2014 10-21-sbc322

QMPlus

Blog post authoring• Decide your groups for authoring

• (must pair with different people than presentation!) • cannot present and blog as part of same "theme" • https://etherpad.mozilla.org/obVAlZUq5D

• Editors: • determine who is responsible for which papers • alert authors when their stuff is due • determine who receives which review task when

• Web people need to decide on platform (e.g. tumblr & color scheme etc), name, potential guidelines, and set up…

https://etherpad.mozilla.org/obVAlZUq5D

RAD? CNV? FST? WTF?

• New papers - specific question session

• Next Tuesday 9a.m. (all welcome)

• Additional needed?

• maybe: this Thursday 16:30 - fogg 5.03A

Genomic dynamics

www.sciencemag.org SCIENCE VOL 331 25 FEBRUARY 2011 1067

REPORTS

on

Mar

ch 1

2, 2

013

ww

w.s

cien

cem

ag.o

rgD

ownl

oade

d fro

m

Solenopsis invicta fire ants are a big problem!very well studied!

Ascunce et al 2011

Solenopsis invicta fire ant: two social forms

!

•1 large queen •Independent founding •Highly territorial •Many sizes of workers

!

•2-100 smaller queens •Dependent founding •No inter-colony aggression •All workers similar size

Single-queen form: Multiple-queen form:

Fire ants+

Population genetics: Allozyme screen

Ken Ross L. Keller

“starch gel”+

1 2 3

Allozyme screen Social form associated to Gp-9 locus

Frequency of the most

common allele!

Locus!

0.3!0.4!0.5!0.6!0.7!0.8!0.9!1.0!

Single queen!Multiple queen!

Est-6!Est-4!G3pdh-1!Ca-4!Pgm-4!Ddh-1!Pro-5!

Pgm-3!

Acoh-5!

acoh-1!

Acy-1!

Pgm-1!

Aat-2!

Gp-9!

Ken Ross and colleagues Laurent Keller and colleagues

Single queen form Multiple queen form


Social form completely associated to Gp-9 locus

bbbbBB BB Bb bb




bbBB BB Bb

x

Gp-9 bb females rareKen Ross and colleagues

Laurent Keller and colleagues



BB BB Bb




BB BB Bb

xKen Ross and colleagues




BB BB Bb

x xKen Ross and colleagues




BB BB Bb

x x xKen Ross and colleagues


Single queen form Multiple queen form(>15% ) (< 5% )


•Is this gene the single überregulator?


Hypothesis: influences queen odor & how workers « smell » queens

Gp-9 is an odorant binding protein

Krieger & Ross

dN/dS = infinityKrieger & Ross

•Is this gene the single überregulator?


maybe 1/14th of the genome?•Only 14 allozyme markers were used

Locus!

0.3!0.4!0.5!0.6!0.7!0.8!0.9!1.0!

Single queen!Multiple queen!

Est-6!Est-4!G3pdh-1!Ca-4!Pgm-4!Ddh-1!Pro-5!

Pgm-3!

Acoh-5!

acoh-1!

Acy-1!

Pgm-1!

Aat-2!

Gp-9!

This changes everything.454

Illumina Solid...

Any lab can sequence anything!

Are other genes linked to Gp-9?

Sequenced: •a Gp-9 B ♂ genome

!

!

Single ♂:

His brothers:

11×

4×

(330bp-insert paired reads) (normal single-end reads)45× +

Sequencing from haploid males (for easier assembly):

(8,000 & 20,000bp-insert paired reads)

B 20x

The genome of a Gp-9 B ♂ fire ant

Single ♂:

His brothers:

11×

4×

(330bp-insert paired reads) (normal single-end reads)45× +

Sequencing from haploid males (for easier assembly):

(8,000 & 20,000bp-insert paired reads)

B 20x

The genome of a Gp-9 B ♂ fire ant

Assembly approach:1. Assemble short Illumina reads with SOAPdenovo→N50: 3600 bp 2. Chop assembly into “fake 454 reads” (300bp) 3. Assemble fake + real 454 reads with Newbler→N50: 720,000 bp

10,000 scaffolds (100 biggest scaffolds: 50% of genome)

Total: 350,000,000 bp assembled. The rest: repeats→

Wurm et al 2011

★ Expansion of lipid-processing gene families (for Cuticular Hydrocarbons?)

★ 420 putative olfactory receptors (more than any other insect!)

The genome of the fire antSome findings:

0.05

SiOR

04648+10

SiOR01968+4

SiOR0

0899+7

SiOR02814+3

SiOR04171+6

SiO

R04609+4

SiOR00330+28

SiO

R02

694+25

SiO

R04609+20

SiOR05285+6

SiOR00330+25

SiOR04510+15

SiOR00330+18

SiOR04609+23

SiOR01

968+23

SiO

R03

952+4

SiOR0

4648+16

SiO

R05

901+2

SiO

R02

944+4

SiOR01968+5

SiOR04171+19 SiOR

04648+5

SiOR10535+3

SiOR0

1224+3

SiOR06723+2

SiOR01968+9

SiOR02883+1

SiOR0

0899+3

SiOR01629+1

SiOR04171+1

SiOR01629+11

SiOR04171+10

SiOR04171+13

SiOR02694+3

SiOR04171+20

SiO

R02694+35

SiOR04171+15

SiOR04609+7

SiOR05118+2

SiOR07

837+2

SiO

R02

694+27

SiOR01968+10

SiOR04648+17

SiOR0196

8+19

SiO

R02

694+17

SiOR04648+13

SiOR01968+6

SiOR

00899+1

2

SiOR0

5901+1

SiOR00330+20

SiO

R02

648+2

SiO

R02

659+2

SiOR01968+1

6

SiOR0

0899+11

SiO

R02974

SiOR04171+2

SiOR03952+2

SiOR06792+2

SiOR04510+4

SiOR04171+28

SiOR05285+5

SiOR05285+9

SiOR

00899+1

5

SiOR

04648+3

SiO

R02694+36

SiOR10535+1

SiO

R02

694+19

SiO

R02

694+23

SiOR02694+1

SiO

R04609+14

SiOR01122

SiOR

04648+9

SiO

R02694+34

SiOR01629+8

SiOR

04648+8

SiOR04510+8

SiOR06573

SiO

R02

944+1

SiOR00330+26

SiO

R00330+1S

iOR02

694+15

SiO

R03038

SiOR05285+7

SiOR0

0899+5

SiOR04609+10

SiO

R04609+3

SiOR04339

SiOR0

8068

SiOR

04648+6

SiOR04510+2

SiOR05285+8

SiOR01

573+4

SiOR0366

3

SiOR04171+8

SiOR01858+2

SiOR01968+2SiOR01968+1

SiOR02694+5

SiOR01968+3

SiOR06723+3

SiOR01968+15

SiOR05285+1

SiOR0

0899+4

SiOR04609+22

SiOR04171+9

SiOR02694+9

SiO

R02

648+1

SiOR06792+3

SiOR0

1573+2

SiO

R02

694+20

SiOR1

0542

SiO

R04609+15

SiOR02694+8

SiOR00330+16

SiOR0

0899+2

SiOR02694+10

SiOR04510+9

SiOR05285+3

SiOR04171+24

SiO

R04609+2

SiOR05285+11

SiOR02

694+14

SiOR01

573+1

SiOR05285+2

SiOR00613

SiOR0196

8+22

SiOR

00899+9

SiO

R06

843+2

SiO

R02694+37

SiOR0

0899+1

SiOR04609+9

SiOR05431+2

SiOR10535+2

SiOR00330+15

SiO

R02

694+18

SiOR0

1224+2

SiOR04510+11

SiOR00330+23

SiO

R02

694+29

SiOR05416

SiOR05285+10

SiOR02694+2

SiOR01629+9

SiOR05431+1

SiOR02883+2

SiOR04171+14

SiO

R08

341

SiO

R02

694+22

SiOR01

224+1

SiOR01968+12

SiOR05431+3

SiOR04510+6

SiOR02694+7

SiOR04609+5

SiO

R02

944+2

SiOR03

952+3

SiOR01968+8

SiOR04609+24

SiO

R02

694+30

SiOR01629+10

SiOR04510+14

SiOR00565

SiOR05118+3

SiO

R01321

SiO

R04609+19

SiOR00330+14

SiO

R02694+38

SiOR04609+8

SiOR04171+16

SiOR10455

SiO

R04609+16

SiO

R04609+21

SiO

R02

694+28

SiO

R02

659+1

SiOR04171+5

SiOR00330+29

SiOR01968+14

SiOR03983

SiOR00330+27

SiOR05285+4

SiOR04510+1

SiOR

00899+8

SiOR

04648+7

SiO

R04609+17

SiO

R00330+5

SiO

R02

694+21

SiOR02814+4

SiOR00330+7

SiOR01

629+3

SiOR01

968+26

SiO

R02694+31

SiOR0

4648+2

SiO

R02694+39

SiOR04171+25

SiOR06577

SiOR01

968+25

SiOR04171+21

SiOR06792+6

SiO

R04609+11

SiOR02694+11

SiOR06792+1

SiOR04171+4

SiOR01629+5

SiOR00330+21

SiOR0

4648+15

SiO

R00330+6

SiO

R02

694+16

SiOR

04648+11

SiOR

04648+4

SiO

R00330+3

SiO

R06

535

SiOR04171+7

SiOR10493

SiO

R02694+32

SiOR06792+4

SiOR04510+3

SiO

R06

890

SiOR0196

8+20

SiO

R04609+12

SiOR04171+3

SiOR01968

+18

SiOR01968+11

SiO

R04609+13

SiOR01629+12

SiOR00330+22

SiO

R02694+33

SiOR00330+13

SiOR01

573+3

SiOR04510+16

SiOR04171+12

SiOR04510+13

SiOR

05118+1

SiO

R02

944+3

SiOR04171+26

SiOR04171+17

SiOR

00899+14

SiOR04171+29

SiOR

00899+1

3

SiOR02694+13

SiOR00330+24

SiOR00330+19

SiOR04171+27

SiO

R02

694+24

SiOR04510+5

SiOR07090

SiOR03952+1

SiOR04510+10

SiOR00330+17

SiO

R02

694+26

SiO

R04609+1

SiOR0

0899+6

SiOR04648+12

SiOR02814+2

SiOR00330+11

SiOR04171+18

SiOR01968+1

7

SiOR00330+10

SiOR00330+9

SiOR0162

9+2

SiOR04171+11

SiOR04510+12

SiOR0196

8+21

SiOR00330+8

SiOR01858+1

SiOR04510+7

SiOR05431+4

SiOR02694+6

SiOR01968+13

SiO

R00330+4

SiO

R04609+18

SiOR0

0899+10

SiOR00330+12

SiOR00330+31

SiO

R06

843+1

SiOR07

837+1

SiO

R00330+2

SiOR01629+4

SiOR0

4648+1

SiOR01

968+24

SiOR04171+23

SiOR0162

9+7

SiOR0

4648+14

SiO

R06

843+3

SiOR06792+5

SiOR01968+7

SiOR01629

+6

SiOR02883+3

SiOR02694+12

SiOR

05118+4

SiOR04171+22

SiO

R01080

SiOR04609+6

SiOR02814+1

SiOR00330+30

SiOR05285+12

SiOR06723+1

SiOR02694+4

Wurm et al 2011

> 400Harvester ant 399 344Nasonia wasp 225honey bee 165

6510

★ Expansion of lipid-processing gene families (for Cuticular Hydrocarbons)


★ Functional DNA-methylation system


Wurm et al 2011

0.05

SiOR

04648+10

SiOR01968+4

SiOR0

0899+7

SiOR02814+3

SiOR04171+6

SiO

R04609+4

SiOR00330+28

SiO

R02

694+25

SiO

R04609+20

SiOR05285+6

SiOR00330+25

SiOR04510+15

SiOR00330+18

SiOR04609+23

SiOR01

968+23

SiO

R03

952+4

SiOR0

4648+16

SiO

R05

901+2

SiO

R02

944+4

SiOR01968+5

SiOR04171+19 SiOR

04648+5

SiOR10535+3

SiOR0

1224+3

SiOR06723+2

SiOR01968+9

SiOR02883+1

SiOR0

0899+3

SiOR01629+1

SiOR04171+1

SiOR01629+11

SiOR04171+10

SiOR04171+13

SiOR02694+3

SiOR04171+20

SiO

R02694+35

SiOR04171+15

SiOR04609+7

SiOR05118+2

SiOR07

837+2

SiO

R02

694+27

SiOR01968+10

SiOR04648+17

SiOR0196

8+19

SiO

R02

694+17

SiOR04648+13

SiOR01968+6

SiOR

00899+1

2

SiOR0

5901+1

SiOR00330+20

SiO

R02

648+2

SiO

R02

659+2

SiOR01968+1

6

SiOR0

0899+11

SiO

R02974

SiOR04171+2

SiOR03952+2

SiOR06792+2

SiOR04510+4

SiOR04171+28

SiOR05285+5

SiOR05285+9

SiOR

00899+1

5

SiOR

04648+3

SiO

R02694+36

SiOR10535+1

SiO

R02

694+19

SiO

R02

694+23

SiOR02694+1

SiO

R04609+14

SiOR01122

SiOR

04648+9

SiO

R02694+34

SiOR01629+8

SiOR

04648+8

SiOR04510+8

SiOR06573

SiO

R02

944+1

SiOR00330+26

SiO

R00330+1S

iOR02

694+15

SiO

R03038

SiOR05285+7

SiOR0

0899+5

SiOR04609+10

SiO

R04609+3

SiOR04339

SiOR0

8068

SiOR

04648+6

SiOR04510+2

SiOR05285+8

SiOR01

573+4

SiOR0366

3

SiOR04171+8

SiOR01858+2

SiOR01968+2SiOR01968+1

SiOR02694+5

SiOR01968+3

SiOR06723+3

SiOR01968+15

SiOR05285+1

SiOR0

0899+4

SiOR04609+22

SiOR04171+9

SiOR02694+9

SiO

R02

648+1

SiOR06792+3

SiOR0

1573+2

SiO

R02

694+20

SiOR1

0542

SiO

R04609+15

SiOR02694+8

SiOR00330+16

SiOR0

0899+2

SiOR02694+10

SiOR04510+9

SiOR05285+3

SiOR04171+24

SiO

R04609+2

SiOR05285+11

SiOR02

694+14

SiOR01

573+1

SiOR05285+2

SiOR00613

SiOR0196

8+22

SiOR

00899+9

SiO

R06

843+2

SiO

R02694+37

SiOR0

0899+1

SiOR04609+9

SiOR05431+2

SiOR10535+2

SiOR00330+15

SiO

R02

694+18

SiOR0

1224+2

SiOR04510+11

SiOR00330+23

SiO

R02

694+29

SiOR05416

SiOR05285+10

SiOR02694+2

SiOR01629+9

SiOR05431+1

SiOR02883+2

SiOR04171+14

SiO

R08

341

SiO

R02

694+22

SiOR01

224+1

SiOR01968+12

SiOR05431+3

SiOR04510+6

SiOR02694+7

SiOR04609+5

SiO

R02

944+2

SiOR03

952+3

SiOR01968+8

SiOR04609+24

SiO

R02

694+30

SiOR01629+10

SiOR04510+14

SiOR00565

SiOR05118+3

SiO

R01321

SiO

R04609+19

SiOR00330+14

SiO

R02694+38

SiOR04609+8

SiOR04171+16

SiOR10455

SiO

R04609+16

SiO

R04609+21

SiO

R02

694+28

SiO

R02

659+1

SiOR04171+5

SiOR00330+29

SiOR01968+14

SiOR03983

SiOR00330+27

SiOR05285+4

SiOR04510+1

SiOR

00899+8

SiOR

04648+7

SiO

R04609+17

SiO

R00330+5

SiO

R02

694+21

SiOR02814+4

SiOR00330+7

SiOR01

629+3

SiOR01

968+26

SiO

R02694+31

SiOR0

4648+2

SiO

R02694+39

SiOR04171+25

SiOR06577

SiOR01

968+25

SiOR04171+21

SiOR06792+6

SiO

R04609+11

SiOR02694+11

SiOR06792+1

SiOR04171+4

SiOR01629+5

SiOR00330+21

SiOR0

4648+15

SiO

R00330+6

SiO

R02

694+16

SiOR

04648+11

SiOR

04648+4

SiO

R00330+3

SiO

R06

535

SiOR04171+7

SiOR10493

SiO

R02694+32

SiOR06792+4

SiOR04510+3

SiO

R06

890

SiOR0196

8+20

SiO

R04609+12

SiOR04171+3

SiOR01968

+18

SiOR01968+11

SiO

R04609+13

SiOR01629+12

SiOR00330+22

SiO

R02694+33

SiOR00330+13

SiOR01

573+3

SiOR04510+16

SiOR04171+12

SiOR04510+13

SiOR

05118+1

SiO

R02

944+3

SiOR04171+26

SiOR04171+17

SiOR

00899+14

SiOR04171+29

SiOR

00899+1

3

SiOR02694+13

SiOR00330+24

SiOR00330+19

SiOR04171+27

SiO

R02

694+24

SiOR04510+5

SiOR07090

SiOR03952+1

SiOR04510+10

SiOR00330+17

SiO

R02

694+26

SiO

R04609+1

SiOR0

0899+6

SiOR04648+12

SiOR02814+2

SiOR00330+11

SiOR04171+18

SiOR01968+1

7

SiOR00330+10

SiOR00330+9

SiOR0162

9+2

SiOR04171+11

SiOR04510+12

SiOR0196

8+21

SiOR00330+8

SiOR01858+1

SiOR04510+7

SiOR05431+4

SiOR02694+6

SiOR01968+13

SiO

R00330+4

SiO

R04609+18

SiOR0

0899+10

SiOR00330+12

SiOR00330+31

SiO

R06

843+1

SiOR07

837+1

SiO

R00330+2

SiOR01629+4

SiOR0

4648+1

SiOR01

968+24

SiOR04171+23

SiOR0162

9+7

SiOR0

4648+14

SiO

R06

843+3

SiOR06792+5

SiOR01968+7

SiOR01629

+6

SiOR02883+3

SiOR02694+12

SiOR

05118+4

SiOR04171+22

SiO

R01080

SiOR04609+6

SiOR02814+1

SiOR00330+30

SiOR05285+12

SiOR06723+1

SiOR02694+4

★ Expansion of lipid-processing gene families (for Cuticular Hydrocarbons)


★ Functional DNA-methylation system ★Ant-specific duplication and subfunctionalization

of vitellogenin (in bees: involved in reproduction & division of labor)


Wurm et al 2011

significance of these duplication events in vitellogenins, odorperception genes, and a family of lipid-processing genes. We alsodiscuss additional features of interest in the fire ant genome rel-evant to the complex social biology of this species, including sexdetermination genes, DNA methylation genes, telomerase, andthe insulin and juvenile hormone pathways.

Vitellogenins. In contrast to other insects that mainly have only oneor two vitellogenins, the fire ant genome harbors four adjacent

copies of vitellogenin (Vg1-4) (Fig. 3A), all of which are at leastpartially supported by EST reads. A phylogenetic analysis revealsthat an ancestral vitellogenin gene first underwent duplication,followed by possibly independent duplications of each of thedaughter vitellogenins, thus giving rise to Vg1 and Vg4 and to Vg2andVg3. All of these duplications occurred after the ancestor offireants split from wasps and bees (Fig. 3B). The single vitellogeninfound inA.mellifera is amultifunctional protein (26) involved in theregulation of life span (27, 28) and division of labor (29). Quanti-tative RT-PCR shows thatVg1 andVg4 are preferentially expressedin workers and Vg2 and Vg3 in queens (Fig. 3C, SI Materials andMethods, and Table S1G). Vitellogenin expression in S. invictaworkers is surprising because they lack ovaries. Given the super-organism properties of ant societies, the expression patterns sug-gest that vitellogenins underwent neo- or subfunctionalizationafter duplication to acquire caste-specific functions.

