How can we identify the genes and molecules responsible for phenotypic differences within and between species?

Quantitative genetics: direct mapping of genes in crosses between species or divergent strains within species

Unbiased and does not require prior knowledgeOffers conclusive proof of a gene's involvementSpecies must be able to hybridizeYou have to develop genetic markers and mapsHard to go from map position to single gene

Comparative developmental biology: using our knowledge of developmental biology and molecular genetics to test evolutionary hypotheses

Most direct route from phenotype to moleculesDoes not require species to be crossable or closely relatedRequires good knowledge of developmentSpecial tools and techniques must often be developedHard to go from correlation to functional proof

Quantitative trait locus (QTL) mapping

Most traits in nature are controlled by multiple genes and therefore vary in a quantitative fashion

To map the genes responsible for this variation, we need:

1. Identify pure-breeding lines (or species) that differ in the trait of interest

2. Develop a large number of molecular markers throughout the genome

3. Cross these lines, then cross F1 offspring to each other or to one of the parental strains

4. Genotype F2 progeny for each marker5. Determine which markers are linked to the trait, and how

strongly6. Infer QTL positions based on this information

1. Identify pure-breeding lines (or species) that differ in the trait of interest2. Develop a large number of molecular markers throughout the genome3. Cross these lines, then cross F1 offspring to each other or to one of the parental strains4. Genotype F2 progeny for each marker5. Determine which markers are linked to the trait, and how strongly6. Infer QTL positions based on this information

What does it take to map QTLs with high resolution?

Highest resolution is required for identification and cloning

Mapping procedure:

Factors affecting power and precision:

Lines should differ as much as possibleLines (species) should be able to hybridize

Marker density should be very highNumber of progeny should be very large

For polygenic traits, very sophisticated math & software are required

Mapping agriculturally important traits in tomatoes

!

Mapping agriculturally important traits in tomatoes

What can QTL mapping tell us?

Easy (relatively):How many genes contribute to phenotypic differences?What are the contributions of individual genes?

Key question: are evolutionary changes due to many genes of small effect, or to few genes of large effect?100 genes that contribute 1% each, or 4 genes that contribute 25% each?

Very hard:What are these genes??? (TFs, enzymes, etc.)What are their normal developmental/biochemical functions?Why do changes in these genes cause phenotypic differences?What are these changes at the molecular level? (coding or non-

coding, how many mutations per gene, etc.)

How many genes are responsible for phenotypic differences between species?

These are sibling species!

How many genes are responsible for phenotypic differences between species?

These are sibling species!

How strong are phenotypic effects of individual genes?

In practice, we are only likely to find these

Allen Orr's theoretical prediction

Conclusion: genes of large effect do play an important role in phenotypic evolution, but our technical limitations may lead us to overestimate this role

Note the log scale

Why is it so hard to identify QTLs?

Factors affecting power and precision:

Lines should differ as much as possibleLines (species) should be able to hybridizeMarker density should be very high (expensive)Number of progeny should be very large (expensive)

Often incompatible

There have been hundreds of QTL mapping studies in a variety of organisms

Hundreds or thousands of QTLs have been detected and roughly mapped

But, only a handful of genes (< 20 total?) have ever been identified at the molecular level

So what can we do about this?

So what can we do about this?

Identification of QTLs can be made much easier if we already know something about the developmental and biochemical basis of the trait

OK, so we know the responsible gene is somewhere in the red interval. Now, how do we identify it?

1. Map it with ever-increasing resolution until we reach single-gene density (positional cloning - long and expensive but certain)

2. Test specific genes that are located in that interval and that might be responsible for the trait based on their molecular function (candidate gene approach - fast and cheap but uncertain).

Combining QTL and developmental-genetic approaches

Pelvic spine reduction in sticklebacks

Spined (marine) Spineless (freshwater)

This transition has occurred many times in stickleback evolution during independent transitions from marine to freshwater habitats

Many changes have occurred in the last 10,000 years (after glaciation)

QTL mapping of pelvic spine reduction

Candidate genes known to be involved in appendage development in all vertebrates

Note that the mapping is very crude

Pitx1 expression in the pelvic spine region is lost in freshwater sticklebacks

Combining experimental and comparative information

We don't know the mutant phenotype of Pitx1 in sticklebacks

But, we know what its phenotype is in mice, and it is consistent with its expression pattern in sticklebacks

The combination of these two lines of evidence suggests that changes in Pitx1 contribute to phenotypic differences

What happened to Pitx1?

Pitx1 is expressed in multiple tissues

Its expression is controlled by separate tissue-specific regulatory sequences

The coding sequence of Pitx1 is unchanged

But it is no longer expressed in the pelvic girdle in spineless fish

Its expression in other tissues is unchanged

Hypothesis: Mutations in the tissue-specific regulatory sequence are the cause of phenotypic divergence

The tb1 gene underlies a major difference in growth pattern between corn and teosinte

Wild-type teosinte

Wild-type maize

Maize tb1 mutant

Last 10,000 years!

