1. CHARACTERS USED IN RECONSTRUCTING PHYLOGENETIC TREES 1. Morphological “ Tiktaalik is the sister group of Acanthostega + Ichthyostega in one of the two most parsimonious trees, and clades with Elpistostege as sister to the tetrapods in the other. “Consistency index excluding uninformative characters = 0.7966”.” “Consensus tree from a phylogenetic analysis of 114 characters and nine taxa” Daeschler et al. Nature 440:757, 2006 Discovery of a Devonian fossil fish that is intermediate between fish with fins and tetrapods with limbs

2. 2. Protein analysis Allozymes - variant enzyme forms encoded by different alleles at a single locus Electrophoretic profile of allozymic forms of six esterases from six individuals in Drosophila virilis population Forms 2 & 4 show slow (S) and fast (F) migration Avers Fig. 6.16

3. Mitochondrial DNA from 18 different eels cleaved with EcoRI M M = size marker Avise,JC Fig. 3.8 (i) RFLPs (restriction fragment length polymorphisms) Restriction maps of primate nuclear rRNA spacer region (iii) AFLP (amplified-fragment length polymorphisms) PCR based methods 3. DNA analysis

4. How to choose which type of DNA sequence to use? If comparing very divergent organisms: - need ones “universally” present and highly conserved otherwise possible errors in alignment, multiple sub… eg. ribosomal RNAs translation elongation factors, ribosomal proteins glycolytic pathway enzymes (ii) DNA sequencing – align sequences and score nt substitutions

5. Nature of nt substitutions in structural RNA genes E.coli 16S rRNA Brown Fig.11.11 - different functional constraints than for protein genes RNA folding – 2 o and 3 o structure, interactions with proteins in ribosome… - conservation of helical structure important (but not nt sequence in some cases) C - G A - U U - A G - C C - G G - C U - A G - C compensatory base changes

6. If comparing very closely related organisms: need rapidly evolving sequences nuclear pseudogenes mitochondrial genes in animals Red – protein & rRNA genes Blue – tRNA genes Lewin Fig. 25.5 Human mitochondrial genome

7. Advantages of using animal mitochondrial sequences High rate nt substitution Maternally inherited, no recombination Easy to isolate & assay - many identical copies per cell Different regions evolve at different rates ribosomal RNA respiratory genes replication origin (D-loop) Biodiversity survey – mt DNA (COI) barcodes (P. Hebert) Small, well-characterized genome, no repetitive DNA Lewin Fig. 25.5 relatively slow relatively fast

8. Fig. 5.37 Dusky seaside sparrow 1966 - A.m. nigrescens subspecies on Atlantic coast of Florida endangered species 1980 - only 6 individuals (males) left - artificial breeding program - chose A.m. peninsulae (female) on Gulf coast based on morphological & behavioral characters

9. 1989 – mitochondrial DNA restriction analysis for 39 individuals, 11 subspecies - Atlantic coast ones more similar to each other than to any Gulf Coast subspecies - estimated divergence time consistent with change in sea level creating Florida peninsula ~ 0.5 Mya – reproductive barrier 1987 – last purebred sparrow died Fig. 5.38

10. Moore Fig. 37.8 PHYLOGENETIC RELATIONSHIPS OF HUMANS AND APES 1950’s - morphological data Human – gorilla split believed to be ~ 20 Mya … and chimp – gorilla split much later

11. Human – gorilla – chimp trichotomy 1960’s – 1980’s Protein & early molecular data Freeman Fig. 14.2 Human-gorilla divergence time ~ 5 Mya

12. Freeman Fig. 14.4 1990’s – molecular data Mitochondrial DNA sequences Y chromosome gene autosomal pseudogenes & nuclear non-coding Human – chimp split more recent than gorilla split “statistically, this tree is about 10 23 times as likely as the alternative tree…”

13. Mitochondrial COII gene - haplotypes cluster together by species Branch lengths = minimum number of inferred changes Maximum parsimony tree Locke, Nature 469:529, 2011 Freeman Fig. 14.6 Scally, Nature 483:169, 2012

14. Langergraber PNAS 109:15706, 2012 Divergence times based on generation times of wild chimps & gorillas... (ie. independent of fossil record or molecular data) “Paler shading indicates the range of split times inferred in this study” - compared average generation times among H, C, & G and applied “recent estimates of the human mutation rate per generation to derive estimates of split times of great apes and humans that are independent of fossil calibration.”

15. K ij = # sub per 100 sites between species i and j ** difference significant at 1% level Are sequences from different primates evolving at same rate? 23