2. 3.2.9 Originally, classification systems were based on observable features but more recent approaches draw on a wider range of evidence to clarify relationships between organisms. Genetic comparisons Genetic comparisons can be made between different species by direct examination of their DNA or of the proteins encoded by this DNA. DNA Comparison of DNA base sequences is used to elucidate relationships between organisms. These comparisons have led to new classification systems in plants. Similarities in DNA may be determined by DNA hybridisation. Proteins Comparisons of amino acid sequences in specific proteins can be used to elucidate relationships between organisms. Immunological comparisons may be used to compare variations in specific proteins. Candidates should be able to interpret data relating to similarities and differences in base sequences in DNA and in amino acid sequences in proteins to suggest relationships between different organisms. Behaviour Courtship behaviour as a necessary precursor to successful mating. The role of courtship in species recognition.

3. Classification systems Classification keys are available to enable identification of known living organisms. Usually the kingdom and phylum are obvious to amateurs and experience soon enables further identification. Sometimes a specialist is necessary to identify the exact genus and species. Due to modern advances in genetic sequencing, it is now possible to analyse gene sequences and calculate the percentage homology (matching sequences) between DNA cell samples. As proteins result from gene expression similarities of the protein primary sequence of common polypeptide between many species can be compared. Immunological comparisons can be made for protein tertiary structure.

4. Genetic comparisons using DNA and Proteins Originally classification systems were based on observable features of organisms. As technology has developed, so have the methods to determine evolutionary relationships. We know now that an organism’s observable features are a result of specific sequences of DNA nucleotides being encoded into specific sequences of amino acids to make proteins. Thus: Changes in DNA (mutations) ultimately lead to changes in observable features and thus different species. Similar species will share more similar (conserved) DNA nucleotide sequences AND protein amino acid sequences Distantly related species will have greater differences in DNA sequence/ protein sequences

5. Comparing DNA sequence between species We have the technology to sequence DNA (i.e. sequencing DNA enables us to know the specific sequence of nucleotides). We can use computers to align and compare such sequences as shown in the diagram below. During evolution, when one species gives rise to another, the DNA sequences of the new and the original species will be very similar to each other. Over time, the number of mutations that have occurred in the new species will increase and therefore there will be more differences in the DNA sequences of the two species. As a result, you would expect species that are more closely related (diverged recently) to have more similarities in their DNA base sequences than species that are distantly related.

6. Homology of DNA.

7. DNA comparison of DNA base sequences is used to elucidate evolutionary relationships between organisms. One method of comparing is to simply decode equivalent DNA portions and compare sequences.

8. The product of gene expression is protein. Genetic comparisons Genetic comparisons can be made between different species by direct examination of their DNA or of the proteins (including ENZYMES) encoded by this DNA.

9. Questions Suggest and explain your reasons as for what might be a possible gene product (i.e. protein) from this comparative strip of DNA?

10. Use the sequences shown to calculate the % homology in this comparative sequence between: Homo sapiens and Methanococcus Homo sapiens and E.coli Methanococcus sp. and E.coli What conclusions can you draw from this data?

11. Answers Two best examples include DNA synthesis (as all living organisms need to synthesis DNA) e.g. Ribosomes and respiration as all living cells need to produce energy e.g. proteins involved in ATP synthesis (e.g. Cytochrome C). Could be others – as long as you can argue that they are in both species. 2a) 83/ 110 x 100 = 75.5% 2b) 75/110 x 100 = 68.2% 2c) 85/110 x 100 = 77.3%

12. Although a clear difference in similarities, as you would expect, (i.e. bacteria more related to each other than to human), it should be a surprise how similar bacterial DNA and human DNA are to each other. This must be a piece of DNA essential to BOTH human and bacterial life. E.g. Ribosomal DNA/Cytochrome C – i.e. very little difference and so essential it has not been allowed to diverge/mutate.

13. DNA Hybridisation – a means of determining DNA similarity Another method used to compare the DNA of two species is that of DNA hybridisation. Bear in mind that when DNA is heated the double strands separate into 2 single stands. When cooled they recombine. If enough time is given then all strands in a mixture of DNA will pair up with their partners.