The difference in branching architecture between maize and teosinte maps near the tb1 locus

tb1 is expressed at a higher level in maize than in teosinte

Maize and teosinte alleles were introgressed into the same genetic background

tb1 expression in axillary meristems and stamens of the ear primordia is required to suppress branching

Hypothesis: increased expression of tb1 in maize is responsible for the architectural differences between maize and teosinte

(Northern blots, normalized by ubiquitin)

Selection has been limited to the non-coding region

Directional selection has removed most variation in maize

No evidence of selection

Different species of Danio (zebrafish)

Parallels between mutant phenotypes and natural diversity

How the zebrafish got its stripes

Pigment patterns in interspecific Danio hybrids

An evolutionary complementation test

Are candidate genes responsible for interspecific differences?

Are candidate genes responsible for interspecific differences?

ovo/shavenbaby controls denticle pattern in the larval epidermis

Divergence of svb expression is responsible for differences between D. melanogaster and D. sechellia

D. melanogaster D. sechellia

D. sechellia D.mel svb x D.sec

D.mel x D.sec D.mel svb

Genetic analysis and mapping of divergent phenotype

svb expression correlates with morphological differences

svb expression and function in the virilis species group

A very simple trait: Excretory system in C. elegans

excretory duct cell lin-48 expression

wild type lin-48 mutant

longer duct

ces-2 mutant

Genetic control of excretory duct development in C. elegans

wild-type

wild-type

C. elegans mutants

C. elegans coding sequence controlled by C. elegans

regulatory sequence

C. elegans coding sequence controlled by C. briggsae

regulatory sequence

Length of ED

Derived excretory system morphology in C. elegans

derived morphology!

Length of EDspecies strain

Derived excretory system function in C. elegans

Salt resistance

Derived function

Divergence of lin-48 cis-regulatory regions

known regulator of lin-48

Evolution of lin-48 regulation: alleles from different species introduced into C. elegans

The function of regulatory sequences of lin-48 has diverged!

Functional tests: Necessity and Sufficiency

Is C. elegans lin-48 necessary for elegans-specific morphology?

Yes

Is C. elegans lin-48 sufficient for elegans-specific morphology?

Yes

Functional sufficiency tests: Morphology

Conclusion: changes map to regulatory, not coding, sequences

C. briggsae coding sequence controlled by C. elegans regulatory sequence

C. briggsae coding sequence controlled by C. elegans regulatory sequence

Functional sufficiency tests: Physiology

Necessary…

… but not sufficient

From observation to testable hypothesis

Comparative analysis can suggest candidate genes that may be responsible for phenotypic changes

However, candidate gene hypotheses cannot be tested without functional

assays

These assays can take the form of either genetic crosses (where hybridization is possible) or transgenic tests

Changes in development need not result in morphological changes

Changes in development need not result from genetic changes

Dobzhansky - Muller incompatibilities and developmental drift

Reproductive isolation and speciation are caused by accumulation of genetic differences among independently evolving populations.

Accumulation of genetic differences does not necessarily result in phenotypic divergence

“Co-adaptation” of accumulated genetic changes is disrupted in hybrids, leading to inviability or sterility

Speciation is due to the evolution of developmental pathways

Xiphophorus helleri

Xiphophorus maculatus x X. helleri

Developmental incompatibility in Xyphophorus hybrids

Tu consists of two RTK genes, Xmrk1 and Xmrk2

Xmrk1 is found in all Xyphophorus species

Xmrk2 has evolved by exchange/fusion of Xmrk1 and D (donor) locus

Xmrk2 is present only in some species, including X. Maculatus

The two Xmrk genes are very similar in sequence, but Xmrk is controlled by a D-derived enhancer

Xmrk1 is expressed at a low level in all tissues; Xmrk2 is expressed at a high level only in hybrid melanomas

Xmrk2 overexpression in non-hybrid fish causes melanic tumors

In X. maculatus strains that do not induce melanomas, Xmrk2 is disrupted

r = cdk2 ???

Genetic basis of reproductive isolation in Xyphophorus

Developmental defects in interspecific hybrids

D. melanogaster C(1)RM/Y females x wild males

Bristle loss in hybrids between D. melanogaster and D. simulans

The extent of bristle loss depends of parental phenotype

neuralized-lacZ

Early neuralized expression is normal

cut

Late loss of sensory organ markers

Bristle loss in hybrids is sensitive to asc dosage

Expression of many enhancers is altered in interspecific hybrids

If it ain’t broke, fix it anyway:Cryptic divergence of developmental pathways

Developmental pathways can diverge rapidly among closely related species

This divergence may occur without any overt phenotypic consequences (“developmental drift”)

Cryptic divergence of developmental pathways is revealed by hybrid breakdown

Speciation is a consequence of developmental changes

Polyphenic development in termites

Pheidole castes

Continuous and discontinuous environments

Environmental control of development - and developmental adaptations to environment

Genetic control of wing growth and patterning

Photoperiod, temperature

Diet

Polyphenism in Pheidole morrisi

Different gene expression in genetically identical individuals

Different gene expression in genetically identical individuals

Different gene expression in genetically identical individuals

Polyphenism and the evolution of genetic networks