The

Origins

of

Eukaryotic

Gene

Structure

Michael

Lynch

Department

of

Biology

Indiana

University

Bloomington

Most

of

the

phenotypic

diversity

that

we

perceive

in

the

natural

world

is

directly

attributable

to

the

peculiar

structure

of

the

eukaryotic

gene

which

harbors

numerous

embellishments

relative

to

the

situation

in

prokaryotes

The

most

profound

changes

include

introns

that

must

be

spliced

out

of

precursor

mRNAs

transcribed

but

untranslated

leader

and

trailer

sequences

-LRB-

untranslated

regions

-RRB-

modular

regulatory

elements

that

drive

patterns

of

gene

expression

and

expansive

intergenic

regions

that

harbor

additional

diffuse

control

mechanisms

Explaining

the

origins

of

these

features

is

difficult

because

they

each

impose

an

intrinsic

disadvantage

by

increasing

the

genic

mutation

rate

to

defective

alleles

To

address

these

issues

a

general

hypothesis

for

the

emergence

of

eukaryotic

gene

structure

is

provided

here

Extensive

information

on

absolute

population

sizes

recombination

rates

and

mutation

rates

strongly

supports

the

view

that

eukaryotes

have

reduced

genetic

effective

population

sizes

relative

to

prokaryotes

with

especially

extreme

reductions

being

the

rule

in

multicellular

lineages

The

resultant

increase

in

the

power

of

random

genetic

drift

appears

to

be

sufficient

to

overwhelm

the

weak

mutational

disadvantages

associated

with

most

novel

aspects

of

the

eukaryotic

gene

supporting

the

idea

that

most

such

changes

are

simple

outcomes

of

semi-neutral

processes

rather

than

direct

products

of

natural

selection

However

by

establishing

an

essentially

permanent

change

in

the

population-genetic

environment

permissive

to

the

genome-wide

repatterning

of

gene

structure

the

eukaryotic

condition

also

promoted

a

reliable

resource

from

which

natural

selection

could

secondarily

build

novel

forms

of

organismal

complexity

Under

this

hypothesis

arguments

based

on

molecular

cellular

and/or

physiological

constraints

are

insufficient

to

explain

the

disparities

in

gene

genomic

and

phenotypic

complexity

between

prokaryotes

and

eukaryotes

Introduction

Although

full-genome

sequencing

has

revealed

numerous

patterns

of

variation

in

genomic

architecture

among

major

taxonomic

groups

a

formidable

remaining

challenge

is

to

transform

the

descriptive

field

of

comparative

genomics

into

a

more

mechanistic

theory

of

evolutionary

genomics

Such

an

enterprise

does

not

have

to

start

from

scratch

Nearly

a

century

of

mathematical

derivation

has

resulted

in

a

formal

theory

for

evolution

based

on

the

expected

dynamics

of

gene-frequency

changes

Initially

dubbed

the

Modern

Synthesis

by

Huxley

in

and

having

experienced

further

enhancements

since

then

this

theory

has

survived

so

much

empirical

scrutiny

that

the

credibility

of

any

proposed

scenario

for

genome

evolution

must

remain

in

doubt

until

shown

to

be

consistent

with

basic

population-genetic

principles

In

turn

if

a

mechanistic

understanding

of

genome

evolution

is

to

be

achieved

population-genetic

theory

will

need

to

go

beyond

its

reliance

on

algebraic

formulations

involving

selection

mutation

recombination

and

random

genetic

drift

to

incorporate

the

DNA-level

constraints

that

are

now

known

to

define

the

evolutionary

playing

field

Ever

since

Darwin

the

vast

majority

of

biologists

have

invoked

natural

selection

as

the

primary

and

in

many

cases

the

only

explanation

for

observed

patterns

of

variation

at

most

levels

of

organization

This

greatly

oversimplifies

the

evolutionary

process

For

example

Kimura

Ohta

and

several

contemporaries

showed

why

numerous

aspects

of

DNA

sequence

evolution

can

not

be

explained

entirely

in

terms

of

adaptive

processes

-LRB-

reviewed

in

Kimura

Ohta

-RRB-

The

neutral

-LRB-

or

nearly

neutral

-RRB-

theory

that

emerged

from

Key

words

complexity

gene

networks

gene

regulation

gene

structure

genome

evolution

genetic

draft

introns

modularity

mutation

natural

selection

population

size

pleiotropy

random

genetic

drift

recombination

subfunctionalization

transcription

factors

UTR

E-mail

milynch@indiana.edu

Mol

Biol

Evol

-LRB-

-RRB-

doi

Advance

Access

publication

November

The

Author

Published

by

Oxford

University

Press

on

behalf

of

the

Society

for

Molecular

Biology

and

Evolution

All

rights

reserved

For

permissions

please

e-mail

journals.permissions@oxfordjournals.org

this

work

still

enjoys

a

central

place

in

the

field

of

molecular

evolution

and

has

been

applied

to

some

aspects

of

evolutionary

genomics

-LRB-

Force

et

al.

Lynch

et

al.

Lynch

Lynch

and

Conery

Lynch

Scofield

and

Hong

-RRB-

The

goal

of

this

paper

is

to

expand

on

these

previous

results

to

demonstrate

the

plausibility

of

the

hypothesis

that

many

of

the

unique

complexities

of

the

eukaryotic

gene

arose

by

semi-neutral

processes

with

little

if

any

direct

involvement

of

positive

selection

Although

eukaryotes

share

many

basic

aspects

of

transcription

translation

and

replication

with

their

prokaryotic

ancestors

there

are

profound

differences

at

the

level

of

gene

architecture

Prokaryotic

genes

are

often

organized

into

operons

that

are

transcribed

into

polycistronic

units

whereas

with

few

exceptions

eukaryotic

genes

are

transcribed

as

single-gene

units

Unlike

prokaryotic

genes

eukaryotic

genes

often

have

complex

regulatory

regions

and

in

multicellular

species

such

regions

often

have

a

modular

structure

that

helps

facilitate

tissue-specific

expression

Eukaryotic

protein-coding

genes

also

often

contain

introns

whereas

prokaryotic

genes

do

not

and

eukaryotic

transcripts

generally

contain

longer

untranslated

leader

and

terminal

sequences

-LRB-

untranslated

regions

-LSB-

UTRs

-RSB-

-RRB-

than

do

those

of

prokaryotes

Three

general

observations

have

encouraged

the

view

that

that

these

kinds

of

increases

in

gene

complexity

were

necessary

prerequisites

to

the

origin

of

organisms

with

multiple

cell

types

-LRB-

i

-RRB-

most

aspects

of

gene

architecture

are

much

more

elaborate

in

multicellular

than

unicellular

eukaryotes

-LRB-

ii

-RRB-

similar

forms

of

genomic

architecture

are

found

in

the

two

major

and

independently

evolved

multicellular

lineages

animals

and

land

plants

and

-LRB-

iii

-RRB-

more

complex

genes

can

often

carry

out

more

complex

sets

of

tasks

-LRB-

Raff

Gerhart

and

Kirschner

Davidson

Carroll

Grenier

and

Weatherbee

-RRB-

However

despite

these

clear

associations

the

direction

of

causality

in

the

link

between

genome

and

organismal

complexity

is

far

from

certain

There

is

no

direct

evidence

that

Origins

of

Eukaryotic

Gene

Structure

multicellularity

itself

was

promoted

by

adaptive

processes

and

the

fact

that

many

prokaryotes

are

capable

of

cell

differentiation

reminds

us

that

the

evolution

of

multicellularity

need

not

have

awaited

the

emergence

of

eukaryotes

The

key

point

to

be

made

below

is

that

the

types

of

genomic

evolution

that

can

occur

within

a

species

are

not

so

much

dependent

on

aspects

of

cell

biology

as

on

the

constraints

imposed

by

population-level

processes

most

notably

by

population

size

itself

The

arguments

underlying

this

hypothesis

will

be

laid

out

in

three

sections

First

I

will

review

the

role

that

chance

plays

in

evolution

and

why

this

depends

on

population

size

Second

I

will

summarize

several

sets

of

empirical

data

that

show

that

the

efficiency

of

natural

selection

declines

dramatically

between

prokaryotes

unicellular

eukaryotes

and

multicellular

eukaryotes

Third

I

will

demonstrate

that

theory

and

empirical

observations

are

mutually

consistent

in

pointing

to

a

central

role

for

nonadaptive

processes

in

the

origins

of

many

of

aspects

of

eukaryotic

gene

structure

The

Role

of

Chance

in

Evolution

Evolution

is

an

inherently

stochastic

process

starting

from

the

chance

events

that

produce

single

mutations

and

proceeding

through

a

series

of

fortuitous

steps

that

gradually

lead

to

the

spread

of

some

mutations

to

every

member

of

the

descendant

population

The

usual

conceptual

point

of

departure

here

is

the

classic

Wright-Fisher

model

which

assumes

a

population

of

diploid

individuals

each

contributing

equally

and

synchronously

to

an

effectively

infinite

gamete

pool

The

idealized

mating

system

consists

of

randomly

mating

hermaphrodites

and

ignores

complexities

associated

with

overlapping

generations

separate

sexes

spatial

structure

nonrandom

variation

in

family

sizes

and

so

on

However

most

of

these

complications

can

be

dealt

with

by

equating

the

genetic

effective

size

of

a

population

-LRB-

Ne

-RRB-

to

the

size

of

an

ideal

Wright-Fisher

population

yielding

equivalent

gene-frequency

dynamics

The

effective

size

of

a

population

is

a

fundamental

determinant

of

nearly

all

aspects

of

evolution

as

it

determines

the

probability

of

-LRB-

and

times

to

-RRB-

fixation

or

removal

of

mutant

alleles

Most

deviations

from

the

assumptions

of

the

WrightFisher

model

cause

Ne

to

be

less

than

the

total

number

of

adult

individuals

-LRB-

N

-RRB-

-LRB-

Caballero

Whitlock

and

Barton

Rousset

-RRB-

For

example

if

adults

differ

in

the

number

of

gametes

produced

either

because

of

selection

or

chance

ecological

events

Ne

will

be

reduced

simply

because

some

individuals

contribute

little

or

nothing

to

the

following

generation

A

sex

ratio

that

deviates

from

reduces

Ne

because

the

rarer

sex

-LRB-

which

necessarily

contributes

half

of

the

genes

to

the

next

generation

-RRB-

acts

as

a

population

bottleneck

Population-size

fluctuations

reduce

long-term

Ne

relative

to

the

arithmetic

average

because

the

losses

of

variation

during

population

bottlenecks

exceed

the

preservational

effects

of

equal

population-size

expansions

A

number

of

procedures

have

been

developed

to

estimate

Ne

from

pedigree

data

or

by

relating

temporal

fluctuations

in

allele

frequencies

to

the

expectations

from

sampling

gametes

Studies

of

this

sort

mostly

confined

to

vertebrates

suggest

an

average

Ne/N

of

-LRB-

Frankham

-RRB-

To

see

how

population

size

influences

the

long-term

rate

of

evolution

consider

a

newly

arisen

mutation

in

a

diploid

population

containing

gene

copies

at

each

locus

If

the

mutation

is

neutral

with

no

external

forces

favoring

one

allele

over

another

the

probability

of

eventual

fixation

is

always

equal

to

the

initial

frequency

-LRB-

-RRB-

Because

this

fraction

is

inversely

proportional

to

N

one

might

expect

neutral

changes

to

accumulate

more

slowly

in

larger

populations

However

the

expected

number

of

new

mutations

arising

at

a

locus

per

generation

is

where

l

is

the

rate

of

origin

of

neutral

mutations

per

gene

and

the

long-term

rate

of

evolution

is

equal

to

the

product

of

the

rate

of

origin

of

mutations

and

their

probability

of

fixation

Thus

the

rate

of

neutral

evolution

reduces

to

the

genic

mutation

rate

l

which

is

entirely

independent

of

the

effective

and

absolute

population

size

-LRB-

Kimura

-RRB-

Intuition

suggests

that

the

fixation

probability

of

a

beneficial

allele

must

exceed

the

neutral

expectation

-LRB-

-RRB-

whereas

that

of

a

detrimental

allele

must

be

-LRB-

-RRB-

but

how

much

so

To

see

that

a

beneficial

mutation

is

never

guaranteed

to

go

to

fixation

no

matter

how

favorable

consider

a

new

mutation

that

improves

the

fitness

of

its

initial

carrier

by

a

fraction

s

-LRB-

the

selection

coefficient

-RRB-

Such

an

allele

has

expected

frequency

-LRB-

s

-RRB-

-LRB-

-RRB-

in

the

gamete

pool

leading

to

the

next

generation

and

the

probability

that

it

is

not

successfully

inherited

by

at

least

one

offspring

-LRB-

-RRB-

is

closely

approximated

by

-LRB-

s

-RRB-

e

for

small

s.

