1. Molecular evolution of rDNA in sturgeons: features and mechanisms Constantine V. Rozhkovan Galina N. Chelomina

2. 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.

3. 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 blackened or cross-hatched boxes). mutation intrachromosomal homogenization interchromosomal gene conversion (rate-limiting step) interchromosomal homogenization

4. TaxanShHdPik Acipenser fulvescens* 1831120.9350.011385.510 Acipenser schrenckii 2220110.7140.008784.251 Huso dauricus 3813160.8630.009794.649 Total 7844340.8710.011235.335 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. TaxanShHdPik A. schrenckii × H. dauricus2642210.9780.016668.031 A. schrenckii × A. baerii1418100.8900.012586.088 Total4050290.9550.015307.373

5. “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

6. 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 B C

7. 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.

8. Groups comparednShHdPik Genes Species Hybrids 31 12 9 16 6969 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 comparedZnS (C  T) + (G  A) : other substitution La-LbZ Species Genes Pseudogenes 0.182 0.082 1 : 2.5 1 : 1 -0.0108362.540 Hybrids Genes Pseudogenes 0.226 0.276 1 : 2 1 : 1.5 -0.0105872.083 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 Z  1.96, than hypothesis of constant evolutionary rate is rejected.

9. Recombination events TaxaRgRas4 gtRmSd A. fulvescens*13.70.028310231483.22 A. schrenckii0.500.001012220483.77 H. dauricus5.800.012030413483.39 A. schrenckii × H. dauricus12.500.028520218483.57 A. schrenckii × A. baerii23.600.048822542483.69 TaxaRg (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.

10. 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 TaxaNgcPsiNisConversion tracts A. fulvescens*20.007237391-451 – Af-05; 235-259 – Af-11 A. schrenckii20.0082611259-274 – As-12; 68-274 – As-14 H. dauricus80.011749235-274 – 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. dauricus30.0167611236-275 – SH-05; 435-436 – SH-07; 68-236 – SH-16 A. schrenckii × A. baerii30.01609968-275 – SB-01; 435-436 – SB-07; 435-452 – SB-11

11. TaxaSites 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 disequilibrium Species 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 68 236 260 376 381 396 435 436 452 392 Acipenser fulvescens* 68 236 260 372 392 435 436 452 Acipenser schrenckii 68 236 260 275 372 392 435 436 452 Huso dauricus 35 68 236 260 295 333 392 435 436 452 68 236 260 275 372 392 435 436 452 SD SB F=48.8%; B=18.2%F=89.3%; B=32% F=100%; B=86.1%F=61.1%; B=22.7%

12. TaxaSites 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 disequilibrium Pseudogenes 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 366 380 395 435 68 235 259 371 391 434 435 451 68 235 259 274 371 391 434 435 451 68 35 236 260 295 333 392 435 436 452 68 236 260 275 372 392 435 436 452 Af* AsHd SDSB 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%

13. -0.2 -0.1 0.0 0.2 0.4 0 100 200300 400 -0.2 -0.1 0.0 0.2 0.4 0 100 200 300400 -0.2 -0.1 0.0 0.2 0.4 0 100 200300 400 -0.2 -0.1 0.0 0.2 0.4 0 100 200 300 400 Nucleotide distance Acipenser schrenckii Huso dauricus Acipenser schrenckii × Huso dauricus Acipenser schrenckii × Acipenser baerii D D D D Nucleotide distance Linkage disequilibrium