Ultraconserved

Elements

in

the

Human

Genome

Gill

Bejerano

et

al.

Science

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L.

Shen

K.

L.

Rock

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

S.

P.

Schoenberger

et

al.

J.

Immunol

-LRB-

-RRB-

M.

L.

Albert

B.

Sauter

N.

Bhardwaj

Nature

-LRB-

-RRB-

M.

Bellone

et

al.

J.

Immunol

-LRB-

-RRB-

J.

W.

Yewdell

C.

C.

Norbury

J.

R.

Bennink

Adv.

Immunol

-LRB-

-RRB-

A.

Serna

M.

C.

Ramirez

A.

Soukhanova

L.

J.

Sigal

J.

Immunol

-LRB-

-RRB-

N.

P.

Restifo

et

al.

J.

Immunol

-LRB-

-RRB-

We

thank

B.

Buschling

D.

Tokarchick

and

A.

Schell

for

technical

assistance

We

are

grateful

to

M.

Epler

and

S.

Tevethia

for

their

generous

gift

of

tetramers

This

work

was

supported

in

part

by

a

Wellcome

Prize

Traveling

Fellowship

and

U.S.

Public

Health

Service

grants

and

NIH

grant

to

C.C.N.

Supporting

Online

Material

Materials

and

Methods

Figs

and

References

and

Notes

February

accepted

April

Ultraconserved

Elements

in

the

Human

Genome

Gill

Bejerano

Michael

Pheasant

Igor

Makunin

Stuart

Stephen

W.

James

Kent

John

S.

Mattick

David

There

are

segments

longer

than

base

pairs

-LRB-

bp

-RRB-

that

are

absolutely

conserved

-LRB-

identity

with

no

insertions

or

deletions

-RRB-

between

orthologous

regions

of

the

human

rat

and

mouse

genomes

Nearly

all

of

these

segments

are

also

conserved

in

the

chicken

and

dog

genomes

with

an

average

of

and

identity

respectively

Many

are

also

significantly

conserved

in

fish

These

ultraconserved

elements

of

the

human

genome

are

most

often

located

either

overlapping

exons

in

genes

involved

in

RNA

processing

or

in

introns

or

nearby

genes

involved

in

the

regulation

of

transcription

and

development

Along

with

more

than

sequences

of

over

bp

that

are

absolutely

conserved

among

the

three

sequenced

mammals

these

represent

a

class

of

genetic

elements

whose

functions

and

evolutionary

origins

are

yet

to

be

determined

but

which

are

more

highly

conserved

between

these

species

than

are

proteins

and

appear

to

be

essential

for

the

ontogeny

of

mammals

and

other

vertebrates

Although

only

about

of

the

human

genome

appears

to

code

for

proteins

-LRB-

-RRB-

it

has

been

estimated

that

as

much

as

is

more

conserved

than

would

be

expected

from

neutral

evolution

since

the

split

with

rodents

and

hence

may

be

under

negative

or

purifying

selection

-LRB-

-RRB-

Several

studies

have

found

specific

noncoding

segments

in

the

human

genome

that

appear

to

be

under

selection

using

a

threshold

for

conservation

of

or

identity

with

the

mouse

over

more

than

base

pairs

-LRB-

bp

-RRB-

-LRB-

-RRB-

A

study

of

these

elements

on

human

chromosome

found

that

those

that

were

very

highly

conserved

in

multiple

species

contained

significant

numbers

of

noncoding

elements

-LRB-

-RRB-

Similar

results

were

found

when

comparing

the

human

mouse

and

rat

genomes

-LRB-

-RRB-

in

a

study

of

the

megabase

-LRB-

Mb

-RRB-

CFTR

region

-LRB-

-RRB-

and

in

a

functional

study

of

the

locus

in

a

number

of

mammalian

species

-LRB-

-RRB-

We

determined

the

longest

segments

of

the

human

genome

that

are

maximally

Department

of

Biomolecular

Engineering

Hughes

Medical

Institute

University

of

California

Santa

Cruz

Santa

Cruz

CA

USA

Special

Research

Centre

for

Functional

and

Applied

Genomics

Institute

for

Molecular

Bioscience

University

of

Queensland

Brisbane

QLD

Australia

To

whom

correspondence

should

be

addressed

Email

jill@soe.ucsc.edu

-LRB-

G.B.

-RRB-

haussler@soe.ucsc.edu

-LRB-

D.H.

-RRB-

served

with

orthologous

segments

in

rodents

those

showing

identity

and

with

no

insertions

or

deletions

in

their

alignment

with

the

mouse

and

rat

Exclusive

of

ribosomal

RNA

-LRB-

rRNA

-RRB-

regions

there

are

such

segments

longer

than

bp

that

we

call

ultraconserved

elements

-LRB-

table

-RRB-

They

are

widely

distributed

in

the

genome

-LRB-

on

all

chromosomes

except

chromosomes

and

Y

-RRB-

and

are

often

found

in

clusters

-LRB-

Fig.

-RRB-

The

probability

is

less

than

of

finding

even

one

such

element

in

billion

bases

under

a

simple

model

of

neutral

evolution

with

independent

substitutions

at

each

site

using

the

slowest

neutral

substitution

rate

that

is

observed

for

any

region

of

the

genome

-LRB-

supporting

text

section

-RRB-

Nearly

all

of

these

elements

also

exhibit

extremely

high

levels

of

conservation

with

orthologous

regions

in

the

chicken

genome

-LSB-

out

of

-LRB-

-RRB-

of

the

elements

aligning

at

an

average

of

identity

at

identity

-RSB-

and

about

two-thirds

of

them

with

the

fugu

genome

as

well

-LRB-

of

the

elements

aligning

at

an

average

of

identity

-RRB-

despite

the

fact

that

only

about

of

the

human

genome

can

be

reliably

aligned

to

the

chicken

genome

-LRB-

at

an

average

of

identity

where

an

alignment

is

found

-RRB-

and

less

than

of

the

human

genome

aligns

to

fugu

-LRB-

at

an

average

of

identity

-RRB-

In

addition

nearly

all

exhibit

extremely

high

levels

of

conservation

with

the

dog

genome

which

was

estimated

using

reads

from

the

National

Center

for

Biotechnology

Information

-LRB-

NCBI

-RRB-

trace

archive

-LRB-

of

the

elements

aligning

at

an

average

of

identity

-RRB-

Thus

it

appears

that

nearly

all

of

these

ultraconserved

elements

may

have

been

under

extreme

negative

selection

in

many

species

for

more

than

million

years

and

some

of

them

for

at

least

million

years

As

expected

the

ultraconserved

elements

exhibit

almost

no

natural

variation

in

the

human

population

Only

out

of

bases

examined

in

the

ultraconserved

elements

-LRB-

excluding

the

first

and

last

bases

in

each

element

-RRB-

are

at

validated

singlenucleotide

polymorphisms

-LRB-

SNPs

-RRB-

in

NCBI

's

SNP

database

-LRB-

dbSNP

-RRB-

-LRB-

table

-RRB-

For

this

much

DNA

we

would

have

expected

validated

sites

so

validated

SNPs

are

underrepresented

by

-LRB-

P

-RRB-

The

unvalidated

SNPs

we

found

revealed

many

likely

errors

in

the

unvalidated

portion

of

dbSNP

-LRB-

table

-RRB-

These

same

bases

exhibit

very

few

differences

with

the

chimp

genome

as

well

showing

only

single

base

changes

where

the

chimp

base

has

a

Phred

quality

score

at

least

whereas

the

expected

number

would

be

-LRB-

roughly

a

reduction

P

supporting

text

section

-RRB-

This

low

level

of

variation

within

the

human

population

and

in

comparison

with

the

chimp

suggests

that

these

elements

are

currently

changing

at

a

rate

that

is

roughly

times

slower

than

the

average

for

the

genome

Only

of

the

bases

are

different

in

the

chicken

which

is

also

consistent

with

a

roughly

reduction

from

neutral

substitution

rates

-LRB-

supporting

text

section

-RRB-

Of

the

ultraconserved

elements

overlap

the

mRNA

of

a

known

human

protein-coding

gene

-LSB-

including

the

untranslated

regions

-LRB-

UTRs

-RRB-

-RSB-

show

no

evidence

of

transcription

from

any

matching

expressed

sequence

tag

-LRB-

EST

-RRB-

or

mRNA

from

any

species

and

for

the

remaining

the

evidence

for

transcription

is

inconclusive

We

call

these

partly

exonic

-LRB-

or

exonic

for

short

-RRB-

nonexonic

and

possibly

exonic

ultraconserved

elements

respectively

A

hundred

nonexonic

elements

are

located

in

introns

of

known

genes

and

the

rest

are

intergenic

The

non-exonic

elements

both

intronic

and

intergenic

tend

to

congregate

in

clusters

near

transcription

factors

and

developmental

genes

whereas

the

exonic

and

possibly

SCIENCE

VOL

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from

on

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REPORTS

exonic

elements

are

more

randomly

distributed

along

the

chromosomes

-LRB-

Fig.