Odor Perception. Consistent with studies in other insects, we finda single S. invicta ortholog to DmOr83b, a broadly expressed ol-factory receptor (OR) required to interact with other ORs forDrosophila and Tribolium castaneum olfaction (30–32). BeyondOR83b, OR number varies greatly between insect species. Blastsearches and GeneWise searches using an HMM profile con-structed with aligned ORs from N. vitripennis (33) and Pogono-myrmex barbatus identified more than 400 loci in the S. invictagenome with significant sequence similarity to ORs. Preliminarywork on gene model reconstruction identified 297 intact full-length proteins. Many S. invicta ORs are in tandem arrays (Fig.S2A) and derive from recent expansions. S. invicta may thus har-bor the largest identified insect OR repertoire because there are10 ORs in Pediculus humanus (34), 60 in Drosophila, 165 inA. mellifera, 225 in N. vitripennis (33), and 259 in T. castaneum(32). The large numbers of N. vitripennis and T. castaneum ORsare thought to be due to current or past difficulties in host andfood finding. As has been suggested for A. mellifera (35), the largenumber of S. invicta ORs may result from the importance ofchemical communication in ants. The odorant-binding proteins(OBPs) are another family of genes also known to play roles inchemosensation in Drosophila (36). Intriguingly, the social orga-nization of S. invicta colonies is completely associated with se-

Eum

etaz

oa

No hits 3424

Not assigned 274

Cnidaria 100

Art

hro

po

da

Ne

op

tera

Coe

lom

ata

Bila

teri

a

Nematoda 25

Deuterostomia 173

Arachnida 50

Paraneoptera 577

End

opte

rygo

ta

Hym

en

op

tera

Apoidea 7398

Chalcidoidea 3271

Ichneumonoidea 94

Diptera 404

Lepidoptera 29

Coleoptera 713

Fig. 2. Taxonomic distribution of best blastp hits of S. invicta proteins to thenonredundant (nr) protein database (E < 10−5). Results were first plottedusing MEGAN software (22) and then branches with fewer than 20 hits wereremoved, branch lengths were reduced for compactness, and tree topologywas adjusted to reflect consensus phylogenies (23, 24).

Vg1Vg4 Vg3 Vg2

2,330,000 bp 2,360,000 bpA

B CSolenopsis Vg1Solenopsis Vg4Solenopsis Vg2Solenopsis Vg3

Apis VgBombus Vg

Nasonia Vg1

Nasonia Vg2Pteromalus Vg

Encarsia VgPimpla Vg

Athalia Vg

Apocrita

Tenthedinoidea

Vespoidea

Apoidea

Aculeata

Chalcidoidea

Ichneumonoidea 0

5000

10000

15000

20000

25000 Vg3Vg2

142 389 17820 1.4 9269 0.61 40WQ WQW Q WQ

*** ***Vg1 Vg4* ***

0

100

200

300

400

500

600

Fig. 3. S. invicta vitellogenins. (A) Four vitellogenins are located within a single 40,000-bp region of the S. invicta genome. (B) Parsimony tree of known hy-menopteran vitellogenin protein sequences suggests that two rounds of vitellogenin duplication occurred after the split between ants and other hyme-nopterans including bees and wasps. (C) Quantitative RT-PCR of the four putative S. invicta vitellogenins on whole bodies of major workers (W) and matedqueens (Q) (n = 10). The y axis indicates mRNA concentrations for the different vitellogenins. Values depicted by each bar are shown below the x-axis labels.Error bars represent SEs. Expression differences between queens and workers were significant (Bonferroni-corrected two-tailed t tests: *P < 0.05, ***P < 10−10).

Wurm et al. PNAS Early Edition | 3 of 6

EVOLU

TION

0.05

SiOR

04648+10

SiOR01968+4

SiOR0

0899+7

SiOR02814+3

SiOR04171+6

SiO

R04609+4

SiOR00330+28

SiO

R02

694+25

SiO

R04609+20

SiOR05285+6

SiOR00330+25

SiOR04510+15

SiOR00330+18

SiOR04609+23

SiOR01

968+23

SiO

R03

952+4

SiOR0

4648+16

SiO

R05

901+2

SiO

R02

944+4

SiOR01968+5

SiOR04171+19 SiOR

04648+5

SiOR10535+3

SiOR0

1224+3

SiOR06723+2

SiOR01968+9

SiOR02883+1

SiOR0

0899+3

SiOR01629+1

SiOR04171+1

SiOR01629+11

SiOR04171+10

SiOR04171+13

SiOR02694+3

SiOR04171+20

SiO

R02694+35

SiOR04171+15

SiOR04609+7

SiOR05118+2

SiOR07

837+2

SiO

R02

694+27

SiOR01968+10

SiOR04648+17

SiOR0196

8+19

SiO

R02

694+17

SiOR04648+13

SiOR01968+6

SiOR

00899+1

2

SiOR0

5901+1

SiOR00330+20

SiO

R02

648+2

SiO

R02

659+2

SiOR01968+1

6

SiOR0

0899+11

SiO

R02974

SiOR04171+2

SiOR03952+2

SiOR06792+2

SiOR04510+4

SiOR04171+28

SiOR05285+5

SiOR05285+9

SiOR

00899+1

5

SiOR

04648+3

SiO

R02694+36

SiOR10535+1

SiO

R02

694+19

SiO

R02

694+23

SiOR02694+1

SiO

R04609+14

SiOR01122

SiOR

04648+9

SiO

R02694+34

SiOR01629+8

SiOR

04648+8

SiOR04510+8

SiOR06573

SiO

R02

944+1

SiOR00330+26

SiO

R00330+1S

iOR02

694+15

SiO

R03038

SiOR05285+7

SiOR0

0899+5

SiOR04609+10

SiO

R04609+3

SiOR04339

SiOR0

8068

SiOR

04648+6

SiOR04510+2

SiOR05285+8

SiOR01

573+4

SiOR0366

3

SiOR04171+8

SiOR01858+2

SiOR01968+2SiOR01968+1

SiOR02694+5

SiOR01968+3

SiOR06723+3

SiOR01968+15

SiOR05285+1

SiOR0

0899+4

SiOR04609+22

SiOR04171+9

SiOR02694+9

SiO

R02

648+1

SiOR06792+3

SiOR0

1573+2

SiO

R02

694+20

SiOR1

0542

SiO

R04609+15

SiOR02694+8

SiOR00330+16

SiOR0

0899+2

SiOR02694+10

SiOR04510+9

SiOR05285+3

SiOR04171+24

SiO

R04609+2

SiOR05285+11

SiOR02

694+14

SiOR01

573+1

SiOR05285+2

SiOR00613

SiOR0196

8+22

SiOR

00899+9

SiO

R06

843+2

SiO

R02694+37

SiOR0

0899+1

SiOR04609+9

SiOR05431+2

SiOR10535+2

SiOR00330+15

SiO

R02

694+18

SiOR0

1224+2

SiOR04510+11

SiOR00330+23

SiO

R02

694+29

SiOR05416

SiOR05285+10

SiOR02694+2

SiOR01629+9

SiOR05431+1

SiOR02883+2

SiOR04171+14

SiO

R08

341

SiO

R02

694+22

SiOR01

224+1

SiOR01968+12

SiOR05431+3

SiOR04510+6

SiOR02694+7

SiOR04609+5

SiO

R02

944+2

SiOR03

952+3

SiOR01968+8

SiOR04609+24

SiO

R02

694+30

SiOR01629+10

SiOR04510+14

SiOR00565

SiOR05118+3

SiO

R01321

SiO

R04609+19

SiOR00330+14

SiO

R02694+38

SiOR04609+8

SiOR04171+16

SiOR10455

SiO

R04609+16

SiO

R04609+21

SiO

R02

694+28

SiO

R02

659+1

SiOR04171+5

SiOR00330+29

SiOR01968+14

SiOR03983

SiOR00330+27

SiOR05285+4

SiOR04510+1

SiOR

00899+8

SiOR

04648+7

SiO

R04609+17

SiO

R00330+5

SiO

R02

694+21

SiOR02814+4

SiOR00330+7

SiOR01

629+3

SiOR01

968+26

SiO

R02694+31

SiOR0

4648+2

SiO

R02694+39

SiOR04171+25

SiOR06577

SiOR01

968+25

SiOR04171+21

SiOR06792+6

SiO

R04609+11

SiOR02694+11

SiOR06792+1

SiOR04171+4

SiOR01629+5

SiOR00330+21

SiOR0

4648+15

SiO

R00330+6

SiO

R02

694+16

SiOR

04648+11

SiOR

04648+4

SiO

R00330+3

SiO

R06

535

SiOR04171+7

SiOR10493

SiO

R02694+32

SiOR06792+4

SiOR04510+3

SiO

R06

890

SiOR0196

8+20

SiO

R04609+12

SiOR04171+3

SiOR01968

+18

SiOR01968+11

SiO

R04609+13

SiOR01629+12

SiOR00330+22

SiO

R02694+33

SiOR00330+13

SiOR01

573+3

SiOR04510+16

SiOR04171+12

SiOR04510+13

SiOR

05118+1

SiO

R02

944+3

SiOR04171+26

SiOR04171+17

SiOR

00899+14

SiOR04171+29

SiOR

00899+1

3

SiOR02694+13

SiOR00330+24

SiOR00330+19

SiOR04171+27

SiO

R02

694+24

SiOR04510+5

SiOR07090

SiOR03952+1

SiOR04510+10

SiOR00330+17

SiO

R02

694+26

SiO

R04609+1

SiOR0

0899+6

SiOR04648+12

SiOR02814+2

SiOR00330+11

SiOR04171+18

SiOR01968+1

7

SiOR00330+10

SiOR00330+9

SiOR0162

9+2

SiOR04171+11

SiOR04510+12

SiOR0196

8+21

SiOR00330+8

SiOR01858+1

SiOR04510+7

SiOR05431+4

SiOR02694+6

SiOR01968+13

SiO

R00330+4

SiO

R04609+18

SiOR0

0899+10

SiOR00330+12

SiOR00330+31

SiO

R06

843+1

SiOR07

837+1

SiO

R00330+2

SiOR01629+4

SiOR0

4648+1

SiOR01

968+24

SiOR04171+23

SiOR0162

9+7

SiOR0

4648+14

SiO

R06

843+3

SiOR06792+5

SiOR01968+7

SiOR01629

+6

SiOR02883+3

SiOR02694+12

SiOR

05118+4

SiOR04171+22

SiO

R01080

SiOR04609+6

SiOR02814+1

SiOR00330+30

SiOR05285+12

SiOR06723+1

SiOR02694+4


BB BB Bb

x x x

Single queen form Multiple queen form(>15% ) (< 5% )



Sequenced: •a Gp-9 B ♂ genome

!

!•a Gp-9 b ♂ genome

“Next Generation Genotyping.”RAD sequencing

“Next Generation Genotyping.”

Bb

unfertilised eggs

haploid ♂

Gp-9 B Gp-9 b Gp-9 B Gp-9 b Gp-9 b Gp-9 B

RAD sequencing

38 B♂ & 38 b♂

EcoR1 EcoR1 EcoR1

RAD sequencing of haploid ♂ for SNP discovery & genotypingGp-9 B

EcoR1 EcoR1 EcoR1

AACTG

AACTG

AACTG

AACTG

Gp-9 B

RAD sequencing of haploid ♂ for SNP discovery & genotypingGp-9 B

AACTGGp-9 B

Gp-9 B

GGCCT

Gp-9 B

Gp-9 B

AAGGTGp-9 B

Gp-9 B

CCAGTGp-9 b

Gp-9 b

TAAATGp-9 b

Gp-9 b

GGAATGp-9 b

Gp-9 b

38 Gp-9 B males

38 Gp-9 b males

RAD sequencing of haploid ♂ for SNP discovery & genotyping

Identify polymorphismindividual x locus

genotype table

RADseq: sequencing the same 0.01% of the genome in many individuals

A B C D E FL1 A C A A C CL2 G G T - T GL3 - A G A - GL4 C - - G G CL5 T T C T C -L6 G A A - - G

2419

loci

38 B♂ & 38 b♂

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20+

Amount of variance explained per principal component

Principal Component

% V

aria

nce

Exp

lain

ed

05

1015

2025

30

12.7%

6.1% 5.4% 4.8% 4.7% 3.9% 3.5% 3.2% 3.1% 2.9% 2.8% 2.6% 2.4% 2.3% 2.2% 2.0% 1.9% 1.7% 1.6%

30.2%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20+

Amount of variance explained per principal component

Principal Component

% V

aria

nce

Exp

lain

ed

05

1015

2025

30

PCA: Principal Component Analysis

pc: 2 % variance: 6.073

pc: 3

%

var

ianc

e: 5

.441

-0.2

-0.1

0.0

0.1

0.2

-0.2 -0.1 0.0 0.1 0.2pc: 2 % variance: 6.073

pc: 3

%

var

ianc

e: 5

.441

-0.2

-0.1

0.0

0.1

0.2

-0.2 -0.1 0.0 0.1 0.2

Principal Components: PC2 vs PC3

Gp-9 B ♂Gp-9 b ♂


pc: 2

%

var

ianc

e: 6

.073

-0.2

-0.1

0.0

0.1

0.2

-0.10 -0.05 0.00 0.05 0.10 0.15

Principal Components: PC1 vs PC2


pc: 2

%

var

ianc

e: 6

.073

-0.2

-0.1

0.0

0.1

0.2

-0.10 -0.05 0.00 0.05 0.10 0.15

Gp-9 B ♂Gp-9 b ♂

Amount of sequence linked to Gp-9

Confirmed in 8 additional families (sons of Bb queens) with RAD (3 families) and PCR (5 families). BBqueens: normal recombination throughout (RAD 3 families) ⟹ Gp-9 is not a “recombination cold-spot”

brc_m013_0001..brc_m013_0005brc_m013_0006..brc_m013_0014brc_m013_0015..brc_m013_0017brc_m013_0018brc_m013_0019..brc_m013_0020brc_m013_0021..brc_m013_0029brc_m013_0030..brc_m013_0031brc_m013_0032..brc_m013_0034brc_m013_0035..brc_m013_0036brc_m013_0037..brc_m013_0038brc_m013_0039..brc_m013_0043brc_m013_0044brc_m013_0045brc_m013_0046..brc_m013_0048brc_m013_0049brc_m013_0050..brc_m013_0051brc_m013_0052..brc_m013_0056brc_m013_0057..brc_m013_0061

brc_m013_0062..brc_m013_0075brc_m013_0076..brc_m013_0078brc_m013_0079..brc_m013_0081

brc_m013_0082..brc_m013_0088

brc_m013_0089..brc_m013_0092

brc_m013_0093..brc_m013_0096brc_m013_0097brc_m013_0098..brc_m013_0113brc_m013_0114..brc_m013_0119brc_m013_0120..brc_m013_0130brc_m013_0131brc_m013_0132..brc_m013_0134brc_m013_0135..brc_m013_0136brc_m013_0137..brc_m013_0139brc_m013_0140..brc_m013_0142brc_m013_0143brc_m013_0144..brc_m013_0146brc_m013_0147..brc_m013_0154brc_m013_0155

brc_m013_0156brc_m013_0157..brc_m013_0180brc_m013_0181brc_m013_0182brc_m013_0183..brc_m013_0188brc_m013_0189..brc_m013_0208brc_m013_0209brc_m013_0210..brc_m013_0211brc_m013_0212..brc_m013_0215brc_m013_0216brc_m013_0217brc_m013_0218..brc_m013_0224brc_m013_0225..brc_m013_0228brc_m013_0229..brc_m013_0237brc_m013_0238..brc_m013_0245

brc_m013_0246..brc_m013_0270

brc_m013_0271..brc_m013_0274brc_m013_0275brc_m013_0276..brc_m013_0278

brc_m013_0279

brc_m013_0280

brc_m013_0281..brc_m013_0294

brc_m013_0295..brc_m013_0303brc_m013_0304brc_m013_0305..brc_m013_0307brc_m013_0308brc_m013_0309..brc_m013_0314brc_m013_0315..brc_m013_0317brc_m013_0318..brc_m013_0320brc_m013_0321..brc_m013_0326brc_m013_0327brc_m013_0328brc_m013_0329..brc_m013_0330brc_m013_0331..brc_m013_0333brc_m013_0334..brc_m013_0339brc_m013_0340brc_m013_0341..brc_m013_0343brc_m013_0344brc_m013_0345..brc_m013_0349brc_m013_0350

brc_m013_0351..brc_m013_0354brc_m013_0355brc_m013_0356brc_m013_0357..brc_m013_0361brc_m013_0362..brc_m013_0376brc_m013_0377..brc_m013_0390brc_m013_0391..brc_m013_0393brc_m013_0394..brc_m013_0400brc_m013_0401..brc_m013_0439brc_m013_0440..brc_m013_0478brc_m013_0479..brc_m013_0480

0

20

40

60

80

100

120

140

160

180

LG1brc_m013_0481brc_m013_0482..brc_m013_0484brc_m013_0485..brc_m013_0488brc_m013_0489..brc_m013_0502brc_m013_0503brc_m013_0504..brc_m013_0519brc_m013_0520..brc_m013_0532brc_m013_0533..brc_m013_0534brc_m013_0535..brc_m013_0537brc_m013_0538..brc_m013_0540brc_m013_0541..brc_m013_0543brc_m013_0544..brc_m013_0545brc_m013_0546..brc_m013_0549

brc_m013_0550..brc_m013_0554

brc_m013_0555..brc_m013_0560

brc_m013_0561..brc_m013_0562brc_m013_0563..brc_m013_0565

brc_m013_0566brc_m013_0567brc_m013_0568..brc_m013_0570brc_m013_0571..brc_m013_0573

brc_m013_0574..brc_m013_0575brc_m013_0576..brc_m013_0578brc_m013_0579..brc_m013_0580brc_m013_0581brc_m013_0582..brc_m013_0584brc_m013_0585brc_m013_0586brc_m013_0587brc_m013_0588..brc_m013_0591brc_m013_0592brc_m013_0593brc_m013_0594..brc_m013_0612brc_m013_0613..brc_m013_0614brc_m013_0615..brc_m013_0632brc_m013_0633brc_m013_0634..brc_m013_0648brc_m013_0649..brc_m013_0655brc_m013_0656brc_m013_0657..brc_m013_0694brc_m013_0695..brc_m013_0703brc_m013_0704brc_m013_0705brc_m013_0706..brc_m013_0707brc_m013_0708brc_m013_0709..brc_m013_0711brc_m013_0712..brc_m013_0715brc_m013_0716..brc_m013_0721brc_m013_0722brc_m013_0723brc_m013_0724..brc_m013_0728brc_m013_0729brc_m013_0730brc_m013_0731brc_m013_0732..brc_m013_0735brc_m013_0736..brc_m013_0769brc_m013_0770..brc_m013_0771brc_m013_0772..brc_m013_0773brc_m013_0774..brc_m013_0775brc_m013_0776..brc_m013_0782brc_m013_0783brc_m013_0784..brc_m013_0795brc_m013_0796..brc_m013_0798brc_m013_0799..brc_m013_0801brc_m013_0802..brc_m013_0805brc_m013_0806..brc_m013_0809brc_m013_0810..brc_m013_0811brc_m013_0812..brc_m013_0824brc_m013_0825..brc_m013_0826brc_m013_0827..brc_m013_0829brc_m013_0830..brc_m013_0831brc_m013_0832..brc_m013_0842brc_m013_0843..brc_m013_0854brc_m013_0855..brc_m013_0861brc_m013_0862brc_m013_0863..brc_m013_0864brc_m013_0865..brc_m013_0867brc_m013_0868..brc_m013_0883brc_m013_0884..brc_m013_0893brc_m013_0894brc_m013_0895..brc_m013_0897brc_m013_0898..brc_m013_0906brc_m013_0907..brc_m013_0910brc_m013_0911..brc_m013_0925brc_m013_0926..brc_m013_0928brc_m013_0929..brc_m013_0931

