class Reptilia contains lizards, snakes, turtles and crocodiles, while birds get a class of their own, Aves. But crocodiles share a more recent common ancestor with birds than with lizards. A group that contains crocodiles and lizards but not birds leaves out some descendants of its own common ancestor, so it does not reflect evolution.

Nature of science: a paradigm shift. Cladistics does not repair the ranks; it replaces them with unranked clades. A clade is defined by a branch point, not by being "a class" or "an order". Moving from a fixed ladder to a branching tree is an example of the paradigm shift that sometimes happens in science, when the framework itself changes rather than the details within it.

4Why classify by evolutionary relationships

The ideal classification follows ancestry, so that every member of a group has evolved from one common ancestor. The advantage is prediction. Characteristics are inherited from ancestors, so they are shared within a clade. Discover a new species of bat and, before you have examined it, you can predict hair, milk production and a four-chambered heart, because every bat inherited them from the ancestor of mammals.

This has practical value. The anti-cancer drug paclitaxel was first found in the bark of the Pacific yew. The next place to look for the same or similar compounds was obviously other yews, its closest relatives, and a precursor of the drug is now extracted from the needles of the European yew, so the tree survives the harvest.

A clade is a common ancestor and all of its descendants. Because features are inherited, members of a clade share them, and that makes classification predictive.

5Clades, and the evidence that defines them

A clade is a group of organisms that consists of a common ancestor and all its descendants, and nothing else. Its members share characteristics inherited from that ancestor.

What evidence places organisms in the same clade?

• Base sequences of genes, or amino acid sequences of proteins. These are the most objective evidence. Every organism has DNA, the sequences can be compared position by position, and the count of differences is a number rather than an opinion. Mutations accumulate over time, so the fewer the differences, the more recently two species shared an ancestor.
• Morphological traits, meaning the structure of the body. These are still used, and are the only evidence for fossils. The risk is convergence: two unrelated organisms can evolve similar structures, so morphology is most trusted when it agrees with the molecules.

6Analysing cladograms

A cladogram is read by its branch points, and the guide names the parts you must use. Figure 2 labels them.

• The root is the base of the tree: the common ancestor of everything on it.
• A node is a branch point. Each node represents a hypothetical common ancestor: hypothetical because it is inferred from the living species, not found as a fossil.
• A terminal branch leads to a single species at a tip, living or extinct.
• A clade is any node together with every branch and tip beyond it. In Figure 2, A, B and C form a clade; so do A and B; so do D and E; and so does the whole tree.

How to deduce relationships. Two species are more closely related the more recent their most recent common ancestor, which means the further from the root the node where their lineages meet. In Figure 2, A is more closely related to B than to C, because A and B meet at a later node. C is equally related to A and to B, because it meets both at the same node.

The trap: do not read the order of the tips. Branches can be swung round any node without changing the tree, just as a mobile can turn on its strings. Figure 3 draws one cladogram two ways.

In (a) the frog sits next to the salmon; in (b) it sits next to the lamprey. Neither placement means anything. What matters is the nodes: salmon and shark meet first, the frog joins them next, the lamprey last. Two tips that are drawn side by side may be distantly related.

One more point to state in an answer. No living species on a cladogram is the ancestor of another. Humans did not evolve from chimpanzees; the two share a common ancestor at a node, and both have evolved since.

7The molecular clock

Mutations in a gene accumulate gradually. If they build up at a roughly steady rate, the number of sequence differences between two species is a measure of how long ago they diverged from their common ancestor. This is the molecular clock.

The clock has to be calibrated, usually with a pair of species whose split is dated from fossils. Figure 4 plots a calibration line from invented data.

Worked example. Two species known from fossils to have diverged 20 million years ago differ at 30 bases in a 1,000-base gene. A new pair differs at 48 bases in the same gene. When did the new pair diverge?

Why it gives only estimates. The guide lists the reasons, and each is worth a mark.

• Generation time. Most mutations arise when DNA is copied. A species with short generations copies its DNA more often per year, so its clock runs faster.
• Population size. Chance plays a larger part in which mutations spread in a small population, so the rate at which differences become fixed varies with population size.
• Intensity of selection. A gene doing an essential job tolerates few changes, so its clock runs slowly; a gene under little selective pressure changes faster. Different genes tick at different rates.
• Other factors, including uncertainty in the fossil dates used for calibration, and the same site mutating more than once, which hides earlier changes.

So a molecular clock answer should always say "about" and should compare pairs using the same gene.

8Building a cladogram from sequences: parsimony

Given sequences, how do you turn them into a tree? The guide asks for simple examples, so here is one worked in full. Four invented species, P, Q, R and S, and an outgroup O are compared at ten positions of a gene. An outgroup is a species known from other evidence to have branched off before the rest. It roots the tree, and it tells you which base is probably the ancestral one.

Step 1: count differences between each pair. This alone often gives the answer.

P and Q differ at only one site, so they share the most recent common ancestor. R is next closest to them. S is the most different, and it is also the closest to the outgroup (3 differences), so it branched off first.

