1. More molecular phylogenetics applications
Xuhua Xia

2. “Barcode of life”
Quick, inexpensive way to identify species using short DNA sequences that are universally present among organisms of interest
For animal kingdom, part of mitochondrial COI gene is PCR-amplified with primers that map to conserved coding region & then sequenced
“[The authors] imagine a day when a handheld scanner (similar to a GPS device) will link to a database of the barcodes of all species. Then, by inserting a snippet of tissue into the scanner, anyone can get an instant identification of a creature or plant.”
Paul Hebert
U. Guelph
Stoekle & Hebert Scient Amer Oct. 2008

3. Bucklin Ann Rev Marine Sci 3:471, 2011
“DNA barcoding” using mitochondrial cytochrome oxidase subunit I (COI) gene
Unrooted tree
Bucklin Ann Rev Marine Sci 3:471, 2011
“Distance-based analysis of barcodes [COI] for marine zooplankton from Sargasso Sea. Tree was determined using the Neighbor Joining algorithm and Kimura-2-Parameter (K2P) genetic distances and was bootstrapped 1,000 times. A total of 328 individuals of 207 species were barcoded.
Kimura-2-parameter model corrects for multiple hits taking into consideration transition vs. transversion sub rates
Slight potential pitfall with COI barcode: distinguishing “true” mitochondrial gene from pseudogene copies in nucleus (NucMt)

4. Human-gorilla-chimp divergence time ~ 5 Mya
Phylogenetic Relationships of Humans and Apes
Prior to 1960’s, human-gorilla split believed to be ~ 20 Mya, and gorilla-chimp split much later
1960’s – 1980’s
Protein & early molecular data
Apes
Man
13 Mya
25 Mya
trichotomy
Human-gorilla-chimp divergence time ~ 5 Mya
Old textbook
Freeman Fig. 14.2

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

6. More recently, data from many individuals...
... and from whole genome sequencing
Gorilla
Human
Chimps
4-6 Mya
6-8 Mya
Locke, Nature 469:529, 2011
Mitochondrial COII gene
Gorilla genome sequenced (2012)
“Branch lengths = minimum number of
inferred changes”
~ 10 Mya
Maximum parsimony tree
~ 6 Mya
- haplotypes cluster together by species
Average % nt divergence of genomes
Freeman Fig. 14.6
Scally, Nature 483:169, 2012

7. (ie. independent of fossil record or molecular data)
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.”
Langergraber PNAS 109:15706, 2012

8. ** difference significant at 1% level
Are sequences from different primates evolving at same rate? 23
Kij = # sub per 100 sites between species i and j
** difference significant at 1% level
Nt substitution rate higher in African
monkeys than in humans
Human 2
African
monkey 1
New World
monkey 3
“hominoid slowdown” ?
Partly due to generation time effect?
Old World monkeys (11.4 years), chimpanzees (22 years), humans (28 years)
relative rates test
DNA repair efficiency…?

9. “Loss and recovery of wings in stick insects”
Were the ancestors of these insects likely winged or wingless?
Whiting Nature 421: 264, 2003
Male
Female
... based on status of early-diverging lineages, they had wings
Last common ancestor
Winged
Loss of wings
= derived trait
... because all insects examined from those lineages have wings
occurred after that lineage diverged from “pink” common ancestor
Blueprint for wing formation still present
but genes “silenced” in some cases?
...dramatic D phenotype because of changes in gene regulation or signalling pathways?
Partially
winged
Selective pressures due to lifestyle or
environment can lead to reactivation...?
Were the ancestors of insects in this clade likely winged or wingless?
Male vs. female
differences
but “re-evolution” of wings in some cases!!
Very close
relatives differ
in wing status
Wingless
Phylogeny based on molecular data
Morphological data
18S rDNA & 28S rDNA (ribosomal RNAs) & histone sequences