0

20

40

60

80

100

120

140

LG2brc_m013_0932..brc_m013_0941brc_m013_0942..brc_m013_0943brc_m013_0944..brc_m013_0945brc_m013_0946..brc_m013_0949brc_m013_0950..brc_m013_0952

brc_m013_0953..brc_m013_0975

brc_m013_0976..brc_m013_1019brc_m013_1020brc_m013_1021brc_m013_1022..brc_m013_1061brc_m013_1062brc_m013_1063

brc_m013_1064..brc_m013_1065

brc_m013_1066..brc_m013_1068brc_m013_1069brc_m013_1070

brc_m013_1071..brc_m013_1074

brc_m013_1075

brc_m013_1076..brc_m013_1081

brc_m013_1082..brc_m013_1086brc_m013_1087..brc_m013_1088brc_m013_1089..brc_m013_1098brc_m013_1099..brc_m013_1106brc_m013_1107..brc_m013_1116brc_m013_1117brc_m013_1118..brc_m013_1121brc_m013_1122..brc_m013_1127brc_m013_1128brc_m013_1129..brc_m013_1136brc_m013_1137..brc_m013_1138brc_m013_1139..brc_m013_1141brc_m013_1142..brc_m013_1144brc_m013_1145..brc_m013_1156brc_m013_1157brc_m013_1158..brc_m013_1170brc_m013_1171..brc_m013_1181brc_m013_1182..brc_m013_1185brc_m013_1186brc_m013_1187..brc_m013_1205brc_m013_1206..brc_m013_1218brc_m013_1219..brc_m013_1220brc_m013_1221..brc_m013_1224brc_m013_1225..brc_m013_1228brc_m013_1229brc_m013_1230..brc_m013_1236brc_m013_1237brc_m013_1238..brc_m013_1247brc_m013_1248..brc_m013_1251

brc_m013_1252brc_m013_1253..brc_m013_1268brc_m013_1269..brc_m013_1270brc_m013_1271..brc_m013_1273brc_m013_1274brc_m013_1275..brc_m013_1280brc_m013_1281

brc_m013_1282..brc_m013_1286brc_m013_1287..brc_m013_1298brc_m013_1299..brc_m013_1307brc_m013_1308brc_m013_1309..brc_m013_1313brc_m013_1314..brc_m013_1317brc_m013_1318..brc_m013_1319brc_m013_1320..brc_m013_1326brc_m013_1327..brc_m013_1340brc_m013_1341..brc_m013_1362brc_m013_1363..brc_m013_1385

0

20

40

60

80

100

120

140

LG3brc_m013_1386..brc_m013_1388brc_m013_1389..brc_m013_1398brc_m013_1399..brc_m013_1406brc_m013_1407..brc_m013_1411brc_m013_1412..brc_m013_1413brc_m013_1414..brc_m013_1416

brc_m013_1417brc_m013_1418..brc_m013_1420brc_m013_1421..brc_m013_1424brc_m013_1425..brc_m013_1432brc_m013_1433..brc_m013_1442brc_m013_1443brc_m013_1444..brc_m013_1450brc_m013_1451brc_m013_1452brc_m013_1453..brc_m013_1455brc_m013_1456..brc_m013_1467brc_m013_1468..brc_m013_1469brc_m013_1470brc_m013_1471..brc_m013_1474brc_m013_1475brc_m013_1476brc_m013_1477brc_m013_1478..brc_m013_1482brc_m013_1483

brc_m013_1484brc_m013_1485..brc_m013_1487brc_m013_1488..brc_m013_1490


brc_m013_1497..brc_m013_1500brc_m013_1501brc_m013_1502..brc_m013_1513brc_m013_1514..brc_m013_1562brc_m013_1563..brc_m013_1565brc_m013_1566..brc_m013_1567

brc_m013_1568..brc_m013_1580brc_m013_1581..brc_m013_1587brc_m013_1588..brc_m013_1591brc_m013_1592..brc_m013_1593brc_m013_1594..brc_m013_1604brc_m013_1605..brc_m013_1607brc_m013_1608..brc_m013_1609brc_m013_1610..brc_m013_1611brc_m013_1612..brc_m013_1616brc_m013_1617..brc_m013_1618brc_m013_1619..brc_m013_1620brc_m013_1621..brc_m013_1629brc_m013_1630..brc_m013_1633brc_m013_1634..brc_m013_1638brc_m013_1639..brc_m013_1647brc_m013_1648..brc_m013_1649brc_m013_1650..brc_m013_1656brc_m013_1657..brc_m013_1665brc_m013_1666..brc_m013_1672brc_m013_1673..brc_m013_1674brc_m013_1675..brc_m013_1678brc_m013_1679..brc_m013_1682brc_m013_1683brc_m013_1684brc_m013_1685..brc_m013_1686brc_m013_1687..brc_m013_1700brc_m013_1701..brc_m013_1702brc_m013_1703brc_m013_1704..brc_m013_1707brc_m013_1708..brc_m013_1709brc_m013_1710..brc_m013_1714brc_m013_1715..brc_m013_1728brc_m013_1729..brc_m013_1742

0

20

40

60

80

100

120

140

LG4brc_m013_1743..brc_m013_1750


brc_m013_1769..brc_m013_1772

brc_m013_1773..brc_m013_1779brc_m013_1780brc_m013_1781brc_m013_1782..brc_m013_1783brc_m013_1784brc_m013_1785..brc_m013_1786

brc_m013_1787

brc_m013_1788brc_m013_1789..brc_m013_1790brc_m013_1791..brc_m013_1793brc_m013_1794..brc_m013_1797brc_m013_1798..brc_m013_1800brc_m013_1801..brc_m013_1804brc_m013_1805brc_m013_1806..brc_m013_1808brc_m013_1809brc_m013_1810..brc_m013_1813brc_m013_1814..brc_m013_1818brc_m013_1819..brc_m013_1820brc_m013_1821..brc_m013_1822brc_m013_1823..brc_m013_1824brc_m013_1825brc_m013_1826..brc_m013_1840brc_m013_1841..brc_m013_1842brc_m013_1843brc_m013_1844..brc_m013_1848brc_m013_1849brc_m013_1850brc_m013_1851..brc_m013_1853brc_m013_1854..brc_m013_1858brc_m013_1859..brc_m013_1866brc_m013_1867..brc_m013_1868brc_m013_1869brc_m013_1870..brc_m013_1874brc_m013_1875..brc_m013_1876brc_m013_1877..brc_m013_1878brc_m013_1879..brc_m013_1883brc_m013_1884brc_m013_1885brc_m013_1886..brc_m013_1888brc_m013_1889..brc_m013_1895brc_m013_1896..brc_m013_1899brc_m013_1900..brc_m013_1913brc_m013_1914brc_m013_1915..brc_m013_1922brc_m013_1923brc_m013_1924..brc_m013_1928brc_m013_1929..brc_m013_1942brc_m013_1943..brc_m013_1946brc_m013_1947..brc_m013_1948brc_m013_1949brc_m013_1950..brc_m013_1953brc_m013_1954..brc_m013_1955brc_m013_1956brc_m013_1957..brc_m013_1963brc_m013_1964..brc_m013_1966brc_m013_1967..brc_m013_1968brc_m013_1969..brc_m013_1970brc_m013_1971brc_m013_1972brc_m013_1973..brc_m013_1980brc_m013_1981..brc_m013_1983brc_m013_1984..brc_m013_1990brc_m013_1991..brc_m013_1993brc_m013_1994..brc_m013_1996brc_m013_1997..brc_m013_2009

0

20

40

60

80

100

120

LG5brc_m013_2010..brc_m013_2028

brc_m013_2029..brc_m013_2038brc_m013_2039

brc_m013_2040..brc_m013_2041brc_m013_2042..brc_m013_2047brc_m013_2048..brc_m013_2050brc_m013_2051..brc_m013_2053brc_m013_2054..brc_m013_2062brc_m013_2063brc_m013_2064..brc_m013_2065brc_m013_2066..brc_m013_2067brc_m013_2068brc_m013_2069..brc_m013_2071brc_m013_2072..brc_m013_2081brc_m013_2082

brc_m013_2083brc_m013_2084brc_m013_2085..brc_m013_2099brc_m013_2100brc_m013_2101..brc_m013_2102brc_m013_2103..brc_m013_2107brc_m013_2108brc_m013_2109..brc_m013_2112

brc_m013_2113..brc_m013_2114brc_m013_2115..brc_m013_2123brc_m013_2124..brc_m013_2131brc_m013_2132brc_m013_2133brc_m013_2134..brc_m013_2136

brc_m013_2137

brc_m013_2138..brc_m013_2139

brc_m013_2140..brc_m013_2142brc_m013_2143..brc_m013_2150brc_m013_2151..brc_m013_2152brc_m013_2153..brc_m013_2161brc_m013_2162..brc_m013_2163

brc_m013_2164..brc_m013_2165brc_m013_2166..brc_m013_2170brc_m013_2171..brc_m013_2172brc_m013_2173brc_m013_2174..brc_m013_2182brc_m013_2183..brc_m013_2186brc_m013_2187..brc_m013_2190brc_m013_2191..brc_m013_2193brc_m013_2194brc_m013_2195..brc_m013_2201brc_m013_2202..brc_m013_2203brc_m013_2204..brc_m013_2220brc_m013_2221..brc_m013_2232brc_m013_2233..brc_m013_2239brc_m013_2240..brc_m013_2261brc_m013_2262..brc_m013_2267brc_m013_2268..brc_m013_2269brc_m013_2270..brc_m013_2271brc_m013_2272..brc_m013_2282brc_m013_2283..brc_m013_2284brc_m013_2285..brc_m013_2299brc_m013_2300..brc_m013_2301brc_m013_2302..brc_m013_2305brc_m013_2306..brc_m013_2307brc_m013_2308..brc_m013_2330brc_m013_2331..brc_m013_2337brc_m013_2338..brc_m013_2352

0

20

40

60

80

100

120

LG6brc_m013_2353..brc_m013_2365brc_m013_2366..brc_m013_2369

brc_m013_2370..brc_m013_2372brc_m013_2373..brc_m013_2378brc_m013_2379..brc_m013_2386brc_m013_2387brc_m013_2388..brc_m013_2394brc_m013_2395..brc_m013_2397brc_m013_2398brc_m013_2399brc_m013_2400brc_m013_2401brc_m013_2402..brc_m013_2407brc_m013_2408..brc_m013_2411brc_m013_2412..brc_m013_2416brc_m013_2417brc_m013_2418brc_m013_2419..brc_m013_2436brc_m013_2437..brc_m013_2441brc_m013_2442brc_m013_2443..brc_m013_2444brc_m013_2445brc_m013_2446..brc_m013_2453brc_m013_2454brc_m013_2455..brc_m013_2460brc_m013_2461brc_m013_2462..brc_m013_2470brc_m013_2471..brc_m013_2474brc_m013_2475..brc_m013_2482brc_m013_2483brc_m013_2484..brc_m013_2487brc_m013_2488brc_m013_2489..brc_m013_2492brc_m013_2493..brc_m013_2496brc_m013_2497brc_m013_2498..brc_m013_2504brc_m013_2505brc_m013_2506..brc_m013_2510brc_m013_2511..brc_m013_2523brc_m013_2524..brc_m013_2531brc_m013_2532..brc_m013_2536brc_m013_2537..brc_m013_2555brc_m013_2556..brc_m013_2571brc_m013_2572..brc_m013_2573brc_m013_2574..brc_m013_2579brc_m013_2580..brc_m013_2581brc_m013_2582..brc_m013_2587brc_m013_2588brc_m013_2589..brc_m013_2594

brc_m013_2595

brc_m013_2596..brc_m013_2597brc_m013_2598..brc_m013_2604brc_m013_2605brc_m013_2606..brc_m013_2616brc_m013_2617..brc_m013_2619brc_m013_2620..brc_m013_2623


brc_m013_2629..brc_m013_2630

0

20

40

60

80

100

LG7brc_m013_2631..brc_m013_2632brc_m013_2633brc_m013_2634..brc_m013_2635brc_m013_2636..brc_m013_2642brc_m013_2643..brc_m013_2657brc_m013_2658..brc_m013_2659brc_m013_2660..brc_m013_2661brc_m013_2662

brc_m013_2663

brc_m013_2664brc_m013_2665..brc_m013_2666

brc_m013_2667..brc_m013_2668brc_m013_2669..brc_m013_2670brc_m013_2671..brc_m013_2680brc_m013_2681..brc_m013_2682brc_m013_2683brc_m013_2684..brc_m013_2685brc_m013_2686..brc_m013_2694


brc_m013_2714..brc_m013_2725brc_m013_2726..brc_m013_2727brc_m013_2728..brc_m013_2731brc_m013_2732brc_m013_2733..brc_m013_2753brc_m013_2754..brc_m013_2758brc_m013_2759..brc_m013_2763brc_m013_2764..brc_m013_2779brc_m013_2780brc_m013_2781brc_m013_2782..brc_m013_2784brc_m013_2785..brc_m013_2787brc_m013_2788..brc_m013_2791brc_m013_2792..brc_m013_2797brc_m013_2798..brc_m013_2799brc_m013_2800brc_m013_2801..brc_m013_2804brc_m013_2805..brc_m013_2809

brc_m013_2810..brc_m013_2811

brc_m013_2812..brc_m013_2813brc_m013_2814..brc_m013_2817brc_m013_2818..brc_m013_2827brc_m013_2828brc_m013_2829..brc_m013_2832brc_m013_2833brc_m013_2834..brc_m013_2840brc_m013_2841..brc_m013_2846brc_m013_2847brc_m013_2848..brc_m013_2852brc_m013_2853brc_m013_2854..brc_m013_2856brc_m013_2857..brc_m013_2862brc_m013_2863..brc_m013_2868brc_m013_2869..brc_m013_2874brc_m013_2875..brc_m013_2896

0

20

40

60

80

100

LG8

brc_m013_2897..brc_m013_2920brc_m013_2921..brc_m013_2928brc_m013_2929..brc_m013_2931brc_m013_2932

brc_m013_2933

brc_m013_2934..brc_m013_2935brc_m013_2936brc_m013_2937..brc_m013_2943brc_m013_2944brc_m013_2945..brc_m013_2946brc_m013_2947brc_m013_2948brc_m013_2949..brc_m013_2950brc_m013_2951..brc_m013_2957brc_m013_2958..brc_m013_2961brc_m013_2962..brc_m013_2970brc_m013_2971..brc_m013_2980brc_m013_2981..brc_m013_2992brc_m013_2993..brc_m013_2996brc_m013_2997..brc_m013_2998brc_m013_2999..brc_m013_3000brc_m013_3001brc_m013_3002..brc_m013_3003brc_m013_3004brc_m013_3005brc_m013_3006..brc_m013_3010brc_m013_3011..brc_m013_3014brc_m013_3015brc_m013_3016..brc_m013_3019brc_m013_3020brc_m013_3021..brc_m013_3030brc_m013_3031..brc_m013_3032brc_m013_3033..brc_m013_3034brc_m013_3035..brc_m013_3036brc_m013_3037..brc_m013_3045brc_m013_3046..brc_m013_3052brc_m013_3053brc_m013_3054..brc_m013_3061brc_m013_3062..brc_m013_3066brc_m013_3067..brc_m013_3068brc_m013_3069..brc_m013_3076brc_m013_3077..brc_m013_3084brc_m013_3085..brc_m013_3087brc_m013_3088..brc_m013_3089brc_m013_3090..brc_m013_3096brc_m013_3097..brc_m013_3100brc_m013_3101..brc_m013_3104brc_m013_3105brc_m013_3106..brc_m013_3112brc_m013_3113..brc_m013_3122brc_m013_3123..brc_m013_3124brc_m013_3125..brc_m013_3127brc_m013_3128..brc_m013_3145brc_m013_3146..brc_m013_3159brc_m013_3160..brc_m013_3172

0

20

40

60

80

100

LG9brc_m013_3173..brc_m013_3175brc_m013_3176..brc_m013_3180brc_m013_3181..brc_m013_3189brc_m013_3190..brc_m013_3198brc_m013_3199brc_m013_3200..brc_m013_3201brc_m013_3202..brc_m013_3203brc_m013_3204brc_m013_3205..brc_m013_3206brc_m013_3207..brc_m013_3211brc_m013_3212..brc_m013_3214brc_m013_3215..brc_m013_3227brc_m013_3228..brc_m013_3230brc_m013_3231..brc_m013_3235brc_m013_3236..brc_m013_3238brc_m013_3239..brc_m013_3242brc_m013_3243..brc_m013_3244brc_m013_3245brc_m013_3246..brc_m013_3247brc_m013_3248..brc_m013_3249brc_m013_3250..brc_m013_3252brc_m013_3253..brc_m013_3257brc_m013_3258brc_m013_3259brc_m013_3260..brc_m013_3261brc_m013_3262..brc_m013_3263brc_m013_3264brc_m013_3265..brc_m013_3269brc_m013_3270..brc_m013_3274brc_m013_3275..brc_m013_3276brc_m013_3277..brc_m013_3281brc_m013_3282..brc_m013_3284brc_m013_3285brc_m013_3286..brc_m013_3289brc_m013_3290..brc_m013_3296brc_m013_3297brc_m013_3298..brc_m013_3300brc_m013_3301..brc_m013_3302brc_m013_3303..brc_m013_3305brc_m013_3306..brc_m013_3308brc_m013_3309..brc_m013_3314brc_m013_3315..brc_m013_3317brc_m013_3318..brc_m013_3329brc_m013_3330..brc_m013_3331brc_m013_3332..brc_m013_3338brc_m013_3339..brc_m013_3340brc_m013_3341..brc_m013_3344brc_m013_3345..brc_m013_3349brc_m013_3350..brc_m013_3357brc_m013_3358..brc_m013_3359brc_m013_3360brc_m013_3361..brc_m013_3368brc_m013_3369..brc_m013_3372brc_m013_3373..brc_m013_3376brc_m013_3377brc_m013_3378..brc_m013_3386brc_m013_3387..brc_m013_3388brc_m013_3389..brc_m013_3395brc_m013_3396..brc_m013_3399

0

20

40

60

80

LG10brc_m013_3400..brc_m013_3411brc_m013_3412brc_m013_3413..brc_m013_3424brc_m013_3425brc_m013_3426brc_m013_3427..brc_m013_3429

brc_m013_3430

brc_m013_3431..brc_m013_3432brc_m013_3433..brc_m013_3435brc_m013_3436brc_m013_3437..brc_m013_3439

brc_m013_3440..brc_m013_3441brc_m013_3442

brc_m013_3443..brc_m013_3445brc_m013_3446..brc_m013_3447brc_m013_3448..brc_m013_3449brc_m013_3450..brc_m013_3454brc_m013_3455brc_m013_3456..brc_m013_3462brc_m013_3463..brc_m013_3464brc_m013_3465brc_m013_3466..brc_m013_3467brc_m013_3468..brc_m013_3472brc_m013_3473brc_m013_3474..brc_m013_3476brc_m013_3477..brc_m013_3487brc_m013_3488brc_m013_3489..brc_m013_3491brc_m013_3492..brc_m013_3500brc_m013_3501..brc_m013_3512brc_m013_3513..brc_m013_3514brc_m013_3515..brc_m013_3524brc_m013_3525..brc_m013_3527brc_m013_3528..brc_m013_3531brc_m013_3532..brc_m013_3547brc_m013_3548..brc_m013_3557brc_m013_3558..brc_m013_3566brc_m013_3567..brc_m013_3568brc_m013_3569..brc_m013_3570brc_m013_3571..brc_m013_3574brc_m013_3575..brc_m013_3582brc_m013_3583..brc_m013_3592brc_m013_3593..brc_m013_3605brc_m013_3606..brc_m013_3616brc_m013_3617..brc_m013_3618brc_m013_3619..brc_m013_3622brc_m013_3623..brc_m013_3624brc_m013_3625..brc_m013_3628brc_m013_3629..brc_m013_3635

