1 Ancient DNA: would the real Neandertal please stand up? Eur. - - PDF document

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Ancient DNA: would the real Neandertal please stand up?

Multiregional continuity model:

• Transition between archaic and modern forms took place within a single evolutionary lineage.
• The single lineage is composed of geographic sub-populations connected by gene flow.
• Gene flow prevents independent evolution in the sub-populations
• The lineage originated in Africa about 2 million years ago in Homo erectus
• H. erectus left Africa and disperses into other parts of the world
• Regional variation reflects natural selection for local adaptations
• H. sapiens emerged from a lineage-wide process of evolution
• Archaic forms of Homo are subspecies (e.g., H. sapiens neanderthalensis)

Replacement model:

• H. sapiens evolved as a new species in a sub-population (probably Africa)
• The H. sapiens lineage originated about 150-200, thousand years ago
• H. sapiens lefts Africa and dispersed to other parts of the world.
• H. sapiens displaces the pre-existing hominids; no interbreeding occurs
• Under this model the preexisting populations of Homo in Europe and elsewhere are species

(e.g., H. erectus; H. ergaster; H. heidelbergensis; H. neanderthalensis)

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Ancient DNA: would the real Neandertal please stand up?

• H. sapiens

(modern)

• H. sapiens

(archaic)

• H. erectus
• H. habilis

1,800,000 ybp 500,000 ybp 100,000 ybp

Afr. Asia Eur. Afr. Asia Eur.

Modern human “Out of Africa” dispersal

Multiregional continuity model Replacement model

Ancient DNA: would the real Neandertal please stand up?

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Gibraltar 2 Neandertal child

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Ancient DNA: would the real Neandertal please stand up? Loads of data are relevant to the controversy:

• Anatomical structures
• Archeological evidence
• Genetics (Modern Humans and Neandertals; we will focus on these…)

Anatomical structures indicate substantial differences:

Neandertal morphology:

• evolved over a period of 220,000 years (350k to130k ybp).
• final morphological form reached at 130,000 ybp.
• Neandertals are first hominids to adapt to cold mid-latitude climates.

Modern Human morphology:

• appear in fossil record about 140,000 ybp
• Appear elsewhere around 60,000 ybp
• Arrive in Europe 40,000 ybp and begin to displace Neandertals

1. Some argue that coexistence of morphologically distinct forms, and subsequent displacement, supports the “replacement” model. 2. Others point out that coexistence in Europe could have lasted as long as 10,000 years and that some early modern humans exhibit a mosaic of archaic and modern features. [But, discrete Neandertals and modern humans coexisted in Middle East for about 55,000 years] [A morphologically intermediate skeleton was found in Portugal, having a Neandertal-like skeleton and more modern skull. The conclusions that this might be a hybrid is

• controversial. The date of the skeleton (24,500) is very late, and there are issues about

what a hybrid anatomy should look like]

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Archeological evidence :

Neandertals and modern humans:

• very similar up to 40-50,000 ybp
• After 40-50,000 ybp anatomically modern humans undergo a “creative explosion”
• The “creative explosion” appears to have begun in Africa and spread outward.

1. Supporters of the replacement model argue that this period of innovation has a genetic basis and represents gene flow as well as cultural flow

• 2. Supporters of the continuity model argue that transmission could have been purely by

social mechanisms and point out that there are no changes in skull morphology during the period of the outward spreading of the “creative explosion” Sites in southern France reveal populations of Neandertals using (mimicking) the culture

• f modern humans associated with the creative explosion. No clearly intermediate

morphologies at this site.

Extant human mtDNA polymorphism supports “Out of Africa”

Africans dominate the root of the human mtDNA tree Consistent with the “Out of Africa model”; i.e, that Africa was the source for contemporary human mtDNA diversity

African populations have the most polymorphism. Some argue that they must be oldest, because they have accumulated the most mutations.

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Krings et al. (1997) conclude:

• Neandertals went extinct without

contributing mtDNA to modern humans

• Used as outgroup, the Neandertal

sequence supports the “Out of Africa” hypothesis of modern humans

• Modern human and Neandertal mtDNA

coalesce at 500,000 ybp Ancient DNA:

• single neandertal
• 30,000 years old (max 100,000)
• HVI of mt control region: 387 bp
• Great effort to authenticate the ancient

DNA (aDNA)

There are now 8 Neandertal aDNA sequences

The shaded area indicates the known range of Neanderthals. Mezmaiskaya is the location where the baby Neanderthal whose DNA was sequenced was found. An earlier Neanderthal DNA sequence was determined from bones found in Feldhofer Cave in Germany.

