Divergence and population traits in evolution of the genus Pisum L. as reconstructed using genes of two histone H1 subtypes showing different phylogenetic resolution

GENE-40112; No. of pages: 10; 4C: Gene xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Gene journal homepage: www.elsevier.com/locate/gene

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Olga O. Zaytseva a,b, Konstantin V. Gunbin a, Anatoliy V. Mglinets a, Oleg E. Kosterin a,b,⁎ a

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Article history: Received 31 July 2014 Received in revised form 7 November 2014 Accepted 29 November 2014 Available online xxxx

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Keywords: Phylogenetic trees Effective population size Evolution Vavilovia Peas

a b s t r a c t

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Institute of Cytology and Genetics SB RAS, Acad. Lavrentyev ave. 10, Novosibirsk 630090, Russia Novosibirsk State University, Pirogova str. 2, Novosibirsk 630090, Russia

Two histone H1 subtype genes, His7 and His5, were sequenced in a set of 56 pea accessions. Phylogenetic reconstruction based on concatenated His5 and His7 sequences had three main clades. First clade corresponded to Pisum fulvum, the next divergence separated a clade inside Pisum sativum in the broad sense that did not correspond strictly to any proposed taxonomical subdivisions. According to our estimations, the earliest divergence separating P. fulvum occurred 1.7 ± 0.4 MYA. The other divergence with high bootstrap support that separated two P. sativum groups took place approximately 1.3 ± 0.3 MYA. Thus, the main divergences in the genus took place either in late Pliocene or in early Pleistocene, the time of onset of the profound climate cooling in the northern hemisphere. The ω = K(a) / K(s) ratio was 2.5 times higher for His5 sequences than for His7. Thus, His7 gene, coding for a unique subtype specific for actively growing tissues, might have evolved under stricter evolutionary constraints than His5, that codes for a minor H1 subtype with less specific expression pattern. For this reason phylogenetic reconstructions separately obtained from His5 sequences resolved tree topology much better than those obtained from His7 sequences. Computational estimation of population dynamic parameters in the genus Pisum L. from His5-His7 sequences using IMa2 software revealed a decrease of effective population size on the early stage of Pisum evolution. © 2014 Published by Elsevier B.V.

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Several non-allelic H1 subtypes are present in the chromatin of many eukaryotic organisms (Ponte et al., 1998; Brown, 2001; Happel and Doenecke, 2009). These subtypes differ in their ability to condense DNA, stability (Brown, 2001; De et al., 2002) and distribution in chromatin (Th'ng et al., 2005; Izzo et al., 2013), turnover rate and posttranslational modifications (Wisniewski et al., 2007; Weiss et al., 2010). Some of histone H1 subtypes are tissue-specific or act as replacement subtypes in differentiated cells (Lin et al., 1999; Happel and Doenecke, 2009). Recent studies showed that H1 is a highly flexible component of chromatin (Misteli et al., 2000; Happel and Doenecke, 2009) that interacts with specific regulators of transcription (Lee et al., 2004). Therefore, it defines the transcriptional state of chromatin and creates

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1. Introduction

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Divergence and population traits in evolution of the genus Pisum L. as reconstructed using genes of two histone H1 subtypes showing different phylogenetic resolution

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Abbreviations:His5, pea histone H1 subtype 5 gene; His7, pea histone H1 subtype 7 gene; ICG, Institute of Cytology and Genetics; K(a), the number of nonsynonymous substitutions per nonsynonymous site; K(s), the number of synonymous substitutions per synonymous site; MP, maximum parsimony; Ne, effective population size; Ne*, 4Neμ, where μ is a constant being the product of mutation rate and the time span; SB RAS, Siberian Branch of Russian Academy of Sciences; SD, standard deviation. ⁎ Corresponding author at: Institute of Cytology and Genetics SB RAS, Acad. Lavrentyev ave. 10, Novosibirsk 630090, Russia. E-mail address: [email protected] (O.E. Kosterin).