Thus

relative

to

the

situation

for

a

neutral

allele

-LRB-

s

-RRB-

selection

only

reduces

the

probability

of

a

rapid

initial

exit

for

a

beneficial

allele

by

a

fraction

s

This

shows

that

until

a

favorable

mutation

has

avoided

chance

elimination

in

the

first

few

generations

and

increased

its

frequency

in

doing

so

there

is

little

assurance

that

it

will

successfully

go

to

fixation

For

mutations

with

additive

effects

on

fitness

-LRB-

increasing

homozygote

fitness

by

-RRB-

the

probability

of

fixation

is

pf

N

e

s

-LRB-

Kimura

-RRB-

Thus

even

in

very

large

populations

-LRB-

Ne

N

-RRB-

the

high

degree

of

stochasticity

in

the

early

phase

of

mutation

establishment

still

restricts

the

probability

of

fixation

to

an

upper

limit

of

Given

the

arguments

presented

above

this

means

that

the

probability

of

fixation

of

a

favorable

mutation

is

almost

always

s.

Letting

lb

be

the

beneficial-mutation

rate

per

gene

and

be

the

rate

at

the

population

level

the

above

results

imply

that

the

upper

limit

to

the

rate

of

incorporation

of

beneficial

mutations

is

which

unlike

the

situation

for

neutral

mutations

increases

with

Ne

In

contrast

because

detrimental

mutations

with

Nes

have

fixation

probabilities

at

least

half

as

great

as

the

neutral

expectation

if

the

rate

of

origin

of

mutations

in

this

range

of

effects

is

sufficiently

high

a

considerable

load

of

mildly

deleterious

mutations

can

accumulate

in

populations

of

sufficiently

small

size

-LRB-

Ohta

-RRB-

Modifications

to

this

theory

for

situations

in

which

populations

are

subdivided

or

changing

in

size

do

not

change

these

basic

scaling

properties

-LRB-

Otto

and

Whitlock

Whitlock

-RRB-

These

results

yield

the

robust

prediction

that

the

ability

of

a

population

to

incorporate

beneficial

mutations

and

to

purge

deleterious

mutations

should

scale

positively

with

population

size

assuming

that

Ne

scales

positively

with

N.

However

something

beyond

the

demographic

features

Lynch

of

a

population

the

physical

structure

of

the

genome

itself

will

generally

limit

the

growth

of

Ne

with

N

in

the

largest

of

populations

Because

tightly

linked

nucleotide

sites

are

transmitted

across

generations

as

a

unit

to

a

degree

that

depends

on

the

rate

of

recombination

the

fate

of

any

new

mutation

depends

on

the

selective

forces

operating

on

all

linked

loci

On

average

this

causes

the

fixation

rates

of

beneficial

mutations

to

be

lower

and

detrimental

mutations

to

be

higher

than

the

single-locus

predictions

suggested

above

-LRB-

Hill

and

Robertson

-RRB-

For

example

a

beneficial

mutation

that

rapidly

sweeps

through

a

population

will

necessarily

drag

along

any

deleterious

alleles

at

tightly

linked

loci

with

which

it

is

associated

at

the

time

of

origin

whereas

the

selective

removal

of

deleterious

alleles

can

impede

adaptive

evolution

at

linked

loci

Even

mutually

advantageous

alleles

will

interfere

with

each

other

's

fixation

when

linked

Consider

a

beneficial

allele

A

segregating

at

one

locus

with

a

second

beneficial

mutation

B

arising

at

a

tightly

linked

locus

on

an

a-bearing

chromosome

If

the

advantages

of

each

mutation

were

the

same

then

the

Ab

and

aB

linkage

groups

would

compete

with

each

other

in

the

fixation

process

with

one

eventually

excluding

the

other

Linkage

need

not

be

absolute

for

these

effects

to

be

important

but

the

stronger

the

degree

of

linkage

the

greater

the

degree

of

selective

interference

Gillespie

-LRB-

-RRB-

presented

an

elegant

argument

relating

the

influence

of

linkage

to

the

effective

population

size

of

a

chromosomal

region

The

key

issue

is

that

the

effective

size

of

a

population

defines

the

variance

of

allele-frequency

change

from

generation

to

generation

which

for

a

neutral

locus

is

p

-LRB-

p

-RRB-

-LRB-

-RRB-

where

p

is

the

current

allele

frequency

and

is

the

effective

number

of

genes

sampled

per

locus

Now

imagine

a

neutrally

evolving

site

completely

linked

to

another

site

that

is

experiencing

selective

sweeps

at

rate

d.

On

average

selective

sweeps

do

not

influence

which

alleles

go

to

fixation

at

linked

neutral

sites

because

the

probability

that

a

beneficial

mutation

destined

to

fixation

will

arise

in

association

with

a

particular

allele

is

simply

equal

to

that

allele

's

frequency

However

selective

sweeps

do

magnify

the

fluctuations

of

allele

frequencies

at

linked

neutral

sites

Assuming

as

a

first

approximation

that

sweeps

cleanse

a

population

of

linked

variation

essentially

instantaneously

then

conditional

on

a

sweep

occurring

the

variance

in

allele-frequency

change

at

the

neutral

locus

is

p

-LRB-

p

-RRB-

Thus

for

a

neutral

locus

in

an

ideal

randomly

mating

population

the

average

variance

of

allele-frequency

change

is

approximately

p

-LRB-

p

-RRB-

f

-LSB-

-LRB-

d

-RRB-

-LRB-

-RRB-

-RSB-

dg

Equating

the

right-hand

quantity

to

-LRB-

-RRB-

the

long-term

effective

population

size

is

Nl

Ne

-LRB-

-RRB-

where

Ne

is

now

defined

to

be

the

short-term

effective

size

during

phases

free

of

selective

sweeps

-LRB-

Maruyama

and

Birky

-LSB-

-RSB-

obtained

essentially

the

same

result

by

a

different

method

-RRB-

Recombination

reduces

the

likelihood

that

a

selective

sweep

will

completely

purge

the

variation

at

a

linked

locus

but

Gillespie

-LRB-

-RRB-

showed

that

this

simply

modifies

the

preceding

expression

to

Nl

Ne

-LRB-

-RRB-

where

C

is

the

average

squared

frequency

of

the

neutral

hitchhiking

allele

after

the

completion

of

a

sweep

-LRB-

previously

assumed

to

be

equal

to

one

-RRB-

An

unresolved

issue

is

the

way

in

which

the

rate

of

selective

sweeps

in

a

tightly

linked

region

Cd

scales

with

population

size

If

Cd

were

completely

independent

of

population

size

then

Nl

would

increase

with

Ne

at

a

decreasing

rate

eventually

reaching

an

upper

limit

of

-LRB-

-RRB-

Because

larger

populations

contain

more

targets

for

rare

beneficial

mutations

and

also

experience

more

recombination

events

the

rate

of

sweeps

-LRB-

d

-RRB-

is

expected

to

increase

and

the

breadth

of

sweeps

-LRB-

C

-RRB-

to

decrease

with

increasing

Ne

so

in

principle

these

opposite

patterns

of

scaling

might

fortuitously

balance

such

that

the

product

Cd

is

indeed

independent

of

Ne

However

in

the

absence

of

any

direct

observations

on

this

matter

a

more

general

approximation

is

to

treat

Cd

as

a

power

function

of

Ne

yielding

a

relationship

of

the

form

Nl

Ne

-LRB-

aNeb

-RRB-

Here

b

describes

the

case

in

which

Cd

is

independent

of

Ne

whereas

with

b

the

rate

of

selective

sweeps

is

proportional

to

Ne

as

in

the

single-locus

result

described

above

The

latter

condition

is

clearly

too

high

to

be

biologically

realistic

as

the

rate

of

selective

sweeps

per

locus

eventually

exceeds

one

per

generation

at

large

Ne

Thus

b

is

likely

to

fall

in

the

range

of

although

this

remains

to

be

formally

demonstrated

Assuming

that

short-term

Ne

scales

linearly

with

absolute

population

size

-LRB-

N

-RRB-

these

qualitative

arguments

suggest

that

long-term

Nl

will

also

scale

linearly

with

N

for

small

to

moderate

N

where

random

genetic

drift

is

the

predominant

stochastic

force

However

the

degree

of

scaling

is

expected

to

be

progressively

reduced

at

larger

N

with

an

asymptotic

limit

to

Nl

possibly

being

reached

at

very

large

N

where

stochastic

fluctuations

in

allele

frequencies

are

primarily

a

function

of

the

chromosomal

nature

of

the

genome

-LRB-

Gillespie

's

genetic

draft

-RRB-

To

gain

an

appreciation

of

the

power

of

random

genetic

drift

and

draft

to

compromise

the

efficiency

of

natural

selection

it

is

useful

to

consider

the

ratio

of

the

single-locus

fixation

probability

pf

and

the

neutral

expectation

-LRB-

-RRB-

This

is

a

simple

function

of

s

hf

ffi

s

e

Nl

s

where

Nl

is

defined

by

the

function

in

the

preceding

paragraph

-LRB-

fig.

-RRB-

If

the

strength

of

selection

is

sufficiently

large

relative

to

the

power

of

random

genetic

drift

-LRB-

-RRB-

the

fixation

probability

for

an

advantageous

allele

is

inflated

by

a

factor

of

relative

to

the

neutral

expectation

whereas

the

fixation

probability

of

a

deleterious

allele

asymptotically

approaches

zero

as

N.

However

if

the

probability

of

fixation

is

within

of

the

neutral

expectation

and

if

the

deviation

from

neutrality

is

no

more

than

Thus

for

any

long-term

effective

population

size

there

exists

a

range

of

deleterious

mutations

whose

selective

disadvantages

are

overwhelmed

by

stochastic

forces

Such

alleles

are

said

to

be

effectively

neutral

The

Three

Genomic

Perils

of

Increased

Organism

Size

A

central

premise

of

this

paper

is

that

there

is

a

general

reduction

in

the

efficiency

of

selection

between

prokaryotes

unicellular

eukaryotes

and

multicellular

species

We

now

take

a

more

empirical

look

at

this

issue

showing

that

all

three

major

factors

responsible

for

reductions

in

Nl

small

population

size

tight

linkage

and

high

background

mutational

activity

are

jointly

exacerbated

as

organisms

increase

in

size

producing

a

synergism

that

causes

substantial

reductions

in

the

efficiency

of

natural

selection

Origins

of

Eukaryotic

Gene

Structure

FIG.

The

probability

of

fixation

of

a

new

mutant

allele

relative

to

the

neutral

expectation

of

-LRB-

-RRB-

given

as

a

function

of

the

product

of

the

short-term

-LRB-

drift

determining

-RRB-

effective

population

size

-LRB-

Ne

-RRB-

and

the

selection

coefficient

-LRB-

s

-RRB-

For

the

dashed

and

dotted

curves

the

long-term

effective

population

size

-LRB-

Nl

-RRB-

is

defined

by

the

function

in

the

text

with

a

whereas

the

solid

line

denotes

Nl

Ne

which

assumes

an

absence

of

selective

sweeps

-LRB-

a

-RRB-

The

horizontal

dotted

line

denotes

the

neutral

expectation

Negative

values

of

Nes

denote

deleterious

mutations

FIG.

The

negative

relationship

between

recombination

rate

per

physical

distance

-LRB-

c

-RRB-

and

total

genome

size

-LRB-

G

-RRB-

in

eukaryotes

scaling

as

c

G

for

animals

and

land

plants

with

the

exponent

having

a

standard

error

-LRB-

SE

-RRB-

of

and

as

c

G

for

fungi

and

other

unicellular/oligocellular

species

with

the

exponent

having

a

SE

of

In

the

two

respective

cases

genome

size

accounts

for

and

of

the

variance

in

recombination

rate

-LRB-

For

further

details

see

Supplementary

Table

Supplementary

Material

online

-RRB-

Body

Size

and

Population

Size

A

typical

prokaryote

is

five

to

seven

orders

of

magnitude

smaller

than

the

average

single-celled

eukaryote

with

a

similar

disparity

existing

between

unicellular

and

multicellular

eukaryotes

-LRB-

Bonner

-RRB-

Such

massive

differences

in

size

impose

numerous

ecological

and

physiological

constraints

and

opportunities

but

the

implications

for

the

population-genetic

environment

are

equally

pronounced

All

other

things

being

equal

the

genetic

effective

size

of

a

population

should

generally

increase

with

the

actual

number

of

breeding

adults

-LRB-

N

-RRB-

and

one

of

the

few

wellestablished

laws

in

ecology

is

that

a

primary

determinant

of

N

is

the

average

size

of

members

of

the

population

Eukaryotes

generally

show

an

inverse

relationship

between

population

density

per

unit

area

and

average

individual

body

mass

within

a

species

with

the

extreme

values

ranging

from

for

the

largest

vertebrates

to

for

the

smallest

unicellular

eukaryotes

-LRB-

Damuth

Schmid

Tokeshi

and

Schmid-Araya

Enquist

and

Niklas

Carbone

and

Gittleman

Finlay

-RRB-

Ecological

factors

unique

to

individual

species

can

cause

local

deviations

around

this

pattern

and

an

inverse

scaling

between

population

density

and

organism

size

need

not

reflect

the

pattern

for

total

population

size

as

it

does

not

account

for

total

species

ranges

However

the

geographic

area

occupied

by

vertebrate

species

is

negligibly

to

weakly

positively

correlated

with

average

body

size

-LRB-

Gaston

and

Blackburn

Diniz

and

Torres

Housworth

Martins

and

Lynch

-RRB-

and

the

geographic

ranges

of

unicellular

species

appear

to

be

substantially

greater

than

those

for

multicellular

taxa

-LRB-

Finlay

et

al.