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The oldest modern human DNA is not from Africa

Neandertal Oldest Modern Human DNA [LM3: > 55,000 ybp]

LM3 is the oldest reliably dated modern human. LM3 lived >55,000 ybp LM3 was Australian The LM3 mtDNA lineage diverged before the MRCA of living humans, but has gone extinct Contemporary diversity at a single locus cannot be used to infer the pattern of human evolution

All the phylogenetic analyses that include Neandertal DNA indicate a substantial divergence of the mtDNA lineages BUT, what about genetic drift? Random sampling from one generation to the next means that some lineages will become extinct by chance alone; i.e., stochastic lineage sorting will occur

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So, divergent lineages is not evidence on its own. Coalescent models can be used to answer the question: “How much introgression could have occurred without leaving any evidence of Neandertal mtDNA in the modern human population?”

Species 1 Species 2 Species 3 Species 4

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Published this week in PLOS!

Assumptions:

• Complete displacement along a

narrow front of a spatially expanding human population.

• Population growth is logistic. This

means that introgressed neandertal genes are not lost by drift; rather they are amplified.

If we assume a different model, as much as 25% introgression could occur and we would not see any Neandertal mtDNA lineages within the human population

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Some alternative views:

A robust modern human mandible Discovered in 2002 (pub. 2003) Dated to 34-36,000 ybp “presents a mosaic of archaic, early modern human, and possible Neandertal morphological features” Reanalysis of Neandertal data Published in 2002 Results sensitive to substitution model Divergence of Neandertal lineage is not supported under more sophisticated substitution models

Best-fit model Best-fit model Suboptimal model

The controversies continue. The replacement model seems to have greater support. Would the real Neandertal please stand up?

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Paleomolecular biochemistry Paleomolecular biochemistry: the scientific discipline devoted to the “resurrection” of an ancestral protein for the purpose of studying how its biophysical properties evolved, or to make inferences about the evolution of the

• rganisms that expressed the protein.

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Paleomolecular biochemistry: allows us to work beyond the limits of ancient DNA

Ancient DNA Paleomolecular biochemistry

Paleomolecular biochemistry: can’t do it without ancestral reconstruction Ancestral reconstruction: the inference of the ancestral character states of a gene or protein sequence for the most recent common ancestor of a given set of descendent sequences.

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1. Identify a period of rapid genetic change, or episode of adaptive evolution (dN/dS). 2. Now determine if this episode in the molecules history is correlated with events in the geological, paleanotological or phylogenetic record 3. Resurrect proteins from points before and after the molecular episode. 4. Examine changes in phenotypes between the two proteins.

B- PR G - PR B- PR G - PR

Ancestral protein 1 Ancestral protein 2

Paleomolecular biochemistry: can’t do it without ancestral reconstruction

1. Obtain DNA/protein sequences 2. Resolve 3D structure of protein 3. Measure phenotype of modern proteins 4. Infer a phylogeny for the gene 5. Identify which of many genetic changes are adaptive (or rapid) 6. Map sites with adaptive changes to 3D structure 7. Construct hypotheses about the affect of adaptive changes on 3D structure and phenotype 8. Site-directed mutagenesis to reconstruct “ancient genes” 9. Experimental test of hypothesis by comparing function/phenotype of ancient genes with modern genes

Paleomolecular biochemistry: a generalized protocol

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Case 1: Resurrecting ancestral coral pigments

Reef-building corals exhibit an amazing variety of colours:

• Proteins related to the green florescent proteins (GFPs) are

responsible for the colour diversity.

• GFPs in high density protect endosymbiotic algae from

excessive solar irradiation.

• Function in low density, as in colour-morphs is unclear.

Paleomolecular biochemistry of GFP-like proteins: Questions: What is the evolutionary mechanism of colour diversity? Is color diversity tuned by natural selection? Is there a relationship between colour and endosymbiotic algae? Why is there colour diversity in species with low density GFPs?