-RRB-

There

are

known

genes

that

overlap

with

exonic

ultraconserved

elements

we

call

these

type

I

genes

The

genes

that

are

near

the

non-exonic

elements

we

call

type

II

genes

-LRB-

methods

in

supporting

text

section

-RRB-

We

looked

for

categories

of

biological

process

and

molecular

function

defined

in

the

Gene

Ontology

-LRB-

GO

-RRB-

database

-LRB-

-RRB-

that

are

significantly

enriched

in

type

I

and

II

genes

and

also

searched

InterPro

-LRB-

-RRB-

for

enrichment

in

particular

structural

domains

-LRB-

Fig.

-RRB-

The

type

I

genes

show

significant

functional

enrichment

for

RNA

binding

and

regulation

of

splicing

-LRB-

P

and

respectively

against

all

GO

annotated

human

genes

-RRB-

and

are

uniquely

abundant

in

the

RNA

recognition

motif

RRM

-LRB-

P

against

all

InterPro

annotated

human

genes

-RRB-

In

contrast

the

type

II

genes

are

devoid

of

enrichment

for

RNA

binding

or

splicing

or

the

RRM

-LRB-

P

and

respectively

-RRB-

However

type

II

genes

are

strongly

enriched

for

regulation

of

transcription

and

DNA

binding

-LRB-

P

and

respectively

-RRB-

as

well

as

DNA

binding

motifs

in

particular

the

Homeobox

domain

-LRB-

P

-RRB-

These

three

attributes

are

enriched

in

type

I

genes

as

well

but

and

orders

of

magnitude

less

significantly

respectively

This

suggests

that

exonic

ultraconserved

elements

may

be

specifically

associated

with

RNA

processing

and

non-exonic

elements

with

regulation

of

transcription

at

the

DNA

level

Non-exonic

ultraconserved

elements

are

often

found

in

gene

deserts

that

extend

more

than

a

megabase

In

particular

of

the

non-exonic

elements

there

are

that

are

more

than

kilobases

-LRB-

kb

-RRB-

away

from

any

known

gene

and

that

are

more

than

kb

away

The

set

of

annotated

genes

that

FIGN

HOXD

HNRPK

MNAB

OAZ

LRBA

EHZF

HOXB

orthopedia-ortholog

HOXC

chrX

chrY

DACH

POLA/ARX

Base

Position

Chromosome

Band

POLA

ARX

Ultra-conserved

Elements

Fig.

Locations

of

the

ultraconserved

elements

on

the

human

chromosomes

Each

partly

exonic

element

is

represented

by

a

thin

blue

tick

mark

extending

above

the

chromosome

each

non-exonic

element

by

a

green

tick

mark

extending

below

the

chromosome

and

each

possibly

exonic

element

by

a

black

tick

mark

centered

on

the

chromosome

Purple

boxes

represent

centromeres

By

joining

two

elements

into

a

cluster

when

they

are

separated

by

less

than

kb

we

obtained

local

clusters

of

two

or

more

elements

each

of

which

is

boxed

and

named

Names

are

taken

from

a

prominent

gene

or

gene

family

co-located

with

the

cluster

or

from

a

Drosophila

ortholog

or

mRNA

entry

if

no

Human

Genome

Organization

-LRB-

HUGO

-RRB-

named

gene

was

available

Among

the

cluster

representatives

there

is

a

distinct

enrichment

for

non-exonic

elements

and

for

developmental

genes

suggesting

that

many

of

these

clusters

may

be

part

of

distal

enhancers

or

global

control

loci

analogous

to

those

studied

in

association

with

HOXD

-LRB-

-RRB-

or

DACH

-LRB-

-RRB-

One

possible

such

cluster

near

the

ARX

gene

is

shown

in

more

detail

in

the

inset

at

the

bottom

of

the

figure

There

known

genes

are

shown

in

blue

-LRB-

tall

boxes

for

coding

exons

shorter

boxes

for

UTRs

and

hatched

lines

for

introns

-RRB-

and

ultraconserved

elements

are

shown

below

them

MAY

VOL

SCIENCE

Downloaded

from

on

May

castor-ortholog

HNRPU

HOXA

HIC/TFEC

COCH

REPORTS

flank

intergenic

ultraconserved

elements

is

significantly

enriched

for

developmental

genes

-LRB-

P

-RRB-

and

in

particular

for

genes

involved

in

early

developmental

tasks

-LRB-

P

-RRB-

suggesting

that

many

of

the

associated

ultraconserved

elements

may

be

distal

enhancers

of

these

early

developmental

genes

Indeed

one

of

these

elements

-LRB-

uc

in

table

-RRB-

is

contained

in

an

enhancer

situated

about

kb

upstream

of

DACH

-LRB-

homolog

of

the

Drosophila

dachshund

gene

known

to

be

involved

in

the

development

of

brain

limbs

and

sensory

organs

-RRB-

which

has

been

shown

to

reproducibly

drive

expression

in

the

retina

when

cloned

upstream

of

a

mouse

heat

shock

protein

minimal

promoter

coupled

to

galactosidase

and

injected

into

a

mouse

oocyte

-LRB-

-RRB-

Non-exonic

ultraconserved

elements

that

lie

in

introns

are

also

often

associated

with

developmental

genes

These

include

the

neuroretina-specific

enhancer

in

the

fourth

intron

of

-LRB-

uc

-RRB-

investigated

in

quail

but

shown

to

also

be

functionally

conserved

in

mouse

-LRB-

-RRB-

Type

I

genes

-LRB-

harboring

exonic

elements

-RRB-

include

many

genes

encoding

well-known

RNA-binding

proteins

such

as

HNRPK

HNRPU

HNRPDL

HNRPM

and

All

of

the

above

are

among

the

type

I

genes

annotated

by

GO

that

exhibit

clear

mRNA

EST

evidence

of

alternative

splicing

overlapping

the

ultraconserved

element

-LSB-

out

of

elements

in

all

from

a

total

of

exonic

elements

-LRB-

table

-RRB-

-RSB-

Many

of

the

above

including

the

six

members

of

the

SFRS

family

contain

the

RNA

recognition

motif

The

ultraconserved

elements

associated

with

alternative

splicing

events

often

contain

small

coding

exons

that

are

skipped

in

the

mRNA

in

some

tissues

but

the

elements

extend

well

into

the

flanking

intronic

regions

on

one

or

both

sides

of

the

exon

Such

is

the

case

for

one

explicitly

studied

ultraconserved

element

-LRB-

uc

-RRB-

in

a

polypyrimidine

tract

binding

protein

-LRB-

-RRB-

contains

a

ultraconserved

segment

that

is

mostly

intronic

but

includes

a

small

-LRB-

-RRB-

exon

that

is

included

in

the

mRNA

only

in

brain

tissue

The

bases

at

the

end

of

the

element

including

the

exon

are

conserved

in

the

chicken

as

well

The

element

may

form

an

RNA

structure

in

the

pre-mRNA

that

participates

in

the

regulation

of

splicing

through

interactions

with

the

spliceosome

-LRB-

-RRB-

We

used

the

program

RNAfold

-LRB-

-RRB-

to

further

assess

the

potential

of

this

and

other

ultraconserved

elements

to

form

an

RNA

secondary

structure

comparing

the

energy

of

the

best

folded

structure

for

both

the

positive

and

negative

strand

element

to

that

of

random

permutations

of

the

same

sequence

-LRB-

table

-RRB-

No

statistically

significant

structure

was

found

for

the

element

but

the

energy

of

the

fold

for

the

ultraconserved

self-regulated

alternatively-spliced

UTR

element

-LRB-

uc

-RRB-

in

arginine/serine-rich

splicing

factor

-LRB-

-RRB-

was

lower

than

that

of

all

but

one

of

the

randomized

versions

of

this

sequence

indicating

that

it

may

form

an

important

RNA

secondary

structure

-LRB-

fig

-RRB-

In

addition

to

alternative

splicing

the

exonic

ultraconserved

elements

also

include

the

consecutive

mutually

exclusive

flop

and

flip

exons

-LRB-

uc

-RRB-

from

the

glutamate

receptor

-LRB-

-RRB-

which

exhibits

RNA

editing

as

well

as

alternate

splicing

-LRB-

-RRB-

The

flop

ultraconserved

element

extends

into

the

intron

of

the

gene

At

the

other

end

-LRB-

adjacent

to

the

previous

exon

-RRB-

intron

contains

a

much

shorter

highly

conserved

RNA

hairpin

structure

that

guides

the

essential

and

highly

regulated

editing

of

adenosine

to

inosine

-LRB-

-RRB-

Although

the

element

containing

the

flop

exon

does

not

have

detectable

RNA

secondary

structure

preferences

the

minimal

energy

of

the

secondary

structure

of

the

element

containing

the

flip

is

less

than

that

of

out

of

permuted

versions

indicating

possible

structure

Although

the

minimal

region

of

conservation

between

human

mouse

and

rat

that

was

required

to

be

included

in

the

ultraconserved

set

was

bp

many

elements

were

considerably

longer

The

longest

elements

-LRB-

and

bp

-RRB-

all

lie

in

the

last

three

introns

in

the

portion

of

POLA

the

DNA

polymerase

alpha

catalytic

subunit

-LRB-

EC

-RRB-

on

chromosome

X

along

with

other

shorter

ultraconserved

elements

-LRB-

Fig.

