Molecular evolution of rDNA in sturgeons: features and

mechanisms

Constantine V. RozhkovanGalina N. Chelomina

Diagram of the rDNA array of eukaryotes

NTS – non-transcribed spacer; ETS – external transcribed spacer; ITS – internal transcribed spacer. The detail of the small subunit (18S) rRNA gene shows PCR primers (and their orientations) used for amplification and sequencing of the gene. Cross-hatched box indicates the cloned and sequenced 486 bp part of the entire gene.

Model for concerted evolution of tandemly repeated multi-gene families (Liao, 1999)

The coding sequences are depicted as unblackened arrows, and the

intergenic spacers are depicted as lines. The flanking

sequences of the two arrays are differently labeled (as either

blackenedor cross-hatched boxes).

mutation

intrachromosomal homogenization

interchromosomal gene conversion (rate-limiting step)

interchromosomal homogenization

Taxa n S h Hd Pi k

Acipenser fulvescens* 18 31 12 0.935 0.01138 5.510

Acipenser schrenckii 22 20 11 0.714 0.00878 4.251

Huso dauricus 38 13 16 0.863 0.00979 4.649

Total 78 44 34 0.871 0.01123 5.335

18S rDNA diversity in sturgeon species and hybrids

n – sample size; S – number of polymorphic (segregating) sites; h – number of haplotypes; Hd – haplotypic diversity; Pi – nucleotide diversity; k – average number of nucleotide differences.

Taxa n S h Hd Pi k

A. schrenckii × H. dauricus 26 42 21 0.978 0.01666 8.031

A. schrenckii × A. baerii 14 18 10 0.890 0.01258 6.088

Total 40 50 29 0.955 0.01530 7.373

Hd-30 Hd-13

Hd-9 SD-11

Hd-20 Hd-3 SD-24

SB-12 Hd-10 As-22 Hd-26

SD-6 Hd-37 As-19 SD-18 As-17

SB-6 As-20 SB-5 As-11 Hd-38

Hd-22 As-9 As-16 Hd-17

As-3 Hd-15 As-5 SD-25

As-7 As-2 As-8

SD-14 Hd-4 Af-D**

SB-10 SD-2

Af-13** As-13 As-10 Af-7** SD-9

Hd-23 Af-11*

Hd-6 SB-7 As-6

SD-12 Hd-32

SB-8 Hd-1 SD-23 SD-10 Hd-12

Hd-34 Hd-19 Hd-24 As-21 Hd-5

SD-19 Af-5*

Hd-2 As-12

SB-11 Hd-35

Hd-28 Hd-36

Hd-14 As-14 SB-1

SD-15 SD-3

Af-29* SD-26 SD-17

SD-8 SD-4

As-18 SD-16

SD-7 Af-18*

Af-9* Af-3* Af-2* Af-1*

Af-31* Af-19* Af-10* Af-26* Af-6*

Af-33* Af-17*

SD-20 SB-2

Hd-21 Hd-27 SD-22 SB-3 Hd-8 Hd-11 Hd-29 Hd-18 SD-13 SB-9 Hd-7 Hd-25 SD-1 SB-14 SB-13 Hd-16 As-1 Hd-31 Hd-33 SB-4

As-15 As-4

SD-21 SD-5

S.salar* S.trutta*

78

75

75

64

63

63

5358

62

59

56

55

0,005

“G

E

N

E

S”

“P

S

E

U

D

O

G

E

N

E

S”

Af – Acipenser fulvescens;

As – Acipenser schrenckii;

Hd – Huso dauricus;

SD – A. schrenckii × H. dauricus;

SB – A. schrenckii × A. baerii;

* - here and further – Genebank data

Af-7,Af-d, Af-13,

As (n=12), Hd (n=11), A. sturio

As (n=1) Hd (n=2) Hd-12

Hd-22

As-7 As-3

Hd (n=2)