Step 2: read the sites that group species.

• Sites 1 and 9 are the same everywhere. They tell you nothing.
• Site 2: every ingroup species has C where the outgroup has T. One change, on the stem leading to all four.
• Sites 3 and 8: P, Q and R share a new base. That supports a clade (P, Q, R).
• Sites 4 and 5: P and Q share a new base. That supports a clade (P, Q).
• Sites 6 and 10: a change in one species only. Each costs one change on any tree, so they cannot tell trees apart.
• Site 7: Q and S share C. That seems to say Q and S are related, and it conflicts with sites 3, 4, 5 and 8.

Step 3: choose by parsimony. Parsimony analysis selects the cladogram that explains the observed differences with the smallest number of sequence changes. Figure 5 counts them for two candidates.

Tree (a) needs 9 changes. It explains site 7 by the same change happening twice, once in Q and once in S. Tree (b) groups Q with S to explain site 7 with one change, but then sites 3, 4, 5 and 8 each need two changes, and the total rises to 12. Tree (a) is preferred. In fact, of all fifteen possible trees for four species rooted on this outgroup, tree (a) is the only one with 9 changes.

Nature of science: criteria shape the hypothesis. Parsimony is a criterion for judgement, not a proof. It assumes that changes are rare, so the simplest explanation is the most probable. A different criterion, such as a statistical model that allows some sites to change faster than others, can pick a different tree from the same data. The cladogram is therefore a hypothesis about relationships, and different criteria for judgement can lead to different hypotheses.

9Testing a classification: the figwort family

Cladistics can check whether a traditional group corresponds to evolutionary relationships. The guide suggests the figwort family, Scrophulariaceae, as a case study. You do not need to memorise its details, but you do need to be able to explain what it shows.

Traditionally the family was large and held together by look-alike flowers: tubular, two-lipped, with similar fruits. It included figworts and mulleins, foxgloves, snapdragons and speedwells, part-parasitic plants such as yellow-rattles, and monkeyflowers. When DNA sequences from many of these plants were compared, they did not form one clade. They fell into several separate clades, some more closely related to other families than to each other. Figure 6 shows the outcome.

Foxgloves, snapdragons and speedwells were moved to the plantain family, Plantaginaceae; the part-parasites to the broomrape family, Orobanchaceae; monkeyflowers to Phrymaceae. Scrophulariaceae kept the figworts and mulleins, and gained some plants from elsewhere, and is now a much smaller family.

Nature of science: falsification. The old family was a scientific claim: "these plants share a common ancestor that others do not". Cladistics tested it and showed it to be false. The similar flowers had evolved separately, by convergent evolution, not by shared ancestry. Theories and other knowledge claims can always be falsified by new evidence, and this is what that looks like in practice. A Paper 2 answer that names convergent evolution as the cause of the false grouping, and sequence evidence as what exposed it, has the central marks.

In an exam, either the common name or the scientific name of an organism is accepted.

10Three domains, from rRNA

Until the 1970s, life was split first into prokaryotes and eukaryotes, by cell structure. In 1977 Carl Woese and George Fox compared the base sequences of ribosomal RNA (rRNA) from the small subunit of the ribosome, taken from many organisms. rRNA suits the job: every organism has it, it has the same essential function in all of them, and it changes slowly, so it can reveal very ancient splits.

The result was revolutionary. The prokaryotes did not form one group. They fell into two, as different from each other in rRNA sequence as either was from the eukaryotes. One group became the Bacteria. The other, first called archaebacteria, became the Archaea. A new taxonomic level was added above the kingdoms to hold them: the domain. Every organism belongs to one of three: Bacteria, Archaea or Eukarya. Figure 7 shows the change.

Two things to know about Archaea. They look like bacteria under the microscope, small with no nucleus, which is why they were missed; their rRNA, their membrane lipids and several of their enzymes are different. And many first known from extreme places such as hot springs, very salty lakes and oxygen-free mud, where some produce methane, although archaea are now known to be common in ordinary soils and oceans too. The rRNA tree also shows archaea sharing a more recent common ancestor with eukaryotes than with bacteria, as Figure 7 draws.

The debate continues: some recent sequence studies suggest that eukaryotes arose from within the Archaea, which would leave two domains, not three. Learn the three-domain system for the exam; the argument is good material for section 11.

11Linking questions

How can similarities between distantly related organisms be explained? By convergent evolution: similar selection pressures produce similar structures in unrelated lineages (A4.1), which is what misled the classification of the figworts. In prokaryotes, a second answer is horizontal gene transfer (A3.1), which can place the same gene in distantly related species.

What are some examples of ideas over which biologists disagree? Whether there are two domains or three; whether two populations are one species or two (A3.1); how many species a group contains, since "splitters" recognise more than "lumpers" (A4.2); and which of two nearly equal cladograms is correct. Disagreement of this kind is normal science: each is settled, or not, by more data.