Finlay

Monaghan

and

Maberly

Green

et

al.

HornerDevine

et

al.

-RRB-

Thus

in

a

broad

phylogenetic

sense

there

is

little

doubt

that

the

total

number

of

individuals

within

a

species

declines

with

increasing

organism

size

and

the

total

range

in

N

over

all

species

certainly

exceeds

orders

of

magnitude

Assuming

prokaryotic

cells

inhabiting

the

earth

-LRB-

Whitman

Coleman

and

Wiebe

-RRB-

and

being

an

upper-bound

estimate

for

the

number

of

prokaryotic

species

-LRB-

Hammond

-RRB-

N

for

an

average

prokaryote

would

be

Reduced

Recombination

in

Large

Genomes

High-density

genetic

maps

allow

the

estimation

of

the

average

amount

of

meiotic

crossing

over

for

numerous

eukaryotes

The

magnitude

of

recombination

per

physical

distance

scales

negatively

with

genome

size

ranging

from

in

Pinus

sylvestris

to

generation

in

Saccharomyces

cerevisiae

-LRB-

fig.

-RRB-

Such

scaling

is

due

mostly

to

the

simple

fact

that

most

species

experience

between

one

and

two

meiotic

crossover

events

per

chromosome

Because

chromosome

number

is

uncorrelated

with

genome

size

the

intensity

of

recombination

per

nucleotide

position

naturally

increases

in

smaller

genomes

-LRB-

with

smaller

average

chromosome

lengths

-RRB-

Less

clear

is

why

the

recombination

rate

declines

with

increasing

genome

size

twice

as

rapidly

in

unicellular

as

in

multicellular

species

-LRB-

fig.

-RRB-

In

any

event

because

genome

size

increases

with

organism

size

these

results

imply

that

increases

in

organism

size

are

accompanied

by

decreases

in

the

intensity

of

recombination

Not

only

can

a

selective

sweep

in

a

multicellular

eukaryote

drag

along

up

to

more

linked

nucleotide

sites

than

is

likely

in

Lynch

a

unicellular

species

but

species

with

small

genomes

also

experience

increased

levels

of

recombination

on

a

per-gene

basis

For

example

the

rate

of

recombination

over

the

entire

physical

distance

associated

with

an

average

gene

-LRB-

including

intergenic

DNA

-RRB-

is

in

S.

cerevisiae

versus

in

Homo

sapiens

and

the

discrepancy

is

greater

if

one

considers

just

coding

exons

and

introns

versus

The

consequences

of

reduced

recombination

rates

are

particularly

clear

in

the

human

population

which

harbors

numerous

haplotype

blocks

tens

to

hundreds

of

kilobases

in

length

with

little

evidence

of

internal

recombination

-LRB-

Daly

et

al.

Reich

et

al.

Dawson

et

al.

Gabriel

et

al.

Greenwood

Rana

and

Schork

McVean

et

al.

-RRB-

The

Rate

of

Mutation

Because

of

the

rarity

of

mutations

at

individual

nucleotides

sites

there

are

enormous

challenges

to

estimating

the

rate

at

which

mutations

arise

at

the

molecular

level

Most

estimates

are

derived

either

from

surveys

of

visible

mutations

at

reporter

loci

or

of

dominant

genetic

disorders

followed

by

sequence

analysis

of

individuals

exhibiting

a

phenotype

These

approaches

are

not

without

problems

as

corrections

must

be

made

for

the

incidence

of

undetectable

mutations

More

indirect

attempts

to

estimate

the

mutation

rate

are

based

on

comparisons

of

distantly

related

species

using

DNA

sequences

thought

to

be

free

of

natural

selection

and

making

assumptions

about

times

of

interspecific

divergence

and

species-specific

generation

times

-LRB-

e.g.

Keightley

and

Eyre-Walker

Kumar

and

Subramanian

-RRB-

Only

a

single

attempt

has

been

made

to

estimate

the

per-nucleotide

mutation

directly

by

brute-force

assays

of

random

sequences

in

unselected

lines

-LRB-

Denver

et

al.

-RRB-

To

minimize

the

assumptions

involved

the

following

survey

largely

relies

on

data

derived

from

the

more

direct

approaches

Across

a

phylogenetically

diverse

set

of

a

species

there

is

a

strong

correlation

between

the

mutation

rate

per

generation

and

genome

size

-LRB-

fig.

-RRB-

The

range

for

the

base-substitution

mutation

rate

is

approximately

two

orders

of

magnitude

and

again

exhibits

a

gradient

with

organism

size

the

extremes

being

and

for

prokaryotes

and

vertebrates

respectively

Despite

the

uncertainties

in

each

estimate

contributing

to

this

pattern

the

validity

of

the

overall

relationship

is

supported

by

two

observations

First

the

estimate

for

the

nematode

Caenorhabditis

elegans

obtained

by

direct

sequence

analysis

-LRB-

highest

red

point

in

the

plot

-RRB-

is

consistent

with

the

remaining

data

obtained

via

reporter

constructs

Second

estimates

of

the

human

mutation

rate

obtained

from

observations

on

dominant

genetic

disorders

-LRB-

Kondrashov

-RRB-

are

very

similar

to

those

obtained

from

comparisons

of

pseudogene

sequences

in

humans

and

chimpanzees

-LRB-

Nachman

and

Crowell

-RRB-

and

respectively

Based

on

more

limited

data

Drake

-LRB-

-RRB-

concluded

that

the

mutation

rate

per

nucleotide

per

generation

is

inversely

related

with

genome

size

in

microbial

species

but

the

results

in

figure

suggest

the

opposite

pattern

even

within

the

subset

of

unicellular

species

FIG.

The

relationship

between

the

mutation

rate

for

base

substitutions

per

nucleotide

site

per

generation

-LRB-

u

-RRB-

and

genome

size

-LRB-

G

-RRB-

scales

as

u

-LRB-

-RRB-

with

the

exponent

having

a

SE

of

and

genome

size

accounting

for

of

the

variance

in

the

mutation

rate

-LRB-

For

further

details

see

Supplementary

Table

Supplementary

Material

online

-RRB-

The

Global

Effective

Population

Sizes

of

Species

The

preceding

results

show

that

three

factors

-LRB-

low

population

sizes

low

recombination

rates

and

high

mutation

rates

-RRB-

conspire

to

reduce

the

efficiency

of

natural

selection

with

increasing

organism

size

although

it

is

difficult

to

predict

the

magnitude

of

decline

in

Nl

from

these

three

factors

alone

For

example

fluctuations

in

population

size

can

result

in

a

substantial

depression

of

Nl

below

average

N

and

it

is

unclear

whether

the

magnitude

of

such

fluctuations

varies

with

organism

size

In

addition

as

discussed

above

hitchhiking

effects

should

depress

the

Nl/N

ratio

much

more

in

large

populations

but

because

the

recombination

rate

-LRB-

per

meiosis

-RRB-

is

substantially

greater

and

the

mutation

rate

is

substantially

lower

in

species

with

large

N

this

decline

could

be

weaker

than

otherwise

expected

Given

the

many

additional

factors

that

can

influence

Nl

the

degree

to

which

Nl

varies

with

organism

size

is

best

resolved

by

direct

empirical

observation

One

way

to

accomplish

this

task

is

to

consider

the

amount

of

nucleotide-sequence

variation

at

silent

sites

in

protein-coding

genes

within

natural

populations

Under

the

assumption

that

mutations

at

such

sites

escape

the

eyes

of

natural

selection

the

amount

of

silent-site

variation

has

a

simple

interpretation

The

rate

of

introduction

of

new

variation

per

nucleotide

site

in

two

randomly

compared

alleles

is

-LRB-

twice

the

base-substitution

mutation

rate

per

nucleotide

-RRB-

while

the

expected

rate

of

loss

of

variation

by

genetic

drift

is

-LRB-

-RRB-

At

equilibrium

the

average

number

of

nucleotide

substitutions

separating

individual

neutral

sites

in

two

randomly

sampled

alleles

is

the

ratio

of

these

two

rates

For

a

haploid

species

the

rate

of

random

genetic

drift

is

and

the

equilibrium

divergence

among

neutral

nucleotide

sites

becomes

Both

results

have

the

same

meaning

at

mutation-drift

equilibrium

the

amount

of

within-species

nucleotide

variation

at

silent

sites

is

equal

Origins

of

Eukaryotic

Gene

Structure

FIG.

Average

levels

of

within-species

nucleotide

variation

-LRB-

measured

as

the

number

of

substitutions

per

site

-RRB-

for

silent

sites

in

proteincoding

genes

from

a

phylogenetically

diverse

assemblage

of

eukaryotic

and

prokaryotic

species

The

sampling

unit

is

the

average

estimate

for

members

of

a

genus

where

information

for

multiple

species

was

available

Means

standard

deviations

and

number

of

genera

are

given

in

parentheses

horizontal

bars

provide

estimates

of

the

observed

ranges

of

values

using

and

limits

to

reduce

the

effects

of

outliers

resulting

from

sampling

error

Almost

all

data

are

derived

from

geographically

wide

surveys

of

multiple

protein-coding

genes

-LRB-

For

further

details

see

Supplementary

Tables

and

Supplementary

Material

online

-RRB-

to

twice

the

effective

number

of

gene

copies

at

the

locus

times

the

per

nucleotide

mutation

rate

An

estimate

of

this

composite

parameter

is

provided

by

the

observed

level

of

silent-site

variation

within

a

species

-LRB-

hereafter

ps

-RRB-

Although

the

complex

nature

of

the

definition

of

Nl

introduces

some

interpretive

issues

with

ps

-LRB-

Laporte

and

Charlesworth

-RRB-

a

fully

general

definition

can

be

described

in

terms

of

allelic

ancestry

ps

is

equal

to

the

average

age

of

random

pairs

of

sequences

times

twice

the

base-substitutional

mutation

rate

per

nucleotide

site

As

will

be

seen

below

the

fact

that

ps

is

a

function

of

the

product

of

Nl

and

u

is

very

useful

because

many

aspects

of

genome

evolution

depend

directly

on

this

product

Information

on

ps

now

exists

for

a

wide

enough

phylogenetic

range

of

species

that

some

general

statements

can

be

made

Drawing

from

a

substantially

larger

database

than

presented

in

the

earlier

survey

of

Lynch

and

Conery

-LRB-

-RRB-

there

is

a

striking

inverse

relationship

between

organism

size

and

silent-site

variation

-LRB-

fig.

-RRB-

For

prokaryotes

ps

lies

in

the

broad

range

of

with

an

average

value

of

This

is

nearly

twice

the

average

value

for

unicellular

eukaryotes

-LRB-

-RRB-

although

the

range

of

values

among

the

latter

taxa

is

again

very

high

-LRB-

-RRB-

For

the

still

larger

invertebrates

there

is

a

further

reduction

in

average

ps

to

with

a

range

of

The

average

value

of

ps

for

plants

-LRB-

-RRB-

is

still

lower

and

that

for

vertebrates

-LRB-

-RRB-

is

even

lower

A

significant

caveat

with

respect

to

these

data

is

that

the

bulk

of

existing

surveys

on

nuclear

variation

in

unicellular

species

have

focused

on

pathogens

which

because

of

the

demographic

dependence

on

their

host

species

probably

have

lower

Ne

than

free-living

species

-LRB-

Hartl

et

al.