As-6

A.ruth Hd-13

Hd-5

Hd-2

Hd-14 As-14

Hd-36

Hd-34

10

As-12

Af-5

14

A.br-1

Hd-6 Hd-23

Af-11

As-1 Hd (n=9)

Af (n=4) A.br (n=2)

Hd-21 Hd-27

Hd-28

Hd-35

As-15

As-4

Af-29

As-18

Af-17 Af-33 8

Af-1, 2

A.br-2 Af-3

Af-31

Af-18

Af-9

Af – A. fulvescens*, As – A. schrenckii, Hd – H. dauricus, A.br – A. brevirostrum*, A.ruth – A. ruthenus*. Number of bars and numerals on connecting lines corresponds to a number of mutation steps, n – number of clones.

Genes Pseudogenes

A

BC

SD (n=3) SB (n=5) SD-21 SB-11

SD-17 SD-26

SD-7

SD-3

SD-4 SD-8

SD-16

5

10

SD-20

SB-1

SD-5 SB-2

SD (n=3) SB-5

SD-15

SD-19

SD-12 SB-7

SD-23

SD-2

SB-12

SD-10 SB-6

SD-6

SD-14

SD-24

SB-10

SD-11

SB-8

Genes Pseudogenes

SD – A. schrenckii × H. dauricus, SB – A. schrenckii × A. baerii. Number of bars and numerals on connecting lines corresponds to a number of mutation steps, n – number of clones.

Groups compared n S h Hd Pi k

Genes

Species

Hybrids

31

12

9

16

6

9

0.301

0.909

0.00120

0.00578

0.581

2.803

Pseudogenes

Species

Hybrids

47

28

38

36

28

20

0.944

0.923

0.01117

0.01379

5.306

6.648

Groups compared ZnS (CT) + (GA) : other substitution

La-Lb Z

Species

Genes

Pseudogenes

0.182

0.082

1 : 2.5

1 : 1

-0.010836 2.540

Hybrids

Genes

Pseudogenes

0.226

0.276

1 : 2

1 : 1.5

-0.010587 2.083

n – sample size; S – number of polymorphic (segregating) sites; h – number of haplotypes; Hd – haplotypic diversity; Pi – nucleotide diversity; k – average number of nucleotide differences.

ZnS – Kelly’s neutrality test; L – relative evolution rate; La and Lb – the average distances (number of nucleotide substitutions per site) between genes and pseudogenes clusters in comparison with the common ancestor; if Z1.96, than hypothesis of constant evolutionary rate is rejected.

Recombination eventsTaxa Rg Ras 4 gt Rm S d

A. fulvescens* 13.7 0.0283 10 2 31 483.22

A. schrenckii 0.50 0.0010 12 2 20 483.77

H. dauricus 5.80 0.0120 30 4 13 483.39

A. schrenckii × H. dauricus 12.50 0.0285 20 2 18 483.57

A. schrenckii × A. baerii 23.60 0.0488 22 5 42 483.69

Taxa Rg (genes/ψgenes)

Ras (genes/ψgenes)

4 gt (genes/ψgenes)

Rm (genes/ψgenes)

A. fulvescens*

A. schrenckii

H. dauricus

Species

0.000/7.9

0.001/76.4

0.001/10.5

0.001/23.7

0/0.0164

0/0.1580

0/0.0217

0/0.0491

0/2

0/12

0/30

0/24

0/1

0/2

0/4

0/5

A. schrenckii × H. dauricus

A. schrenckii × A. baerii

Hybrids

81.4/31.0

>10 000/6.6

160/23.9

0.1682/0.0641

0/0.0137

0.3306/0.0494

0/22

0/16

0/26

0/5

0/2

0/5

R – recombination parameters: Rg – estimate of R per gene, Ras – estimate of R, between adjacent sites; 4 gt – number of pairs of sites with four gametic types; Rm – minimum number of recombination events; S – number of polymorphic (segregating) sites; d – average nucleotide distance between the most distant sites.