14. DNA hybridisation can be used to compare the DNA of 2 species in the following way: PART I: The DNA from two species is isolated and cut into small pieces One species is labelled by attaching radioactive or fluorescent markers DNA of the 2 species is mixed together and Heated (this splits the double helix into single stranded DNA) The mixture is cooled (so complimentary nucleotides can bind together) The single strands from each species bind together. Some of the double strands that reform will be made up of 1 strand from each species = hybridisation. The new strands are called hybrid strands. They can be identified as 50% of them will be labelled with markers. – Highly similar species will have numerous similar DNA binding nucleotides – Species with low similarity have very few nucleotides that bind together

16. PART 2: The DNA hybrids (i.e. one strand from each species) are isolated Remembering that nucleotides are bound together by HYDROGEN BONDS Thus the more nucleotides in similar, the more hydrogen bonds present HEAT ENERGY is needed to separate the hybrid DNA strands The isolated DNA hybrids have heat increased gradually Hybrids from distantly related species will split at lower temperatures Hybrids from closely related species require high temperatures to split

17. DNA Hybridisation. Similarities in DNA may be determined by DNA hybridisation. DNA becomes single stranded (ssDNA) on heating then recombines on cooling.

19. Results. The temperature is correlated with the degree to which the two strands remain combined. Two species that are closely related will share more complementary nucleotide bases than more distant relatives, thus there will be more hydrogen bonding within the hybrid strand. More bonds – stronger DNA therefore more heat resistant. Higher temperature to form ssDNA from hybrids = closer relationships. Lower temperatures = distant relatives.

20. Questions During the process o DNA hybridisation explain the following: Why DNA is heated Why some hybrid strands require a higher temperature to separate the two strands than others The significance of the differnce, described in b, in determining the relationship between species, What causes the DNA sequences of genes to change over a period of time?

22. Answers 4.a. to separate the 2 complimentary strands of DNA molecule. b. Because more complementary bases are joined together and therefore more energy is needed to break the hydrogen bonds linking them. c. the more complementary bases are joined, the more similar are the DNA strands of the 2 species. The more similar the DNA is the more closely they are related. mutations – changes in the base sequence of DNA a) The temperature at which the hybrid DNA denatures tells us how similar the two strands are. Temperature is just kinetic energy, kinetic energy vibrates molecules ‘pulling apart’ the hydrogen-bonds. The more hydrogen-bonds the more energy needed – thus highest temperature between human/human – most similarity. ai) Orangutan – lowest temperature therefore least similarity. aii) explanation as above for 6a i.e. less similarity means LESS h- bonds thus LESS energy needed to denature hybrid DNA

23. To interpret this data- understand the graph Samples from the Human/other species DNA mixture are taken at each five degree increase in temperature then analysed to see how much (%) of the DNA has become single stranded.

24. Interpretation and analysis. Order the species in closeness to the human DNA. 1 2 3 4 Which two species are closest to each other and explain.

25. Comparison of amino acid sequences in specific proteins You must choose a protein(s) that all of your comparative organisms possess Proteins can be used as well as DNA. Comparisons of amino acid specific sequences in specific proteins can be used to determine relationships between organisms. The specific sequence of amino acids in proteins is referred to as the PROTEIN PRIMARY STRUCTURE and is determined by DNA. The number of similarities and/or differences in amino acid sequences can be calculated. Evolutionary patterns can be determined by mapping the number of similarities in the primary structure of a protein common to all the organisms being mapped.

27. Proteins are the products of gene expression. Proteins can be used too. Comparisons of amino acid sequences in specific proteins can be used to elucidate relationships between organisms. The sequence of amino acids in proteins referred to as the PROTEIN PRIMARY STRUCTURE is determined by DNA. Either the number of similarities or the differences can be calculated.

28. Alpha and beta chains of Haemoglobin (Hb) are varied in structure (between species) as organisms have adapted (through mutations) to different environments, demanding relatively different oxygen carrying capacities of the Hb. The following diagram shows the % homology between haemoglobin alpha chains between 6 species.

31. 8) The sequences of amino acids in haemoglobin molecules have been used to clarify the evolution of primates. The amino acids found in seven specific positions in the haemoglobin molecule of 6 different primates were compared. The results of the study are shown below. Where the amino acid differs from that in human haemoglobin, the letter is shown in red. Use this information to list the evolutionary relationship of humans to the other primates shown, Start with the most closely related primate and end with the most distantly related.

33. 9)

34. What evidence is there from the table to show humans are more closely related to orang-utans than lemurs? Do these data support the evolutionary relationships of these primates suggested by the study above?