molecular environment in which transcription takes place (Alami et al., 2003; Terme et al., 2011). Hence, changes in H1 subtype spectrum that are observed in ontogenesis may contribute to tuning of differential gene expression in the cell (Brown, 2001; Jedrusik and Schulze, 2001; Song and Gorovsky, 2007; Sancho et al., 2008; Terme et al., 2011), for example, there is evidence that one particular H1 subtype in mice is involved in differentiation of skeletal muscles (Lee et al., 2004). In contrast to the core histones which are highly or moderately conserved, histone H1 is highly variable because of its long positively charged C-terminal domain harboring repeats and motifs giving rise to indels (Stein et al., 1984; Trusov et al., 2004; Zaytseva et al., 2012). Therefore histone H1 often exhibits substantial polymorphism even on intra-species level (Kosterin et al., 1994; Sarg et al., 2005). The Pisum species are convenient model organisms for studying effects of histone H1 variation on phenotype. At least seven nonallelic H1 subtypes are known in pea, and each of them has a number of allelic variants. The gene His1 coding for subtype H1-1 is located on linkage group V, the genes of subtypes 2–6 form a tightly linked cluster His2– 6 on linkage group II. Gene His7 of subtype H1-7 is also situated on linkage group II. As alterations in primary structure of histone H1 may affect both its ability to compact chromatin and interact with regulatory factors, they are supposed to contribute to phenotypic variation. It was shown that

http://dx.doi.org/10.1016/j.gene.2014.11.062 0378-1119/© 2014 Published by Elsevier B.V.

Please cite this article as: Zaytseva, O.O., et al., Divergence and population traits in evolution of the genus Pisum L. as reconstructed using genes of two histone H1 subtypes showing different phylogenetic resolution, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.062

51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73

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2.1. Plant material

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The 56 pea accessions analyzed represent the following taxa:

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Pisum fulvum Sibth. et Smith (wild): L95, WL2140, VIR2523, VIR3397, VIR6070, VIR6071, 701, 702, 703, 706, 707, 708; Pisum abyssinicum A. Br. (cultivated): JI1876, WL1446, VIR2759, VIR3567;

108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133

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Pisum sativum subsp. elatius (Bieb.) Schmahl. in the broad sense by Maxted and Ambrose (2001) (wild): CE1, CE2, JI254, JI1092, JI1094, JI1095, JI1096, JI1794, JI2055, JI2724, JI3553, Ps002, Ps008, P012, P015, P017, PI343993, PI344008, PI344537, VIR320, VIR320*, VIR1851, VIR2524, VIR2998, VIR4014, WL2123, 711, 713, 714, 721, 722, 723; P. sativum subsp. sativum L. (cultivated) JI2105, Pa014; VIR4362, VIR7327, WL1238, and two subspecies of dubious validity: P. sativum subsp. transcaucasicum Govorov from Georgia: VIR3249 and P. sativum subsp. jomardii (Schrank) Kosterin from Egypt: VIR3424, VIR3439. Detailed provenance of these accessions is available in Table 1 in Kosterin and Bogdanova (2008), Table 1 in Kosterin et al. (2010) and Materials and Methods section in Zaytseva et al. (2012). Each accession was represented in the analysis by a single plant. Outgroup sequences of Vavilovia formosa (Steven) Fed. His5 and His7 were obtained from a specimen from Turkey generously provided by Dr. Petr Smykałl. A sample of individuals from a natural population of wild pea, P. sativum subsp. elatius was taken by the last author on July 8, 2010 in North Portugal, Trás-os-Montes e Alto Douro Province, Bragança District, Vimioso Municipality, 1.4 km NE of Uva village on the hill slope at the left board of the Angueira River valley. Wild pea plants were winding at Cytisus sp. bushes, oak saplings or, less frequently, in grass at the border of a small mowing outlined by a low stone wall and rows of tall stone oaks (Quercus ilex L.), at an area about 80–100 × 10–20 m. The plants demonstrated the syndrome of characters found only in wild peas: dehiscing pods (Dpo), the seed coat gritty (Gty) and camouflaged with different kinds of spots (brown marbled pattern, M; violet specks, F; long violet strokes, Ust,; darkened micropyle, Rf), large (Him) and black (Pl) hilum. All pea plants were already