0

20

40

60

80

LG11



brc_m013_3672


brc_m013_3683..brc_m013_3685

brc_m013_3686..brc_m013_3688

brc_m013_3689..brc_m013_3693

brc_m013_3694..brc_m013_3698brc_m013_3699

brc_m013_3700brc_m013_3701..brc_m013_3702brc_m013_3703..brc_m013_3704brc_m013_3705..brc_m013_3712brc_m013_3713brc_m013_3714..brc_m013_3716brc_m013_3717..brc_m013_3724brc_m013_3725..brc_m013_3730brc_m013_3731..brc_m013_3752brc_m013_3753..brc_m013_3758brc_m013_3759..brc_m013_3789brc_m013_3790..brc_m013_3801brc_m013_3802..brc_m013_3814brc_m013_3815..brc_m013_3818brc_m013_3819..brc_m013_3822brc_m013_3823..brc_m013_3826brc_m013_3827..brc_m013_3832brc_m013_3833..brc_m013_3837brc_m013_3838brc_m013_3839..brc_m013_3841brc_m013_3842..brc_m013_3847

brc_m013_3848..brc_m013_3853brc_m013_3854..brc_m013_3858brc_m013_3859..brc_m013_3868brc_m013_3869..brc_m013_3871brc_m013_3872..brc_m013_3901brc_m013_3902brc_m013_3903..brc_m013_3909brc_m013_3910brc_m013_3911..brc_m013_3926brc_m013_3927..brc_m013_3931brc_m013_3932..brc_m013_3948

0

20

40

60

80

LG12


brc_m013_3972..brc_m013_3975brc_m013_3976

brc_m013_3977..brc_m013_3985

brc_m013_3986brc_m013_3987..brc_m013_3994brc_m013_3995..brc_m013_3997brc_m013_3998..brc_m013_4004

brc_m013_4005..brc_m013_4006brc_m013_4007..brc_m013_4008brc_m013_4009..brc_m013_4010brc_m013_4011..brc_m013_4013brc_m013_4014brc_m013_4015brc_m013_4016..brc_m013_4019brc_m013_4020..brc_m013_4021brc_m013_4022..brc_m013_4025brc_m013_4026..brc_m013_4032brc_m013_4033..brc_m013_4036brc_m013_4037..brc_m013_4041brc_m013_4042..brc_m013_4043

brc_m013_4044..brc_m013_4046brc_m013_4047..brc_m013_4056brc_m013_4057brc_m013_4058..brc_m013_4063brc_m013_4064..brc_m013_4071brc_m013_4072..brc_m013_4075brc_m013_4076brc_m013_4077brc_m013_4078..brc_m013_4085brc_m013_4086..brc_m013_4089brc_m013_4090..brc_m013_4091brc_m013_4092..brc_m013_4093brc_m013_4094..brc_m013_4095brc_m013_4096..brc_m013_4114brc_m013_4115..brc_m013_4117brc_m013_4118..brc_m013_4131brc_m013_4132..brc_m013_4133brc_m013_4134..brc_m013_4146

0

20

40

60

80

LG13


brc_m013_4168brc_m013_4169

brc_m013_4170brc_m013_4171..brc_m013_4172brc_m013_4173..brc_m013_4175brc_m013_4176brc_m013_4177..brc_m013_4178brc_m013_4179..brc_m013_4183brc_m013_4184..brc_m013_4185brc_m013_4186..brc_m013_4187brc_m013_4188..brc_m013_4191brc_m013_4192brc_m013_4193..brc_m013_4194brc_m013_4195..brc_m013_4206brc_m013_4207..brc_m013_4210brc_m013_4211..brc_m013_4213brc_m013_4214brc_m013_4215..brc_m013_4217brc_m013_4218..brc_m013_4221brc_m013_4222..brc_m013_4223brc_m013_4224..brc_m013_4231brc_m013_4232..brc_m013_4234brc_m013_4235..brc_m013_4239brc_m013_4240..brc_m013_4246brc_m013_4247..brc_m013_4248brc_m013_4249..brc_m013_4258brc_m013_4259..brc_m013_4260brc_m013_4261..brc_m013_4269brc_m013_4270..brc_m013_4271brc_m013_4272..brc_m013_4278brc_m013_4279..brc_m013_4280brc_m013_4281..brc_m013_4284brc_m013_4285brc_m013_4286..brc_m013_4288brc_m013_4289..brc_m013_4294brc_m013_4295..brc_m013_4296brc_m013_4297..brc_m013_4301brc_m013_4302..brc_m013_4313brc_m013_4314..brc_m013_4320brc_m013_4321..brc_m013_4322brc_m013_4323..brc_m013_4345brc_m013_4346..brc_m013_4351

0

20

40

60

LG14brc_m013_4352..brc_m013_4366brc_m013_4367brc_m013_4368brc_m013_4369..brc_m013_4373brc_m013_4374..brc_m013_4381brc_m013_4382..brc_m013_4383brc_m013_4384..brc_m013_4385brc_m013_4386brc_m013_4387..brc_m013_4388brc_m013_4389brc_m013_4390..brc_m013_4404brc_m013_4405brc_m013_4406..brc_m013_4409brc_m013_4410..brc_m013_4411brc_m013_4412..brc_m013_4418brc_m013_4419..brc_m013_4434brc_m013_4435..brc_m013_4442brc_m013_4443..brc_m013_4448brc_m013_4449..brc_m013_4451brc_m013_4452..brc_m013_4461brc_m013_4462..brc_m013_4471brc_m013_4472..brc_m013_4475brc_m013_4476..brc_m013_4477brc_m013_4478brc_m013_4479brc_m013_4480..brc_m013_4485brc_m013_4486brc_m013_4487..brc_m013_4491brc_m013_4492brc_m013_4493brc_m013_4494..brc_m013_4495brc_m013_4496..brc_m013_4501brc_m013_4502..brc_m013_4510brc_m013_4511..brc_m013_4531brc_m013_4532brc_m013_4533..brc_m013_4534brc_m013_4535..brc_m013_4541brc_m013_4542..brc_m013_4543brc_m013_4544..brc_m013_4545brc_m013_4546..brc_m013_4548brc_m013_4549..brc_m013_4551brc_m013_4552..brc_m013_4555brc_m013_4556..brc_m013_4561

0

20

40

60

LG15

brc_m013_4562..brc_m013_4577brc_m013_4578..brc_m013_4594brc_m013_4595..brc_m013_4599brc_m013_4600..brc_m013_4625brc_m013_4626..brc_m013_4638brc_m013_4639..brc_m013_4642brc_m013_4643..brc_m013_4644brc_m013_4645..brc_m013_4650brc_m013_4651..brc_m013_4663brc_m013_4664..brc_m013_4668brc_m013_4669..brc_m013_4670brc_m013_4671..brc_m013_4674brc_m013_4675..brc_m013_4679brc_m013_4680..brc_m013_4681brc_m013_4682brc_m013_4683..brc_m013_4688brc_m013_4689..brc_m013_4692brc_m013_4693..brc_m013_4695brc_m013_4696..brc_m013_4701brc_m013_4702brc_m013_4703..brc_m013_4712brc_m013_4713..brc_m013_4717brc_m013_4718..brc_m013_4720brc_m013_4721brc_m013_4722..brc_m013_4726brc_m013_4727..brc_m013_4728brc_m013_4729..brc_m013_4742brc_m013_4743brc_m013_4744..brc_m013_4746brc_m013_4747brc_m013_4748..brc_m013_4749brc_m013_4750..brc_m013_4752

brc_m013_4753..brc_m013_4756brc_m013_4757..brc_m013_4759brc_m013_4760..brc_m013_4762brc_m013_4763brc_m013_4764..brc_m013_4766brc_m013_4767..brc_m013_4769brc_m013_4770brc_m013_4771..brc_m013_4774brc_m013_4775..brc_m013_4776brc_m013_4777..brc_m013_4778brc_m013_4779..brc_m013_4780brc_m013_4781..brc_m013_4793brc_m013_4794brc_m013_4795..brc_m013_4798brc_m013_4799..brc_m013_4802brc_m013_4803..brc_m013_4806brc_m013_4807brc_m013_4808..brc_m013_4814brc_m013_4815..brc_m013_4819brc_m013_4820brc_m013_4821brc_m013_4822..brc_m013_4823brc_m013_4824brc_m013_4825..brc_m013_4855brc_m013_4856..brc_m013_4858brc_m013_4859..brc_m013_4863brc_m013_4864..brc_m013_4865brc_m013_4866..brc_m013_4875brc_m013_4876..brc_m013_4881brc_m013_4882..brc_m013_4891brc_m013_4892..brc_m013_4895brc_m013_4896..brc_m013_4911brc_m013_4912..brc_m013_4938brc_m013_4939brc_m013_4940..brc_m013_4957brc_m013_4958..brc_m013_4959brc_m013_4960..brc_m013_4972brc_m013_4973..brc_m013_4981brc_m013_4982..brc_m013_4983

0

20

40

60

80

100

120

LGSB

Gp-9

Figure 1a b

Si_gnF.scaffold00779_nt2778431.7

Si_gnF.scaffold00779_nt1255229Si_gnF.scaffold02684_nt10884.2


Si_gnF.scaffold00779_nt3746833Si_gnF.scaffold00779_nt374687927.3Si_gnF.scaffold00779_nt382158728.5




Si_gnF.scaffold05266_nt63430678.4Si_gnF.scaffold05266_nt65952779.7





Si_gnF.scaffold03404_nt128925Si_gnF.scaffold03404_nt228606Si_gnF.scaffold03404_nt241461

104.4


Si_gnF.scaffold00413_nt1035856109.6Si_gnF.scaffold01573_nt108462110.8Si_gnF.scaffold01573_nt447618112.1Si_gnF.scaffold00899_nt377419Si_gnF.scaffold00899_nt686574114.4Si_gnF.scaffold00899_nt236146Si_gnF.scaffold00899_nt332715Si_gnF.scaffold00899_nt335756

115.8

Si_gnF.scaffold00469_nt794118.0Si_gnF.scaffold00690_nt229012Si_gnF.scaffold00690_nt415290119.3



LGS Bfrom M013




22.2



Si_gnF.scaffold01573_nt10846280.7Si_gnF.scaffold00255_nt314067Si_gnF.scaffold00255_nt407778Si_gnF.scaffold00413_nt1035856Si_gnF.scaffold00413_nt676115Si_gnF.scaffold00469_nt794Si_gnF.scaffold00690_nt229012Si_gnF.scaffold00690_nt415290Si_gnF.scaffold00899_nt236146Si_gnF.scaffold00899_nt332715Si_gnF.scaffold00899_nt335756Si_gnF.scaffold00899_nt377419Si_gnF.scaffold00899_nt686574Si_gnF.scaffold01573_nt447618Si_gnF.scaffold01957_nt412242Si_gnF.scaffold03404_nt128925Si_gnF.scaffold03404_nt228606Si_gnF.scaffold03404_nt241461Si_gnF.scaffold05266_nt634306Si_gnF.scaffold05266_nt659527Si_gnF.scaffold05266_nt733643Si_gnF.scaffold05266_nt753644Si_gnF.scaffold06914_nt297673Si_gnF.scaffold07090_nt1051771Si_gnF.scaffold07090_nt710010Si_gnF.scaffold09758_nt222732

81.8


LGS B/bfrom P034

Gp-9

Total285 non-recombiningmarkersbrc_m013_0001..brc_m013_0005

brc_m013_0006..brc_m013_0014brc_m013_0015..brc_m013_0017brc_m013_0018brc_m013_0019..brc_m013_0020brc_m013_0021..brc_m013_0029brc_m013_0030..brc_m013_0031brc_m013_0032..brc_m013_0034brc_m013_0035..brc_m013_0036brc_m013_0037..brc_m013_0038brc_m013_0039..brc_m013_0043brc_m013_0044brc_m013_0045brc_m013_0046..brc_m013_0048brc_m013_0049brc_m013_0050..brc_m013_0051brc_m013_0052..brc_m013_0056brc_m013_0057..brc_m013_0061

brc_m013_0062..brc_m013_0075brc_m013_0076..brc_m013_0078brc_m013_0079..brc_m013_0081

brc_m013_0082..brc_m013_0088

brc_m013_0089..brc_m013_0092

brc_m013_0093..brc_m013_0096brc_m013_0097brc_m013_0098..brc_m013_0113brc_m013_0114..brc_m013_0119brc_m013_0120..brc_m013_0130brc_m013_0131brc_m013_0132..brc_m013_0134brc_m013_0135..brc_m013_0136brc_m013_0137..brc_m013_0139brc_m013_0140..brc_m013_0142brc_m013_0143brc_m013_0144..brc_m013_0146brc_m013_0147..brc_m013_0154brc_m013_0155

brc_m013_0156brc_m013_0157..brc_m013_0180brc_m013_0181brc_m013_0182brc_m013_0183..brc_m013_0188brc_m013_0189..brc_m013_0208brc_m013_0209brc_m013_0210..brc_m013_0211brc_m013_0212..brc_m013_0215brc_m013_0216brc_m013_0217brc_m013_0218..brc_m013_0224brc_m013_0225..brc_m013_0228brc_m013_0229..brc_m013_0237brc_m013_0238..brc_m013_0245

brc_m013_0246..brc_m013_0270


brc_m013_0279

brc_m013_0280

brc_m013_0281..brc_m013_0294

brc_m013_0295..brc_m013_0303brc_m013_0304brc_m013_0305..brc_m013_0307brc_m013_0308brc_m013_0309..brc_m013_0314brc_m013_0315..brc_m013_0317brc_m013_0318..brc_m013_0320brc_m013_0321..brc_m013_0326brc_m013_0327brc_m013_0328brc_m013_0329..brc_m013_0330brc_m013_0331..brc_m013_0333brc_m013_0334..brc_m013_0339brc_m013_0340brc_m013_0341..brc_m013_0343brc_m013_0344brc_m013_0345..brc_m013_0349brc_m013_0350

brc_m013_0351..brc_m013_0354brc_m013_0355brc_m013_0356brc_m013_0357..brc_m013_0361brc_m013_0362..brc_m013_0376brc_m013_0377..brc_m013_0390brc_m013_0391..brc_m013_0393brc_m013_0394..brc_m013_0400brc_m013_0401..brc_m013_0439brc_m013_0440..brc_m013_0478brc_m013_0479..brc_m013_0480

0

20

40

60

80

100

120

140

160

180

LG1brc_m013_0481brc_m013_0482..brc_m013_0484brc_m013_0485..brc_m013_0488brc_m013_0489..brc_m013_0502brc_m013_0503brc_m013_0504..brc_m013_0519brc_m013_0520..brc_m013_0532brc_m013_0533..brc_m013_0534brc_m013_0535..brc_m013_0537brc_m013_0538..brc_m013_0540brc_m013_0541..brc_m013_0543brc_m013_0544..brc_m013_0545brc_m013_0546..brc_m013_0549

brc_m013_0550..brc_m013_0554

brc_m013_0555..brc_m013_0560


brc_m013_0566brc_m013_0567brc_m013_0568..brc_m013_0570brc_m013_0571..brc_m013_0573

brc_m013_0574..brc_m013_0575brc_m013_0576..brc_m013_0578brc_m013_0579..brc_m013_0580brc_m013_0581brc_m013_0582..brc_m013_0584brc_m013_0585brc_m013_0586brc_m013_0587brc_m013_0588..brc_m013_0591brc_m013_0592brc_m013_0593brc_m013_0594..brc_m013_0612brc_m013_0613..brc_m013_0614brc_m013_0615..brc_m013_0632brc_m013_0633brc_m013_0634..brc_m013_0648brc_m013_0649..brc_m013_0655brc_m013_0656brc_m013_0657..brc_m013_0694brc_m013_0695..brc_m013_0703brc_m013_0704brc_m013_0705brc_m013_0706..brc_m013_0707brc_m013_0708brc_m013_0709..brc_m013_0711brc_m013_0712..brc_m013_0715brc_m013_0716..brc_m013_0721brc_m013_0722brc_m013_0723brc_m013_0724..brc_m013_0728brc_m013_0729brc_m013_0730brc_m013_0731brc_m013_0732..brc_m013_0735brc_m013_0736..brc_m013_0769brc_m013_0770..brc_m013_0771brc_m013_0772..brc_m013_0773brc_m013_0774..brc_m013_0775brc_m013_0776..brc_m013_0782brc_m013_0783brc_m013_0784..brc_m013_0795brc_m013_0796..brc_m013_0798brc_m013_0799..brc_m013_0801brc_m013_0802..brc_m013_0805brc_m013_0806..brc_m013_0809brc_m013_0810..brc_m013_0811brc_m013_0812..brc_m013_0824brc_m013_0825..brc_m013_0826brc_m013_0827..brc_m013_0829brc_m013_0830..brc_m013_0831brc_m013_0832..brc_m013_0842brc_m013_0843..brc_m013_0854brc_m013_0855..brc_m013_0861brc_m013_0862brc_m013_0863..brc_m013_0864brc_m013_0865..brc_m013_0867brc_m013_0868..brc_m013_0883brc_m013_0884..brc_m013_0893brc_m013_0894brc_m013_0895..brc_m013_0897brc_m013_0898..brc_m013_0906brc_m013_0907..brc_m013_0910brc_m013_0911..brc_m013_0925brc_m013_0926..brc_m013_0928brc_m013_0929..brc_m013_0931

0

20

40

60

80

100

120

140

LG2brc_m013_0932..brc_m013_0941brc_m013_0942..brc_m013_0943brc_m013_0944..brc_m013_0945brc_m013_0946..brc_m013_0949brc_m013_0950..brc_m013_0952

brc_m013_0953..brc_m013_0975

brc_m013_0976..brc_m013_1019brc_m013_1020brc_m013_1021brc_m013_1022..brc_m013_1061brc_m013_1062brc_m013_1063

brc_m013_1064..brc_m013_1065


brc_m013_1071..brc_m013_1074

brc_m013_1075

brc_m013_1076..brc_m013_1081

brc_m013_1082..brc_m013_1086brc_m013_1087..brc_m013_1088brc_m013_1089..brc_m013_1098brc_m013_1099..brc_m013_1106brc_m013_1107..brc_m013_1116brc_m013_1117brc_m013_1118..brc_m013_1121brc_m013_1122..brc_m013_1127brc_m013_1128brc_m013_1129..brc_m013_1136brc_m013_1137..brc_m013_1138brc_m013_1139..brc_m013_1141brc_m013_1142..brc_m013_1144brc_m013_1145..brc_m013_1156brc_m013_1157brc_m013_1158..brc_m013_1170brc_m013_1171..brc_m013_1181brc_m013_1182..brc_m013_1185brc_m013_1186brc_m013_1187..brc_m013_1205brc_m013_1206..brc_m013_1218brc_m013_1219..brc_m013_1220brc_m013_1221..brc_m013_1224brc_m013_1225..brc_m013_1228brc_m013_1229brc_m013_1230..brc_m013_1236brc_m013_1237brc_m013_1238..brc_m013_1247brc_m013_1248..brc_m013_1251

brc_m013_1252brc_m013_1253..brc_m013_1268brc_m013_1269..brc_m013_1270brc_m013_1271..brc_m013_1273brc_m013_1274brc_m013_1275..brc_m013_1280brc_m013_1281

brc_m013_1282..brc_m013_1286brc_m013_1287..brc_m013_1298brc_m013_1299..brc_m013_1307brc_m013_1308brc_m013_1309..brc_m013_1313brc_m013_1314..brc_m013_1317brc_m013_1318..brc_m013_1319brc_m013_1320..brc_m013_1326brc_m013_1327..brc_m013_1340brc_m013_1341..brc_m013_1362brc_m013_1363..brc_m013_1385