Gene conversion

Ngc – number of gene conversion tracts identified; Psi – average number of informative nucleotide sites per site; Nis – number of sites with information. First nucleotide of 486 bp fragment corresponds to 960 nucleotide of entire gene sequence

Taxa Ngc Psi Nis Conversion tracts

A. fulvescens* 2 0.00723 7 391-451 – Af-05; 235-259 – Af-11

A. schrenckii 2 0.00826 11 259-274 – As-12; 68-274 – As-14

H. dauricus 8 0.01174 9 235-274 – Hd-02; 68-371 – Hd-06; 371-391 – Hd-13; 235-274 – Hd-14; 235-274 – Hd-23; 434-435 – Hd-28; 434-451 – Hd-35; 235-371 – Hd-36

A. schrenckii × H. dauricus 3 0.01676 11 236-275 – SH-05; 435-436 – SH-07; 68-236 – SH-16

A. schrenckii × A. baerii 3 0.01609 9 68-275 – SB-01; 435-436 – SB-07; 435-452 – SB-11

Taxa Sites with linkage disequilibrium

Af*

As

Hd

SD

SB

68 236 260 367 381 392 396 435 436 452

68 236 260 372 392 435 436 452

68 236 260 275 372 392 435 436 452

35 68 236 260 295 333 392 435 436 452

68 236 260 275 372 392 435 436 452

Linkage disequilibriumSpecies and hybrids

Af – A. fulvescens, As – A. schrenckii, Hd – H. dauricus, SD – A. schrenckii × H. dauricus, SB – A. schrenckii × A. baerii;

Light grey rectangle – 0.01<P<0.05; dark grey rectangle – 0.001<P<0.01; black rectangle – P<0.001; F – sites, significant by Fisher’s exact test, B – sites, significant with Bonferroni correction procedure.

First nucleotide of 486 bp fragment corresponds to 960 nucleotide of entire gene sequence

68236

260

376381

396

435436

452

392

Acipenser fulvescens*

68

236260

372392

435436

452

Acipenser schrenckii

68

236260

275372

392

435436

452

Huso dauricus

3568

236260

295333

392435

436452

68236

260275

372392

435436

452

SD SB

F=48.8%; B=18.2% F=89.3%; B=32% F=89.3%; B=32% F=100%; B=86.1% F=61.1%; B=22.7%

Taxa Sites with linkage disequilibrium

Af*

As

Hd

SD

SB

68 366 380 395 435

68 235 259 371 391 434 435 451

68 235 259 274 371 391 434 465 451

35 68 236 260 295 333 372 392 435 452

68 236 260 275 392 435 436 452

Linkage disequilibriumPseudogenes

Af – A. fulvescens, As – A. schrenckii, Hd – H. dauricus, SD – A. schrenckii × H. dauricus, SB – A. schrenckii × A. baerii;

Light grey rectangle – 0.01<P<0.05; dark grey rectangle – 0.001<P<0.01; black rectangle – P<0.001; F – sites, significant by Fisher’s exact test, B – sites, significant with Bonferroni correction procedure.

First position of 486 bp fragment corresponds to 960 position of entire gene sequence

366

380395

435

68235

259

371391

434435

451

68235

259274

371391

434435

451

68

35

236

260295

333392

435436

452

68236

260

275372

392

435436

452

Af* As Hd SD SB

F=16.6%; B=0%

F=10.7%; B=0% F=63.8%; B=56.5% F=35.5%; B=31.2% F=13.9%; B=0%

-0.2

-0.1

0.0

0.2

0.4

0 100 200 300 400

-0.2

-0.1

0.0

0.2

0.4

0 100 200 300 400

-0.2

-0.1

0.0

0.2

0.4

0 100 200 300 400

-0.2

-0.1

0.0

0.2

0.4

0 100 200 300 400

Nucleotide distance Nucleotide distance

Nucleotide distance

Acipenser schrenckii Huso dauricus

Acipenser schrenckii × Huso dauricus Acipenser schrenckii × Acipenser baerii

D D

D D

Nucleotide distance

Linkage disequilibrium