35. Answers 7 (a) Because all organisms in this study have this same protein. 7 (b) From this data at LEAST 29 x 3 = 87 base pairs (as maximum number of amino acid differences between Human/fruit fly) 7 (c) because Cytochrome C is essential to all organisms in study, likely that there will be much conservation of sequence between species 8) BASED ON AMINO ACIDS: Chimpanzee identical according to THIS SEQUENCE of amino acids Then – Gorilla – 2x differences Then – Orang-utan – 4x differences Then – Lemur – 5x differences Then – Gibbon – 6x differences (or 1x ONLY in common) 9a) Note figures are DIFFERENCES. Thus less differences (29) between Human and Orang-utan than (48) between Lemur and Human Less differences means MORE CLOSELY RELATED 9b) There are some similarities and some differences. Gorilla/chimpanzee relatedness to humans – in DNA Gorillas MOST related to humans Amino Acid shows that Chimpanzee MORE related to humans. Lemur LEAST related to humans in both studies

36. Immunological comparisons of proteins Antibodies responding to Antigens on proteins Idea behind this method is that antibodies of one species will respond to the antigens found on proteins, such as albumin, (Found in the blood serum) of another species. Basically the protein from species A will be injected into species B. Species B will produce antibodies against A These antibodies will then be injected into the species to be compared.

37.  Serum from species B is then mixed with the blood of a third species.  The antibodies respond to the corresponding antigens on the albumin for species C.  The response is the formation of a precipitate  Serum albumin from A is injected into B.  Species B will produce antibodies specific to all the antigen sites on the albumin from species A  Serum is extracted from species B. This serum contains antibodies specific to the antigens for species A

38. The GREATER the number of similar, the MORE precipitate is formed and the more closely the species are related. The FEWER the number of similar antigens, the LESS precipitate is formed and the more distantly the species are related.

40. Hence the more precipitate formed the closer the evolutionary relationship.

41. Ab/Ag interactions – tertiary structure. Immunological comparisons may be used to compare variations in specific proteins. This method exploits antibody/antigen binding interactions between species. Antibodies in one species are created against antigens from another. In the example given the antigen is part of the serum (plasma) protein albumin found in blood plasma (serum).

42. Immunological Comparisons; Compares tertiary 3-D shape of proteins.

43. Questions 10) Using the information in the figure above, state with reasons which 2 species is most closely related to humans. 11) Scientists studied two species of North American seahorse. They thought that these two species were closely related. Describe how comparisons of biological molecules in these two species could be used to find out if they are closely related. 12) A study compared the proteins found in a variety of primates using immunological techniques. The results of this study are shown below. Use these results to answer the following questions: a) Which two primates does this immunological study suggest are the most closely related? Give reasons for your answer. b) Which primate does the study suggest is the nearest relative of the orang-utan? Give reasons for your answer. c) The data on the previous page (Q9) shows the evolutionary relationships between humans and five other primates. In what two ways do these relationships differ from that suggested by the haemoglobin study,

45. Answers 10. The chimpanzee – there is more precipitate formed when the serum (from the human) is added to the chimpanzee serum. This shows that there are more similar antigens and the antibodies have bound to these antigens causing a precipitate to form. 11. Compare) DNA; Sequence of bases/nucleotides; DNA hybridisation; Separate DNA strands / break hydrogen bonds; Mix DNA/strands (of different species); Temperature/heat required to separate (hybrid) strands indicates relationship; Compare same/named protein; Sequence of amino acids /primary structure; Immunological evidence – not a mark Inject (seahorse) protein/serum into animal (Obtain) antibodies/serum; Add protein/serum/plasma from other (seahorse) species; Amount of precipitate indicates relationship 12a. Human / Chimpanzee and Human / Gorilla (both 95% precipitation). 12b. Gibbon. Closest amount of precipitation between Orang-utan and Gibbon. 12c. Haemoglobin suggests 100% between human/chimpanzee, Immunology suggests only 95% Immunology suggests Chimpanzee and Gorilla identical whereas haemoglobin suggests difference. Immunology suggests Lemur is LESS related to human than Gibbon - whereas haemoglobin suggests Lemur is MORE related to human than Gibbon.

46. You should be able to explain how these molecular biology methods can be used to determine evolutionary relationships. 1. Comparison of DNA base sequences. 2. DNA hybridisation. 3. Comparisons of amino acid sequences. 4. Immunological comparisons.

47. Courtship behaviour Similarities and difference in the physical and biochemical make up of organisms helps them to distinguish between members of their own species from those of other species. The same is true of behaviour. The behaviour of members of the same species is more similar than that of members of different species. This allows individuals to be able to identify members of their own species by the way that they act. As with physical features, behaviour is determined by DNA and it influences the chances of individuals surviving. Therefore, in order for a species to survive courtship and mating are required.