-RRB-

Two

of

the

three

prokaryotes

with

ps

are

Serratia

-LRB-

a

human

pathogen

-RRB-

and

Buchnera

-LRB-

an

obligate

endosymbiont

of

aphids

-RRB-

The

six

lowest

estimates

of

ps

for

unicellular

eukaryotes

are

all

derived

from

pathogens

-LRB-

Candida

Coccidioides

Encephalitozoon

Fusarium

Phytophthora

and

Plasmodium

-RRB-

all

other

taxa

-LRB-

including

some

pathogens

-RRB-

having

ps

From

these

estimates

of

ps

Nl

can

be

disentangled

from

u

by

applying

the

mutation-rate

estimates

described

above

For

example

using

the

average

observed

value

of

u

for

prokaryotes

to

factor

u

out

of

the

estimated

average

Nl

for

prokaryotes

is

After

removal

of

the

six

lowest

values

of

ps

for

eukaryotic

parasites

application

of

the

average

mutation

rate

for

unicellular

eukaryotes

-LRB-

-RRB-

yields

an

average

Nl

of

for

this

group

Similar

analyses

for

invertebrates

and

vertebrates

yield

average

Nl

estimates

of

and

respectively

Finally

a

phylogenetically

based

mutation-rate

estimate

for

plants

of

for

plants

-LRB-

Lynch

-RRB-

yields

an

average

Nl

estimate

of

for

annual

species

and

assuming

a

generation

time

of

years

for

trees

Given

the

rough

nature

of

the

preceding

calculations

they

are

intentionally

reported

to

just

an

order

of

magnitude

Nevertheless

it

is

likely

that

the

range

in

Nl

from

prokaryotes

to

multicellular

eukaryotes

exceeds

the

four

orders

of

magnitude

just

noted

Any

selection

on

silent

sites

associated

with

codon-usage

bias

and/or

mRNA

processing

features

will

bias

ps

below

the

neutral

expectation

and

the

magnitude

of

bias

will

be

greatest

in

large

populations

where

selection

is

most

efficient

Several

observations

suggest

that

this

issue

is

of

significance

-LRB-

Bustamante

Nielsen

and

Hartl

Hellmann

et

al.

Chamary

and

Hurst

Desai

et

al.

Halligan

et

al.

Sharp

et

al.

-RRB-

and

because

the

divergence

rate

of

silent

sites

in

prokaryotes

may

be

at

least

ten

times

lower

than

the

mutation

rate

-LRB-

Ochman

-RRB-

the

prokaryotic

Nl

estimates

given

above

could

be

underestimated

by

at

least

tenfold

Despite

these

uncertainties

it

is

clear

that

the

disparity

in

Nl

across

all

domains

of

life

is

nearly

orders

of

magnitude

less

than

the

disparity

in

absolute

numbers

a

pattern

that

is

consistent

with

a

significant

stochastic

role

of

genetic

draft

in

large

populations

Because

of

their

potential

for

considerable

clonal

structure

prokaryotic

species

may

be

particularly

vulnerable

to

selective

sweeps

but

the

breadth

of

such

sweeps

remains

unclear

Moreover

because

prokaryotes

have

a

number

of

mechanisms

for

uptake

and

exchange

of

exogenous

DNA

the

absence

of

meiosis

need

not

imply

exceptionally

low

levels

of

recombination

Some

insight

into

this

matter

can

be

acquired

by

considering

the

statistical

associations

between

locus-specific

allelic

variants

that

develop

stochastically

as

a

consequence

of

random

genetic

drift

At

drift-recombination

balance

the

amount

of

linkage

disequilibrium

in

a

population

is

a

function

of

the

product

Nlc

where

c

is

the

rate

of

recombination

per

nucleotide

site

-LRB-

Ohta

and

Kimura

Hill

-RRB-

This

quantity

can

be

estimated

by

evaluating

the

rate

at

which

the

level

of

disequilibrium

declines

with

the

physical

distance

between

nucleotide

sites

in

samples

of

gene

sequences

When

joint

estimates

of

Nlu

and

Nlc

are

available

their

ratio

eliminates

Nl

providing

an

estimate

of

the

relative

rates

of

recombination

and

mutation

-LRB-

c/u

-RRB-

Lynch

Table

Estimates

of

c/u

the

Ratio

of

the

Recombination

Rate

to

the

Mutation

Rate

Per

Base

Pair

for

Various

Species

Species

Animals

Homo

sapiens

Chorthippus

parallelus

Drososphila

spp.

Land

plants

Arabidopsis

spp.

Brassica

nigra

Cryptomeria

japonica

Pinus

taeda

Zea

mays

Prokaryotes

Neisseria

gonorrheae

Neisseria

meningitidis

Pseudomonas

syringae

Staphylococcus

aureus

Streptococcus

pneumoniae

c/u

Reference

Ptak

Voepel

and

Przeworski

-LRB-

-RRB-

Ibrahim

Cooper

and

Hewitt

-LRB-

-RRB-

Hey

and

Wakeley

-LRB-

-RRB-

Machado

et

al.

-LRB-

-RRB-

Wright

Lauga

and

Charlesworth

-LRB-

-RRB-

Lagercrantz

Kruskopf

Osterberg

and

Lascoux

-LRB-

-RRB-

Kado

et

al.

-LRB-

-RRB-

Brown

et

al.

-LRB-

-RRB-

Tenaillon

et

al.

-LRB-

-RRB-

Posada

et

al.

-LRB-

-RRB-

Feil

et

al.

-LRB-

-RRB-

Sarkar

and

Guttman

-LRB-

-RRB-

Feil

et

al.

-LRB-

-RRB-

Feil

et

al.

-LRB-

-RRB-

For

eukaryotes

the

results

from

figures

and

can

be

used

to

reveal

c/u

more

directly

yielding

average

relationships

of

c/u

G

for

unicellular

species

and

G

for

multicellular

species

which

implies

expected

values

of

and

for

genomes

and

Mb

in

size

respectively

The

few

available

estimates

for

c/u

for

eukaryotes

from

polymorphism

studies

are

in

rough

accord

with

these

predictions

the

average

being

-LRB-

-RRB-

for

animals

and

-LRB-

-RRB-

for

land

plants

-LRB-

table

-RRB-

whose

genomes

are

generally

in

the

vicinity

of

a

few

hundred

to

several

thousand

megabases

The

few

available

estimates

of

c/u

for

prokaryotes

are

of

the

same

order

of

magnitude

as

those

for

multicellular

eukaryotes

although

not

as

high

as

expected

for

unicellular

eukaryotes

averaging

-LRB-

-RRB-

Thus

relative

to

the

background

rate

of

mutation

recombination

at

the

nucleotide

level

is

not

exceptionally

low

in

prokaryotes

Horizontal

transfer

across

species

boundaries

can

also

expand

the

genomic

resources

available

to

prokaryotes

-LRB-

Ochman

Lawrence

and

Groisman

-RRB-

and

hence

the

efficiency

of

selection

although

this

source

of

diversity

has

been

avoided

in

the

preceding

analyses

In

summary

all

lines

of

evidence

point

to

the

fact

that

the

efficiency

of

selection

is

greatly

reduced

in

eukaryotes

to

a

degree

that

depends

on

organism

size

However

as

suggested

by

Gillespie

-LRB-

-RRB-

the

physical

nature

of

chromosomes

puts

an

ultimate

limit

on

Nl

even

in

species

with

enormous

numerical

abundances

Indeed

the

highest

estimate

of

Nl

derivable

from

the

results

in

figures

and

is

for

Helicobacter

pyogenes

a

highly

recombining

member

of

the

eubacteria

After

accounting

for

the

probable

downward

bias

of

this

estimate

the

upper

limit

to

Nl

for

all

species

dictated

by

the

unavoidable

constraints

of

linkage

and

selective

sweeps

may

be

on

the

order

of

These

numbers

are

relevant

because

as

will

be

shown

below

they

are

just

a

few

orders

of

magnitude

above

the

point

at

which

the

population-genetic

environment

for

genestructure

evolution

becomes

significantly

altered

One

caveat

with

respect

to

estimates

of

Nl

derived

from

polymorphism

data

is

that

they

apply

only

over

the

time

span

necessary

for

the

fixation

of

an

average

neutral

mutation

and

generations

for

diploids

and

haploids

respectively

which

necessarily

increases

in

species

with

larger

Nl

Because

many

of

the

gross

features

of

genomes

may

require

tens

to

hundreds

of

millions

of

years

to

emerge

the

short-term

estimates

of

Nl

for

any

particular

species

are

likely

to

frequently

misrepresent

longer

term

conditions

relevant

to

genome

evolution

For

example

newly

emergent

pathogenic

bacteria

which

often

harbor

almost

no

genetic

variation

-LRB-

Daubin

and

Moran

-RRB-

are

not

expected

to

exhibit

a

signature

of

random

genetic

drift

at

the

level

of

genomic

architecture

On

the

other

hand

averages

of

Nl

over

the

members

of

broad

taxonomic/functional

groups

-LRB-

fig.

-RRB-

eliminate

outliers

resulting

from

sampling

error

and

stochastic

temporal

fluctuations

in

population

size

thereby

providing

more

meaningful

estimates

of

long-term

conditions

Drift

Mutation

Pressure

and

the

Emergence

of

Eukaryotic

Gene

Complexity

Associated

with

reductions

in

Nl

in

eukaryotes

are

dramatic

expansions

in

genome

size

most

of

which

reflect

changes

in

noncoding

regions

introns

mobile

elements

and

their

remnants

and

other

forms

of

intergenic

DNA

-LRB-

fig.

-RRB-

A

notable

feature

of

these

scalings

is

their

continuity

over

all

forms

of

life

even

across

the

prokaryote-eukaryote

boundary

This

strongly

suggests

that

neither

cellular

nor

physiological

changes

associated

with

phylogenetic

transitions

are

major

determinants

of

genome

size

We

have

previously

suggested

that

the

types

of

genomic

evolution

that

are

possible

in

various

lineages

are

instead

largely

defined

by

the

population-genetic

environment

in

particular

by

the

effective

number

of

individuals

within

a

species

-LRB-

Lynch

and

Conery

-RRB-

In

the

remainder

of

this

paper

these

ideas

Origins

of

Eukaryotic

Gene

Structure

FIG.

Scaling

of

genome

content

with

genome

size

in

prokaryotes

-LRB-

inverted

triangles

-RRB-

and

various

eukaryotic

groupings

unicellular

and

oligocellular

species

-LRB-

diamonds

-RRB-

invertebrates

-LRB-

squares

-RRB-

vertebrates

-LRB-

circles

-RRB-

and

land

plants

-LRB-

triangles

-RRB-

Diagonal

dashed

lines

denote

points

of

equal

proportional

contributions

to

total

genome

content

Note

that

mobile-element

associated

DNA

-LRB-

which

includes

retrotransposons

and

DNA-based

transposons

-RRB-

may

be

found

in

introns

as

well

as

intergenic

regions

and

that

the

intronic

DNA

depicted

here

does

not

include

introns

in

noncoding

exons

-LRB-

UTRs

-RRB-

The

data

were

obtained

from

the

various

whole-genome

sequencing

projects

will

be

extended

to

show

how

many

aspects

of

eukaryotic

gene

structure

may

have

arisen

by

nonadaptive

processes

Prokaryotic

genes

generally

have

remarkably

simple

structures

a

single

continuous

coding

region

with

one

or

two

transcription-factor

binding

-LRB-

TFB

-RRB-

sites

residing

just

a

few

nucleotides

upstream

Often

a

single

transcriptioninitiation

site

services

several

downstream

prokaryotic

genes

which

are

jointly

transformed

into

a

single

polycistronic

mRNA

-LRB-

an

operon

-RRB-

In

contrast

the

coding

regions

of

eukaryotic

genes

are

often

dissected

by

introns

which

are

transcribed

into

precursor

mRNAs

and

then

subsequently

eliminated

by

splicing

-LRB-

fig.

-RRB-

In

multicellular

species

dozens

of

introns

may

occupy

a

single

gene

and

each

intron

can

be

many

times

longer

than

its

surrounding

exons

Eukaryotic

genes

also

often

have

complex

sets

of

regulatory

elements

distributed

over

large

distances

upstream

-LRB-

and

sometimes

internally

or

downstream

-RRB-

of

the

coding

region

and

with

few

exceptions

eukaryotic

genes

are

transcribed

as

single

monocistronic

units

Finally

eukaryotic

gene

transcripts

are

generally

flanked

by

extensive

UTRs

which

may

harbor

additional

introns

Understanding

how

such

modifications

of

gene

structure

emerged

is

a

major

challenge

for

evolutionary

genomics

because

each

additional

layer

of

gene

complexity

entails

a

cost

in

terms

of

mutational

vulnerability

Population

geneticists

have

historically

treated

selection

and

mutation

as

separate

forces

in

the

dynamics

of

evolutionary

change

with

mutation

producing

the

variation

upon

which

natural

selection

acts

but

having

no

further

influence

on

the

fates

of

alleles

However

in

the

context

of

gene

architectural

features

there

are

numerous

ways

in

which

mutation

can

act

indirectly

as

a

selective

agent

Consider

a

pair

of

alleles

with

different

forms

of

gene

architecture

but

otherwise

identical

functions

Aside

from

any

energetic

burden

associated

with

the

maintenance

of

larger

numbers

of

nucleotides

as

a

larger

mutational

target

the

more

complex

allele

will

experience

a

greater

rate

of

transformation

to

defective

copies

For

example

intergenic

DNA

has

the

potential

to

incur

mutations

that

produce

spurious

TFB

sites

that

cause

inappropriate

patterns

of

gene

expression

introns

necessitate

the

maintenance

of

localization

signals

at

the

nucleotide

level

to

insure

proper

mRNA

splicing

and

UTRs

can

acquire

premature

translationinitiation

codons

that

cause

downstream

frameshifts

In

this

sense

most

aspects

of

gene-architectural

complexity

impose

an

intrinsic

mutational

burden

The

selective

disadvantage

associated

with

any

single

aspect

of

gene

complexity

need

not

be

very

large

as

it

is

roughly

equivalent

to

the

product

of

the

mutation

rate

per

nucleotide

per

generation

-LRB-

u

-RRB-

and

the

excess

number

of

nucleotide

sites

in

the

more

complex

allele

critical

to

gene

function

-LRB-

n

-RRB-

-LRB-

Lynch

-RRB-

and

arguments

presented

below

suggest

that

n

often

falls

in

the

approximate

range

of

This

implies

that

expansions

of

gene-architectural

complexity

are

unusually

vulnerable

to

fixation

by

random

genetic

drift

in

populations

with

small

genetic

effective

sizes

As

noted

above

if

a

costly

modification

of

gene

architecture

is

to

evolve

in

an

effectively

neutral

manner

must

be

smaller

than

Because

s

nu

and

ps

is

a

function

of

Nlu

this

criterion

is

equivalent

to

psn

Thus

recalling

the

average

estimate

of

ps

for

prokaryotes

-LRB-

fig.