-RRB-

A

similar-sized

conserved

region

bp

formed

by

concatenation

of

uc

and

uc

-LRB-

separated

by

a

single

base

-RRB-

lies

in

the

intergenic

region

between

the

end

of

POLA

and

its

downstream

neighbor

the

ARX

homeobox

gene

ARX

is

involved

in

central

nervous

system

development

and

is

associated

with

a

host

of

X-linked

Mendelian

diseases

including

epilepsy

mental

retardation

autism

and

cerebral

malformations

-LRB-

-RRB-

Because

this

group

of

elements

lies

at

the

end

of

the

POLA

gene

nearer

to

the

end

of

ARX

than

to

the

rest

of

POLA

-LRB-

Fig.

-RRB-

it

is

possible

that

their

function

is

not

related

to

POLA

but

that

they

instead

form

a

cluster

of

enhancers

of

ARX

The

longest

of

these

ultraconserved

elements

bp

is

actually

adjacent

to

a

element

which

together

form

a

region

with

only

one

change

in

rodents

As

a

calibration

note

that

these

POLA/ARX

elements

are

considerably

longer

than

the

ultraconserved

portions

of

the

human

mouse

and

rat

rRNA

genes

which

harbor

six

ultraconserved

segments

three

each

in

the

and

rRNA

genes

the

longest

of

which

is

bp

-LRB-

table

-RRB-

In

sharp

contrast

to

rRNA

and

most

human

coding

regions

there

were

only

out

of

cases

-LRB-

-RRB-

where

an

ortholog

of

an

ultraconserved

element

could

be

partially

traced

back

by

sequence

similarity

search

as

far

as

Ciona

intestinalis

Drosoplila

melanogaster

or

Caenorhabditis

elegans

-LRB-

table

-RRB-

All

of

these

were

among

the

elements

-LRB-

-RRB-

that

overlapped

coding

exons

from

Annotation

Enrichment

in

Type

I

and

Type

II

Genes

RNA

binding

Type

I

Genes

RNA

splicing

RNA

rec

motif

Transcription

reg

DNA

binding

Homeobox

RNA

binding

Type

II

Genes

RNA

splicing

RNA

rec

motif

Transcription

reg

DNA

binding

Homeobox

observed

expected

No

of

Genes

Fig.

Annotation

enrichment

in

type

I

and

type

II

genes

In

the

top

half

of

the

figure

the

maroon

bars

-LRB-

observed

-RRB-

give

the

numbers

of

type

I

genes

that

are

annotated

in

the

GO

database

-LRB-

-RRB-

with

molecular

function

RNA

binding

or

DNA

binding

or

biological

process

RNA

splicing

or

transcription

regulation

or

are

annotated

in

InterPro

-LRB-

-RRB-

as

containing

the

domains

RNA

recognition

motif

or

homeobox

The

blue

bars

-LRB-

expected

-RRB-

give

the

number

of

genes

that

one

would

expect

to

obtain

if

the

same

number

of

genes

-LRB-

genes

for

type

I

-RRB-

were

chosen

at

random

among

all

genes

annotated

in

the

relevant

database

The

bottom

half

of

the

figure

gives

similar

information

for

type

II

genes

It

is

apparent

that

type

I

genes

are

enriched

for

RNA-related

functions

whereas

type

II

genes

are

not

Both

types

are

enriched

for

DNA-related

functions

but

the

type

II

genes

are

more

enriched

SCIENCE

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known

genes

In

of

these

ancient

cases

there

is

clear

mRNA

or

EST

evidence

that

the

coding

region

overlapped

by

the

element

is

alternatively

spliced

in

humans

These

include

alternatively

spliced

exons

of

genes

ZFR

and

as

well

as

In

of

the

other

cases

could

we

find

evidence

that

any

element

that

was

intronic

in

humans

was

coding

in

another

species

although

in

some

cases

there

was

EST

evidence

for

a

retained

intron

that

presumably

has

a

function

other

than

protein

coding

Moreover

indels

of

noncoding

ultraconserved

elements

relative

to

their

alignments

with

the

chicken

and

other

species

are

often

not

in

multiples

of

three

giving

further

evidence

that

these

sequences

are

noncoding

-LRB-

fig

A

and

B

-RRB-

The

ultraconserved

elements

we

found

in

introns

seem

to

have

been

at

one

time

rather

fast-evolving

as

compared

to

the

known

coding

exons

in

their

genes

We

tried

to

map

selected

introns

containing

ultraconserved

intronic

elements

to

more

distant

species

using

protein/translated

DNA

matches

to

their

enclosing

exons

Often

only

a

core

conserved

region

was

recognizable

in

fish

and

this

had

very

different

flanking

DNA

suggesting

that

additional

parts

of

the

ultraconserved

region

were

innovations

after

the

common

ancestor

with

fish

as

observed

in

the

analysis

of

uc

near

HOXD

-LRB-

-RRB-

In

cases

where

we

could

trace

beyond

vertebrates

we

always

found

that

the

orthologous

intron

in

the

more

distant

species

was

either

very

small

with

apparently

unrelated

sequence

or

was

nonexistent

For

example

tracing

the

intron

that

contains

the

first

-LRB-

most

-RRB-

ultraconserved

element

in

POLA

-LRB-

uc

-RRB-

we

find

that

although

it

is

an

approximately

intron

in

humans

its

ortholog

in

Fugu

rubripes

is

only

bp

-LRB-

which

is

still

large

relative

to

most

fugu

introns

-RRB-

only

about

bp

in

C.

intestinalis

and

does

not

exist

-LRB-

the

flanking

exons

abut

-RRB-

in

D.

melanogaster

and

C.

elegans

The

human

element

is

not

recognizably

similar

to

anything

in

the

orthologous

intron

of

Ciona

Yet

like

the

other

POLA

ultraconserved

elements

discussed

above

this

element

is

more

than

identical

between

human

and

chicken

Similar

results

were

found

for

the

three

longest

POLA

intronic

elements

Another

similar

case

was

a

cluster

of

seven

ultraconserved

elements

-LRB-

uc

to

uc

-RRB-

with

sizes

ranging

from

to

bp

all

contained

in

an

intron

of

pre-B

cell

leukemia

transcription

factor

a

member

of

the

TALE/PBX

homeobox

family

This

was

one

of

the

largest

introns

we

found

and

it

contained

one

of

the

largest

collections

of

ultraconserved

elements

in

a

single

intron

The

orthologous

intron

in

F.

rubripes

is

kb

in

C.

intestinalis

it

appears

to

be

kb

in

Drosophila

it

is

bp

-LRB-

ortholog

exd

-RRB-

and

the

flanking

exons

abut

in

C.