48. The role of courtship in species recognition Enables recognition of members of their own species, hence promoting the chance of fertile offspring being produced. Identifying a mate capable of breeding i.e. sexually mature, fertile and receptive. Form a pair bond to lead to successful mating and rearing of the offspring. Synchronise mating to maximise the probability of fertilisation and zygote formation and development of offspring.

49. The females of many species undergo a cycle of sexual activity and they are only able to conceive during a short period of time. They are often only receptive mating around the time when they produce eggs. Courtship behaviour is used by males to determine whether the female is receptive. If the female responds appropriately, courtship continues and is likely to lead to the production of offspring. If she is not receptive, the female exhibits different behaviour and the male ceases to court her and turns his attentions elsewhere.

50. Rituals. The courting pair will undergo a stimulus-response chain ritual that progressively leads them closer to mating. Generally, the male carries out an action which acts as a stimulus to the female. The female responds with an action of her own. The response of the female acts as a stimulus to the male to carry out further action The chain is performed in the same way for all members of the SAME species. Other species will differ in their actions. This allows individuals to recognise members of the same species. The longer the courtship, the more likely mating is to occur. If either one of the pair fails to respond appropriately at any point, the courtship sequence ends.

51. Rituals The courting pair will undergo a stimulus-response chain ritual that progressively leads them closer to mating. The chain is performed in the same way for all members of the same species. Other species will differ in their actions.

52. Mallard Ducks. The male has about 10 different moves to his courtship ritual including tail shaking, bill shaking, grunt whistling, head flicking, nod- swimming, and raising head/tail.

53. Females are often only fertile for short periods of time so to ensure successful mating and that the offspring have a maximum chance of survival courtship behaviour may occur.

54. Mallard ducks courtship. The male has about 10 different moves to his courtship ritual including tail shaking, bill shaking, grunt whistling, head flicking, nod swimming, and raising head/tail. Below the courtship behaviour of six male ducks is summarised.

56. Courtship Behaviour of Mallard Duck Duck A Shakes tailShakes billGrunt whistlesFlicks headRaises head/tailTurns to the female Duck B Shakes tailShakes billGrunt whistlesFlicks headRaises head/tailTurns to the female Duck C Shakes tailShakes billGrunt whistlesFlicks headTurns to the female __________ Duck D Shakes tailShakes billGrunt whistlesNods and swimsRaises head/tailTurns to the female Duck E Shakes tailShakes billFlicks headGrunt whistlesRaises head/tailTurns to the female Duck F Flicks HeadShakes tailNods and swimsGrunt whistlesTurns to the female ______

57. Advantages of courtship. Enables recognition of members of their own species, hence promoting the chance of fertile offspring being produced. Identifying a mate capable of breeding. Partners need to be sexually mature, fertile and receptive. Form a pair bond to lead to successful mating and rearing of the offspring. Synchronise mating to maximise the probability of fertilisation and zygote formation and development of offspring.

58. Questions 13. Why do different species of duck have different courtship displays? 14. How many different species of duck are represented on the table? 15. Suggest why the courtship rituals provide evidence that ducks B and C are closely related? 16. Which duck does the evidence from the table suggest is only distantly related to the others? Give your reasoning. 17. When a female Mallard chooses a male she performs characteristic head movements and calls. Suggest a possible reason for this behaviour.

59. Answers 13. Recognition of own species to provide FERTILE offspring. Identify SEXUAL maturity. Synchronise mating to maximise FERTILITY. Bond pairing. 14. FIVE – note all different except Duck A and B. 15. Every ritual the same except Duck C does not Raise head/tail. This would suggest that one species has either lost a part of a ritual OR one has gained a new part of a ritual. Either way suggests closely related. Sequence or order is important to compare as well as the acts/rituals. 16. Duck F. Its rituals have the LEAST commonality to any of the other duck species shown. 17. Recognition and confirmation of same species. Confirmation of Sexual maturity. Confirmation of fertility to maximise potential of reproducing.

60. Beware Anthropomorphism in Animal Behaviour Descriptions Anthropomorphism is a term coined in the mid 1700s to refer to any attribution of human characteristics (or characteristics assumed or believed by some to belong only to humans) to animals or non-living things, phenomena, material states and objects or abstract concepts. E.g. At the end of successful courtship paired mallard ducks don’t make love, they copulate!

61. Don’t apply human traits to animal Behaviour Behaviour Courtship behaviour as a necessary precursor to successful mating. The role of courtship in species recognition. When is comes to survival of the species (rather than individuals) courtship and mating are essential. For a species to survive DNA must be passed on as no individual lives forever.