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DNA was isolated from pea following Bogdanova et al. (2009). DNA from a freeze-dried Vavilovia Fed. sample was isolated following CTAB chloroform-isoamyl alcohol DNA extraction protocol (Wilkie, 1997). We designed 17 primers corresponding to the published nucleotide sequence of the His7 gene with adjacent non-coding regions (GenBank accession L34578), their sequences are provided in supplementary Table S1. These primers were used in different combinations to specifically amplify His7 but not other paralogous H1 genes. Seven primers derived from the coding region were used as additional ones for the Sanger reaction. Disposition of the primers is shown in a Supplementary Fig. S1. The primers used for His5 amplification were described in detail in Zaytseva et al. (2012). The sequences for His5 and His7 genes were amplified from genomic DNA of the same plant, that allowed us to analyze recombination in concatenated sequences of His7 and His5. PCR-amplification was carried out using BIS (produced by BIS-N, Koltsovo) and BIO-RAD MyCycler thermal cyclers under the following conditions: denaturation at 94 °C for 2 min 30 s followed by 5 cycles including denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, elongation at 70 °C for 1 min 30 s, followed by 35 cycles, including denaturation at 93 °C for 20 s, annealing at 56 °C for 30 s, elongation at 72 °C for 1 min 30 s. PCR reagents (Smart-Taq polymerase, dNTPs, buffers) were manufactured by Laboratory Medigen, Novosibirsk, and the Taq polymerase used was manufactured in Laboratory of Immunogenetics of ICG SB RAS, Novosibirsk. PCR-products were purified with Invisorb® Spin Filter PCRapid Kit according to provider's instructions. Purified PCR-products were sequenced using Big Dye Terminators version 3.0 or 1.1 ABI PRISM at the SB RAS Genomics Core Facility.

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2.3. Data analysis

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Raw sequence data were processed using the Staden package (Staden et al., 2000). Alignment of homologous nucleotide sequences was performed automatically with ClustalW algorithm (Larkin et al., 2007) incorporated in MEGA 6.06 package (Tamura et al., 2013) and MultAlin software (Corpet, 1988). Alignments were manually optimized: codons were aligned according to the derived amino acid alignment. Diversity data analysis was carried out with MEGA 6.06 and DnaSP v5 packages (Librado and Rozas, 2009). Phylogenetic analysis was based on sequences of His5 and His7 (intron sequences were not included), either separately or concatenated. All trees were rooted with the nucleotide sequences of V. formosa His5 and His7 orthologs as outgroups (sequenced by us for this study, [GenBank: HG934385, GenBank: HF562427], respectively), which were obtained using the same primers as for pea. Phylogenetic analysis was carried out using Maximum Parsimony, Maximum Likelihood and Bayesian methods. The Maximum Parsimony analysis was carried out using MEGA 6.06 package, with Subtree-Purifying-Regrafting method, 10 initial trees, M search level 1. The Maximum Likelihood analysis was carried out using the same package, using the Tamura-Nei model, uniform rates among sites, Nearest-Neighbor Interchange heuristic method, very strong branch swap filter. For these phylogenetic reconstructions, 500 bootstrap replicates were made. The Bayesian analysis was carried out with MrBayes 3.2.1 package (Huelsenbeck and Ronquist, 2001) using the HKY substitution model with two codon partitions corresponding to codon positions 1 & 2 and codon position 3; the discrete-gamma

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dry and their pods dehisced, yet some seeds were found as occasionally captured by the spirally coiled pod walls. This sample was given an internal lab designation PE1 (from “Portuguese elatius”) (= JI3557 in John Innes Centre). The seeds appeared heavily infested by pea weevil (Bruchus pisorum L.) and only minority of them were viable. We obtained 19 plants from the seeds taken from the population and extracted DNA from them.