elegans

Despite

the

inability

to

trace

most

of

the

vertebrate

ultraconserved

elements

to

distant

species

the

possibility

that

processes

similar

to

those

that

produced

ultraconserved

elements

in

vertebrates

also

exist

in

other

classes

of

species

remains

open

In

one

tantalizing

example

it

has

been

observed

that

the

mating

type

gene

in

yeast

shows

conservation

over

bp

in

four

yeast

species

-LRB-

-RRB-

The

mechanism

of

this

conservation

is

not

known

We

found

only

paralogous

sets

each

consisting

of

two

or

three

elements

among

all

ultraconserved

elements

-LRB-

table

-RRB-

Each

paralogous

set

is

consistent

with

the

paralogy

relationship

between

the

enclosing

or

nearby

host

genes

All

paralogs

-LRB-

except

currently

uc

overlapping

-RRB-

have

highly

conserved

matches

in

the

chicken

providing

more

opportunities

for

evolutionary

analysis

of

these

duplication

events

that

predate

the

divergence

from

birds

In

each

of

the

clusters

we

found

significant

divergence

between

the

paralogs

which

must

have

occurred

in

the

early

part

of

their

evolution

-LRB-

fig

-RRB-

because

each

individual

instance

in

a

paralogous

set

has

changed

very

little

in

the

past

million

years

in

birds

and

mammals

This

combined

with

the

above

analysis

suggests

that

the

bulk

of

the

ultraconserved

elements

represent

chordate

innovations

that

evolved

fairly

rapidly

at

first

but

then

slowed

down

considerably

becoming

effectively

frozen

in

birds

and

mammals

A

more

extensive

analysis

of

paralogs

based

on

a

recent

global

clustering

of

highly

conserved

noncoding

human

DNA

-LRB-

-RRB-

reveals

several

further

highly

conserved

intronic

and

intergenic

elements

in

functionally

equivalent

positions

relative

to

paralogous

genes

These

were

not

classified

as

ultraconserved

by

our

stringent

criteria

Indeed

if

we

merge

alignment

blocks

of

bases

each

with

at

least

identical

columns

we

obtain

highly

conserved

elements

up

to

bp

long

in

the

human

Four

of

the

five

longest

elements

are

the

aforementioned

POLA/ARX

elements

along

with

a

element

-LRB-

encompassing

uc

-RRB-

in

an

intron

of

adjacent

to

If

instead

we

demand

at

least

a

exact

match

between

humans

and

rodents

we

get

more

than

highly

conserved

elements

Tens

of

thousands

more

are

found

at

lower

cutoffs

for

example

there

is

a

exactly

conserved

sequence

overlapping

an

alternatively

spliced

exon

of

the

gene

which

is

invariant

in

mammals

and

in

chickens

and

is

largely

conserved

in

fishes

-LRB-

fig

-RRB-

The

percentage

of

the

conserved

elements

that

overlap

with

a

known

coding

region

steadily

rises

from

to

as

the

length

criteria

defining

these

elements

is

reduced

from

to

bp

-LRB-

table

-RRB-

If

experiments

with

less

conserved

elements

in

recent

studies

-LRB-

-RRB-

are

any

indication

many

of

these

shorter

elements

are

also

functional

Compared

to

the

ultraconserved

elements

a

greater

percentage

of

these

shorter

conserved

elements

are

significantly

different

in

birds

but

are

highly

conserved

in

mammals

This

suggests

that

the

process

of

the

evolution

of

new

elements

followed

by

near

freezing

of

their

DNA

sequences

is

probably

still

ongoing

in

vertebrates

Lineage-specific

specializations

of

these

elements

may

reflect

regulatory

changes

that

are

important

to

the

ontogeny

and

physiology

of

the

clade

The

patterns

of

conservation

exhibited

in

the

ultraconserved

elements

must

result

from

the

onset

during

chordate

evolution

of

either

a

highly

elevated

negative

selection

rate

in

these

regions

-LRB-

about

a

times

smaller

chance

of

mutations

becoming

fixed

in

the

population

-RRB-

a

highly

reduced

mutation

rate

-LRB-

about

times

fewer

mutations

-RRB-

or

some

combination

of

these

effects

The

possibility

of

strong

negative

selection

is

intriguing

because

selection

to

maintain

protein

coding

protein-nucleic

acid

interactions

or

RNARNA

interactions

does

not

result

in

near

total

conservation

over

long

stretches

of

bases

unless

multiple

functions

are

overlaid

on

the

same

DNA

such

as

in

regions

of

coding

exons

that

also

bind

splicing

factors

or

in

regions

of

rRNA

that

must

form

RNA

structures

as

well

as

bind

proteins

If

the

exonic

ultraconserved

elements

form

pre-mRNA

structures

that

are

under

selection

to

preserve

interaction

with

the

spliceosome

or

editing

machinery

-LRB-

-RRB-

then

these

interactions

must

be

extremely

constraining

over

hundreds

of

bases

of

DNA

much

like

those

of

the

anciently

derived

rRNAs

making

them

potentially

quite

novel

objects

for

molecular

study

The

same

holds

true

if

the

conservation

in

the

non-exonic

elements

is

associated

with

selection

for

molecular

interactions

involved

in

the

regulation

of

transcription

which

could

be

in

cis

over

long

genomic

distances

or

in

trans

perhaps

also

involving

RNA

-LRB-

-RRB-

On

the

other

hand

if

reduced

mutation

rates

are

the

explanation

then

the

existence

of

regions

of

a

few

hundred

bases

with

reduced

mutation

rates

would

itself

be

quite

novel

Although

neutral

mutation

rates

may

vary

depending

on

chromosomal

location

on

a

megabase

scale

-LRB-

-RRB-

there

is

to

our

knowledge

no

evidence

or

precedent

for

the

existence

of

short

hypomutable

or

hyperrepaired

neutral

regions

Finally

the

answer

could

also

be

a

combination

of

negative

selection

and

better

repair

in

these

regions

owing

to

some

vital

role

that

these

elements

play

such

as

self-regulating

networks

of

RNA

processing

control

in

the

case

of

exonic

elements

and

self-regulatory

networks

of

transcriptional

control

for

non-exonic

ele

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from

on

May

REPORTS

ments

In

any

case

the

questions

remain

What

kind

of

elements

associated

with

these

processes

would

have

arrived

relatively

early

in

chordate

evolution

and

then

become

practically

frozen

in

birds

and

mammals

And

what

mechanisms

would

underlie

this

allowing

them

to

resist

virtually

all

further

change

Note

added

in

proof

We

recently

became

aware

of

related

observations

made

by

Boffelli

et

al.

-LRB-

-RRB-

peat

sites

for

windows

and

S.

Walton

for

help

in

preparing

the

manuscript

G.B.

W.J.K.

and

D.H.

were

supported

by

National

Human

Genome

Research

Institute

grant

and

National

Cancer

Institute

contract

and

D.H.

was

additionally

supported

by

the

Howard

Hughes

Medical

Institute

S.S.

M.P.

I.M.

and

J.S.M.

were

supported

by

the

Australian

Research

Council

and

the

Queensland

State

Government

Supporting

Online

Material

SOM

Text

Figs

to

Tables

to

References

and

Notes

March

accepted

April

Published

online

May

science

Include

this

information

when

citing

this

paper

References

and

Notes

E.

S.

Lander

et

al.

Nature

-LRB-

-RRB-

J.

C.

Venter

et

al.

Science

-LRB-

-RRB-

Human

Genome

Sequencing

Consortium

in

preparation

R.

H.

Waterston

et

al.

Nature

-LRB-

-RRB-

K.

M.

Roskin

M.

Diekhans

D.

Haussler

in

Proceedings

of

the

Annual

International

Conference

on

Research

in

Computational

Molecular

Biology

-LRB-

ACM

New

York

NY

-RRB-

pp.

F.

Chiaromonte

et

al.

Cold

Spring

Harbor

Symp

Quant

Biol

-LRB-

-RRB-

R.

C.

Hardison

Trends

Genet

-LRB-

-RRB-

G.

G.

Loots

et

al.

Science

-LRB-

-RRB-

L.

A.

Pennacchio

E.

M.

Rubin

Nature

Rev.

Genet

-LRB-

-RRB-

K.

A.

Frazer

et

al.

Genome

Res

-LRB-

-RRB-

U.

DeSilva

et

al.

Genome

Res

-LRB-

-RRB-

E.

T.

Dermitzakis

et

al.

Nature

-LRB-

-RRB-

E.

T.

Dermitzakis

et

al.

Science

-LRB-

-RRB-

Rat

Genome

Sequencing

Consortium

Nature

-LRB-

-RRB-

G.

M.

Cooper

et

al.

Genome

Res

-LRB-

-RRB-

J.

W.

Thomas

et

al.

Nature

-LRB-

-RRB-

E.

H.

Margulies

M.

Blanchette

D.

Haussler

E.

D.

Green

Genome

Res

-LRB-

-RRB-

K.

A.

Frazer

et

al.

Genome

Res

-LRB-

-RRB-

M.

Ashburner

et

al.

Nature

Genet

-LRB-

-RRB-

N.

J.

Mulder

et

al.

Nucleic

Acids

Res

-LRB-

-RRB-

M.

A.

Nobrega

I.

Ovcharenko

V.

Afzal

E.

M.

Rubin

Science

-LRB-

-RRB-

S.

Plaza

C.

Dozier

M.

C.

Langlois

S.

Saule

Mol

Cell

Biol

-LRB-

-RRB-

L.

Rahman

V.

Bliskovski

F.

J.

Kaye

M.

Zajac-Kaye

Genomics

-LRB-

-RRB-

I.

L.

Hofacker

Nucleic

Acids

Res

-LRB-

-RRB-

H.

Jumaa

P.

J.

Nielsen

EMBO

J.

-LRB-

-RRB-

B.

Sommer

et

al.

Science

-LRB-

-RRB-

P.

J.

Aruscavage

B.

L.

Bass

RNA

-LRB-

-RRB-

E.

H.

Sherr

Curr

Opin

Pediatr

-LRB-

-RRB-

C.

Sabarinadh

S.

Subramanian

R.

Mishra

Genome

Biol

-LRB-

-RRB-

M.

Kellis

N.

Patterson

M.

Endrizzi

B.

Birren

E.

S.

Lander

Nature

-LRB-

-RRB-

G.

Bejerano

D.

Haussler

M.

Blanchette

Bioinformatics

-LRB-

Suppl

-RRB-

in

press

J.

S.

Mattick

M.

J.

Gagen

Mol

Biol

Evol

-LRB-

-RRB-

E.

T.

Dermitzakis

et

al.

Genome

Res

-LRB-

-RRB-

K.

H.

Wolfe

P.

M.

Sharp

W.

H.

Li

Nature

-LRB-

-RRB-

R.

C.

Hardison

et

al.

Genome

Res

-LRB-

-RRB-

J.

H.

Chuang

H.

Li

PLoS

Biol

-LRB-

-RRB-

D.

Boffelli

M.

Nobrega

E.

M.

Rubin

Nature

Rev.

Genet.

in

press

F.

Spitz

F.

Gonzalez

D.

Duboule

Cell

-LRB-

-RRB-

We

thank

the

Genome

Sequencing

Consortia

for

the

human

mouse

rat

dog

chicken

and

other

genome

sequences

we

used

in

this

analysis

W.

Miller

M.

Diekhans

A.

Hinrichs

K.

Rosenbloom

D.

Thomas

and

the

members

of

the

University

of

California

Santa

Cruz

-LRB-

UCSC

-RRB-

browser

team

for

providing

the

genome

alignments

and

other

tracks

of

genome

annotation

available

on

the

UCSC

genome

browser

M.

Blanchette

S.

Salama

T.

Lowe

M.

Ares

K.

Pollard

and

B.

Cohen

for

helpful

discussions

A.

Siepel

for

the

neutral

substitution

rate

analysis

involving

chicken

and

chimp

K.

Roskin

for

the

calculation

of

the

percent

identity

in

ancestral

re

A

Family

with

Severe

Insulin

Resistance

and

Diabetes

Due

to

a

Mutation

in

Downloaded

from

on

May

Stella

George

Justin

J.