0

20

40

60

80

100

120

140

LG3brc_m013_1386..brc_m013_1388brc_m013_1389..brc_m013_1398brc_m013_1399..brc_m013_1406brc_m013_1407..brc_m013_1411brc_m013_1412..brc_m013_1413brc_m013_1414..brc_m013_1416

brc_m013_1417brc_m013_1418..brc_m013_1420brc_m013_1421..brc_m013_1424brc_m013_1425..brc_m013_1432brc_m013_1433..brc_m013_1442brc_m013_1443brc_m013_1444..brc_m013_1450brc_m013_1451brc_m013_1452brc_m013_1453..brc_m013_1455brc_m013_1456..brc_m013_1467brc_m013_1468..brc_m013_1469brc_m013_1470brc_m013_1471..brc_m013_1474brc_m013_1475brc_m013_1476brc_m013_1477brc_m013_1478..brc_m013_1482brc_m013_1483



brc_m013_1497..brc_m013_1500brc_m013_1501brc_m013_1502..brc_m013_1513brc_m013_1514..brc_m013_1562brc_m013_1563..brc_m013_1565brc_m013_1566..brc_m013_1567

brc_m013_1568..brc_m013_1580brc_m013_1581..brc_m013_1587brc_m013_1588..brc_m013_1591brc_m013_1592..brc_m013_1593brc_m013_1594..brc_m013_1604brc_m013_1605..brc_m013_1607brc_m013_1608..brc_m013_1609brc_m013_1610..brc_m013_1611brc_m013_1612..brc_m013_1616brc_m013_1617..brc_m013_1618brc_m013_1619..brc_m013_1620brc_m013_1621..brc_m013_1629brc_m013_1630..brc_m013_1633brc_m013_1634..brc_m013_1638brc_m013_1639..brc_m013_1647brc_m013_1648..brc_m013_1649brc_m013_1650..brc_m013_1656brc_m013_1657..brc_m013_1665brc_m013_1666..brc_m013_1672brc_m013_1673..brc_m013_1674brc_m013_1675..brc_m013_1678brc_m013_1679..brc_m013_1682brc_m013_1683brc_m013_1684brc_m013_1685..brc_m013_1686brc_m013_1687..brc_m013_1700brc_m013_1701..brc_m013_1702brc_m013_1703brc_m013_1704..brc_m013_1707brc_m013_1708..brc_m013_1709brc_m013_1710..brc_m013_1714brc_m013_1715..brc_m013_1728brc_m013_1729..brc_m013_1742

0

20

40

60

80

100

120

140

LG4brc_m013_1743..brc_m013_1750


brc_m013_1769..brc_m013_1772

brc_m013_1773..brc_m013_1779brc_m013_1780brc_m013_1781brc_m013_1782..brc_m013_1783brc_m013_1784brc_m013_1785..brc_m013_1786

brc_m013_1787

brc_m013_1788brc_m013_1789..brc_m013_1790brc_m013_1791..brc_m013_1793brc_m013_1794..brc_m013_1797brc_m013_1798..brc_m013_1800brc_m013_1801..brc_m013_1804brc_m013_1805brc_m013_1806..brc_m013_1808brc_m013_1809brc_m013_1810..brc_m013_1813brc_m013_1814..brc_m013_1818brc_m013_1819..brc_m013_1820brc_m013_1821..brc_m013_1822brc_m013_1823..brc_m013_1824brc_m013_1825brc_m013_1826..brc_m013_1840brc_m013_1841..brc_m013_1842brc_m013_1843brc_m013_1844..brc_m013_1848brc_m013_1849brc_m013_1850brc_m013_1851..brc_m013_1853brc_m013_1854..brc_m013_1858brc_m013_1859..brc_m013_1866brc_m013_1867..brc_m013_1868brc_m013_1869brc_m013_1870..brc_m013_1874brc_m013_1875..brc_m013_1876brc_m013_1877..brc_m013_1878brc_m013_1879..brc_m013_1883brc_m013_1884brc_m013_1885brc_m013_1886..brc_m013_1888brc_m013_1889..brc_m013_1895brc_m013_1896..brc_m013_1899brc_m013_1900..brc_m013_1913brc_m013_1914brc_m013_1915..brc_m013_1922brc_m013_1923brc_m013_1924..brc_m013_1928brc_m013_1929..brc_m013_1942brc_m013_1943..brc_m013_1946brc_m013_1947..brc_m013_1948brc_m013_1949brc_m013_1950..brc_m013_1953brc_m013_1954..brc_m013_1955brc_m013_1956brc_m013_1957..brc_m013_1963brc_m013_1964..brc_m013_1966brc_m013_1967..brc_m013_1968brc_m013_1969..brc_m013_1970brc_m013_1971brc_m013_1972brc_m013_1973..brc_m013_1980brc_m013_1981..brc_m013_1983brc_m013_1984..brc_m013_1990brc_m013_1991..brc_m013_1993brc_m013_1994..brc_m013_1996brc_m013_1997..brc_m013_2009

0

20

40

60

80

100

120

LG5brc_m013_2010..brc_m013_2028

brc_m013_2029..brc_m013_2038brc_m013_2039

brc_m013_2040..brc_m013_2041brc_m013_2042..brc_m013_2047brc_m013_2048..brc_m013_2050brc_m013_2051..brc_m013_2053brc_m013_2054..brc_m013_2062brc_m013_2063brc_m013_2064..brc_m013_2065brc_m013_2066..brc_m013_2067brc_m013_2068brc_m013_2069..brc_m013_2071brc_m013_2072..brc_m013_2081brc_m013_2082

brc_m013_2083brc_m013_2084brc_m013_2085..brc_m013_2099brc_m013_2100brc_m013_2101..brc_m013_2102brc_m013_2103..brc_m013_2107brc_m013_2108brc_m013_2109..brc_m013_2112

brc_m013_2113..brc_m013_2114brc_m013_2115..brc_m013_2123brc_m013_2124..brc_m013_2131brc_m013_2132brc_m013_2133brc_m013_2134..brc_m013_2136

brc_m013_2137

brc_m013_2138..brc_m013_2139

brc_m013_2140..brc_m013_2142brc_m013_2143..brc_m013_2150brc_m013_2151..brc_m013_2152brc_m013_2153..brc_m013_2161brc_m013_2162..brc_m013_2163

brc_m013_2164..brc_m013_2165brc_m013_2166..brc_m013_2170brc_m013_2171..brc_m013_2172brc_m013_2173brc_m013_2174..brc_m013_2182brc_m013_2183..brc_m013_2186brc_m013_2187..brc_m013_2190brc_m013_2191..brc_m013_2193brc_m013_2194brc_m013_2195..brc_m013_2201brc_m013_2202..brc_m013_2203brc_m013_2204..brc_m013_2220brc_m013_2221..brc_m013_2232brc_m013_2233..brc_m013_2239brc_m013_2240..brc_m013_2261brc_m013_2262..brc_m013_2267brc_m013_2268..brc_m013_2269brc_m013_2270..brc_m013_2271brc_m013_2272..brc_m013_2282brc_m013_2283..brc_m013_2284brc_m013_2285..brc_m013_2299brc_m013_2300..brc_m013_2301brc_m013_2302..brc_m013_2305brc_m013_2306..brc_m013_2307brc_m013_2308..brc_m013_2330brc_m013_2331..brc_m013_2337brc_m013_2338..brc_m013_2352

0

20

40

60

80

100

120

LG6brc_m013_2353..brc_m013_2365brc_m013_2366..brc_m013_2369

brc_m013_2370..brc_m013_2372brc_m013_2373..brc_m013_2378brc_m013_2379..brc_m013_2386brc_m013_2387brc_m013_2388..brc_m013_2394brc_m013_2395..brc_m013_2397brc_m013_2398brc_m013_2399brc_m013_2400brc_m013_2401brc_m013_2402..brc_m013_2407brc_m013_2408..brc_m013_2411brc_m013_2412..brc_m013_2416brc_m013_2417brc_m013_2418brc_m013_2419..brc_m013_2436brc_m013_2437..brc_m013_2441brc_m013_2442brc_m013_2443..brc_m013_2444brc_m013_2445brc_m013_2446..brc_m013_2453brc_m013_2454brc_m013_2455..brc_m013_2460brc_m013_2461brc_m013_2462..brc_m013_2470brc_m013_2471..brc_m013_2474brc_m013_2475..brc_m013_2482brc_m013_2483brc_m013_2484..brc_m013_2487brc_m013_2488brc_m013_2489..brc_m013_2492brc_m013_2493..brc_m013_2496brc_m013_2497brc_m013_2498..brc_m013_2504brc_m013_2505brc_m013_2506..brc_m013_2510brc_m013_2511..brc_m013_2523brc_m013_2524..brc_m013_2531brc_m013_2532..brc_m013_2536brc_m013_2537..brc_m013_2555brc_m013_2556..brc_m013_2571brc_m013_2572..brc_m013_2573brc_m013_2574..brc_m013_2579brc_m013_2580..brc_m013_2581brc_m013_2582..brc_m013_2587brc_m013_2588brc_m013_2589..brc_m013_2594

brc_m013_2595

brc_m013_2596..brc_m013_2597brc_m013_2598..brc_m013_2604brc_m013_2605brc_m013_2606..brc_m013_2616brc_m013_2617..brc_m013_2619brc_m013_2620..brc_m013_2623


brc_m013_2629..brc_m013_2630

0

20

40

60

80

100

LG7brc_m013_2631..brc_m013_2632brc_m013_2633brc_m013_2634..brc_m013_2635brc_m013_2636..brc_m013_2642brc_m013_2643..brc_m013_2657brc_m013_2658..brc_m013_2659brc_m013_2660..brc_m013_2661brc_m013_2662

brc_m013_2663

brc_m013_2664brc_m013_2665..brc_m013_2666

brc_m013_2667..brc_m013_2668brc_m013_2669..brc_m013_2670brc_m013_2671..brc_m013_2680brc_m013_2681..brc_m013_2682brc_m013_2683brc_m013_2684..brc_m013_2685brc_m013_2686..brc_m013_2694


brc_m013_2714..brc_m013_2725brc_m013_2726..brc_m013_2727brc_m013_2728..brc_m013_2731brc_m013_2732brc_m013_2733..brc_m013_2753brc_m013_2754..brc_m013_2758brc_m013_2759..brc_m013_2763brc_m013_2764..brc_m013_2779brc_m013_2780brc_m013_2781brc_m013_2782..brc_m013_2784brc_m013_2785..brc_m013_2787brc_m013_2788..brc_m013_2791brc_m013_2792..brc_m013_2797brc_m013_2798..brc_m013_2799brc_m013_2800brc_m013_2801..brc_m013_2804brc_m013_2805..brc_m013_2809

brc_m013_2810..brc_m013_2811

brc_m013_2812..brc_m013_2813brc_m013_2814..brc_m013_2817brc_m013_2818..brc_m013_2827brc_m013_2828brc_m013_2829..brc_m013_2832brc_m013_2833brc_m013_2834..brc_m013_2840brc_m013_2841..brc_m013_2846brc_m013_2847brc_m013_2848..brc_m013_2852brc_m013_2853brc_m013_2854..brc_m013_2856brc_m013_2857..brc_m013_2862brc_m013_2863..brc_m013_2868brc_m013_2869..brc_m013_2874brc_m013_2875..brc_m013_2896

0

20

40

60

80

100

LG8


brc_m013_2933

brc_m013_2934..brc_m013_2935brc_m013_2936brc_m013_2937..brc_m013_2943brc_m013_2944brc_m013_2945..brc_m013_2946brc_m013_2947brc_m013_2948brc_m013_2949..brc_m013_2950brc_m013_2951..brc_m013_2957brc_m013_2958..brc_m013_2961brc_m013_2962..brc_m013_2970brc_m013_2971..brc_m013_2980brc_m013_2981..brc_m013_2992brc_m013_2993..brc_m013_2996brc_m013_2997..brc_m013_2998brc_m013_2999..brc_m013_3000brc_m013_3001brc_m013_3002..brc_m013_3003brc_m013_3004brc_m013_3005brc_m013_3006..brc_m013_3010brc_m013_3011..brc_m013_3014brc_m013_3015brc_m013_3016..brc_m013_3019brc_m013_3020brc_m013_3021..brc_m013_3030brc_m013_3031..brc_m013_3032brc_m013_3033..brc_m013_3034brc_m013_3035..brc_m013_3036brc_m013_3037..brc_m013_3045brc_m013_3046..brc_m013_3052brc_m013_3053brc_m013_3054..brc_m013_3061brc_m013_3062..brc_m013_3066brc_m013_3067..brc_m013_3068brc_m013_3069..brc_m013_3076brc_m013_3077..brc_m013_3084brc_m013_3085..brc_m013_3087brc_m013_3088..brc_m013_3089brc_m013_3090..brc_m013_3096brc_m013_3097..brc_m013_3100brc_m013_3101..brc_m013_3104brc_m013_3105brc_m013_3106..brc_m013_3112brc_m013_3113..brc_m013_3122brc_m013_3123..brc_m013_3124brc_m013_3125..brc_m013_3127brc_m013_3128..brc_m013_3145brc_m013_3146..brc_m013_3159brc_m013_3160..brc_m013_3172

0

20

40

60

80

100

LG9brc_m013_3173..brc_m013_3175brc_m013_3176..brc_m013_3180brc_m013_3181..brc_m013_3189brc_m013_3190..brc_m013_3198brc_m013_3199brc_m013_3200..brc_m013_3201brc_m013_3202..brc_m013_3203brc_m013_3204brc_m013_3205..brc_m013_3206brc_m013_3207..brc_m013_3211brc_m013_3212..brc_m013_3214brc_m013_3215..brc_m013_3227brc_m013_3228..brc_m013_3230brc_m013_3231..brc_m013_3235brc_m013_3236..brc_m013_3238brc_m013_3239..brc_m013_3242brc_m013_3243..brc_m013_3244brc_m013_3245brc_m013_3246..brc_m013_3247brc_m013_3248..brc_m013_3249brc_m013_3250..brc_m013_3252brc_m013_3253..brc_m013_3257brc_m013_3258brc_m013_3259brc_m013_3260..brc_m013_3261brc_m013_3262..brc_m013_3263brc_m013_3264brc_m013_3265..brc_m013_3269brc_m013_3270..brc_m013_3274brc_m013_3275..brc_m013_3276brc_m013_3277..brc_m013_3281brc_m013_3282..brc_m013_3284brc_m013_3285brc_m013_3286..brc_m013_3289brc_m013_3290..brc_m013_3296brc_m013_3297brc_m013_3298..brc_m013_3300brc_m013_3301..brc_m013_3302brc_m013_3303..brc_m013_3305brc_m013_3306..brc_m013_3308brc_m013_3309..brc_m013_3314brc_m013_3315..brc_m013_3317brc_m013_3318..brc_m013_3329brc_m013_3330..brc_m013_3331brc_m013_3332..brc_m013_3338brc_m013_3339..brc_m013_3340brc_m013_3341..brc_m013_3344brc_m013_3345..brc_m013_3349brc_m013_3350..brc_m013_3357brc_m013_3358..brc_m013_3359brc_m013_3360brc_m013_3361..brc_m013_3368brc_m013_3369..brc_m013_3372brc_m013_3373..brc_m013_3376brc_m013_3377brc_m013_3378..brc_m013_3386brc_m013_3387..brc_m013_3388brc_m013_3389..brc_m013_3395brc_m013_3396..brc_m013_3399

0

20

40

60

80

LG10brc_m013_3400..brc_m013_3411brc_m013_3412brc_m013_3413..brc_m013_3424brc_m013_3425brc_m013_3426brc_m013_3427..brc_m013_3429

brc_m013_3430

brc_m013_3431..brc_m013_3432brc_m013_3433..brc_m013_3435brc_m013_3436brc_m013_3437..brc_m013_3439

brc_m013_3440..brc_m013_3441brc_m013_3442

brc_m013_3443..brc_m013_3445brc_m013_3446..brc_m013_3447brc_m013_3448..brc_m013_3449brc_m013_3450..brc_m013_3454brc_m013_3455brc_m013_3456..brc_m013_3462brc_m013_3463..brc_m013_3464brc_m013_3465brc_m013_3466..brc_m013_3467brc_m013_3468..brc_m013_3472brc_m013_3473brc_m013_3474..brc_m013_3476brc_m013_3477..brc_m013_3487brc_m013_3488brc_m013_3489..brc_m013_3491brc_m013_3492..brc_m013_3500brc_m013_3501..brc_m013_3512brc_m013_3513..brc_m013_3514brc_m013_3515..brc_m013_3524brc_m013_3525..brc_m013_3527brc_m013_3528..brc_m013_3531brc_m013_3532..brc_m013_3547brc_m013_3548..brc_m013_3557brc_m013_3558..brc_m013_3566brc_m013_3567..brc_m013_3568brc_m013_3569..brc_m013_3570brc_m013_3571..brc_m013_3574brc_m013_3575..brc_m013_3582brc_m013_3583..brc_m013_3592brc_m013_3593..brc_m013_3605brc_m013_3606..brc_m013_3616brc_m013_3617..brc_m013_3618brc_m013_3619..brc_m013_3622brc_m013_3623..brc_m013_3624brc_m013_3625..brc_m013_3628brc_m013_3629..brc_m013_3635

0

20

40

60

80

LG11



brc_m013_3672


brc_m013_3683..brc_m013_3685

brc_m013_3686..brc_m013_3688

brc_m013_3689..brc_m013_3693

brc_m013_3694..brc_m013_3698brc_m013_3699

brc_m013_3700brc_m013_3701..brc_m013_3702brc_m013_3703..brc_m013_3704brc_m013_3705..brc_m013_3712brc_m013_3713brc_m013_3714..brc_m013_3716brc_m013_3717..brc_m013_3724brc_m013_3725..brc_m013_3730brc_m013_3731..brc_m013_3752brc_m013_3753..brc_m013_3758brc_m013_3759..brc_m013_3789brc_m013_3790..brc_m013_3801brc_m013_3802..brc_m013_3814brc_m013_3815..brc_m013_3818brc_m013_3819..brc_m013_3822brc_m013_3823..brc_m013_3826brc_m013_3827..brc_m013_3832brc_m013_3833..brc_m013_3837brc_m013_3838brc_m013_3839..brc_m013_3841brc_m013_3842..brc_m013_3847

brc_m013_3848..brc_m013_3853brc_m013_3854..brc_m013_3858brc_m013_3859..brc_m013_3868brc_m013_3869..brc_m013_3871brc_m013_3872..brc_m013_3901brc_m013_3902brc_m013_3903..brc_m013_3909brc_m013_3910brc_m013_3911..brc_m013_3926brc_m013_3927..brc_m013_3931brc_m013_3932..brc_m013_3948

0

20

40

60

80

LG12


brc_m013_3972..brc_m013_3975brc_m013_3976

brc_m013_3977..brc_m013_3985

brc_m013_3986brc_m013_3987..brc_m013_3994brc_m013_3995..brc_m013_3997brc_m013_3998..brc_m013_4004

brc_m013_4005..brc_m013_4006brc_m013_4007..brc_m013_4008brc_m013_4009..brc_m013_4010brc_m013_4011..brc_m013_4013brc_m013_4014brc_m013_4015brc_m013_4016..brc_m013_4019brc_m013_4020..brc_m013_4021brc_m013_4022..brc_m013_4025brc_m013_4026..brc_m013_4032brc_m013_4033..brc_m013_4036brc_m013_4037..brc_m013_4041brc_m013_4042..brc_m013_4043

brc_m013_4044..brc_m013_4046brc_m013_4047..brc_m013_4056brc_m013_4057brc_m013_4058..brc_m013_4063brc_m013_4064..brc_m013_4071brc_m013_4072..brc_m013_4075brc_m013_4076brc_m013_4077brc_m013_4078..brc_m013_4085brc_m013_4086..brc_m013_4089brc_m013_4090..brc_m013_4091brc_m013_4092..brc_m013_4093brc_m013_4094..brc_m013_4095brc_m013_4096..brc_m013_4114brc_m013_4115..brc_m013_4117brc_m013_4118..brc_m013_4131brc_m013_4132..brc_m013_4133brc_m013_4134..brc_m013_4146