-RRB-

and

its

likely

downward

bias

the

population-genetic

environment

of

prokaryotic

species

may

only

rarely

be

conducive

to

expansions

in

gene-architectural

complexity

In

contrast

the

extremely

low

levels

of

ps

for

multicellular

eukaryotes

create

situations

that

are

highly

permissive

to

the

accumulation

of

gene

architectural

changes

with

weak

mutational

disadvantages

which

are

easily

overwhelmed

by

the

power

of

random

genetic

drift

With

their

wide

ranges

of

Nl

the

various

lineages

of

unicellular

eukaryotes

are

expected

to

fall

between

these

two

extremes

It

should

be

noted

however

that

although

organism

size

appears

to

be

the

primary

determinant

of

Nl

it

is

the

latter

that

ultimately

governs

the

genetic

properties

of

populations

It

is

conceivable

that

some

unicellular

species

may

reside

at

a

sufficiently

low

Nl

for

long

enough

periods

to

promote

genomic

expansion

but

exceedingly

unlikely

that

any

Lynch

FIG.

Generalized

structures

of

prokaryotic

and

eukaryotic

genes

and

transcription

units

Note

that

the

lengths

of

various

gene

parts

are

not

necessarily

to

scale

for

example

eukaryotic

genes

can

harbor

as

many

as

several

dozens

of

introns

each

of

which

greatly

exceeds

the

length

of

its

surrounding

exons

and

TFB

sites

-LRB-

small

ovals

-RRB-

can

be

much

more

numerous

and

widely

distributed

than

illustrated

TATA

denotes

one

of

the

possible

eukaryotic

core

promoter

elements

to

which

transcription

factors

convey

information

and

poly

-LRB-

A

-RRB-

denotes

the

posttranscriptional

addition

of

a

poly

-LRB-

A

-RRB-

tail

Black

bars

denote

coding

DNA

open

bars

denote

transcribed

but

untranslated

DNA

and

thin

lines

within

transcribed

regions

denote

introns

multicellular

species

ever

achieves

prokaryote-like

levels

of

Nl

It

is

clear

that

the

emergence

of

the

complexities

of

eukaryotic

gene

structure

offered

novel

opportunities

for

the

evolution

of

organismal

diversity

many

of

which

have

been

exploited

by

multicellular

eukaryotes

for

example

increased

regulatory-region

complexity

and

alternative

splicing

associated

with

introns

Less

certain

is

whether

multiple

cell

types

and

mechanisms

of

cell

signaling

are

advantageous

in

a

formal

fitness

sense

In

any

event

because

any

such

adaptive

modifications

are

highly

unlikely

to

have

arisen

de

novo

alternative

explanations

are

needed

for

the

first

steps

in

the

retailoring

of

the

eukaryotic

genome

Specific

examples

are

now

given

on

how

three

aspects

of

eukaryotic

gene

complexity

may

have

emerged

despite

their

initial

intrinsic

disadvantages

Each

scenario

discussed

is

quantitatively

consistent

with

the

theory

presented

above

and

shows

how

a

reduction

in

Nl

can

passively

promote

the

evolution

of

gene

architectural

changes

that

ultimately

facilitate

the

evolution

of

organismal

complexity

by

descent

with

modification

Introns

As

noted

above

introns

impose

a

burden

on

their

host

genes

in

that

specific

nucleotide

signatures

must

be

reserved

to

insure

precise

recognition

of

each

exon-intron

junction

by

the

spliceosome

The

most

conserved

nucleotide

sites

are

located

at

the

ends

of

introns

and

at

internal

intronic

branch

points

-LRB-

Burge

Tuschl

and

Sharp

Lorkovic

et

al.

Bon

et

al.

-RRB-

However

this

information

is

often

insufficient

for

proper

spliceosomal

recognition

particularly

in

the

case

of

large

introns

containing

numerous

spurious

recognition

sites

-LRB-

Mount

et

al.

Burge

Tuschl

and

Sharp

Long

and

Deutsch

-RRB-

Supplemental

information

often

resides

within

the

surrounding

exons

in

the

form

of

exon

splicing

enhancers

and

exon

splicing

silencers

-LRB-

ESSs

-RRB-

each

typically

four

to

ten

nucleotides

in

length

-LRB-

Liu

Zhang

Krainer

Schaal

and

Maniatis

Blencowe

-RRB-

In

mammals

of

exonic

sequences

match

the

signatures

of

known

ESSs

with

such

clumps

per

exon

-LRB-

Fairbrother

et

al.

-RRB-

and

the

maintenance

of

such

motifs

by

transcription-related

processes

is

supported

by

the

significantly

different

frequencies

of

various

oligomers

in

intron-containing

versus

intron-free

genes

-LRB-

Federov

et

al.

-RRB-

The

most

direct

evidence

for

the

increased

mutational

vulnerability

associated

with

introns

derives

from

the

observation

that

about

a

third

of

human

genetic

disorders

is

attributable

to

mutations

causing

defective

splice-site

recognition

-LRB-

Culbertson

Frischmeyer

and

Dietz

Philips

and

Cooper

-RRB-

many

of

which

are

located

in

exons

-LRB-

including

substitutions

at

synonymous

sites

-RRB-

-LRB-

Cooper

and

Mattox

Nissim-Rafinia

and

Kerem

-RRB-

Based

on

the

known

molecular

requirements

for

spliceosomal

recognition

one

may

surmise

that

the

equivalent

of

n

to

nucleotide

sites

are

required

for

the

precise

removal

of

each

intron

-LRB-

Lynch

-RRB-

and

indirect

estimates

based

on

the

incidence

of

splicing-defective

alleles

among

new

mutations

are

consistent

with

this

prediction

-LRB-

Lynch

Hong

and

Scofield

-RRB-

Recalling

the

theory

presented

above

a

permissive

environment

for

intron

colonization

requires

that

Nls

Nlnu

be

smaller

than

or

equivalently

ps

Thus

as

a

first-order

approximation

populations

with

silent-site

nucleotide

diversities

greater

than

are

expected

to

be

nearly

immune

to

intron

colonization

Essentially

the

full

range

of

variation

in

observed

ps

for

animals

and

land

plants

is

well

below

this

Origins

of

Eukaryotic

Gene

Structure

threshold

value

-LRB-

fig.

-RRB-

and

all

members

of

these

groups

have

an

average

of

four

to

seven

introns

per

protein-coding

gene

-LRB-

Lynch

and

Conery

-RRB-

In

contrast

the

average

value

of

ps

for

unicellular

species

-LRB-

-RRB-

slightly

exceeds

the

expected

threshold

yielding

the

prediction

that

the

demographic

features

of

such

species

often

place

them

in

close

proximity

to

the

barrier

to

intron

colonization

-LRB-

and

maintenance

-RRB-

Consistent

with

this

prediction

is

the

broad

range

of

variation

in

intron

numbers

in

unicellular

eukaryotes

ranging

from

a

few

dozen

or

less

in

the

entire

genomes

of

some

species

-LRB-

e.g.

trypanosomes

the

diplomonad

Giardia

the

red

alga

Cyanidioschyzon

and

some

fungi

-RRB-

to

numbers

approaching

those

in

animals

and

land

plants

in

other

fungi

-LRB-

fig.

-RRB-

For

prokaryotes

which

are

devoid

of

spliceosomal

introns

average

ps

is

more

than

twice

the

threshold

for

intron

colonization

The

phylogenetic

distribution

of

introns

and

the

components

of

the

spliceosome

make

it

quite

clear

that

the

stem

eukaryote

harbored

introns

-LRB-

Lynch

and

Richardson

Collins

and

Penny

-RRB-

and

perhaps

a

substantial

number

of

them

-LRB-

an

average

of

up

to

three

per

protein-coding

gene

being

plausible

-RRB-

-LRB-

Rogozin

et

al.

Roy

and

Gilbert

see

Qiu

Schisler

and

Stoltzfus

for

an

alternative

view

-RRB-

Thus

because

there

is

no

evidence

of

the

prior

existence

of

a

spliceosome

in

any

prokaryote

the

stem

eukaryote

must

have

provided

a

highly

permissive

environment

for

intron

colonization

with

some

subsequent

lineages

then

experiencing

conditions

that

favored

intron

loss

and

others

favoring

further

intron

gain

Could

the

stem

eukaryote

have

had

a

sufficiently

small

population

size

to

allow

the

accumulation

of

a

substantial

intron

population

via

effectively

neutral

processes

alone

Two

different

approaches

have

led

to

the

conclusion

that

the

birth

rate

of

introns

within

the

past

Myr

is

nucleotide

site/Byr

in

invertebrates

-LRB-

Lynch

and

Richardson

Roy

and

Gilbert

-RRB-

At

this

rate

Byr

would

be

required

since

the

origin

of

the

spliceosome

for

the

protein-coding

genes

of

the

stem

eukaryote

to

acquire

an

average

of

introns

assuming

an

average

coding

length

of

kb

as

in

common

in

today

's

eukaryotes

Thus

because

the

time

span

between

the

origin

of

life

and

the

origin

of

eukaryotes

is

Byr

-LRB-

Knoll

Furnes

et

al.

-RRB-

the

passive

acquisition

of

more

than

one

intron

per

protein-coding

gene

in

the

stem

eukaryote

is

just

barely

plausible

unless

the

physical

rate

of

intron

birth

was

substantially

higher

than

in

today

's

species

A

more

rapid

early

proliferation

of

introns

could

have

occurred

if

some

form

of

positive

selection

offset

the

intrinsic

disadvantages

associated

with

elevated

mutational

vulnerability

as

this

would

increase

the

rate

of

fixation

beyond

the

neutral

expectation

One

possibility

involves

the

nonsense-mediated

decay

-LRB-

NMD

-RRB-

pathway

an

mRNA

surveillance

mechanism

for

detecting

and

eradicating

transcripts

harboring

premature

termination

codons

-LRB-

PTCs

-RRB-

The

details

of

this

process

have

been

worked

out

in

only

a

few

organisms

but

at

least

in

mammals

NMD

often

uses

a

protein

complex

laid

down

at

splicing

junctions

-LRB-

the

exon

junction

complex

-LSB-

EJC

-RSB-

-RRB-

to

discriminate

PTCs

from

proper

termination

codons

If

a

termination

codon

is

detected

upstream

of

an

EJC

the

transcript

is

generally

targeted

for

destruction

a

process

that

works

so

long

as

the

true

termination

codon

generally

resides

in

the

final

exon

as

is

usually

the

case

in

mammals

-LRB-

Maquat

-RRB-

Aside

from

the

transcription

of

mutant

alleles

there

are

many

different

routes

to

the

stochastic

production

of

PTC-containing

transcripts

including

base

misincorporation

sloppy

points

of

transcription

initiation

and

erroneous

splicing

-LRB-

reviewed

in

Lynch

Hong

and

Scofield

-RRB-

Thus

most

cells

are

regularly

confronted

with

the

need

to

eliminate

transcripts

that

could

lead

to

harmful

truncated

proteins

and

the

benefits

of

doing

so

via

NMD

are

well

documented

-LRB-

Hodgkin

et

al.

Leeds

et

al.

Dahlseid

et

al.

Mendell

et

al.

Medghalchi

et

al.