Rochford

Christian

Wolfrum

Sarah

L.

Gray

Sven

Schinner

Jenny

C.

Wilson

Maria

A.

Soos

Peter

R.

Murgatroyd

Rachel

M.

Williams

Carlo

L.

Acerini

David

B.

Dunger

David

Barford

A.

Margot

Umpleby

Nicholas

J.

Wareham

Huw

Alban

Davies

Alan

J.

Schafer

Markus

Stoffel

Stephen

O'Rahilly

Ines

Inherited

defects

in

signaling

pathways

downstream

of

the

insulin

receptor

have

long

been

suggested

to

contribute

to

human

type

diabetes

mellitus

Here

we

describe

a

mutation

in

the

gene

encoding

the

protein

kinase

in

a

family

that

shows

autosomal

dominant

inheritance

of

severe

insulin

resistance

and

diabetes

mellitus

Expression

of

the

mutant

kinase

in

cultured

cells

disrupted

insulin

signaling

to

metabolic

end

points

and

inhibited

the

function

of

coexpressed

wild-type

AKT

These

findings

demonstrate

the

central

importance

of

AKT

signaling

to

insulin

sensitivity

in

humans

Most

forms

of

diabetes

are

likely

to

be

polygenic

in

origin

although

a

number

of

monogenic

forms

are

being

recognized

-LRB-

-RRB-

Although

rare

these

monogenic

examples

offer

insight

into

the

function

of

the

affected

gene

in

humans

as

well

as

offering

important

clues

to

understanding

more

common

forms

We

have

been

screening

genomic

DNA

from

unrelated

subjects

with

severe

insulin

resistance

for

mutations

in

genes

that

are

implicated

in

insulin

signaling

We

idenDepartment

of

Clinical

Biochemistry

of

Paediatrics

University

of

Cambridge

Addenbrooke

's

Hospital

Hills

Road

Cambridge

UK

of

Metabolic

Diseases

Rockefeller

University

New

York

NY

USA

of

Structural

Biology

Institute

of

Cancer

Research

Chester

Beatty

Laboratories

London

UK

of

Diabetes

Endocrinology

and

Internal

Medicine

Guy

's

King

's

and

St.

Thomas

School

of

Medicine

London

UK

Research

Council

Epidemiology

Unit

Strangeways

Research

Laboratory

Worts

Causeway

Cambridge

UK

Valley

Hospital

Darenth

Wood

Road

Dartford

Kent

UK

Porter

Drive

Palo

Alto

CA

USA

Trust

Sanger

Institute

Wellcome

Trust

Genome

Campus

Hinxton

Cambridge

UK

These

authors

contributed

equally

to

this

work

To

whom

correspondence

should

be

addressed

Email

sorahill@hgmp.mrc.ac.uk

tified

a

missense

mutation

in

the

serine

threonine

kinase

gene

in

one

Caucasian

proband

-LRB-

also

known

as

PKB

-RRB-

is

highly

expressed

in

insulin-sensitive

tissues

and

is

activated

in

response

to

growth

factors

and

related

stimuli

-LRB-

-RRB-

a

process

that

requires

its

phosphorylation

by

the

phosphate-dependent

kinase

activities

designated

and

-LRB-

-RRB-

The

proband

-LRB-

Fig.

-RRB-

is

a

nonobese

female

who

developed

diabetes

mellitus

at

years

of

age

The

proband

her

nonobese

mother

her

maternal

grandmother

and

a

maternal

uncle

were

all

heterozygous

for

a

G-to-A

substitution

predicted

to

result

in

an

R-to-H

substitution

at

amino

acid

-LRB-

Fig.

A

and

B

-RRB-

-LRB-

-RRB-

All

were

markedly

hyperinsulinemic

-LRB-

table

-RRB-

and

the

mother

and

maternal

grandmother

developed

diabetes

mellitus

in

their

late

thirties

Three

other

first-degree

relatives

available

for

study

were

all

clinically

normal

with

normal

fasting

glucose

and

insulin

and

were

homozygous

for

the

wild-type

sequence

-LRB-

Fig.

and

table

-RRB-

This

mutation

was

not

found

in

the

genomic

DNA

of

Caucasian

control

subjects

from

the

United

Kingdom

forms

part

of

an

RD

sequence

motif

within

the

catalytic

loop

of

the

kinase

SCIENCE

VOL

MAY

B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

humans
selection
evolution
dna
ultraconserved
genomics
human genome


Vol

May

doi

LETTERS

Drought

sensitivity

shapes

species

distribution

patterns

in

tropical

forests

Bettina

M.

J.

Liza

S.

Richard

Thomas

A.

Melvin

T.

Benjamin

L.

Stephen

P.

Although

patterns

of

tree

species

distributions

along

environmental

gradients

have

been

amply

documented

in

tropical

mechanisms

causing

these

patterns

are

seldom

known

Efforts

to

evaluate

proposed

mechanisms

have

been

hampered

by

a

lack

of

comparative

data

on

species

reactions

to

relevant

axes

of

environmental

Here

we

show

that

differential

drought

sensitivity

shapes

plant

distributions

in

tropical

forests

at

both

regional

and

local

scales

Our

analyses

are

based

on

experimental

field

assessments

of

drought

sensitivity

of

species

of

trees

and

shrubs

and

on

their

local

and

regional

distributions

within

a

network

of

inventory

sites

spanning

a

rainfall

gradient

across

the

Isthmus

of

Panama

Our

results

suggest

that

niche

differentiation

with

respect

to

soil

water

availability

is

a

direct

determinant

of

both

local

and

regional-scale

distributions

of

tropical

trees

Changes

in

soil

moisture

availability

caused

by

global

climate

change

and

forest

fragmentation

are

therefore

likely

to

alter

tropical

species

distributions

community

composition

and

diversity

Ecologists

have

long

recognized

correlations

between

water

availability

and

species

Indeed

the

most

consistent

patterns

of

species

distributions

in

tropical

forests

worldwide

as

well

as

in

many

non-tropical

systems

are

regional

and

continental

correlations

with

and

local

associations

with

topography

which

frequently

drives

variation

in

soil

water

Most

tropical

forests

experience

regular

dry

seasons

once

or

twice

per

year

and

even

in

many

aseasonal

equatorial

areas

extended

dry

spells

occur

during

which

plants

may

suffer

drought

Variation

in

water

availability

especially

during

dry

periods

mediated

through

rainfall

soil

characteristics

or

topography

is

therefore

an

important

candidate

for

niche

partitioning

in

tropical

forest

species

However

other

factors

that

may

co-vary

with

water

availability

have

also

been

proposed

to

lead

to

the

observed

patterns

namely

light

and

nutrient

availability

or

herbivore

and

pathogen

Furthermore

species

distributions

can

result

from

limited

dispersal

coupled

with

speciation

delayed

responses

to

climate

change

or

other

historical

To

advance

our

understanding

beyond

pattern

description

and

determine

which

mechanisms

are

important

we

need

experimental

assessments

of

the

responses

of

species

to

potentially

limiting

factors

and

a

linking

of

species-specific

responses

to

species

distributions

at

different

We

have

previously

shown

experimentally

that

seedlings

of

tropical

woody

plants

in

central

Panama

vary

considerably

in

their

drought

Other

studies

have

documented

nonrandom

species

distributions

with

respect

to

water

availability

at

both

regional

and

local

scales

across

the

Isthmus

of

Here

we

set

out

to

test

the

hypothesis

that

variation

in

drought

sensitivity

explains

these

distribution

patterns

We

link

our

extensive

comparative

data

set

on

seedling

drought

sensitivity

to

quantitative

data

on

the

regional

distribution

of

species

across

the

rainfall

gradient

in

central

Panama

and

to

their

local

distribution

with

respect

to

wet

and

dry

habitats

in

a

permanent

forest

plot

We

further

test

whether

correlations

km

a

Isthmus

of

Panama

Atlantic

Pacific

N

b

BCI

plot

m

Figure

Maps

of

the

study

areas

and

distribution

of

one

tree

species

as

an

example

a

Location

of

the

study

plots

along

the

Panama

Canal

The

Cocoli

plot

on

the

dry

Pacific

side

-LRB-

upright

filled

triangle

-RRB-

the

Sherman

plot

at

the

wet

Atlantic

side

-LRB-

inverted

filled

triangle

-RRB-

the

forest

dynamics

plot

on

BCI

-LRB-

filled

square

-RRB-

and

the

additional

inventory

sites

-LRB-

circles

-RRB-

are

shown

Xylopia

macrantha

-LRB-

Annonaceae

-RRB-

a

relatively

drought-sensitive

species

-LRB-

Supplementary

Information

-RRB-

occurs

on

the

wet

Caribbean

but

not

on

the

drier

Pacific

side

of

the

isthmus

-LRB-

filled

circles

present

open

circles

absent

-RRB-

b

Topographic

map

of

the

forest

dynamics

plot

-LRB-

contour

interval

m

-RRB-

Xylopia

macrantha

-LRB-

black

circles

-RRB-

shows

a

higher

density

on

wet

slopes

than

in

dry

plateau

habitats

Smithsonian

Tropical

Research

Institute

Apartado

Balboa

Ancon

Panama

of

Plant

Ecology

and

Systematics

University

of

Kaiserslautern

Kaiserslautern

Germany

of

Plant

Biology

University

of

Georgia

Athens

Georgia

USA

of

Biology

University

of

Utah

Salt

Lake

City

Utah

USA

Forest

Service

PO

Box

Burlington

Vermont

USA

of

Renewable

Resources

University

of

Alberta

Edmonton

Alberta

Canada

Nature

Publishing

Group

NATURE

Vol

May

LETTERS

n

P

adults

n

P

-RRB-

Drought

sensitivity

explained

more

than

twice

the

variation

in

density

in

dry

versus

wet

sites

for

adult

trees

than

it

did

for

seedlings

-LRB-

versus

Fig.