0

20

40

60

80

LG13


brc_m013_4168brc_m013_4169

brc_m013_4170brc_m013_4171..brc_m013_4172brc_m013_4173..brc_m013_4175brc_m013_4176brc_m013_4177..brc_m013_4178brc_m013_4179..brc_m013_4183brc_m013_4184..brc_m013_4185brc_m013_4186..brc_m013_4187brc_m013_4188..brc_m013_4191brc_m013_4192brc_m013_4193..brc_m013_4194brc_m013_4195..brc_m013_4206brc_m013_4207..brc_m013_4210brc_m013_4211..brc_m013_4213brc_m013_4214brc_m013_4215..brc_m013_4217brc_m013_4218..brc_m013_4221brc_m013_4222..brc_m013_4223brc_m013_4224..brc_m013_4231brc_m013_4232..brc_m013_4234brc_m013_4235..brc_m013_4239brc_m013_4240..brc_m013_4246brc_m013_4247..brc_m013_4248brc_m013_4249..brc_m013_4258brc_m013_4259..brc_m013_4260brc_m013_4261..brc_m013_4269brc_m013_4270..brc_m013_4271brc_m013_4272..brc_m013_4278brc_m013_4279..brc_m013_4280brc_m013_4281..brc_m013_4284brc_m013_4285brc_m013_4286..brc_m013_4288brc_m013_4289..brc_m013_4294brc_m013_4295..brc_m013_4296brc_m013_4297..brc_m013_4301brc_m013_4302..brc_m013_4313brc_m013_4314..brc_m013_4320brc_m013_4321..brc_m013_4322brc_m013_4323..brc_m013_4345brc_m013_4346..brc_m013_4351

0

20

40

60

LG14brc_m013_4352..brc_m013_4366brc_m013_4367brc_m013_4368brc_m013_4369..brc_m013_4373brc_m013_4374..brc_m013_4381brc_m013_4382..brc_m013_4383brc_m013_4384..brc_m013_4385brc_m013_4386brc_m013_4387..brc_m013_4388brc_m013_4389brc_m013_4390..brc_m013_4404brc_m013_4405brc_m013_4406..brc_m013_4409brc_m013_4410..brc_m013_4411brc_m013_4412..brc_m013_4418brc_m013_4419..brc_m013_4434brc_m013_4435..brc_m013_4442brc_m013_4443..brc_m013_4448brc_m013_4449..brc_m013_4451brc_m013_4452..brc_m013_4461brc_m013_4462..brc_m013_4471brc_m013_4472..brc_m013_4475brc_m013_4476..brc_m013_4477brc_m013_4478brc_m013_4479brc_m013_4480..brc_m013_4485brc_m013_4486brc_m013_4487..brc_m013_4491brc_m013_4492brc_m013_4493brc_m013_4494..brc_m013_4495brc_m013_4496..brc_m013_4501brc_m013_4502..brc_m013_4510brc_m013_4511..brc_m013_4531brc_m013_4532brc_m013_4533..brc_m013_4534brc_m013_4535..brc_m013_4541brc_m013_4542..brc_m013_4543brc_m013_4544..brc_m013_4545brc_m013_4546..brc_m013_4548brc_m013_4549..brc_m013_4551brc_m013_4552..brc_m013_4555brc_m013_4556..brc_m013_4561

0

20

40

60

LG15

brc_m013_4562..brc_m013_4577brc_m013_4578..brc_m013_4594brc_m013_4595..brc_m013_4599brc_m013_4600..brc_m013_4625brc_m013_4626..brc_m013_4638brc_m013_4639..brc_m013_4642brc_m013_4643..brc_m013_4644brc_m013_4645..brc_m013_4650brc_m013_4651..brc_m013_4663brc_m013_4664..brc_m013_4668brc_m013_4669..brc_m013_4670brc_m013_4671..brc_m013_4674brc_m013_4675..brc_m013_4679brc_m013_4680..brc_m013_4681brc_m013_4682brc_m013_4683..brc_m013_4688brc_m013_4689..brc_m013_4692brc_m013_4693..brc_m013_4695brc_m013_4696..brc_m013_4701brc_m013_4702brc_m013_4703..brc_m013_4712brc_m013_4713..brc_m013_4717brc_m013_4718..brc_m013_4720brc_m013_4721brc_m013_4722..brc_m013_4726brc_m013_4727..brc_m013_4728brc_m013_4729..brc_m013_4742brc_m013_4743brc_m013_4744..brc_m013_4746brc_m013_4747brc_m013_4748..brc_m013_4749brc_m013_4750..brc_m013_4752

brc_m013_4753..brc_m013_4756brc_m013_4757..brc_m013_4759brc_m013_4760..brc_m013_4762brc_m013_4763brc_m013_4764..brc_m013_4766brc_m013_4767..brc_m013_4769brc_m013_4770brc_m013_4771..brc_m013_4774brc_m013_4775..brc_m013_4776brc_m013_4777..brc_m013_4778brc_m013_4779..brc_m013_4780brc_m013_4781..brc_m013_4793brc_m013_4794brc_m013_4795..brc_m013_4798brc_m013_4799..brc_m013_4802brc_m013_4803..brc_m013_4806brc_m013_4807brc_m013_4808..brc_m013_4814brc_m013_4815..brc_m013_4819brc_m013_4820brc_m013_4821brc_m013_4822..brc_m013_4823brc_m013_4824brc_m013_4825..brc_m013_4855brc_m013_4856..brc_m013_4858brc_m013_4859..brc_m013_4863brc_m013_4864..brc_m013_4865brc_m013_4866..brc_m013_4875brc_m013_4876..brc_m013_4881brc_m013_4882..brc_m013_4891brc_m013_4892..brc_m013_4895brc_m013_4896..brc_m013_4911brc_m013_4912..brc_m013_4938brc_m013_4939brc_m013_4940..brc_m013_4957brc_m013_4958..brc_m013_4959brc_m013_4960..brc_m013_4972brc_m013_4973..brc_m013_4981brc_m013_4982..brc_m013_4983

0

20

40

60

80

100

120

LGSB

Gp-9

Figure 1a b




Si_gnF.scaffold00779_nt3746833Si_gnF.scaffold00779_nt374687927.3Si_gnF.scaffold00779_nt382158728.5




Si_gnF.scaffold05266_nt63430678.4Si_gnF.scaffold05266_nt65952779.7






104.4


Si_gnF.scaffold00413_nt1035856109.6Si_gnF.scaffold01573_nt108462110.8Si_gnF.scaffold01573_nt447618112.1Si_gnF.scaffold00899_nt377419Si_gnF.scaffold00899_nt686574114.4Si_gnF.scaffold00899_nt236146Si_gnF.scaffold00899_nt332715Si_gnF.scaffold00899_nt335756

115.8




LGS Bfrom M013




22.2



Si_gnF.scaffold01573_nt10846280.7Si_gnF.scaffold00255_nt314067Si_gnF.scaffold00255_nt407778Si_gnF.scaffold00413_nt1035856Si_gnF.scaffold00413_nt676115Si_gnF.scaffold00469_nt794Si_gnF.scaffold00690_nt229012Si_gnF.scaffold00690_nt415290Si_gnF.scaffold00899_nt236146Si_gnF.scaffold00899_nt332715Si_gnF.scaffold00899_nt335756Si_gnF.scaffold00899_nt377419Si_gnF.scaffold00899_nt686574Si_gnF.scaffold01573_nt447618Si_gnF.scaffold01957_nt412242Si_gnF.scaffold03404_nt128925Si_gnF.scaffold03404_nt228606Si_gnF.scaffold03404_nt241461Si_gnF.scaffold05266_nt634306Si_gnF.scaffold05266_nt659527Si_gnF.scaffold05266_nt733643Si_gnF.scaffold05266_nt753644Si_gnF.scaffold06914_nt297673Si_gnF.scaffold07090_nt1051771Si_gnF.scaffold07090_nt710010Si_gnF.scaffold09758_nt222732

81.8


LGS B/bfrom P034

Gp-9

Total285 non-recombiningmarkers

No recombination between B and b over 4% of genome: ⅔ of a chromosme!

Sex chromosomes

X Y

Gp-9 B

Gp-9 b

SB Sb

“Social chromosomes”

?

Why non-recombining?

SB

Sb

difficult to detect in sequence alignments

Why non-recombining? Structural differences

Sb SB Gp-9B

genetic map

E3

E17

A22

Gp-9 B male Gp-9 b male Figure 2 a

b Diploid Gp-9 Bb

Sb SB Gp-9B

genetic map

E3

E17

A22


b Diploid Gp-9 Bb

Sb SB Gp-9B

genetic map

E3

E17

A22


b Diploid Gp-9 Bb

Sb SB Gp-9B

genetic map

E3

E17

A22


b Diploid Gp-9 Bb

Why non-recombining? Structural differencesusing Flourescence in situ Hybridization

John Wang @ Taipei

X

ʁ

X X Y

ʂ

Maybe several rearrangements

Predictions: •genes in S are responsible for phenotype?

SBSB SB Sb

Single queen colony Multiple queen colony

Most BB vs Bb gene expression differences map to S

Non-recombing region of S contains 800 genes

polygyne colonies (Table 2 and Table S2; all P,0.05). The 11genes encoding mitochondrial and prefoldin complex (molecularchaperone) proteins were all up-regulated in polygyne comparedto monogyne workers. Increased mitochondrial gene expressionmay reflect increased oxidative metabolism, while increasedprefoldin expression may indicate higher protein synthesis rates,possibly in relation to the relatively smaller size and highermetabolic rates of polygyne workers [42].

The pattern of expression of the six genes in the viral genecategory is consistent with the expectation that differences in socialorganization affect susceptibility to pathogens and parasites. In themonogyne form, there is intense selection against susceptibleinfected individuals during independent colony founding, a stagethat colonies of the polygyne form never display [26]. Accordingly,we found that workers in the polygyne form express moresequences corresponding to viral genes than their counterparts inthe monogyne form, presumably because of relaxed selection andgenerally greater susceptibility in the former (see also [43]). Basedon sequence similarity and correlated expression across ourexperiments, we identified six gene products that likely representthree different viruses, a ssRNA negative-strand (2) virus and twossRNA positive-strand (+) viruses, one of which is the SINV-2 virus[44]. All 20 polygyne study colonies showed evidence of infectionwith at least one virus (mean number of viral types per colony,2.560.67), whereas only three of the 20 monogyne coloniesshowed evidence of infection, in all cases by a single viral type.Finally, the pattern of expression of another socially-regulated

gene, this one encoding a defensin (a class of small proteinantibiotics active against viruses, bacteria, and fungi [45]), also isconsistent with greater selection for resistance in the monogyneform, as this gene was more highly expressed in monogyne thanpolygyne workers.

The numbers of genes differentially expressed in the genotype (39)and social form (91) comparisons are relatively low compared to thenumbers expected based on other published microarray experi-ments. There are several possible explanations for this. First, the useof whole worker bodies as the source of RNA may decrease theprobability of detecting genes whose level of expression varies amongcells or tissues. Second, our comparisons were performed on groupsof workers originating from different colonies, thus adding a colony-level effect to, and thus increasing the total variance in, geneexpression. Finally, workers of alternate genotype or social formapparently exhibit fewer phenotypic differences than queens[20,21,46,47], possibly reflecting the involvement of fewer differen-tially expressed genes in the former caste.

Remarkably, there was almost no overlap between genes whoselevel of expression was influenced by the focal workers’ Gp-9genotypes and genes whose expression was influenced by the socialenvironment, with only one of the 129 differentially expressedgenes appearing in both categories. This demonstrates an almostcomplete decoupling of the direct effects of the genomic regionmarked by Gp-9 and the indirect effects mediated by socialinteractions within colonies. Moreover, there is little indication ofan interaction between these direct and indirect effects; genes

Figure 1. Expression profiles between S. invicta adult workers with the BB and Bb genotypes of Gp-9. Expression profiles for 39differentially expressed genes are depicted (ANOVA, 10% false discovery rate [FDR]). Each row represents data for one gene, and each columnrepresents data for a pool of 7–10 nestmates with the same Gp-9 genotype sampled from each of twenty colonies of each social form. Colonies werecollected from Georgia (2004, checkered peach bar; 2006, solid peach bar) and Louisiana (2006, solid red bar), USA (data from each polygyne colonyare presented in the identical order for the alternate genotype groupings). Expression levels for each gene are depicted relative to the average levelacross all experimental samples (blue, low levels; yellow, high levels). Genes are arranged by hierarchical clustering. See Text S1 and Table S1 forconfirmation of selected gene expression results with quantitative RT-PCR (qRT-PCR).doi:10.1371/journal.pgen.1000127.g001

Gene Expression Patterns for a Social Trait

PLoS Genetics | www.plosgenetics.org 3 July 2008 | Volume 4 | Issue 7 | e1000127

Gene expression: Gp-9 Bb vs BB workers in multiple queen colonies

29 significant genes are in the SB/Sb region

(p<10-10)

20 of

Similar for BB vs Bb queens; & for B vs b males. Wang et al 2008

Predictions: •genes in S are responsible for phenotype?

•Sb is degenerating?

probably!⟹ directional (antagonistic?) selection?

X

ʁ

X X Y

ʂ

Maybe several rearrangements

SBSB SB Sb

Single queen colony Multiple queen colony

Is Sb degenerating?

99.8% of non-gap sequences are identical

SB

Sb

(Almost) no SB or Sb-specific sequenceActually quite similar to SB:

But clearly: relaxation of purifying selectionSb contains more small repeats

genes seem to be intact in Sb

Introns bigger in Sb than SB

Sb is degenerating: repeats cause bad assembly

[b] vs. [c]: p < 10-4

[a] vs. [c]: p < 10-7

Gp-9B male Gp-9b male

Region:

Genome assembly:

Normally recombiningregions from all 16

linkage groups

Normally recombiningregions from all 16

linkage groups

Sb region without recombination

in Gp-9 Bb queens

SB region without recombination

in Gp-9 Bb queens

Sca

ffold

leng

th (b

p)

0

1,000,000

2,000,000

3,000,000

4,000,000

5,000,000

6,000,000

[a] [a], [b] [a] [c]

SB Sb

Is Sb degenerating?

Sb contains more big repeats ⟹ bad assemblydN/dS bigger in S than rest of the genome

Probably ♂ haploidy = strong purifying selection

⟹ slower degeneration

99.8% of non-gap sequences are identical(Almost) no SB or Sb-specific sequence

Actually quite similar to SB:

But clearly: relaxation of purifying selectionSb contains more small repeats

genes seem to be intact in Sb

Introns bigger in Sb than SB

Age of the region based on dS

0

50

100

150

200

250

0.00 0.05 0.10 0.15 0.20 0.25leafcutterDndsSubset$dS

coun

tleafcutterdS

0

50

100

150

0.00 0.05 0.10 0.15 0.20 0.25subset(dndsdata, gp9linked == TRUE)$dS

coun

t

gp9linkedSolenopsisdS

## Min. 1st Qu. Median Mean 3rd Qu. Max.

## 0.00000 0.08160 0.10300 0.10559 0.12600 0.32190

Richard thinks the mega-dS values are likely not orthologs. WARNING: We still have some leafcutterD-ndsSubset dS > 1 here!!!!

1.3.4 Calculating age using mean based on dS

10000000 * median(subset(dndsdata, gp9linked == TRUE)$dS)/median(leafcutterDndsSubset$dS)

## [1] 330097.09


## [1] 264077.67

Richard Nichols says I can use Maximum Likelihood to compare such distributions. See section below.

1.3.5 Calculating age based on dN

What do my dN distributions look like?

multiplot(qplot(x = subset(leafcutterDndsSubset, dN < 0.1)$dN, binwidth = 0.002, main = "leafcutterdNs"), qplot(x = subset(dndsdata,

gp9linked == TRUE & dN < 0.1)$dN, binwidth = 0.0005, main = "gp9linkedSolenopsisdNs"), cols = 1)

3

0

50

100

150

200

250

0.00 0.05 0.10 0.15 0.20 0.25leafcutterDndsSubset$dS

coun

tleafcutterdS

0

50

100

150

0.00 0.05 0.10 0.15 0.20 0.25subset(dndsdata, gp9linked == TRUE)$dS

coun

t

gp9linkedSolenopsisdS

## Min. 1st Qu. Median Mean 3rd Qu. Max.

## 0.00000 0.08160 0.10300 0.10559 0.12600 0.32190

Richard thinks the mega-dS values are likely not orthologs. WARNING: We still have some leafcutterD-ndsSubset dS > 1 here!!!!

1.3.4 Calculating age using mean based on dS


## [1] 330097.09


## [1] 264077.67

Richard Nichols says I can use Maximum Likelihood to compare such distributions. See section below.

1.3.5 Calculating age based on dN

What do my dN distributions look like?

multiplot(qplot(x = subset(leafcutterDndsSubset, dN < 0.1)$dN, binwidth = 0.002, main = "leafcutterdNs"), qplot(x = subset(dndsdata,

gp9linked == TRUE & dN < 0.1)$dN, binwidth = 0.0005, main = "gp9linkedSolenopsisdNs"), cols = 1)

3

Leafcutter common ancestor: 8,000,000-10,000,000 years ago

Maximum Likelihood Estimation of SB/Sb age:280,000-425,000⟹ little time for degeneration

Summary

Solenopsis invicta queen number determined by Gp-9 genotypes: •only BB workers ➔ single BB queen •with Bb workers ➔ multiple Bb queens

Genome sequencing + RAD Genotyping•Gp-9 marks ~4% of genome •social like sex chromosomes: SB is like X; Sb is like Y

some relaxation of purifying selection but haploid males ➔ strong purifying selection

Structural differences between SB and Sb ➔ no recombination

Ants are cool.

SB and Sb stopped recombining ~400,000 years ago.

ARTICLEdoi:10.1038/nature13151

Origins and functional evolution of Ychromosomes across mammalsDiego Cortez1,2, Ray Marin1,2, Deborah Toledo-Flores3, Laure Froidevaux1, Angelica Liechti1, Paul D. Waters4, Frank Grutzner3

& Henrik Kaessmann1,2

Y chromosomes underlie sex determination in mammals, but their repeat-rich nature has hampered sequencing and asso-ciated evolutionary studies. Here we trace Y evolution across 15 representative mammals on the basis of high-throughputgenome and transcriptome sequencing. We uncover three independent sex chromosome originations in mammals andbirds (the outgroup). The original placental and marsupial (therian) Y, containing the sex-determining gene SRY, emergedin the therian ancestor approximately 180 million years ago, in parallel with the first of five monotreme Y chromosomes,carrying the probable sex-determining gene AMH. The avian W chromosome arose approximately 140 million years ago inthe bird ancestor. The small Y/Wgene repertoires, enriched in regulatory functions, were rapidly defined following strati-fication (recombination arrest) and erosion events and have remained considerably stable. Despite expression decreasesin therians, Y/Wgenes show notable conservation of proto-sex chromosome expression patterns, although various Ygenesevolved testis-specificities through differential regulatory decay. Thus, although some genes evolved novel functions throughspatial/temporal expression shifts, most Y genes probably endured, at least initially, because of dosage constraints.

In most mammals, Y chromosomes are required to override the programunderlying development of the default sex, females1. Extant mammalspossess an XY (male heterogametic) sex chromosome system, with rareexceptions that experienced secondary XY loss2, but sex chromosomesevolved from different autosomes in therians (placentals and marsu-pials) and egg-laying monotremes (Fig. 1). Therians share the same XYsystem, whereas monotremes have multiple X and Y chromosomes thatare partially homologous to bird sex chromosomes3–5, where females areheterogametic (ZW system). Sex chromosome differentiation occurredthrough recombination arrests along the Y, leading to reduced selectionand associated gene decay and repeat accumulation6,7. Consequences ofY deterioration for X chromosome evolution were recently investigated8,but the fact that this chromosome is refractory to assembly owing to itsrepeat-rich nature7 have hindered evolutionary studies of the Y itself.Nevertheless, dedicated efforts determined complete Y sequences in threeprimates9–11 (human, chimpanzee, macaque) and large portions of twocarnivore (dog and cat) Y chromosomes12. Together with smaller scalework, these studies provided initial clues to Y evolution, such as the stabili-zation of Y gene content through ampliconic sequence accumulation10,11,13.However, our understanding of mammalian Y chromosome evolutionremains limited owing to the restricted amount and phylogenetic rep-resentation of available Y chromosome data.