-RRB-

Phylogenetic

analysis

suggests

that

both

NMD

and

the

EJC

were

present

in

the

stem

eukaryote

-LRB-

Lynch

Hong

and

Scofield

-RRB-

Thus

an

early

functional

association

of

NMD

with

introns

could

have

elevated

the

rate

of

intron

proliferation

beyond

the

neutral

expectation

-LRB-

Lynch

and

Kewalramani

-RRB-

Under

this

hypothesis

the

first

intron

to

colonize

a

gene

would

provide

a

basis

for

eliminating

transcripts

with

the

subset

of

upstream

PTCs

However

because

the

spatial

locations

of

initially

colonizing

introns

must

be

largely

random

because

introns

themselves

encourage

the

production

of

erroneous

transcripts

via

splicing

errors

and

because

some

PTCs

may

be

unable

to

elicit

NMD

if

the

nearest

EJC

is

too

far

downstream

once

this

coevolutionary

process

initiated

further

colonization

of

introns

would

be

encouraged

In

this

manner

some

of

the

earliest

colonizing

introns

-LRB-

those

in

locations

that

allowed

sufficient

PTC

detection

-RRB-

may

have

had

a

selective

advantage

that

offset

the

cost

of

increased

mutational

susceptibility

The

overdispersed

distributions

of

introns

in

the

genes

of

multicellular

species

support

the

hypothesis

that

selection

favors

a

uniform

coverage

of

coding

regions

with

introns

-LRB-

Lynch

and

Kewalramani

-RRB-

Other

factors

may

encourage

the

colonization

of

introns

-LRB-

Lynch

and

Richardson

-RRB-

but

the

central

point

here

is

that

once

introns

became

a

reliable

aspect

of

a

substantial

fraction

of

eukaryotic

genes

they

served

as

a

natural

substrate

for

secondary

adaptive

evolution

Despite

the

obvious

benefits

of

an

mRNA

surveillance

system

a

few

species

appear

to

have

lost

the

NMD

pathway

-LRB-

Lynch

Hong

and

Scofield

-RRB-

These

include

the

kinetoplastids

Trypanosoma

and

Leishmania

the

unicellular

red

alga

Cyanidioschyzon

the

microsporidian

Encephalitozoon

and

the

diplomonad

Giardia

Remarkably

each

of

these

lineages

is

almost

entirely

devoid

of

introns

and

with

the

exception

of

Leishmania

they

all

appear

to

have

lost

the

EJC

apparatus

It

is

tempting

to

conclude

that

the

loss

of

introns

and

NMD

must

go

hand

in

hand

simply

because

of

the

latter

's

functional

requirement

for

an

EJC

However

because

NMD

operates

on

some

genes

in

an

intronindependent

manner

in

a

phylogenetically

broad

group

of

species

-LRB-

Ruiz-Echevarria

Gonzalez

and

Peltz

Hilleren

and

Parker

Gatfield

et

al.

Amrani

et

al.

-RRB-

it

is

not

clear

that

introns

are

an

absolute

requirement

for

the

maintenance

of

NMD

An

alternative

explanation

for

NMD

losses

is

that

the

species

involved

have

had

very

large

historical

effective

sizes

which

facilitated

the

elimination

of

all

forms

of

extraneous

DNA

including

mobile

elements

and

most

intergenic

DNA

As

the

degree

of

genomic

streamlining

increases

the

production

of

erroneous

Lynch

transcripts

may

eventually

decline

to

the

point

at

which

the

selective

advantage

of

an

intron-based

NMD

system

is

no

longer

sufficient

to

insure

its

evolutionary

stability

-LRB-

Lynch

Hong

and

Scofield

-RRB-

A

central

unresolved

issue

with

respect

to

introns

is

whether

intron

numbers

have

reached

a

steady-state

equilibrium

and

if

so

what

prevents

runaway

intron

colonization

The

equilibrium

occupancy

of

introns

is

a

function

of

the

ratio

of

birth

-LRB-

b

-RRB-

to

death

-LRB-

d

-RRB-

rates

per

coding

nucleotide

site

-LRB-

Lynch

-RRB-

but

no

equilibrium

is

possible

if

b

d

for

all

levels

of

occupancy

The

NMD

hypothesis

provides

a

potential

density-dependent

mechanism

that

could

stabilize

intron

numbers

As

a

sufficiently

well-distributed

population

of

introns

is

established

the

NMD-associated

advantages

of

additional

introns

will

progressively

decline

until

a

point

is

eventually

reached

at

which

further

intron

colonization

imposes

a

net

disadvantage

Such

a

scenario

would

provide

a

natural

barrier

to

runaway

intron

colonization

only

if

the

net

selective

disadvantage

of

each

additional

intron

exceeded

the

power

of

random

genetic

drift

or

if

the

physical

rate

of

intron

removal

somehow

increased

with

intron

number

The

average

number

of

introns

per

protein-coding

gene

in

vertebrates

ranges

from

-LRB-

Fugu

-RRB-

to

-LRB-

Gallus

-RRB-

whereas

the

range

for

invertebrates

is

nearly

nonoverlapping

-LRB-

Drosophila

-RRB-

to

-LRB-

Bombyx

-RRB-

Thus

it

is

clear

that

the

animal

lineage

experienced

a

basal

increase

in

intron

number

although

it

is

an

open

question

as

to

whether

the

numbers

in

vertebrates

continued

to

expand

-LRB-

perhaps

even

today

-RRB-

as

a

consequence

of

the

reduced

efficiency

of

selection

associated

with

low

Nl

One

analysis

is

consistent

with

the

latter

interpretation

-LRB-

Rogozin

et

al.

-RRB-

Finally

it

is

worth

considering

the

origin

of

the

complex

molecular

machine

that

makes

introns

possible

the

spliceosome

The

most

credible

hypothesis

involves

descent

from

a

group

II

intron

-LRB-

Sharp

Cech

Lambowitz

and

Zimmerly

-RRB-

Although

these

selfsplicing

introns

have

never

been

found

in

nuclear

genes

their

presence

in

eubacteria

archaea

and

the

organelles

of

plants

fungi

and

numerous

protists

-LRB-

Bonen

and

Vogel

Dai

and

Zimmerly

Rest

and

Mindell

-RRB-

makes

plausible

the

idea

that

they

were

present

in

the

stem

eukaryote

Deriving

further

support

from

the

numerous

structural

and

functional

similarities

between

the

excision

mechanisms

for

group

II

introns

and

spliceosome-dependent

introns

-LRB-

Michel

and

Ferat

Hetzer

et

al.

Burge

Tuschl

and

Sharp

Sontheimer

Gordon

and

Piccirilli

Shukla

and

Padgett

Valadkhan

and

Manley

-RRB-

the

group

II

seed

hypothesis

postulates

that

the

five

small

RNAs

at

the

heart

of

the

spliceosome

are

direct

descendants

of

the

major

subunits

of

the

catalytic

core

of

a

group

II

intron

However

the

transition

from

a

self-splicing

group

II

intron

to

a

large

population

of

eukaryotic

spliceosome-dependent

introns

would

have

involved

a

number

of

evolutionary

challenges

-LRB-

Stoltzfus

Lynch

and

Richardson

-RRB-

not

the

least

of

which

is

the

reassignment

of

functional

fragments

from

group

II

introns

associated

with

specific

genes

to

a

more

generalized

splicing

mechanism

servicing

hundreds

to

perhaps

thousands

of

genes

The

proposed

evolutionary

pathway

to

group

II

intron

fragmentation

involves

a

series

of

effec

tively

neutral

steps

-LRB-

Cavalier-Smith

Stoltzfus

-RRB-

that

are

intrinsic

to

the

subfunctionalization

process

-LRB-

Force

et

al.

-RRB-

However

subfunctionalization

is

exceedingly

unlikely

in

large

populations

which

impose

difficulties

for

the

establishment

of

functional

fragments

without

mutational

deterioration

during

the

long

time

required

for

fixation

-LRB-

Lynch

and

Richardson

-RRB-

Thus

if

the

group

II

seed

hypothesis

is

correct

it

reinforces

the

view

that

the

emergence

of

eukaryotes

was

accompanied

by

a

reduction

in

Nl

UTRs

Like

introns

the

untranslated

leader

sequences

of

mRNAs

are

liabilities

for

genes

because

they

increase

the

size

of

the

mutational

target

Most

notably

the

UTR

serves

as

substrate

for

the

mutational

appearance

of

premature

translation

start

codons

-LRB-

PSCs

-RRB-

which

because

of

the

scanning

mechanism

for

translation

initiation

in

eukaryotes

-LRB-

Kozak

-RRB-

can

lead

to

N-terminal

expansion

of

the

protein

product

-LRB-

in

of

cases

-RRB-

and

a

shift

in

the

reading

frame

and

protein

truncation

-LRB-

in

of

cases

-RRB-

A

deficit

of

ATG

triplets

in

the

exons

of

UTRs

but

much

less

so

in

their

introns

provides

compelling

evidence

of

the

negative

translation-associated

consequences

of

such

mutations

-LRB-

Rogozin

et

al.

Lynch

Scofield

and

Hong

-RRB-

as

does

the

incidence

of

human

genetic

disorders

associated

with

the

appearance

of

PSCs

-LRB-

Kozak

-RRB-

This

raises

questions

not

only

as

to

why

UTRs

are

present

but

why

they

are

so

long

Contrary

to

the

situation

for

introns

which

vary

in

average

size

by

over

two

orders

of

magnitude

in

different

phylogenetic

groups

the

average

UTR

lengths

of

most

eukaryotic

lineages

are

remarkably

constant

falling

in

the

narrow

range

of

bp

-LRB-

Lynch

Scofield

and

Hong

-RRB-

Messenger

RNAs

are

unlikely

to

require

leader

sequences

as

physical

landing

pads

for

the

ribosome

For

example

although

archaebacteria

employ

transcriptional

mechanisms

similar

to

those

of

eukaryotes

-LRB-

Bell

and

Jackson

-RRB-

their

UTRs

are

generally

no

more

than

a

dozen

nucleotides

in

length

and

in

some

cases

are

completely

absent

-LRB-

Slupska

et

al.

-RRB-

Similar

situations

are

observed

in

eubacteria

-LRB-

Weiner

Herrmann

and

Browning

Moll

et

al.

-RRB-

and

in

mitochondria

-LRB-

Gillham

Taanman

-RRB-

Moreover

UTRs

in

the

diplomonad

Giardia

often

consist

of

just

a

single

nucleotide

-LRB-

Iwabe

and

Miyata

-RRB-

and

a

substantial

fraction

of

those

in

a

variety

of

other

unicellular

eukaryotes

are

bp

including

those

in

the

ciliate

Euplotes

crassus

-LRB-

Ghosh

et

al.

-RRB-

the

amoeba

Entamoeba

histolytica

-LRB-

Singh

et

al.

-RRB-

and

the

trichomonad

Trichomonas

vaginalis

-LRB-

Liston

and

Johnson

-RRB-

Experimental

evidence

suggests

that

such

diminutive

leader

sequences

are

sufficient

to

support

translation

in

mammals

and

yeast

although

the

efficiency

of

translation

can

be

reduced

with

UTRs

shorter

than

bp

-LRB-

van

den

Heuvel

et

al.

Maicas

Shago

and

Friesen

Hughes

and

Andrews

-RRB-

To

evaluate

whether

the

expansion

of

eukaryotic

#UTR

lengths

might

be

a

simple

consequence

of

the

reduced

efficiency

of

selection

associated

with

small

Nl

a

simple

model

for

the

stochastic

growth

and

contraction

of

Origins

of

Eukaryotic

Gene

Structure

UTRs

based

on

mutational

gains

and

losses

of

PSCs

and

transcription-initiation

signals

-LRB-

TISs

e.g.

the

TATA

box

-RRB-

has

been

developed

-LRB-

Lynch

Scofield

and

Hong

-RRB-

Under

this

model

all

alleles

are

assumed

to

be

effectively

neutral

with

respect

to

each

other

with

the

exception

of

two

defective

classes

alleles

containing

a

harmful

PSC

between

the

TIS

and

the

true

translation

start

codon

and

alleles

for

which

the

TIS

has

moved

so

close

to

the

coding

region

that

transcription

initiates

beyond

the

translationstart

point

As

ATG

triplets

are

free

to

accumulate

upstream

of

currently

utilized

transcription-initiation

sites

this

process

results

in

a

natural

barrier

to

excessive

growth

of

UTRs

which

can

only

expand

in

a

direction

if

the

extension

is

devoid

of

harmful

PSCs

Over

time

the

stochastic

winking

on

and

off

of

PSCs

and

TISs

upstream

of

the

true

translation-start

site

results

in

an

equilibrium

L-shaped

distribution

of

UTR

lengths

with

a

mean

and

variance

that

are

quite

similar

to

those

observed

within

a

wide

variety

of

species

largely

independent

of

the

assumed

length

of

the

TIS

-LRB-

Lynch

Scofield

and

Hong

-RRB-

This

result

suggests

that

the

reduced

mutational

burden

of

short

UTRs

may

be

inconsequential

in

comparison

to

the

power

of

random

genetic

drift

in

most

eukaryotes

To

understand

the

mechanistic

underpinnings

of

such

a

condition

it

is

necessary

to

consider

the

long-term

excess

mutation

rate

to

defective

alleles

for

an

allelic

lineage

in

which

UTR

lengths

simply

stochastically

grow

and

contract

-LRB-

as

in

the

above

model

-RRB-

without

selective

discrimination

among

functional

alleles

in

contrast

to

the

situation

in

which

minimal

length

UTRs

are

maintained

by

positive

selection

The

mutational

disadvantage

of

stochastically

generated

UTRs

increases

with

the

length

of

the

TIS

-LRB-

n

-RRB-

not

only

because

of

the

larger

target

size

of

the

TIS

itself

but

also

because

a

larger

n

implies

that

in

the

event

of

a

TIS

loss

the

nearest

upstream

alternative

TIS

will

be

increasingly

likely

to

reside

beyond

a

harmful

PSC

-LRB-

because

of

the

rarity

of

complex

sequences

-RRB-

This

is

a

potential

concern

because

of

the

limited

information

available

on

the

length

of

core

promoter

sequences

However

the

key

result

is

fairly

robust

to

the

length

of

the

TIS

for

n

in

the

range

of

-LRB-

e.g.