d

-RRB-

In

addition

more

species

showed

associations

with

the

wetter

slope

habitat

as

adults

than

as

juveniles

in

the

BCI

This

implies

that

at

local

scales

seeds

arrive

and

seedlings

are

able

to

establish

in

sites

with

suboptimal

soil

moisture

conditions

and

differential

mortality

across

species

and

habitats

subsequently

enhances

associations

with

favourable

habitats

Although

tropical

plant

species

reactions

to

environmental

factors

namely

light

and

nutrients

have

been

experimentally

assessed

in

numerous

studies

only

a

few

have

quantitatively

linked

these

data

to

distribution

These

studies

were

restricted

to

a

small

number

of

species

precluding

analysis

of

the

importance

of

environmental

factors

across

the

community

Thus

the

findings

presented

here

represent

the

most

thorough

study

so

far

linking

tropical

plant

species

distribution

patterns

with

species

reactions

to

an

environmental

factor

at

the

community

level

Our

results

suggest

that

drought

sensitivity

has

a

direct

role

in

determining

species

distributions

with

respect

to

local

and

regional

water

availability

However

significant

correlations

between

drought

sensitivity

and

distributions

might

arise

indirectly

as

a

result

of

differences

in

the

shade

tolerance

of

species

if

there

is

a

trade-off

between

shade

and

drought

tolerance

-LRB-

as

proposed

in

ref

-RRB-

and

if

forest

light

availability

decreases

as

water

availability

increases

We

found

no

correlation

between

light

requirements

and

species

densities

-LRB-

or

probability

of

occurrence

-RRB-

in

dry

versus

wet

habitats

at

the

local

or

regional

scale

-LRB-

see

Supplementary

Information

-RRB-

In

addition

we

found

no

correlation

between

the

drought

sensitivity

of

species

and

their

light

requirements

-LRB-

Supplementary

Information

-RRB-

This

is

in

contrast

to

the

results

of

a

recent

that

found

a

trade-off

between

drought

tolerance

and

shade

tolerance

for

temperate

species

On

the

basis

of

our

data

we

can

rule

out

the

possibility

that

the

observed

correlations

between

tropical

species

distributions

and

drought

sensitivity

indirectly

result

from

differential

species

light

requirements

This

is

despite

the

obvious

importance

that

species

responses

to

light

have

for

the

distribution

of

species

in

young

successional

versus

mature

forests

and

in

canopy

gap

versus

understorey

sites

Observed

correlations

between

species

distributions

and

drought

sensitivity

might

also

be

driven

by

correlations

between

water

and

nutrient

availability

coupled

with

a

correlation

between

species

drought

sensitivity

and

nutrient

requirements

Sufficient

experimental

data

on

the

nutrient

requirements

of

species

in

this

system

are

lacking

However

preliminary

data

on

soil

nutrients

and

pH

suggest

that

water

and

nutrient

availability

are

not

significantly

correlated

-LRB-

see

Supplementary

Information

-RRB-

This

does

not

rule

out

the

possibility

that

nutrient

availability

is

involved

in

regulating

the

distributions

of

species

in

tropical

but

indicates

that

it

is

unlikely

that

the

significant

correlations

between

species

distribution

patterns

and

drought

sensitivity

are

driven

by

variation

in

nutrient

availability

The

results

presented

here

emphasize

the

sensitivity

of

tropical

forests

to

water

and

suggest

that

future

changes

in

hydrological

processes

and

precipitation

patterns

will

have

direct

consequences

for

species

ranges

tropical

forest

community

composition

and

ecosystem

function

Such

changes

in

rainfall

patterns

have

been

observed

in

parts

of

the

tropics

over

the

past

decades

and

are

projected

to

continue

and

intensify

as

a

consequence

of

forest

fragmentation

and

global

climate

Current

vegetation

climate

models

particularly

for

tropical

regions

suffer

from

a

lack

of

ecological

data

and

mechanistic

understanding

of

the

factors

shaping

current

species

distributions

The

knowledge

that

water

availability

together

with

the

sensitivity

of

species

to

drought

is

a

causal

mechanistic

factor

influencing

species

distribution

patterns

in

tropical

forests

will

help

to

improve

the

accuracy

and

specificity

of

predictions

of

vegetation

shifts

under

global

change

scenarios

Nature

Publishing

Group

between

drought

sensitivity

and

species

distributions

might

arise

indirectly

as

a

result

of

differences

in

the

shade

tolerance

of

species

due

to

a

trade-off

between

drought

and

shade

To

assess

drought

sensitivity

we

conducted

irrigation

experiments

on

transplanted

seedlings

of

native

tree

and

shrub

species

The

difference

in

survival

between

dry

and

irrigated

conditions

ranged

from

to

-LRB-

see

Supplementary

Information

-RRB-

confirming

that

species

vary

widely

and

continuously

in

drought

sensitivity

In

separate

studies

we

showed

that

variation

in

seedling

drought

sensitivity

is

due

to

species

differences

in

the

effectiveness

of

physiological

mechanisms

of

drought

tolerance

-LRB-

that

is

their

differential

ability

to

survive

despite

low

leaf

water

-RRB-

-LRB-

T.A.K.

et

al.

unpublished

data

-RRB-

To

assess

regional

species

distributions

we

first

examined

the

densities

of

species

in

two

large

plots

-LRB-

ha

-RRB-

on

opposite

sides

of

the

Isthmus

of

Panama

-LRB-

Fig.

-RRB-

We

found

that

species

density

at

the

dry

Pacific

side

relative

to

the

wet

Atlantic

side

correlated

negatively

with

experimentally

assessed

drought

sensitivity

-LRB-

Fig.

n

P

-RRB-

Analysis

of

an

independent

data

set

of

species

occurrence

in

a

network

of

sites

across

the

isthmus

-LRB-

Fig.

-RRB-

also

showed

that

experimental

drought

sensitivity

was

a

significant

predictor

of

the

probability

of

occurrence

of

the

species

on

the

dry

relative

to

the

wet

side

-LRB-

Fig.

n

P

-RRB-

species

exhibiting

high

sensitivity

to

drought

in

the

field

experiments

occurred

more

often

towards

the

wet

end

of

the

climatic

gradient

At

the

local

scale

we

assessed

species

densities

on

dry

plateau

versus

wet

slope

sites

within

the

Barro

Colorado

Island

-LRB-

BCI

-RRB-

plot

-LRB-

Fig.

-RRB-

Both

for

seedlings

and

trees

densities

on

the

dry

plateaux

relative

to

wet

slopes

were

significantly

and

negatively

correlated

with

drought

sensitivity

-LRB-

Fig.

d

seedlings

Dry

log

-LSB-

-LRB-

density

dry

-RRB-

-LRB-

density

wet

-RRB-

-RSB-

a

Regional

b

-LRB-

density

dry/density

wet

-RRB-

Local

Density

Seedlings

Wet

Drought

sensitivity

-LRB-

-RRB-

Drought

sensitivity

-LRB-

-RRB-

c

Probability

of

occurrence

d

-LRB-

density

dry/density

wet

-RRB-

Drought

sensitivity

-LRB-

-RRB-

Adults

Wet

Dry

log

-LSB-

prob

dry/prob

wet

-RSB-

Drought

sensitivity

-LRB-

-RRB-

Figure

Significant

relationships

between

drought

sensitivity

of

seedlings

and

regional

and

local

distributions

of

species

a

c

Regional

distributions

of

tree

and

shrub

species

were

assessed

as

the

density

in

a

plot

at

the

dry

Pacific

relative

to

the

wet

Atlantic

side

of

the

Isthmus

of

Panama

-LRB-

a

-RRB-

and

as

the

probability

of

occurrence

on

the

dry

side

relative

to

the

wet

side

of

the

isthmus

based

on

data

from

inventory

sites

-LRB-

c

-RRB-

-LRB-

Note

that

results

were

similar

when

omitting

the

two

outlying

species

in

c.

-RRB-

b

d

Local

habitat

affinities

with

topographic

units

within

the

forest

dynamics

plot

on

BCI

were

assessed

for

seedlings

-LRB-

b

-RRB-

and

adults

-LRB-

d

-RRB-

as

the

density

of

a

species

in

dry

plateau

sites

relative

to

wet

slope

and

streamside

habitats

Drought

sensitivity

was

experimentally

assessed

as

the

relative

survival

difference

between

dry

and

irrigated

conditions

-LRB-

see

Supplementary

Information

-RRB-

LETTERS

NATURE

Vol

May

METHODS

SUMMARY

We

assessed

regional

species

distributions

across

the

Isthmus

of

Panama

-LRB-

Fig.