Mammalian Y gene repertoiresTo explore Y evolution, we developed a subtraction approach that directlytargets and assembles exons of the male-specific (non-pseudoautosomal)Y chromosome (MSY) on the basis of high-throughput sequencing oftranscriptomes and genomes from both sexes (Extended Data Fig. 1and Methods). In brief, male transcripts were assembled from male-specific RNA sequencing (RNA-seq) reads not mapping onto femalereference genomes. Y identity was confirmed using the whole-genomesequence data; that is, true Y transcripts are supported by genomic readsunique to males. Genes with no or low expression were detected by screening

male-specific genomic data with Y orthologues from other species. Thegenomic data also served to support the absence of ancestral Y genes (thatis, their evolutionary loss). We validated our approach using large-scalePCR/Sanger sequencing-based screening of male/female genomic DNAand published Y chromosomes, thus confirming that complete codingsequences of all Y genes (with the potential exception of those not expressedin the sampled tissues and lacking a known Y orthologue) could be deducedfor a given species.

We applied our procedure to sequencing data that we collected forten mammals (Supplementary Tables 1–4 and Methods). Together withavailable Y sequences9–12, we could thus investigate Y evolution in 15species covering all major lineages of the class Mammalia14 (placentalsor eutherians, marsupials, monotremes), all but one (Xenarthra) of theeutherian superorders14, the two marsupial superorders (American andAustralian marsupials), both extant monotreme families (platypus,echidna), and all major groups of ‘higher primates’ (that is, simians:the great apes, Old World monkeys (OWMs) and New World mon-keys (NWMs)). For comparison, we produced similar data for the bird(chicken) W chromosome.

We identified a total of 134 different Y protein-coding genes in theten new species, thus approximately doubling the number of previouslyknown Y genes (Fig. 1 and Extended Data Fig. 3; note that the letter Yis added at the end of gene symbols for all genes for which Y-linkage isdetected, following common practice), and 214 distinct pseudogenesand noncoding RNAs (Supplementary Tables 5–17 and SupplementaryData 1). Our read coverage analysis (Methods) predicts 0–6 multi-copyprotein-coding genes per species, some of which are shared across speciesand thus presumably belong to conserved ampliconic Y regions11 (Fig. 1and Supplementary Tables 5–17). Most of the 155 distinct noncodingRNAs seem to stem from ampliconic regions, with two or more ident-ical copies per locus (Supplementary Tables 5–17). We recovered all ofthe recently identified chicken W protein-coding genes15,16 and added11 distinct noncoding RNAs (Fig. 1 and Supplementary Table 18).

1Center for Integrative Genomics, University of Lausanne, 1015 Lausanne, Switzerland. 2Swiss Institute of Bioinformatics, 1015 Lausanne, Switzerland. 3The Robinson Research Institute, School ofMolecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia. 4School of Biotechnology and Biomolecular Sciences, UNSW Australia, Sydney, New South Wales 2052,Australia.

4 8 8 | N A T U R E | V O L 5 0 8 | 2 4 A P R I L 2 0 1 4

Macmillan Publishers Limited. All rights reserved©2014

LETTER OPENdoi:10.1038/nature12326

Genomic evidence for ameiotic evolution in thebdelloid rotifer Adineta vagaJean-François Flot1,2,3,4,5,6, Boris Hespeels1,2, Xiang Li1,2, Benjamin Noel3, Irina Arkhipova7, Etienne G. J. Danchin8,9,10,Andreas Hejnol11, Bernard Henrissat12, Romain Koszul13, Jean-Marc Aury3, Valerie Barbe3, Roxane-Marie Barthelemy14,Jens Bast15, Georgii A. Bazykin16,17, Olivier Chabrol14, Arnaud Couloux3, Martine Da Rocha8,9,10, Corinne Da Silva3,Eugene Gladyshev7, Philippe Gouret14, Oskar Hallatschek6,18, Bette Hecox-Lea7,19, Karine Labadie3, Benjamin Lejeune1,2,Oliver Piskurek20, Julie Poulain3, Fernando Rodriguez7, Joseph F. Ryan11, Olga A. Vakhrusheva16,17, Eric Wajnberg8,9,10,Benedicte Wirth14, Irina Yushenova7, Manolis Kellis21, Alexey S. Kondrashov16,22, David B. Mark Welch7, Pierre Pontarotti14,Jean Weissenbach3,4,5, Patrick Wincker3,4,5, Olivier Jaillon3,4,5,21* & Karine Van Doninck1,2*

Loss of sexual reproduction is considered an evolutionary dead endfor metazoans, but bdelloid rotifers challenge this view as theyappear to have persisted asexually for millions of years1. Neithermale sex organs nor meiosis have ever been observed in thesemicroscopic animals: oocytes are formed through mitotic divi-sions, with no reduction of chromosome number and no indica-tion of chromosome pairing2. However, current evidence does notexclude that they may engage in sex on rare, cryptic occasions. Herewe report the genome of a bdelloid rotifer, Adineta vaga (Davis,1873)3, and show that its structure is incompatible with conven-tional meiosis. At gene scale, the genome of A. vaga is tetraploidand comprises both anciently duplicated segments and less diver-gent allelic regions. However, in contrast to sexual species, theallelic regions are rearranged and sometimes even found on thesame chromosome. Such structure does not allow meiotic pairing;instead, we find abundant evidence of gene conversion, which maylimit the accumulation of deleterious mutations in the absence ofmeiosis. Gene families involved in resistance to oxidation, car-bohydrate metabolism and defence against transposons are signifi-cantly expanded, which may explain why transposable elements coveronly 3% of the assembled sequence. Furthermore, 8% of the genes arelikely to be of non-metazoan origin and were probably acquiredhorizontally. This apparent convergence between bdelloids and pro-karyotes sheds new light on the evolutionary significance of sex.

With more than 460 described species4, bdelloid rotifers (Fig. 1)represent the highest metazoan taxonomic rank in which males, her-maphrodites and meiosis are unknown. Such persistence and diver-sification of an ameiotic clade of animals are in contradiction with thesupposed long-term disadvantages of asexuality, making bdelloids an‘evolutionary scandal’5. Another unusual feature of bdelloid rotifers istheir extreme resistance to desiccation at any stage of their life cycle6,enabling these microscopic animals to dwell in ephemeral freshwaterhabitats such as mosses, lichens and forest litter; this ability is presum-ably the source of their extreme resistance to ionizing radiation7.

We assembled the genome of a clonal A. vaga lineage into separatehaplotypes with a N50 of 260 kilobases (kb) (that is, half of the assemblywas composed of fragments longer than 260 kb). Assembly size was218 megabases (Mb) but 26 Mb of the sequence had twice the averagesequencing coverage, suggesting that some nearly identical regionswere not resolved during assembly (Supplementary Fig. 3); hence,the total genome size is likely to be 244 Mb, which corresponds tothe estimate obtained independently using fluorometry (Supplemen-tary Note C2). Annotation of the complete assembly (including allhaplotypes) yielded 49,300 genes. Intragenomic sequence comparisonsrevealed numerous homologous blocks with conserved gene order(colinear regions). For each such block we computed the per-site syn-onymous divergence (Ks) and a colinearity metric defined as the frac-tion of colinear genes. Colinear blocks fell into two groups (Fig. 2a): agroup characterized by high colinearity and low average synonymousdivergence, and a group characterized by lower colinearity and highersynonymous divergence. The presence of two classes of colinear blocksis consistent with a tetraploid structure comprised of alleles (recenthomologues) and ohnologues (ancient homologues formed by genomeduplication). Allelic pairs of coding sequences are on average 96.2%

*These authors contributed equally to this work.

1University of Namur, Department of Biology, URBE, Laboratory of Evolutionary Genetics and Ecology, 5000 Namur, Belgium. 2Namur Research Institute for Life Sciences (NARILIS), 5000 Namur, Belgium.3CEA-Institut de Genomique, GENOSCOPE, Centre National de Sequençage, 2 rue Gaston Cremieux, CP5706, 91057 Evry Cedex, France. 4Universite d’Evry, UMR 8030, CP5706, 91057 Evry Cedex, France.5Centre National de la Recherche Scientifique (CNRS), UMR 8030, CP5706, 91057 Evry Cedex, France. 6Max Planck Institute for Dynamics and Self-Organization, Biological Physics and EvolutionaryDynamics, Bunsenstraße 10, 37073 Gottingen, Germany. 7Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, Massachusetts 02543,USA. 8INRA, UMR 1355 ISA, Institut Sophia Agrobiotech, 400 route des Chappes, 06903 Sophia-Antipolis, France. 9CNRS, UMR 7254 ISA, Institut Sophia Agrobiotech, 400 route des Chappes, 06903Sophia-Antipolis, France. 10Universite de Nice Sophia-Antipolis, UMR ISA, Institut Sophia Agrobiotech, 400 route des Chappes, 06903, Sophia-Antipolis, France. 11Sars International Centre for MarineMolecular Biology, University of Bergen, 5008 Bergen, Norway. 12Architecture et Fonction des Macromolecules Biologiques, Aix-Marseille University, CNRS UMR 7257, 13288 Marseille, France. 13GroupeSpatial regulation of genomes, CNRS UMR 3525, Department of Genomes and Genetics, Institut Pasteur, 75724 Paris, France. 14LATP UMR-CNRS 7353, Evolution Biologique et Modelisation, Aix-MarseilleUniversity, 13331 Marseille cedex 3, France. 15J.F. Blumenbach Institute of Zoology and Anthropology, University of Gottingen, 37073 Gottingen, Germany. 16Department of Bioengineering andBioinformatics, M.V. Lomonosov Moscow State University, Leninskye Gory 1-73, Moscow, 119991, Russia. 17Institute for Information Transmission Problems of the Russian Academy of Sciences(Kharkevich Institute), Bolshoi Karetny pereulok 19, Moscow, 127994, Russia. 18Department of Physics, University of California, Berkeley, California 94720, USA. 19Department of Biology, NortheasternUniversity, Boston, Massachusetts 02115, USA. 20Courant Research Centre Geobiology, Georg-August-Universitat Gottingen, Goldschmidtstraße 3, Gottingen 37077, Germany. 21MIT Computer Scienceand Artificial IntelligenceLaboratory, Broad Institute ofMIT and Harvard,Cambridge,Massachusetts02139, USA. 22Life Sciences Institute and Departmentof Ecologyand Evolutionary Biology,University ofMichigan, Ann Arbor, Michigan 48109-2216, USA.

DeuterostomiaChordataHemichordata

Protostomia

Ecdysozoa

Adineta vaga (Rotifera: Bdelloidea)

Lophotrochozoa ChaetognathaEchinodermata

Figure 1 | Position of bdelloid rotifers among metazoans. Bdelloid rotifers(‘leech-like wheel-bearers’) are a clade of microscopic animals (scale bar,100 mm) within the phylum Rotifera. Photographs of Hemichordata(Saccoglossus), Chordata (Homo) and Ecdysozoa (Drosophila) courtesy ofDavid Remsen (MBL), John van Wyhe (http://darwin-online.org.uk) andAndre Karwath, respectively.

2 2 A U G U S T 2 0 1 3 | V O L 5 0 0 | N A T U R E | 4 5 3


LETTER OPENdoi:10.1038/nature12326

Genomic evidence for ameiotic evolution in thebdelloid rotifer Adineta vagaJean-François Flot1,2,3,4,5,6, Boris Hespeels1,2, Xiang Li1,2, Benjamin Noel3, Irina Arkhipova7, Etienne G. J. Danchin8,9,10,Andreas Hejnol11, Bernard Henrissat12, Romain Koszul13, Jean-Marc Aury3, Valerie Barbe3, Roxane-Marie Barthelemy14,Jens Bast15, Georgii A. Bazykin16,17, Olivier Chabrol14, Arnaud Couloux3, Martine Da Rocha8,9,10, Corinne Da Silva3,Eugene Gladyshev7, Philippe Gouret14, Oskar Hallatschek6,18, Bette Hecox-Lea7,19, Karine Labadie3, Benjamin Lejeune1,2,Oliver Piskurek20, Julie Poulain3, Fernando Rodriguez7, Joseph F. Ryan11, Olga A. Vakhrusheva16,17, Eric Wajnberg8,9,10,Benedicte Wirth14, Irina Yushenova7, Manolis Kellis21, Alexey S. Kondrashov16,22, David B. Mark Welch7, Pierre Pontarotti14,Jean Weissenbach3,4,5, Patrick Wincker3,4,5, Olivier Jaillon3,4,5,21* & Karine Van Doninck1,2*

Loss of sexual reproduction is considered an evolutionary dead endfor metazoans, but bdelloid rotifers challenge this view as theyappear to have persisted asexually for millions of years1. Neithermale sex organs nor meiosis have ever been observed in thesemicroscopic animals: oocytes are formed through mitotic divi-sions, with no reduction of chromosome number and no indica-tion of chromosome pairing2. However, current evidence does notexclude that they may engage in sex on rare, cryptic occasions. Herewe report the genome of a bdelloid rotifer, Adineta vaga (Davis,1873)3, and show that its structure is incompatible with conven-tional meiosis. At gene scale, the genome of A. vaga is tetraploidand comprises both anciently duplicated segments and less diver-gent allelic regions. However, in contrast to sexual species, theallelic regions are rearranged and sometimes even found on thesame chromosome. Such structure does not allow meiotic pairing;instead, we find abundant evidence of gene conversion, which maylimit the accumulation of deleterious mutations in the absence ofmeiosis. Gene families involved in resistance to oxidation, car-bohydrate metabolism and defence against transposons are signifi-cantly expanded, which may explain why transposable elements coveronly 3% of the assembled sequence. Furthermore, 8% of the genes arelikely to be of non-metazoan origin and were probably acquiredhorizontally. This apparent convergence between bdelloids and pro-karyotes sheds new light on the evolutionary significance of sex.

With more than 460 described species4, bdelloid rotifers (Fig. 1)represent the highest metazoan taxonomic rank in which males, her-maphrodites and meiosis are unknown. Such persistence and diver-sification of an ameiotic clade of animals are in contradiction with thesupposed long-term disadvantages of asexuality, making bdelloids an‘evolutionary scandal’5. Another unusual feature of bdelloid rotifers istheir extreme resistance to desiccation at any stage of their life cycle6,enabling these microscopic animals to dwell in ephemeral freshwaterhabitats such as mosses, lichens and forest litter; this ability is presum-ably the source of their extreme resistance to ionizing radiation7.

We assembled the genome of a clonal A. vaga lineage into separatehaplotypes with a N50 of 260 kilobases (kb) (that is, half of the assemblywas composed of fragments longer than 260 kb). Assembly size was218 megabases (Mb) but 26 Mb of the sequence had twice the averagesequencing coverage, suggesting that some nearly identical regionswere not resolved during assembly (Supplementary Fig. 3); hence,the total genome size is likely to be 244 Mb, which corresponds tothe estimate obtained independently using fluorometry (Supplemen-tary Note C2). Annotation of the complete assembly (including allhaplotypes) yielded 49,300 genes. Intragenomic sequence comparisonsrevealed numerous homologous blocks with conserved gene order(colinear regions). For each such block we computed the per-site syn-onymous divergence (Ks) and a colinearity metric defined as the frac-tion of colinear genes. Colinear blocks fell into two groups (Fig. 2a): agroup characterized by high colinearity and low average synonymousdivergence, and a group characterized by lower colinearity and highersynonymous divergence. The presence of two classes of colinear blocksis consistent with a tetraploid structure comprised of alleles (recenthomologues) and ohnologues (ancient homologues formed by genomeduplication). Allelic pairs of coding sequences are on average 96.2%

*These authors contributed equally to this work.

1University of Namur, Department of Biology, URBE, Laboratory of Evolutionary Genetics and Ecology, 5000 Namur, Belgium. 2Namur Research Institute for Life Sciences (NARILIS), 5000 Namur, Belgium.3CEA-Institut de Genomique, GENOSCOPE, Centre National de Sequençage, 2 rue Gaston Cremieux, CP5706, 91057 Evry Cedex, France. 4Universite d’Evry, UMR 8030, CP5706, 91057 Evry Cedex, France.5Centre National de la Recherche Scientifique (CNRS), UMR 8030, CP5706, 91057 Evry Cedex, France. 6Max Planck Institute for Dynamics and Self-Organization, Biological Physics and EvolutionaryDynamics, Bunsenstraße 10, 37073 Gottingen, Germany. 7Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, Massachusetts 02543,USA. 8INRA, UMR 1355 ISA, Institut Sophia Agrobiotech, 400 route des Chappes, 06903 Sophia-Antipolis, France. 9CNRS, UMR 7254 ISA, Institut Sophia Agrobiotech, 400 route des Chappes, 06903Sophia-Antipolis, France. 10Universite de Nice Sophia-Antipolis, UMR ISA, Institut Sophia Agrobiotech, 400 route des Chappes, 06903, Sophia-Antipolis, France. 11Sars International Centre for MarineMolecular Biology, University of Bergen, 5008 Bergen, Norway. 12Architecture et Fonction des Macromolecules Biologiques, Aix-Marseille University, CNRS UMR 7257, 13288 Marseille, France. 13GroupeSpatial regulation of genomes, CNRS UMR 3525, Department of Genomes and Genetics, Institut Pasteur, 75724 Paris, France. 14LATP UMR-CNRS 7353, Evolution Biologique et Modelisation, Aix-MarseilleUniversity, 13331 Marseille cedex 3, France. 15J.F. Blumenbach Institute of Zoology and Anthropology, University of Gottingen, 37073 Gottingen, Germany. 16Department of Bioengineering andBioinformatics, M.V. Lomonosov Moscow State University, Leninskye Gory 1-73, Moscow, 119991, Russia. 17Institute for Information Transmission Problems of the Russian Academy of Sciences(Kharkevich Institute), Bolshoi Karetny pereulok 19, Moscow, 127994, Russia. 18Department of Physics, University of California, Berkeley, California 94720, USA. 19Department of Biology, NortheasternUniversity, Boston, Massachusetts 02115, USA. 20Courant Research Centre Geobiology, Georg-August-Universitat Gottingen, Goldschmidtstraße 3, Gottingen 37077, Germany. 21MIT Computer Scienceand Artificial IntelligenceLaboratory, Broad Institute ofMIT and Harvard,Cambridge,Massachusetts02139, USA. 22Life Sciences Institute and Departmentof Ecologyand Evolutionary Biology,University ofMichigan, Ann Arbor, Michigan 48109-2216, USA.

DeuterostomiaChordataHemichordata

Protostomia

Ecdysozoa

Adineta vaga (Rotifera: Bdelloidea)

Lophotrochozoa ChaetognathaEchinodermata

Figure 1 | Position of bdelloid rotifers among metazoans. Bdelloid rotifers(‘leech-like wheel-bearers’) are a clade of microscopic animals (scale bar,100 mm) within the phylum Rotifera. Photographs of Hemichordata(Saccoglossus), Chordata (Homo) and Ecdysozoa (Drosophila) courtesy ofDavid Remsen (MBL), John van Wyhe (http://darwin-online.org.uk) andAndre Karwath, respectively.

2 2 A U G U S T 2 0 1 3 | V O L 5 0 0 | N A T U R E | 4 5 3


NATURE GENETICS VOLUME 46 | NUMBER 10 | OCTOBER 2014 1081

Insect pollination is necessary for one-third of our food and is a vital part of the ecosystem. The honeybee A. mellifera is a key pollinator, with its services to agriculture valued at >$200 billion per year world-wide1. It is therefore a major cause of concern that honeybees have faced huge and largely unexplained colony losses in recent decades2. However, little is known about global patterns of genomic variation in this species, which hold the key to an understanding of its evolution-ary history, the biological basis of adaptation to different climates and mechanisms governing resistance to disease.

The native distribution of A. mellifera encompasses Africa, Europe and western Asia3–8, and molecular dating suggests that the population expanded into this range around 1 million years ago3,4. Conflicting hypotheses have been proposed for the origin of this expansion8, with analyses of limited numbers of genetic and morphometric markers supporting an origin in the Middle East3–5 and a study of nuclear SNPs arguing for an African origin7,9. Honeybees show substantial pheno-typic variation across their extensive geographic range. European bees exhibit morphological and behavioral adaptations to survive colder winters, whereas African colonies are more aggressive and show a greater tendency to swarm. African bees are also reported to have greater resistance to the pathogenic mite Varroa destructor10–12, a

major honeybee pathogen13,14. The genetic basis of this phenotypic variation is largely unknown.