TATA

-RRB-

to

-LRB-

which

is

beyond

the

complexity

of

known

TISs

-RRB-

the

long-term

mutational

advantage

of

a

minimallength

UTR

is

in

the

narrow

range

of

u

to

-LRB-

Lynch

Scofield

and

Hong

-RRB-

substantially

smaller

than

the

mutational

penalty

associated

with

an

intron

Again

following

the

logic

outlined

above

this

result

implies

that

a

permissive

environment

for

the

stochastic

expansion

of

UTRs

is

ps

Thus

because

all

estimates

of

ps

for

multicellular

eukaryotes

are

well

below

-LRB-

fig.

-RRB-

the

power

of

genetic

drift

in

all

such

species

is

far

too

great

for

the

maintenance

of

minimal-length

UTRs

by

positive

selection

The

estimates

of

ps

for

essentially

all

unicellular

eukaryotes

are

also

below

the

threshold

value

of

although

as

discussed

above

some

of

these

estimates

are

likely

to

be

downwardly

biased

raising

the

possibility

that

the

long-term

Nl

of

some

microbial

eukaryotes

and

certainly

many

prokaryotes

is

adequate

to

allow

the

selective

promotion

of

alleles

with

UTRs

of

minimal

length

Although

the

data

are

limited

a

few

protist

lineages

do

appear

to

have

average

UTR

lengths

too

small

to

be

accommodated

by

the

model

-LRB-

Ghosh

et

al.

Singh

et

al.

Liston

and

Johnson

Yee

et

al.

Adam

-RRB-

If

the

preceding

hypothesis

is

correct

then

selection

for

gene-specific

regulatory

features

need

not

be

invoked

to

explain

either

the

expansion

of

eukaryotic

UTRs

relative

to

the

situation

in

prokaryotes

or

the

thousandfold

range

of

UTR

lengths

among

genes

within

species

Nevertheless

once

permanently

established

expanded

UTRs

may

have

provided

novel

substrate

for

the

evolution

of

posttranscriptional

mechanisms

for

regulating

gene

expression

For

example

numerous

features

of

UTRs

-LRB-

including

their

lengths

-RRB-

in

today

's

eukaryotes

can

influence

the

rate

of

protein

synthesis

by

modifying

the

efficiency

of

translation

-LRB-

de

Moor

and

Richter

Pickering

and

Willis

-RRB-

and

some

have

argued

that

upstream

open

reading

frames

-LRB-

uORFs

-RRB-

serve

an

adaptive

function

by

causing

the

ribosome

to

terminate

and/or

reinitiate

thereby

slowing

the

rate

of

translation

-LRB-

Morris

and

Geballe

Meijer

and

Thomas

Vilela

and

McCarthy

-RRB-

However

the

extent

to

which

any

features

of

UTRs

are

adaptive

remains

an

open

question

For

example

uORFs

are

generally

codons

in

length

approximately

what

is

expected

by

chance

and

these

may

simply

exist

because

their

stop

codons

have

neutralized

the

effects

of

a

PSC

enabling

the

downstream

coding

domain

to

perform

at

normal

levels

Modularization

of

Regulatory

Regions

The

ability

to

regulate

the

expression

of

genes

in

tissue

developmental

stage

and/or

environment-specific

manners

is

the

hallmark

of

organisms

with

multiple

cell

types

To

accomplish

such

tasks

genes

generally

harbor

numerous

cis-acting

regulatory

elements

that

cooperatively

interact

with

multiple

trans-acting

factors

to

finely

tune

levels

of

transcription

These

regulatory

elements

are

often

organized

into

modular

units

such

that

mutations

to

individual

gene

subfunctions

have

restricted

phenotypic

effects

-LRB-

Force

et

al.

Prince

and

Pickett

-RRB-

It

has

often

been

argued

that

this

particulate

form

of

gene

regulation

was

a

fundamental

prerequisite

for

the

origin

of

developmental

modules

capable

of

independent

evolutionary

trajectories

and

some

have

suggested

that

such

organization

enhances

the

evolvability

of

lineages

-LRB-

Raff

Wagner

Wagner

and

Altenberg

Gerhart

and

Kirschner

Hartwell

et

al.

Niehrs

and

Pollet

Carroll

Grenier

and

Weatherbee

Davidson

-RRB-

However

it

is

an

open

question

as

to

whether

evolvability

a

populationlevel

phenomenon

is

ever

promoted

by

natural

selection

Each

essential

regulatory

element

elevates

a

gene

's

susceptibility

to

inactivating

mutations

and

it

is

unclear

whether

adaptive

processes

are

sufficient

to

explain

the

origin

of

modular

gene

structure

and

regulatory

logic

Therefore

an

essential

first

step

in

understanding

the

initial

establishment

of

modular

gene-regulatory

structure

is

a

consideration

of

the

situation

in

which

selection

is

a

negligible

force

-LRB-

Zuckerkandl

Force

et

al.

-RRB-

The

following

discussion

outlines

how

the

modular

organization

of

regulatory

regions

can

arise

passively

in

response

to

drift

and

mutation

processes

alone

To

simplify

the

presentation

new

subfunctions

will

be

assumed

to

be

defined

by

TFB

Lynch

sites

-LRB-

or

integrated

regions

of

such

sites

-RRB-

that

are

potentially

separable

from

other

such

sites

both

mutationally

and

functionally

-LRB-

Yuh

Bolouri

and

Davidson

Force

et

al.

Arnosti

-RRB-

However

the

principles

outlined

below

are

broadly

generalizable

as

a

gene

subfunction

may

also

correspond

to

alternative

splice

sites

transcriptioninitiation

sites

polyadenylation

signals

etc.

Before

exploring

the

population-genetic

issues

a

brief

overview

of

eukaryotic

transcriptional

control

will

be

useful

Transcription-factor

genes

are

generally

expressed

at

specific

stages

of

development

at

a

hierarchy

of

levels

of

organization

Some

transcription

factors

are

organ

specific

some

are

specific

to

individual

tissues

within

an

organ

and

still

others

are

expressed

in

specific

cell

types

within

a

tissue

By

virtue

of

their

ability

to

bind

DNA

and/or

to

act

as

cofactors

in

binding

each

other

together

in

functionally

significant

ways

the

full

suite

of

transcription

factors

and

their

combinatorial

expression

provides

a

heterogeneous

informational

network

within

the

organism

A

gene

's

pattern

of

expression

is

then

defined

by

the

match

between

its

TFB

sites

and

the

local

transcription-factor

environment

Although

the

entire

regulatory

domain

of

a

gene

can

sometimes

rival

the

length

of

the

coding

region

and

individual

genes

can

harbor

dozens

of

regulatory

elements

individual

TFB

sites

are

usually

no

more

than

a

dozen

base

pairs

in

length

and

such

sites

are

often

partially

overlapping

or

entirely

embedded

Because

small

regulatory

elements

can

arise

by

a

fortuitous

series

of

de

novo

base

substitutions

and/or

by

segmental

insertions

there

are

many

potential

ways

in

which

the

numbers

locations

and

types

of

TFB

sites

for

a

gene

can

become

modified

-LRB-

Brosius

von

Dassow

and

Monro

Edelman

et

al.

Stone

and

Wray

Rockman

and

Wray

Jordan

et

al.

Wray

et

al.

MacArthur

and

Brookfield

-RRB-

However

the

evolution

of

a

new

form

of

gene

regulatory

architecture

will

generally

require

a

period

of

redundancy

as

this

will

allow

the

modification

of

one

set

of

TFB

sites

before

the

other

is

abandoned

thereby

insuring

overall

constancy

of

gene

expression

during

the

transition

phase

Because

the

origin

of

tandem

duplicated

stretches

of

DNA

is

quite

high

especially

for

small

segments

-LRB-

Lynch

and

Conery

Katju

and

Lynch

-RRB-

such

situations

appear

to

be

very

common

in

eukaryotes

and

various

permutations

of

TFB

sites

often

yield

functionally

equivalent

patterns

of

gene

expression

-LRB-

Bonneton

et

al.

Hancock

et

al.

Ludwig

et

al.

Dermitzakis

and

Clark

Shaw

et

al.

Arnosti

Dermitzakis

Bergman

and

Clark

-RRB-

Before

proceeding

it

is

worth

emphasizing

that

the

goal

here

is

not

to

explain

the

expression

of

a

gene

in

a

new

temporal

or

spatial

context

but

to

understand

how

a

gene

that

is

initially

under

control

of

a

ubiquitously

expressed

transcription

factor

comes

to

be

regulated

by

spatially

and/or

temporally

restricted

transcription

factors

while

initially

retaining

the

same

overall

expression

pattern

The

process

envisioned

subfunction

fission

invokes

gradual

structural

modifications

of

preexisting

enhancers

within

a

gene

-LRB-

descent

with

modification

-RRB-

rather

than

the

saltatory

appearance

of

entirely

new

regulatory

modules

-LRB-

Force

et

al.

-RRB-

Subfunction

fission

involves

consecutive

phases

of

regulatory-region

expansion

and

contraction

-LRB-

fig.

-RRB-

The

first

step

toward

the

modularization

of

a

gene

's

regulatory

apparatus

accretion

and

degeneration

involves

the

acquisition

of

new

semi-redundant

TFB

sites

combined

with

the

degeneration

of

one

or

more

ancestral

sites

This

converts

a

universally

used

set

of

TFB

sites

to

a

semiindependent

enhancer

with

a

pair

of

partially

overlapping

regulatory

elements

-LRB-

series

a

d

upper

left

of

fig.

-RRB-

If

the

alternative

TFB

sites

are

subject

to

both

mutational

gain

and

loss

there

is

not

necessarily

a

permanent

allelic

state

under

this

model

as

the

alternative

alleles

are

free

to

mutate

back

and

forth

-LRB-

hence

the

two-way

arrows

in

fig.

-RRB-

Nevertheless

it

is

instructive

to

know

the

population-genetic

conditions

under

which

the

semi-independently

regulated

allele

-LRB-

d

-RRB-

is

most

likely

to

accumulate

as

this

is

an

essential

first

step

in

the

transition

to

an

allele

with

two

entirely

independent

subfunctions

Letting

lb

and

ld

respectively

denote

the

rates

of

birth

and

loss

of

individual

TFB

sites

it

is

possible

to

estimate

the

average

time

that

transpires

between

a

transient

state

fixed

for

an

allele

with

shared

regulatory

sites

-LRB-

a

-RRB-

and

an

allele

with

a

semi-independent

enhancer

-LRB-

d

-RRB-

-LRB-

Force

et

al.

-RRB-

Provided

the

transition

time

to

the

semi-independently

regulated

state

is

independent

of

population

size

because

the

waiting

times

for

mutational

changes

and

are

much

greater

than

the

time

to

drift

to

fixation

In

contrast

as

Nl

approaches

the

point

at

which

the

transition

time

to

the

semi-independently

regulated

allele

begins

to

scale

linearly

with

Nl

Thus

as

in

the

case

of

introns

and

UTRs

the

key

condition

for

maximizing

the

likelihood

of

a

transition

from

a

simple

to

more

complex

-LRB-

semi-independently

regulated

-RRB-

regulatory

region

is

a

function

of

the

ratio

of

the

power

of

a

mutational

force

-LRB-

being

the

rate

of

loss

of

TFB

sites

per

individual

-RRB-

and

the

power

of

drift

-LRB-

-RRB-

Although

the

net

addition

of

a

regulatory

element

to

a

gene

imposes

a

weak

selective

disadvantage

by

increasing

the

mutation

rate

to

defective

alleles

selection

is

ineffective

except

in

very

large

populations

Given

the

small

size

of

typical

TFB

sites

is

expected

to

be

on

the

order

of

-LRB-

or

perhaps

somewhat

higher

after

accounting

for

bias

and

also

allowing

for

inactivation

by

insertion

deletion

mutations

-RRB-

In

any

event

again

recalling

the

results

in

figure

the

condition

for

accretion

and

degeneration

is

probably

fulfilled

in

all

multicellular

eukaryotes

as

well

as

in

numerous

unicellular

eukaryotes

and

probably

violated

in

most

prokaryotes

This

implies

that

that

as

soon

as

transcription

factors

with

spatially

and/or

temporally

restricted

patterns

of

expression

evolved

large

numbers

of

broadly

expressed

genes

in

eukaryotes

would

have

become

subject

to

the

passive

accumulation

of

alleles

with

semiindependent

regulation

In

the

second

step

toward

complete

modularization

-LRB-

duplication

degeneration

and

complementation

-RRB-

the

local

duplication

of

a

semi-independent

enhancer

provides

an

opportunity

for

the

formation

of

two

entirely

independent

regulatory

elements

-LRB-

lower

left

of

fig.