10. Ancient DNA 8 clones of PCR-amplified mtDNA from
26,000 year old cave-bear bone
“Note that direct sequencing would lead to ambiguous results at least at two positions (arrows)”
Forms of DNA damage likely to
affect ancient DNA
C/G to T/A changes due to deamination of C residues?
“Assuming neutral pH, 15oC … take about 100,000 years for hydrolytic damage
to destroy all DNA…. Some environmental conditions could extend this time limit…”
“ … to consider amplification of DNA molecules older than one million years of age
is overly optimistic.” (Svante Paabo lab)
Slide 10
Xuhua Xia
Hofreiter, Nat.Rev.Genet. 2:353, 2001

11. “The future of ancient DNA: Technical advances and conceptual shifts”
150 bp
25bp
100% (ie. not degraded)
< 0.1% left
“heat map” plot
Expected survival of DNA after 10,000 years for 150 bp fragments or 25 bp fragments
DNA “from permafrost-preserved hair, about 4,000 years old, of a male palaeo-Eskimo of the Saqqaq culture, the earliest known settlers in Greenland.”
Rasmussen et al. Nature 463:757, 2010
Cumulative number of ancient genomes published
Hofreiter et al. Bioessays 37:284, 2015

13. Tasmanian wolf
Classified as relative of South American marsupials (based on morphology)
… but PCR of mtDNA from museum
specimen …
Tasmanian wolf (T. cynocephalus)
Aus. tiger cat (D. maculatus)
Tasmanian devil (S. harrisii)
Fig. 5.35
Slide 13

14. Dispersal of modern human population
“Out-of-Africa mitochondrial Eve” hypothesis
Hartl & Jones Fig
Slide 14
Xuhua Xia

15. Otzi the Iceman
5,300 year-old frozen mummy from Italian Alps found in 1991
Tattoos are from charcoal rubbed into cuts on body (healing therapy)
“New insights into the Tyrolean Iceman’s origin and phenotype as inferred by whole-genome sequencing”
Nature Comm 3:698, 2012
“The research helps flesh out a picture of the iceman, who had brown hair, type-O blood, the lactose intolerance that was common among Neolithic agrarian societies.”
Exhibition at South Tyrol Museum of Archaeology

16. “Otzi’s last meals: DNA analysis of the intestinal content…”
“Bootstrapped neighbor-joining tree” (mitochondrial 12S rDNA) of selected animals
“Bootstrapped neighbor-joining tree” (nuclear 18S rDNA spacer) of selected grasses
Did not choose mitochondrial DNA, because nt sub rate is very slow in plants
Intergenic regions in ribosomal RNA gene clusters evolve rapidly (see Practice set #2, question #2)
Rollo Proc Natl Acad Sci 99:12594, 2002

17. Helicobacter pylori bacteria in stomach can cause ulcers
“… arthritis, spinal damage, tooth decay, lactose intolerance…. and a murderous arrow wound…”
“researchers have used geographically distinct bacterial strains to reconstruct human migrations”
“Otzi’s H.pylori is the oldest specimen ever sequenced”
Ottawa Metro Jan.10, 2016

18. divergent because not under functional constraint
Nature,1993
- but in fact this turned out to be contaminating “modern” DNA
In some cases, mistakes were due to organellar DNA fragments (pseudogenes) integrated into “modern” nuclear genome,
divergent because not under functional constraint
“It is now a matter of when, not if someone will produce a genome from an Arctic animal buried in permafrost for longer than 1 million years” (Nature 507: 414, 2014)

19. Macaque
Gorilla
Human
Stedman Nature 428:415, 2004

20. MYH16 (member of myosin multi-gene family) 2 nt deletion
Non-human
Human
stop codon
2 nt deletion
= frameshift mutation
…so MYH16 is a pseudogene in humans
Position of indel
Stedman Nature 428:415, 2004

21. ...compared rates of non-synonymous vs. synonymous nt substitution
To assess degree of functional constraint on MYH16 in different primates...
ie. gene is under strong functional constraint
ie. no non-syn subs
(lack of info)
same rate at syn & non-syn sites
(no selective pressure)
dN / dS
...compared rates of non-synonymous vs. synonymous nt substitution
KA / KS
Estimated time of MYH16 inactivation ~ 2.4 Mya
Stedman Nature 428:415, 2004