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substitutions of allelic variants of subtypes H1-1, H1-6 and H1-7 in nearly isogenic pea lines affect plant growth dynamics (Berdnikov et al., 1999; Bogdanova et al., 2007; Kosterin et al., 2012). Small but significant effects of changes in H1-1 structure on some quantitative traits were observed in nearly isogenic lines of lentil and grass pea (Berdnikov et al., 2003). As histone H1 appeared to be among the most variable nuclear genes in eukaryotes, it was natural to attempt its use as a phylogenetic marker. Earlier we obtained phylogenetic reconstruction of relationships in the genus Pisum L. on both inter- and intraspecies levels based on sequences of one of paralogous pea H1 genes, His5, coding for subtype H1-5 (Zaytseva et al., 2012). In this work, we sequenced His7 gene encoding subtype H1-7 in 56 wild and cultivated pea accessions that represent main taxa in the genus Pisum. We inferred Bayesian phylogenetic reconstructions in the genus from concatenated sequences of His7 genes with earlier obtained sequences of His5 (Zaytseva et al., 2012). As very little is known about pea population parameter dynamics from both molecular studies and paleontological records, we addressed this question as well using histone H1 gene sequences. We made rough estimations of the divergence times and effective population size dynamics in the evolutionary history of the genus Pisum with the same concatenated alignment. We also compared nucleotide variation in two pea H1 subtypes, that presumably have different roles in the cell, to find out whether their evolutionary regimes differ as well.

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Please cite this article as: Zaytseva, O.O., et al., Divergence and population traits in evolution of the genus Pisum L. as reconstructed using genes of two histone H1 subtypes showing different phylogenetic resolution, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.062

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forming 32 His7 alleles [GenBank: HF562372–GenBank: HF562426]. DNA sequences of His7 gene were identical in 10 groups of more than one accession enumerated in Table 1. Nucleotide diversity parameters of His7 sequences are listed in Table 2. Fifteen variants of the amino acid sequence of H1-7 as derived from His7 nucleotide sequences were found, and the alignment is shown in Fig. 1. The overwhelming majority of the derived amino acid sequences (54 of 56) were 186 amino acid residues long (Fig. 1). Deletions were found only in the C-terminal domain, which is normally the less conserved of three domains in H1 molecule. A deletion of 3 amino acid residues corresponding to positions 98–100 in consensus sequence was revealed in the accession JI1092 (P. sativum subsp. elatius) originating from Greece, and a different deletion of 8 amino acid residues starting at position 129 was observed in the testerline WL1238 (P. sativum subsp. sativum). The latter deletion was found earlier in another, probably related testerline WL1688 by Bogdanova et al. (2007). The Nterminal, globular and C-terminal domains in the H1-7 sequences without deletions were 18 (the methionine residue, which is cleaved after translation, not included), 68, 99, amino acid residues long, respectively. On the whole, 14 variable amino acid positions were found in the reconstructed amino acid sequences of His7 products: 2 in the conserved globular domain and 12 in the C-terminal domain. Only one substitution of histidine to tyrosine, located in the globular domain, alters the positive charge of H1-7 molecule. This particular substitution was also revealed earlier in Bogdanova et al. (2007).

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3. Results

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3.1. Sequencing His7 gene

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DNA sequences of the His7 gene obtained from 56 pea lines revealed in total 43 nucleotide substitutions in 40 polymorphic nucleotide sites,

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Table 1 Groups of pea accessions with identical nucleotide sequences of gene His7.

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Number of accessions

t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17

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243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265

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We obtained Maximum Parsimony (MP) and Bayesian phylogenetic reconstructions for His5 (Figs. 2, S3) and His7 (Figs. 3, S4) coding nucleotide sequences derived from the same set of 56 pea accessions. If compared with corresponding trees for His7, the trees based on His5 sequences provided better phylogenetic resolution, that is better recognition of braches and their better support. The His5 MP tree provides three main clades (Fig. 2). The first dichotomy, with bootstrap value of 91%, separates a clade with all P. fulvum accessions (further referred to as clade 1). The second dichotomy, with the support of 89%, separates an unstructured set of long branches (clade 2) with significant bootstrap support combining wild P. sativum subsp. elatius accessions from Anterior Asia and central Mediterranean, with the P. abyssinicum subcluster found among those branches too. The third well-supported clade (bootstrap value 89%), (clade 3), embraces wild P. sativum subsp. elatius from Turkey and Tauro-Caucasian region, accession JI1794 from Holan Heights and cultivated P. sativum subsp. sativum accessions. The substructure of this clade is unresolved, although the first dichotomy inside this clade is well-supported: it separates accessions JI1794, P012 and Ps002 from the rest of accessions in