-RRB-

which

spans

a

strong

gradient

of

annual

and

dry

season

length

-LRB-

Supplementary

Data

and

-RRB-

in

two

ways

First

we

calculated

the

density

of

adults

of

each

species

at

a

dry

site

on

the

Pacific

side

of

the

isthmus

relative

to

a

wet

site

located

on

the

Atlantic

Second

we

analysed

presence/absence

data

from

inventory

sites

across

the

-LRB-

Supplementary

Data

-RRB-

An

index

of

dry

season

response

for

of

the

experimental

species

was

based

on

the

fitted

probability

of

occurrence

towards

the

dry

end

-LRB-

dry

season

of

days

-RRB-

and

towards

the

wet

end

of

the

gradient

-LRB-

days

Supplementary

Data

and

Supplementary

Program

-RRB-

Local-scale

species

distributions

were

assessed

within

a

forest

dynamics

plot

on

at

the

centre

of

the

rainfall

gradient

-LRB-

Fig.

-RRB-

We

analysed

local

associations

with

dry

and

wet

habitats

as

species

density

on

dry

plateaux

relative

to

wet

slopes

for

and

-LRB-

Supplementary

Information

-RRB-

Species

drought

sensitivity

for

seedlings

of

native

tree

and

shrub

species

was

experimentally

assessed

in

the

field

as

the

relative

difference

in

survival

over

the

dry

season

of

transplanted

seedlings

in

dry

versus

irrigated

-LRB-

see

Supplementary

Information

-RRB-

As

an

index

of

the

light

requirements

of

species

we

used

observational

data

on

the

percentage

of

recruits

encountered

in

low

canopy

sites

-LRB-

that

is

high

light

conditions

in

gaps

-RRB-

in

the

BCI

Soil

pH

and

the

concentrations

of

nitrogen

and

phosphorous

were

measured

at

of

the

inventory

sites

across

the

rainfall

gradient

with

standard

procedures

-LRB-

see

Supplementary

Information

-RRB-

Linear

regressions

were

used

to

examine

whether

species

reactions

to

drought

or

light

were

significant

predictors

of

their

densities

-LRB-

or

probability

of

occurrence

-RRB-

in

dry

versus

wet

sites

Full

Methods

and

any

associated

references

are

available

in

the

online

version

of

the

paper

at

Received

November

accepted

March

Veenendaal

E.

M.

Swaine

M.

D.

Limits

to

tree

species

distribution

in

lowland

tropical

rainforests

In

Dynamics

of

Tropical

Communities

-LRB-

eds

Newbery

D.

M.

Prins

H.

H.

T.

Brown

N.

-RRB-

Symp

Brit

Ecol

Soc

-LRB-

Blackwell

Oxford

-RRB-

Webb

C.

O.

Peart

D.

R.

Habitat

associations

of

trees

and

seedlings

in

a

Bornean

rain

forest

J.

Ecol

-LRB-

-RRB-

Harms

K.

E.

Condit

R.

Hubbell

S.

P.

Foster

R.

B.

Habitat

associations

of

trees

and

shrubs

in

a

neotropical

forest

plot

J.

Ecol

-LRB-

-RRB-

Pyke

C.

R.

Condit

R.

Aguilar

S.

Lao

S.

Floristic

composition

across

a

climatic

gradient

in

a

neotropical

lowland

forest

J.

Veg

Sci

-LRB-

-RRB-

Condit

R.

et

al.

Beta-diversity

in

tropical

forest

trees

Science

-LRB-

-RRB-

Bongers

F.

Poorter

L.

Van

Rompaey

R.

S.

A.

R.

Parren

M.

P.

E.

Distribution

of

twelve

moist

forest

canopy

tree

species

in

Liberia

and

Cote

d'Ivoire

response

curves

to

a

climatic

gradient

J.

Veg

Sci

-LRB-

-RRB-

ter

Steege

H.

et

al.

A

spatial

model

of

tree

a-diversity

and

tree

density

for

the

Amazon

Biodiv

Conserv

-LRB-

-RRB-

Schimper

A.

F.

W.

Plant

Geography

upon

a

Physiological

Basis

-LRB-

Clarendon

Oxford

-RRB-

Gentry

A.

H.

Changes

in

plant

community

diversity

and

floristic

composition

on

environmental

and

geographical

gradients

Ann

Mo.

Bot

Gard

-LRB-

-RRB-

Daws

M.

I.

Mullins

C.

E.

Burslem

D.

F.

R.

P.

Paton

S.

R.

Dalling

J.

W.

Topographic

position

affects

the

water

regime

in

a

semideciduous

tropical

forest

in

Panama

Plant

Soil

-LRB-

-RRB-

Walsh

R.

P.

D.

Climate

In

The

Tropical

Rain

Forest

an

Ecological

Study

edn

-LRB-

ed

Richards

P.

W.

-RRB-

-LRB-

Cambridge

Univ.

Press

Cambridge

USA

-RRB-

Givnish

T.

J.

On

the

causes

of

gradients

in

tropical

tree

diversity

J.

Ecol

-LRB-

-RRB-

Hubbell

S.

P

The

Unified

Neutral

Theory

of

Biodiversity

and

Biogeography

-LRB-

Princeton

Univ.

Press

Princeton

-RRB-

Engelbrecht

B.

M.

J.

Kursar

T.

A.

Comparative

drought-resistance

of

seedlings

of

species

of

co-occurring

tropical

woody

plants

Oecologia

-LRB-

-RRB-

Huston

M.

A.

Biological

Diversity

The

Coexistence

of

Species

on

Changing

Landscapes

-LRB-

Cambridge

Univ.

Press

Cambridge

UK

-RRB-

Tyree

M.

T.

Engelbrecht

B.

M.

J.

Vargas

G.

Kursar

T.

A.

Desiccation

tolerance

of

five

tropical

seedlings

in

Panama

Relationship

to

a

field

assessment

of

droughtperformance

Plant

Physiol

-LRB-

-RRB-

Comita

L.

S.

Condit

R.

Hubbell

S.

P.

Developmental

changes

in

habitat

associations

of

tropical

trees

J.

Ecol

-LRB-

-RRB-

Veenendaal

E.

M.

et

al.

Responses

of

West

African

forest

tree

seedlings

to

irradiance

and

soil

fertility

Funct

Ecol

-LRB-

-RRB-

Palmiotto

P.

A.

et

al.

Soil-related

habitat

specialization

in

Dipterocarp

rain

forest

tree

species

in

Borneo

J.

Ecol

-LRB-

-RRB-

Niinemets

U.

Valladares

F.

Tolerance

to

shade

drought

and

waterlogging

of

temperate

northern

hemisphere

trees

and

shrubs

Ecol

Monogr

-LRB-

-RRB-

John

R.

et

al.

Soil

nutrients

influence

spatial

distributions

of

tropical

tree

species

Proc

Natl

Acad

Sci

USA

-LRB-

-RRB-

Potts

M.

D.

Drought

in

a

Bornean

everwet

rain

forest

J.

Ecol

-LRB-

-RRB-

Malhi

Y.

Phillips

O.

L.

Tropical

forests

and

global

atmospheric

change

a

synthesis

Phil

Trans

R.

Soc

Lond

B

-LRB-

-RRB-

Smithsonian

Tropical

Research

Institute

ACP

physical

monitoring

downloads

-LRB-

-RRB-

Condit

R.

et

al.

Tropical

forest

dynamics

across

a

rainfall

gradient

and

the

impact

of

an

El

Nino

dry

season

J.

Trop

Ecol

-LRB-

-RRB-

Perez

R.

et

al.

Tree

species

composition

and

diversity

in

the

Upper

Chagres

River

Basin

Panama

In

The

Rio

Chagres

Panama

A

Multidisciplinary

Profile

of

a

Tropical

Watershed

-LRB-

ed

Harmon

R.

S.

-RRB-

-LRB-

Springer

Dordrecht

The

Netherlands

-RRB-

Condit

R.

Hubbell

S.

P.

Foster

R.

B.

Barro

Colorado

Forest

census

plot

data

-LRB-

-RRB-

Comita

L.

S.

Aguilar

S.

Perez

R.

Lao

S.

Hubbell

S.

P.

Patterns

of

woody

plant

species

abundance

and

diversity

in

the

seedling

layer

of

a

tropical

forest

J.

Veg

Sci

-LRB-

-RRB-

Engelbrecht

B.

M.

J.

Kursar

T.

A.

Tyree

M.

T.

Drought

effects

on

seedling

survival

in

a

tropical

moist

forest

Trees

-LRB-

Berl

-RRB-

-LRB-

-RRB-

Welden

C.

W.

Hewett

S.

W.

Hubbell

S.

P.

Foster

R.

B.

Sapling

survival

growth

and

recruitment

Relationship

to

canopy

height

in

a

neotropical

forest

Ecology

-LRB-

-RRB-

Supplementary

Information

is

linked

to

the

online

version

of

the

paper

at

Acknowledgements

We

thank

the

more

than

two

dozen

field

assistants

who

conducted

the

extensive

censuses

and

drought

experiments

S.

Aguilar

and

R.

Perez

were

instrumental

in

species

identifications

and

S.

Lao

assisted

with

data

management

The

study

was

financially

supported

by

the

Andrew

W.

Mellon

Foundation

-LRB-

T.A.K.

B.M.J.E.

and

M.T.T.

-RRB-

the

National

Science

Foundation

-LRB-

S.P.H.

and

L.S.C.