Humans began harvesting wax and honey from honeybee colonies at least 7,000 years before the present15. Human activity has led to the transportation of honeybee colonies all over the world, artificial selec-tion for desirable traits and gene flow between native subspecies16, including the expansion of hybrid strains of Africanized bees, known for their highly aggressive stinging behavior, across the Americas17 after their introduction to Brazil. The effects of these processes on the levels of genetic variation in honeybees have not been comprehen-sively evaluated. Here we investigate the evolution and genetic basis of adaptation in honeybees by performing whole-genome sequencing of 140 A. mellifera worker bees from 14 separate populations from a worldwide sample.

RESULTSGlobal patterns of variationWe sampled A. mellifera from a total of 14 populations, which included 9 native subspecies chosen from across the native range of the species in addition to managed strains of mixed ancestry from apiaries in Europe and North America and Africanized bees from South America

A worldwide survey of genome sequence variation provides insight into the evolutionary history of the honeybee Apis melliferaAndreas Wallberg1, Fan Han1,10, Gustaf Wellhagen1,10, Bjørn Dahle2, Masakado Kawata3, Nizar Haddad4, Zilá Luz Paulino Simões5, Mike H Allsopp6, Irfan Kandemir7, Pilar De la Rúa8, Christian W Pirk9 & Matthew T Webster1

The honeybee Apis mellifera has major ecological and economic importance. We analyze patterns of genetic variation at 8.3 million SNPs, identified by sequencing 140 honeybee genomes from a worldwide sample of 14 populations at a combined total depth of 634×. These data provide insight into the evolutionary history and genetic basis of local adaptation in this species. We find evidence that population sizes have fluctuated greatly, mirroring historical fluctuations in climate, although contemporary populations have high genetic diversity, indicating the absence of domestication bottlenecks. Levels of genetic variation are strongly shaped by natural selection and are highly correlated with patterns of gene expression and DNA methylation. We identify genomic signatures of local adaptation, which are enriched in genes expressed in workers and in immune system– and sperm motility–related genes that might underlie geographic variation in reproduction, dispersal and disease resistance. This study provides a framework for future investigations into responses to pathogens and climate change in honeybees.

1Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. 2Norwegian Beekeepers Association, Kløfta, Norway. 3Department of Ecology and Evolutionary Biology, Graduate School of Life Sciences, Tohoku University, Sendai, Japan. 4Bee Research Department, National Center for Agricultural Research and Extension, Amman, Jordan. 5Department of Biology, University of São Paulo, São Paulo, Brazil. 6Plant Protection Research Institute, Agricultural Research Council, Stellenbosch, South Africa. 7Department of Biology, Ankara University, Ankara, Turkey. 8Department of Zoology and Physical Anthropology, University of Murcia, Murcia, Spain. 9Department of Zoology and Entomology, University of Pretoria, Pretoria, South Africa. 10These authors contributed equally to this work. Correspondence should be addressed to A.W. ([email protected]) or M.T.W. ([email protected]).

Received 17 January; accepted 1 August; published online 24 August 2014; doi:10.138/ng.3077

A RT I C L E S

Diseases (NIAID) grant R01AI091823 to R.J.; by the NIH grant8P51OD011103-51 (to the New England Primate Research Center),1R56AI097834-01, and 1R01NS080833-01 to M.D.; and by theSwiss Federal Office for Civil Protection BABS #353003325 toA.R. NE-CAT at the Advanced Photon Source (APS) is supportedby a grant from the National Institute of General Medical Sciences(P41 GM103403). Use of the APS, an Office of Science UserFacility operated for the U.S. Department of Energy (DOE) Officeof Science by Argonne National Laboratory, was supported by

the U.S. DOE under contract DE-AC02-06CH11357. The atomiccoordinates and structure factors of the mini-HA–EC1–EC2complex have been deposited in the Protein Data Bank underthe accession code 4QD2. BoNT availability is subject to therestrictions that apply to Centers for Disease Control andPrevention select agents and NIAID Category A pathogens.R.J. is named as an inventor on a U.S. patent applicationapplied for by University of California, Irvine related to oral drugdelivery systems.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/344/6190/1405/suppl/DC1Materials and MethodsFigs. S1 to S9Table S1References (29–42)

24 March 2014; accepted 27 May 201410.1126/science.1253823

ECOLOGICAL GENOMICS

The genomic landscape underlyingphenotypic integrity in the face ofgene flow in crowsJ. W. Poelstra,1* N. Vijay,1* C. M. Bossu,1* H. Lantz,2,3 B. Ryll,4 I. Müller,5,6 V. Baglione,7

P. Unneberg,8 M. Wikelski,5,6 M. G. Grabherr,3 J. B. W. Wolf1†

The importance, extent, and mode of interspecific gene flow for the evolution of specieshas long been debated. Characterization of genomic differentiation in a classic example ofhybridization between all-black carrion crows and gray-coated hooded crows identifiedgenome-wide introgression extending far beyond the morphological hybrid zone. Geneexpression divergence was concentrated in pigmentation genes expressed in grayversus black feather follicles. Only a small number of narrow genomic islands exhibitedresistance to gene flow. One prominent genomic region (<2 megabases) harbored 81of all 82 fixed differences (of 8.4 million single-nucleotide polymorphisms in total)linking genes involved in pigmentation and in visual perception—a genomic signal reflectingcolor-mediated prezygotic isolation. Thus, localized genomic selection can cause markedheterogeneity in introgression landscapes while maintaining phenotypic divergence.

Genomic studies increasingly appreciate theimportance of interspecific gene flow inthe context of species diversification (1, 2),including that of hominids (3). Yet our un-derstanding of the forces generating het-

erogeneity in differentiation across the genomeand their relationship to phenotypic evolutionis limited (3–5). The European hybrid zone be-tween all-black carrion crows [Corvus (corone)corone] and gray-coated hooded crows [C. (corone)cornix] (6) provides ideal conditions for studyingthe evolutionary consequences of introgressionin a phase of early species divergence. The geo-graphical distribution pattern of these speciessuggests a population history shaped by glaci-

ation cycles during the Pleistocene,when periodsof isolation in distinct southern refugia alter-nated with periods of range expansion and sec-ondary contact (7). The near-absence of neutralgenetic differentiation across the hybrid zone (8)strongly contrasts with abrupt spatial segrega-tion of plumage coloration (Fig. 1), which hasremained remarkably stable over the past cen-tury despite successful backcrossing of hybrids(6). Evidence for color-assortative mating (9)and the independent recurrence of pied plu-mage phenotypes in the otherwise all-black genusCorvus has prompted the hypothesis that thiscolor polymorphism is promoting speciation(fig. S1).We assembled a high-quality reference genome

of one hooded crow male sequenced to 152×coverage (table S1). The final assembly consistedof 1298 scaffolds with an N50 size of 16.4Mb (10)and a cumulative length of 1.04 Gb, including3.52% gaps; 95% of the assembly was containedwithin the 100 largest scaffolds (size range: 1 to50 Mb), which approached chromosome size(crow karyotype: 2n = 80; size range of zebrafinch chromosomes: 1 to 156 Mb) (fig. S2, A andB, and table S2). Evidence-based annotation of thegenome with mRNA sequencing data (table S3)identified a set of 20,794 protein-coding genes.We subsequently resequenced 60 genomes of

unrelated individuals from two populations eachof carrion and hooded crows (Fig. 1) to a mean

sequence coverage of 12.2× (range: 7.1× to 28.6×,table S4); 8.44 million single-nucleotide poly-morphisms (SNPs) segregated across all fourpopulations, 5.27 million of which were sharedbetween carrion and hooded crows. Several linesof evidence suggest substantial genome-wide geneflow across the hybrid zone. The major axes ofgenetic variation (accounting for 11.5% of the to-tal variance) coincided with the presumed direc-tion of spatial expansion out of Spain, yet Germancarrion crows clustered more closely with bothhooded crow populations [Fig. 1; for pairwisefixation index (FST) estimates see Fig. 2A, fig. S3,and table S5; see fig. S4 for mitochondrial DNAdivergence]. Extensive gene flow between hoodedcrows and the German carrion crow populationwas further supported by ABBA-BABA tests (fig.S5 and table S6), admixture analyses (fig. S6),and coalescence-based parameter estimates ofan isolation-with-migration model (fig. S7). Con-sistent with a scenario of admixture upon sec-ondary contact, all populations but Spain showedsignatures of expansion and had higher nucleo-tide diversity than that of the presumably refugialSpanish population (table S5).The low genome-wide differentiation across the

hybrid zone was also reflected in gene express-ion divergence as measured by mRNA sequenc-ing (RNA-seq) from 19 individuals and five tissuesunder controlled conditions. The overall propor-tion of differentially expressed genes betweencarrion and hooded crows was low, between 0.03%and 0.41% across tissues [Fig. 3B; see (8); falsediscovery rate <0.05]. The main difference in geneexpression patterns corresponded to phenotypicdivergence in plumage coloration. Differentiallyexpressed genes predominated in growing fea-ther follicles from the torso, where carrion crowsproduce black feathers and hooded crows grayfeathers (Figs. 1 and 3A; c2df=4 = 169.34, P <0.001), with an overrepresentation of genes inthe melanogenesis pigmentation pathway (Fig.3, B and C; n = 21, c2df=4 = 147.16, P < 0.001; seetable S7 for Gene Ontology terms). Nineteen of20 differentially expressed melanogenesis-relatedgenes were underexpressed in the gray hoodedcrow torso (table S8). The pathway-wide patternof reduced expression could not be attributed todifferences in melanocyte density (Fig. 4), butrather to upstream regulatory changes inducingbroad-scale down-regulation in follicles produc-ing gray feathers (Fig. 3C).We surveyed the landscape of genomic diver-

gence with a window-based approach based onpopulation genetic summary statistics derivedfor 22,072 nonoverlapping 50-kb windows (Fig.2A and fig. S8) and a hypothesis-free clustering

1Science for Life Laboratory and Department of EvolutionaryBiology, Evolutionary Biology Centre, Uppsala University,75236 Uppsala, Sweden. 2Bioinformatics Infrastructure forLife Sciences, Uppsala University, 75124 Uppsala, Sweden.3Science for Life Laboratory and Department of MedicalBiochemistry and Microbiology, Uppsala University, 75123Uppsala, Sweden. 4Department of Organismal Biology,Evolutionary Biology Centre, Uppsala University, 75236Uppsala, Sweden. 5Max Planck Institute for Ornithology,78315 Radolfzell, Germany. 6Department of Biology,University of Konstanz, 78464 Konstanz, Germany.7Departamento de Ciencias Agro-Forestales, Campus LaYutera, Universidad de Valladolid, 34004 Palencia, Spain.8Science for Life Laboratory and Department of Biochemistryand Biophysics, Stockholm University, 171 21 Solna, Sweden.*These authors contributed equally to this work. †Correspondingauthor. E-mail: [email protected]

1410 20 JUNE 2014 • VOL 344 ISSUE 6190 sciencemag.org SCIENCE

RESEARCH | REPORTS

occurs. Thus, although cavity-circulation modelsdriven by regional ocean circulation modelscoupled to global climate models might yielddiffering spatiotemporal variations in melt, theyshould produce patterns of retreat similar tothose we have simulated, but with tighter con-straints on the timing.

An important feature of our numerical sim-ulations is that they reveal a strong sensitivityto mechanical and/or rheological weakening ofthe margins, which can accelerate the rate ofcollapse by decades to centuries. Thus, futuremodels will require careful treatment of shearmargins to accurately project sea-level rise. Oursimulations also assume that there is no retreatof the ice-shelf front. Full or partial ice-shelf col-lapse should produce more rapid retreat thanwe have simulated. In addition, we have not mod-eled ocean-driven melt that extends immediatelyupstream of the grounding line, which could alsoaccelerate retreat (32).

Our simulations provide strong evidence thatthe process of marine ice-sheet destabilization isalready under way on Thwaites Glacier, largelyin response to high subshelf melt rates. Althoughlosses are likely to be relatively modest over thenext century (<0.25 mm/year of sle), rapid col-lapse (>1 mm/year of sle) will ensue once thegrounding line reaches the basin’s deeper re-gions, which could occur within centuries. Suchrapid collapse would probably spill over to ad-jacent catchments, undermining much of WestAntarctica (18). Similar behavior also may beunder way on neighboring Pine Island Glacier(12, 33). Unless CDW recedes sufficiently toreduce melt well below present levels, it is dif-

ficult to foresee a stabilization of the Thwaitessystem, even with plausible increases in surfaceaccumulation. Although our simple melt param-eterization suggests that a full-scale collapse ofthis sector may be inevitable, it leaves large un-certainty in the timing. Thus, ice-sheet modelsfully coupled to ocean/climatemodels are requiredto reduce the uncertainty in the chronology of acollapse. Nonetheless, the similarity between ourhighest melt rates and present observations sug-gests that collapse may be closer to a few cen-turies than to a millennium.

References and Notes1. H. D. Pritchard, R. J. Arthern, D. G. Vaughan, L. A. Edwards,

Nature 461, 971–975 (2009).2. A. Shepherd, Geophys. Res. Lett. 29, 1364 (2002).3. E. Rignot, Geophys. Res. Lett. 35, L12505 (2008).4. A. Shepherd et al., Science 338, 1183–1189 (2012).5. S. S. Jacobs, A. Jenkins, C. F. Giulivi, P. Dutrieux,

Nat. Geosci. 4, 519–523 (2011).6. M. Thoma, A. Jenkins, D. Holland, S. Jacobs,

Geophys. Res. Lett. 35, L18602 (2008).7. E. Rignot, S. S. Jacobs, J. Mouginot, B. Scheuchl, Science

341, 266–270 (2013).8. M. A. Depoorter et al., Nature 502, 89–92 (2013).9. H. D. Pritchard et al., Nature 484, 502–505 (2012).10. A. Shepherd, D. Wingham, E. Rignot, Geophys. Res. Lett.

31, L23402 (2004).11. A. Payne, A. Vieli, A. Shepherd, D. Wingham, E. Rignot,

Geophys. Res. Lett. 31, L23401 (2004).12. I. Joughin, B. E. Smith, D. M. Holland, Geophys. Res. Lett.

37, L20502 (2010).13. I. Joughin, R. B. Alley, D. M. Holland, Science 338,

1172–1176 (2012).14. O. Gagliardini, G. Durand, T. Zwinger, R. C. A. Hindmarsh,

E. Le Meur, Geophys. Res. Lett. 37, L14501 (2010).15. B. Medley et al., Cryosphere Discuss. 8, 953–998

(2014).16. J. Mouginot, E. Rignot, B. Scheuchl, Geophys. Res. Lett.

41, 1576–1584 (2014).

17. T. J. Hughes, J. Glaciol. 27, 518–525 (1981).18. J. Holt et al., Geophys. Res. Lett. 33, L09502 (2006).19. C. Schoof, J. Geophys. Res. Earth Surf. 112, F03S28

(2007).20. J. Weertman, J. Glaciol. 13, 3–11 (1974).21. J. H. Mercer, Nature 271, 321–325 (1978).22. D. R. MacAyeal, J. Geophys. Res. Solid Earth 94,

4071–4087 (1989).23. Model details are provided in the supplementary materials.24. I. Joughin et al., J. Glaciol. 55, 245–257 (2009).25. E. Rignot, J. Mouginot, B. Scheuchl, Geophys. Res. Lett.

38, L10504 (2011).26. E. Rignot, S. S. Jacobs, Science 296, 2020–2023 (2002).27. C. Genthon, G. Krinner, H. Castebrunet, Ann. Glaciol. 50,

55–60 (2009).28. E. Larour, E. Rignot, I. Joughin, D. Aubry, Geophys. Res.

Lett. 32, L05503 (2005).29. C. P. Borstad, E. Rignot, J. Mouginot, M. P. Schodlok,

Cryosphere Discuss. 7, 3567–3610 (2013).30. K. J. Tinto, R. E. Bell, Geophys. Res. Lett. 38, L20503

(2011).31. S. S. R. Jamieson et al., Nat. Geosci. 5, 799–802 (2012).32. B. R. Parizek et al., J. Geophys. Res. 118, 638–655

(2013).33. L. Favier et al., Nat. Clim. Change 4, 117–121 (2014).

Acknowledgments: Comments by M. Maki, D. Shapero, andfour anonymous reviewers improved the manuscript. NSFsupported I.J. and B.M.’s contribution (grants ANT-0424589and ANT-0631973), and B.E.S. was supported by NASA(grant NNX09AE47G). We acknowledge NASA’s OperationIceBridge, which provided much of the data used to constrainthe model. The data used to constrain the model arearchived at the University of Washington Library (http://dx.doi.org/10.6069/H5CC0XMK).

Supplementary Materialswww.sciencemag.org/content/344/6185/735/suppl/DC1Materials and MethodsSupplementary TextFigs. S1 and S2References (34–37)

27 November 2013; accepted 21 April 201410.1126/science.1249055

Stick Insect Genomes Reveal NaturalSelection’s Role in Parallel SpeciationVíctor Soria-Carrasco,1* Zachariah Gompert,2* Aaron A. Comeault,1 Timothy E. Farkas,1

Thomas L. Parchman,3 J. Spencer Johnston,4 C. Alex Buerkle,5 Jeffrey L. Feder,6 Jens Bast,7

Tanja Schwander,8 Scott P. Egan,9 Bernard J. Crespi,10 Patrik Nosil1†

Natural selection can drive the repeated evolution of reproductive isolation, but the genomic basisof parallel speciation remains poorly understood. We analyzed whole-genome divergence betweenreplicate pairs of stick insect populations that are adapted to different host plants and undergoingparallel speciation. We found thousands of modest-sized genomic regions of accentuateddivergence between populations, most of which are unique to individual population pairs. We alsodetected parallel genomic divergence across population pairs involving an excess of coding geneswith specific molecular functions. Regions of parallel genomic divergence in nature exhibitedexceptional allele frequency changes between hosts in a field transplant experiment. The resultsadvance understanding of biological diversification by providing convergent observational andexperimental evidence for selection’s role in driving repeatable genomic divergence.

Whether evolution is predictable and re-peatable is difficult to test yet centralto our understanding of biological di-

versification (1–6). Instances of repeated, parallelevolution in response to similar environmentalpressures provide evidence of evolution by natu-

ral selection and can involve repeated divergenceat specific genes (7–9). Indeed, parallel evolutionof individual phenotypic traits has been esti-mated to involve the same genomic regions 30 to50% of the time (8). Parallel evolution can alsoresult in replicate species formation (i.e., paral-

lel speciation) (10), but the genome-wide conse-quences of this process are unclear (7, 8, 11).Although some genomic regions will likely di-verge repeatedly during parallel speciation, manymight show idiosyncratic patterns because ofcontingencies such as the order in which muta-tions arise (7, 8, 10, 11).

Even if repeated divergence occurs for somegenomic regions (7, 12, 13), the underlyingcauses of this parallelism often remain specu-lative because it is difficult to disentangle the

1Department of Animal and Plant Sciences, University ofSheffield, Sheffield S10 2TN, UK. 2Department of Biology,Utah State University, Logan, UT 84322, USA. 3Deparmentof Biology, University of Nevada, Reno, NV 89557, USA.4Department of Entomology, Texas A&M University, CollegeStation, TX 77843, USA. 5Department of Botany, Universityof Wyoming, Laramie, WY 82071, USA. 6Department ofBiology, Notre Dame University, South Bend, IN 46556, USA.7J. F. Blumenbach Institute of Zoology and Anthropology,University of Göttingen, 37073 Göttingen, Germany. 8Depart-ment of Ecology and Evolution, University of Lausanne,Lausanne CH-1015, Switzerland. 9Department of Ecology andEvolutionary Biology, Rice University, Houston, TX 77005, USA.10Department of Biological Sciences, Simon Fraser University,Burnaby, BC V5A 1S6, Canada.

*These authors contributed equally to this work.†Corresponding author. E-mail: [email protected]

16 MAY 2014 VOL 344 SCIENCE www.sciencemag.org738

REPORTS

https://etherpad.mozilla.org/obVAlZUq5D

2014 10-21-sbc322

Science

Transcript of 2014 10-21-sbc322