-RRB-

The

events

during

this

phase

are

conceptually

identical

to

those

underlying

the

subfunctionalization

model

for

the

preservation

of

duplicate

genes

-LRB-

Force

et

al.

Lynch

et

al.

-RRB-

although

in

this

case

the

process

involves

smaller

regulatory

elements

Progression

to

the

modularized

state

during

this

phase

Origins

of

Eukaryotic

Gene

Structure

FIG.

A

hypothetical

scenario

in

which

an

allele

with

two

independently

mutable

subfunctions

can

arise

from

an

allele

with

a

single

generalized

expression

mechanism

Regulatory

regions

are

depicted

on

the

left

with

each

regulatory

element

color

coded

according

to

the

transcription

factor

that

binds

to

it

On

the

right

the

allele-specific

utilizations

of

transcription

factors

are

depicted

Transcription

factors

denoted

by

black

and

white

are

ubiquitously

expressed

whereas

those

denoted

by

two

shades

of

grey

are

each

expressed

in

single

nonoverlapping

tissues

In

the

first

phase

of

allelic

evolution

-LRB-

accretion

and

degeneration

-RRB-

the

regulatory

region

undergoes

sequential

accretion

of

the

dark

and

light

grey

elements

which

together

are

redundant

with

respect

to

the

white

-LRB-

but

not

the

black

-RRB-

element

The

redundant

white

element

is

then

lost

yielding

a

descendant

allele

will

a

semi-independent

mode

of

expression

as

the

black

element

is

still

essential

to

expression

in

both

tissues

In

the

second

phase

-LRB-

duplication

degeneration

and

complementation

-RRB-

the

entire

enhancer

region

is

tandemly

duplicated

with

each

component

then

losing

a

complementary

-LRB-

light/dark

grey

-RRB-

element

The

resultant

allele

has

two

independently

mutable

subfunctions

denoted

by

the

dark

and

light

grey

open

boxes

as

a

mutation

in

either

region

has

effects

that

are

confined

to

a

single

tissue

Note

that

throughout

these

transitions

there

has

been

no

change

in

the

pattern

of

deployment

of

the

gene

which

is

always

expressed

in

both

tissues

only

the

mechanism

of

achieving

expression

is

altered

requires

that

each

semi-independent

enhancer

incurs

a

complementary

loss

of

tissue-specific

function

prior

to

one

of

the

enhancers

being

entirely

silenced

The

probabilities

of

these

alternative

outcomes

depend

on

the

relative

rates

of

loss

of

tissue-specific

-LRB-

two

shades

of

grey

-RRB-

and

shared

-LRB-

black

-RRB-

TFB

sites

but

regardless

of

these

mutational

properties

large

Nl

can

impose

a

complete

barrier

to

enhancer

subfunctionalization

This

population-size

dependence

is

again

a

consequence

of

a

modularized

allele

serving

as

a

larger

mutational

target

-LRB-

four

black

binding

sites

at

the

bottom

left

of

fig.

as

opposed

to

two

for

allele

d

-RRB-

-LRB-

Lynch

et

al.

Force

et

al.

-RRB-

These

results

have

several

implications

First

a

modular

tissue-specific

gene

regulatory

structure

can

emerge

spontaneously

from

an

initial

state

in

which

the

full

expression

breadth

of

a

gene

is

under

unified

control

Not

only

does

this

process

proceed

via

entirely

nonadaptive

processes

-LRB-

random

genetic

drift

and

mutation

-RRB-

but

because

alleles

with

more

complex

regulatory

architectures

have

an

excess

mutation

rate

to

nulls

it

is

less

likely

to

occur

in

situations

where

the

power

of

natural

selection

exceeds

that

of

drift

Thus

contrary

to

popular

belief

not

only

may

natural

selection

be

an

insufficient

mechanism

for

the

origin

of

genotypic

modularity

but

conditions

where

selection

is

most

likely

to

be

efficient

-LRB-

large

populations

-RRB-

promote

the

opposite

situation

alleles

under

unified

transcriptional

control

In

this

sense

the

reduction

in

Nl

that

accompanied

the

origin

of

eukaryotes

may

have

paved

the

way

for

the

emergence

of

modular

gene

architecture

without

any

direct

involvement

of

positive

selection

The

process

will

be

even

more

powerful

in

multicellular

species

because

a

wider

range

of

regulatory-region

structures

with

mildly

deleterious

fitness

effects

-LRB-

beyond

the

mutational

disadvantage

-RRB-

will

be

effectively

neutral

providing

additional

routes

for

the

colonization

of

new

TFB

sites

Second

the

transition

to

modular

gene

structure

may

not

always

require

the

accretion

of

new

regulatory

elements

Due

to

the

recurrent

mutational

production

of

alternative

TFB

sites

large

populations

can

be

expected

to

harbor

high

frequencies

of

semi-modular

alleles

-LRB-

of

the

form

c

in

fig.

-RRB-

which

owing

to

the

presence

of

redundant

TFB

sites

-LRB-

white

and

grey

elements

-RRB-

have

a

weak

mutationally

induced

selective

advantage

relative

to

alleles

of

type

a

-LRB-

Wagner

-RRB-

This

implies

that

a

population

that

has

experienced

a

prolonged

phase

of

large

effective

size

which

inhibits

the

evolution

of

modularity

can

nevertheless

be

poised

to

make

a

transition

to

allelic

state

d

following

a

population-size

reduction

and

the

subsequent

neutral

loss

Lynch

of

the

shared

redundant

-LRB-

white

-RRB-

TFB

site

from

its

precursor

allele

-LRB-

c

-RRB-

Finally

although

the

pattern

of

tissue-specific

gene

expression

remains

unaltered

throughout

the

process

of

subfunction

fission

the

underlying

molecular

mechanisms

for

achieving

the

pattern

are

modified

in

ways

that

can

eliminate

pleiotropic

constraints

associated

with

shared

regulatory

elements

thereby

opening

up

previously

inaccessible

evolutionary

pathways

Moreover

the

spontaneous

emergence

of

modular

gene

organization

by

random

drift

and

mutation

processes

has

a

powerful

secondary

effect

When

a

modularized

gene

is

duplicated

in

a

small

population

which

occurs

frequently

-LRB-

Lynch

and

Conery

-RRB-

the

two

members

of

the

duplicate

pair

will

have

a

high

probability

of

experiencing

complementary

degenerative

mutations

that

result

in

gene

specialization

-LRB-

Force

et

al.

Lynch

and

Force

Lynch

et

al.

-RRB-

eliminating

still

another

layer

of

pleiotropic

constraints

This

joint

operation

of

subfunction

formation

at

the

gene

level

and

subfunctionalization

at

the

genome

level

provides

a

passive

mechanism

for

the

gradual

remodeling

of

entire

developmental

genetic

pathways

without

any

direct

initial

involvement

of

natural

selection

In

this

sense

multicellular

species

may

be

crucibles

for

the

origin

of

complex

patterns

of

cellular

and

developmental

patterning

not

because

such

features

are

directly

advantageous

but

because

they

are

unavoidable

consequences

of

small

population

size

environments

Concluding

Remarks

Despite

the

enormous

progress

in

molecular

genetics

over

the

past

years

no

general

theory

for

the

evolution

of

the

basic

architectural

features

of

genes

has

been

formulated

Many

attempts

have

been

made

to

explain

the

features

of

genes

genomes

and

genetic

networks

in

the

context

of

putatively

adaptive

cellular

and/or

developmental

features

but

few

of

these

efforts

have

been

accompanied

by

a

formal

evolutionary

analysis

Because

evolution

is

a

population-level

process

any

theory

for

the

origins

of

the

genetic

machinery

must

ultimately

be

consistent

with

basic

population-genetic

mechanisms

However

because

natural

selection

is

just

one

of

several

forces

contributing

to

the

evolutionary

process

an

uncritical

reliance

on

adaptive

Darwinian

mechanisms

to

explain

all

aspects

of

organismal

diversity

is

not

greatly

different

than

invoking

an

intelligent

designer

This

paper

represents

a

first

step

toward

the

formal

development

of

a

general

theory

for

the

evolution

of

the

gene

that

incorporates

the

universal

properties

of

random

genetic

drift

and

mutation

pressure

Although

the

ideas

presented

are

unlikely

to

be

correct

in

every

detail

at

a

minimum

they

serve

as

a

model

For

if

verbal

adaptive

arguments

are

to

provide

confident

explanations

for

any

aspect

of

gene

or

genomic

structure

something

must

be

known

about

patterns

expected

in

the

absence

of

selection

This

is

a

significant

challenge

because

at

this

point

it

is

difficult

to

reject

the

hypothesis

that

the

basic

embellishments

of

the

eukaryotic

gene

originated

largely

as

a

consequence

of

nonadaptive

processes

operating

contrary

to

the

expected

direction

of

natural

selection

A

significant

area

of

future

research

will

be

to

take

these

observations

on

gene

and

genome

com

plexity

to

the

next

level

to

evaluate

whether

natural

selection

is

a

necessary

and/or

sufficient

force

to

explain

the

evolution

of

the

cellular

and

developmental

complexities

of

eukaryotes

Finally

an

issue

not

considered

above

is

the

potential

energetic

consequences

of

the

expansion

of

eukaryotic

genes

It

is

commonly

argued

that

a

significant

metabolic

burden

associated

with

excess

DNA

is

responsible

for

the

streamlining

of

prokaryotic

genomes

by

natural

selection

-LRB-

Cavalier-Smith

Giovannoni

et

al.

-RRB-

However

no

direct

estimate

of

the

contribution

of

DNA

replication

to

a

cell

's

total

energy

budget

has

ever

been

made

and

the

lack

of

correlation

between

prokaryotic

cell

division

rate

and

genome

size

does

not

inspire

confidence

in

the

metabolic-cost

hypothesis

-LRB-

Mira

Ochman

and

Moran

-RRB-

Castillo-Davis

et

al.

-LRB-

-RRB-

have

suggested

that

the

negative

correlation

between

intron

size

and

the

level

of

expression

of

genes

in

nematodes

and

humans

reflects

an

adaptive

response

to

the

costs

of

transcription

although

introns

in

highly

transcribed

genes

might

simply

experience

higher

levels

of

deletion

In

the

only

attempt

to

calculate

the

costs

of

transcription

Wagner

-LRB-

-RRB-

concluded

that

a

doubling

of

the

transcription

of

a

yeast

gene

might

cause

a

fractional

reduction

of

fitness

of

Numerous

assumptions

had

to

be

made

to

arrive

at

this

conclusion

and

incremental

modifications

to

the

complexity

of

transcribed

portions

of

genes

may

generally

only

be

of

the

total

transcript

length

which

would

imply

an

incremental

transcriptional

cost

of

or

perhaps

lower

depending

on

the

relationship

between

energetic

cost

and

fitness

A

welcome

addition

to

this

area

would

be

a

formal

evaluation

of

the

costs

of

DNA

and

mRNA

production

relative

to

the

total

energetic

requirements

of

cell

maintenance

and

replication

If

such

costs

can

be

translated

into

a

selection

coefficient

the

ability

of

natural

selection

to

resist

them

can

then

be

evaluated

using

the

same

procedures

discussed

above

Supplementary

Materials

Supplementary

Tables

are

available

at

Molecular

Biology

and

Evolution

online

-LRB-

oxfordjournals.org

-RRB-

Acknowledgments

I

am

very

grateful

to

E.

Koonin

and

F.

Kondrashov

for

important

comments

that

led

to

significant

improvement

of

this

presentation

J.

Conery

helped

in

procuring

the

prokaryotic

genome

content

data

The

work

has

been

supported

by

National

Institutes

of

Health

grant

and

National

Science

Foundation

grant

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Mol

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William

Martin

Associate

Editor

Accepted

October

gene eukaryotic introns evolution structure evolutionary genomics