-RRB-

the

University

of

Utah

-LRB-

T.A.K.

and

B.M.J.E.

-RRB-

the

Center

of

Tropical

Forest

Science

-LRB-

B.M.J.E

and

L.S.C.

-RRB-

and

the

German

Science

Foundation

-LRB-

B.M.J.E.

-RRB-

We

acknowledge

logistical

support

provided

by

the

Smithsonian

Tropical

Research

Center

and

the

opportunity

to

work

in

the

area

granted

by

the

Autoridad

Nacional

del

Ambiente

-LRB-

ANAM

-RRB-

of

Panama

Author

Contributions

B.M.J.E.

and

L.S.C.

analysed

the

compiled

data

sets

and

coordinated

the

writing

B.M.J.E.

T.A.K.

and

M.T.T.

conducted

the

drought

sensitivity

experiments

L.S.C.

and

S.P.H.

managed

the

seedling

census

and

R.C.

and

S.P.H.

the

tree

census

in

the

forest

dynamics

plot

R.C.

coordinated

the

establishment

and

census

of

the

regional

inventory

network

and

modelled

species

occurrence

against

dry

season

length

B.L.T.

conducted

soil

nutrient

analyses

All

authors

discussed

the

results

and

commented

on

the

manuscript

Author

Information

Reprints

and

permissions

information

is

available

at

The

authors

declare

no

competing

financial

interests

Correspondence

and

requests

for

materials

should

be

addressed

to

B.M.J.E.

-LRB-

engelbrb@si.edu

-RRB-

Nature

Publishing

Group

doi

METHODS

Regional

species

distributions

We

assessed

regional

species

distributions

across

the

Isthmus

of

Panama

which

spans

a

strong

rainfall

gradient

from

to

mm

over

a

distance

of

km

-LRB-

ref

-RRB-

Annual

rainfall

is

highly

correlated

with

dry

season

length

-LRB-

n

P

Supplementary

Information

-RRB-

and

minimum

dry

season

gravimetric

soil

water

content

-LRB-

n

P

B.M.J.E.

unpublished

data

-RRB-

Regional

distributions

of

species

were

assessed

two

ways

First

we

examined

the

density

of

each

species

-LRB-

cm

diameter

at

breast

height

d.b.h.

-RRB-

at

a

dry

site

on

the

Pacific

side

of

the

isthmus

-LRB-

Cocoli

ha

Fig.

-RRB-

and

at

a

wet

site

located

on

the

Atlantic

side

-LRB-

Fort

Sherman

ha

-RRB-

using

data

collected

in

We

calculated

the

ratio

of

species

density

in

the

Pacific-side

plot

to

the

Atlantic-side

plot

for

all

species

with

total

individuals

Second

we

analysed

presence/absence

data

from

inventory

sites

across

the

-LRB-

Fig.

and

Supplementary

Information

-RRB-

for

species

occurring

in

of

the

sites

We

quantified

dry

season

duration

based

on

data

from

rainfall

stations

near

the

Panama

and

used

this

to

model

dry

season

length

at

the

sites

-LRB-

Supplementary

Information

-RRB-

The

responses

of

of

the

experimental

tree

species

were

modelled

as

a

function

of

dry

season

duration

fitting

a

kernel

using

an

optimized

This

approach

requires

no

a

priori

assumptions

about

the

shape

of

the

response

and

produces

an

estimated

probability

of

occurrence

for

each

species

at

any

dry

season

duration

An

index

of

dry

season

response

for

each

species

was

based

on

the

fitted

probability

of

occurrence

towards

the

dry

end

-LRB-

dry

season

of

days

-RRB-

and

towards

the

wet

end

of

the

gradient

-LRB-

days

-RRB-

For

details

see

Supplementary

Information

Local

species

distributions

Local-scale

species

distributions

were

assessed

within

a

forest

dynamics

plot

on

Barro

Colorado

Island

-LRB-

BCI

-RRB-

at

the

centre

of

the

rainfall

gradient

-LRB-

Fig.

-RRB-

At

BCI

as

in

other

tropical

forests

dry

season

soil

moisture

is

lower

on

plateaux

than

We

used

data

on

species

densities

from

a

census

of

stems

cm

height

to

cm

d.b.h.

-LRB-

referred

to

as

seedlings

-RRB-

and

from

a

census

of

stems

cm

d.b.h.

-LRB-

referred

to

as

trees

-RRB-

We

analysed

local

associations

with

dry

and

wet

habitats

as

species

density

on

plateaux

-LRB-

high

and

low

plateaux

combined

as

defined

by

ref

-RRB-

relative

to

slopes

-LRB-

slopes

and

streamside

areas

combined

-RRB-

for

all

species

with

individuals

within

the

focal

habitats

for

both

life

stages

-LRB-

Supplementary

Information

-RRB-

Seedling

drought

sensitivity

The

drought

sensitivity

of

species

was

experimentally

assessed

in

the

Barro

Colorado

Nature

Monument

in

the

centre

of

the

isthmus

for

seedlings

of

native

tree

and

shrub

species

from

families

-LRB-

expanded

from

ref

Supplementary

Information

-RRB-

We

transplanted

firstyear

seedlings

to

plots

in

the

forest

understorey

of

which

half

were

kept

dry

with

transparent

rainout-shelters

and

the

other

half

were

irrigated

-LRB-

see

ref

for

experimental

details

-RRB-

As

an

index

of

species

drought

sensitivity

-LRB-

DS

-RRB-

-LRB-

that

is

their

susceptibility

to

periods

of

low

water

availability

-RRB-

we

calculated

the

relative

difference

in

survival

-LRB-

S

-RRB-

over

the

dry

season

in

dry

versus

irrigated

-LRB-

DS

-LRB-

Sirrigated

Sdry

-RRB-

Sirrigated

Supplementary

Information

-RRB-

The

experimental

approach

allowed

for

the

differentiation

of

effects

of

low

water

availability

from

other

factors

potentially

inducing

mortality

during

the

dry

season

such

as

shade

herbivore

damage

or

nutrient

Experiments

were

conducted

in

the

dry

seasons

of

and

with

eight

species

overlapping

between

experiments

After

weeks

in

relative

differential

survival

of

the

overlapping

species

correlated

highly

significantly

-LRB-

n

P

-RRB-

and

closest

to

with

relative

differential

survival

after

weeks

in

-LRB-

regression

not

different

from

t-test

P

-RRB-

Values

of

drought

sensitivity

measured

in

-LRB-

-RRB-

were

adjusted

using

the

equation

of

the

regression

between

relative

survival

in

the

two

years

for

the

eight

overlapping

species

-LRB-

DSadjusted

-RRB-

yielding

a

comparable

data

set

for

all

species

Plant

light

requirements

As

an

index

of

the

light

requirements

of

species

we

used

observational

data

on

the

percentage

of

recruits

encountered

in

low

canopy

sites

-LRB-

that

is

high

light

conditions

in

gaps

-RRB-

in

the

BCI

A

high

proportion

of

recruits

in

low

canopy

sites

suggests

that

the

species

is

lightdemanding

In

contrast

to

the

experimental

data

on

drought

sensitivity

this

data

set

does

not

allow

us

to

differentiate

among

possible

factors

contributing

to

differences

in

recruitment

rates

between

gaps

and

shaded

understorey

sites

-LRB-

for

example

light

requirements

pest

resistance

etc.

-RRB-

Soil

pH

and

nutrient

content

Soil

pH

and

the

concentrations

of

nitrogen

and

phosphorous

were

analysed

in

of

the

plots

across

the

isthmus

for

the

surface

cm

of

soil

sampled

at

systematically

distributed

sites

per

plot

The

cores

from

each

plot

were

combined

before

analysis

Soil

pH

was

measured

in

a

ratio

of

soil

to

deionized

water

Total

nitrogen

was

determined

by

combustion

and

gas

chromatography

using

a

Flash

Soil

Analyser

-LRB-

CE

Elantech

-RRB-

Total

phosphorus

was

determined

by

ignition

and

acid

digestion

with

detection

by

inductively

coupled

plasma

optical

emission

spectrometry

Total

nitrogen

and

phosphorus

concentrations

were

correlated

tightly

with

measures

of

plant-available

nitrogen

and

phosphorus

respectively

-LRB-

B.L.T.

and

B.M.J.E.

unpublished

data

-RRB-

Linking

data

sets

Linear

regressions

were

used

to

examine

whether

species

drought

sensitivity

or

light

requirement

was

a

significant

predictor

of

their

densities

-LRB-

or

probability

of

occurrence

-RRB-

in

dry

versus

wet

sites

Sample

sizes

depended

on

the

number

of

species

overlapping

between

respective

data

sets

Data

were

transformed

to

meet

requirements

of

normal

distribution

and

constant

variance

-LRB-

see

figures

-RRB-

McCune

B.

Non-parametric

habitat

models

with

automatic

interactions

J.

Veg

Sci

-LRB-

-RRB-

Becker

P.

Rabenold

P.

E.

Idol

J.

R.

Smith

A.

P.

Water

potential

gradients

for

gaps

and

slopes

in

a

Panamanian

tropical

moist

forest

's

dry

season

J.

Trop

Ecol

-LRB-

-RRB-

Nature

Publishing

Group

O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O

climate vegetation tropical ecology drought precipitation