Red/blue colour morphs of the great star coal Montastraea cavernosa

Case 1: Resurrecting ancestral coral pigments If intermediate proteins are green: red is convergent If intermediate proteins are red: red evolved only once

Red fluorescence requires much higher level of functional complexity than green or blue. Green and blue can evolve multiple times by loss of function at some sites. This is not too “hard to do” from an evolutionary standpoint.

MRCA of all colours in M. cavernosa MRCA of the RED proteins MRCA of two intermediate proteins Ancestral proteins were resurrected for all four points in the phylogeny

Gene tree for the GFP-like family of proteins

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Case 1: Resurrecting ancestral coral pigments

Bacteria were engineered to express the extant and ancestral GFP-like

• proteins. These bacteria were then

cultured in a pattern that corresponded to the GFP-LIKE gene tree

The MRCA of all colours is clearly green. The higher level of complexity in red evolved more than once. The colour of the intermediate proteins indicates that the process was a stepwise one, with incremental changes from green to red. This is a mode of adaptive evolution that was forecast by Darwin but had not been previously demonstrated in a molecular system!

Case 1: Resurrecting ancestral coral pigments

Unpublished data has revealed strong support for two patterns of adaptive evolution: 1. Episodes of change associated with colour shifts 2. Continuous modification of an area of the protein surface by diversifying selection. Novel Hypotheses:

• A conflict exists between coral host and endosymbiotic algae.
• Host “wants” to regulate the growth rate in its favour; algae “wants” to proliferate
• Complex colour system evolved to achieve some versatility in control.
• Surface is evolving under diversifying selection involved in binding algae-derived

compounds

Microscopic view of endosymbiotic algae Colours are artificially set to maximize contrast between coral soft tissue and the endosymbiotic algae (zooxanthellae). The green colour indicates the soft tissue of the coral polyp, and the red dots indicate the endosymbiotic algal cells

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Example: archosaur rhodopsin protein (Chang and colleagues, 2002)

Data from Chang et al. (2002) MBE 19:1493-1489.

Case 2: Resurrecting an archosaur visual protein Case 2: Resurrecting an archosaur visual protein

[Background] “The photoreceptors in the retina are of two types: rods and cones, so named because of their shapes. These cells are actually specialized neurons that detect light. Embedded in stacks of cell membranes in the distal portions of rods and cones are molecules that absorb certain wavelengths of light. These molecules are called photopigments and are composed of two parts: a large trans- membrane protein, an opsin, and a smaller chromophore, which is a metabolite of Vitamin A called 11-cis-retinal. The chromophore, which is embedded in the opsin, absorbs light; in so doing it undergoes a shape change. This shape change in turn activates the opsin, setting off a cascade of events that leads to a change in the electrical state of a rod or cone cell membrane. This change in the rod or cone cell membrane is then conducted via the rod or cone axon to other neurons in the retina, and from there to the brain.” [From: John Moran Eye Center, Univ of Utah. http://webvision.med.utah.edu]

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Example: archosaur rhodopsin protein (Chang and colleagues, 2002)

Data from Chang et al. (2002) MBE 19:1493-1489.

Case 2: Resurrecting an archosaur visual protein

Archosaur rhodopsins haven't existed for 240 million years: 1. Download existing rhodopsin gene sequences 2. Reconstruct ancestral archosaur sequence 3. Reconstruct the actual gene in lab (site- directed mutagenesis) 4. Put gene into an animal cell commonly cultured in lab 5. Gene instructs those cells to make the archosaur rhodopsin 6. Collect and purify the rhodopsin 7. Assess the properties of the archosaur rhodopsin Big question: Does the protein function as a rhodopsin? Does it bind 11-cis-retinol? Yes. Does it activate in response to light? Yes. Is it sensitive to visible light? Yes. Does it interact with G-protein transducin? Yes. The protein was a functioning rhodopsin! “Cool beans” !

Case 2: Resurrecting an archosaur visual protein

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Rhodopsin triggers the first step in the biochemical cascade required for vision in animals

• rhodopsin has a direct effect on vision
• archosaur rhodopsin was red shifted
• higher than most mammals and fish
• within higher end of range for reptiles and birds
• suggest archosaurs could see at night under

dim light conditions (i.e., nocturnal)

Data from Chang et al. (2002) MBE 19:1493-1489.

Controversial implication: nocturnal, and not daylight, life histories might have been the ancestral state in amniotes (birds reptiles and mammals). Case 2: Resurrecting an archosaur visual protein