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3.2. Phylogenetic resolution of His7 sequences as compared to His5 266 sequences 267

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distribution with 6 categories was used to account for variable substitution rates among sites. The Bayesian trees were visualized with the 198 Archaeopteryx 0.9901 beta software (Han and Zmasek, 2009). 199 Estimations of the divergence times and effective population size 200 were based on concatenated coding sequences of His5 and His7 (intron 201 sequences were not included). We estimated divergence times of three 202 main Pisum clades and their effective population size dynamic using 203 BEAST 1.8.0 software (Drummond et al., 2012) with relaxed clock (log204 normal) and Bayesian Skyline coalescent model (7 000 000 iterations, 205 10 and 15 bins for Bayesian Skyline). Results obtained with BEAST 1.8.0 206 were summed with Tracer 1.6 (Drummond et al., 2012). Additionally ef207 fective population size dynamics was analyzed using IMa2 software (Hey, 208 Q11 2010) with a single migration parameter, and the rest of run parameters 209 as follows: -hfl, -hn4, -q10, -m5, -b100000, -z100, and -l100000. Comput210 er estimations of the divergence times and effective population size dy211 namics were based on divergence time equal to 6.5 ± 0.25 MYA of 212 Pisum and Vavilovia according to (Schaefer et al., 2012). 213 According to the obtained alignment and reconstructed phylogeny 214 we divided the ingroup nucleotide sequences into 3 clades (1/2/3). 215 For each clade we counted the number of clade-specific polymorphic 216 sites and tested statistical uniformity of the distribution of polymorphic 217 sites along coding sequences of His5 and His7 to detect possible con218 served (evolving mainly under stabilizing selection) and variable (neu219 trally evolving) gene regions. To estimate non-uniformity of the 220 polymorphic site distribution, we performed computer simulations 221 where a number of polymorphic sites with different variability were 222 permuted randomly along the sequence. The permutation was repeated 223 105 times, and then the number of random scatters was counted which 224 produced distances between polymorphic sites smaller or equal to the 225 minimum distance observed for actual sequences. 226 In order to analyze the evolutionary regime of His5 and His7 genes we 227 estimated the number and ratio of synonymous and nonsynonymous 228 nucleotide substitutions with DnaSPv5 software (Librado and Rozas, 229 2009). 230 Detection of recombination events in the sequences obtained was 231 performed with RDP5 software (Martin et al., 2010). The RDP, GENCONV, 232 Chimaera, MaxChi, BootScan, SiScan and 3Seq methods were applied 233 separately to His5 and His7 alignment and to concatenated sequences 234 of His5 and His7. Prior to the analysis, the sequence set was optimized 235 to remove sequences with high level of similarity, with the lowest ac236 ceptable pair-wise identity threshold calculated automatically by RDP5.

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Taxonomical attribution, wild or cultivated

Accessions and their provenance

P. P. P. P. P. P. P. P. P. P. P. P. P. P.

JI1876, VIR2759, Ethiopia; VIR3567, Yemen; WL1446, unknown VIR320*, a genotype admixed to accession VIR320, ‘Palestine’ VIR3424, VIR3439, Egypt VIR3249, Georgia CE1, CE2, Crimea PI343993, VIR7327 (taxonomic attribution of this accession uncertain), Turkey VIR6071, Israel VIR2524, Israel Pa014, Turkey, JI2105, Iran JI1094, JI1095, JI1096 — Greece, Mt. Athos Peninsula JI2724, Menorca; PI344008, Greece, Mt. Athos peninsula 721, WL2123, Israel; JI254, Ethiopia (?); JI2055 Italy; VIR2998, Georgia VIR2523, VIR3397, Israel 703, L95, Israel

abyssinicum, cultivated sativum subsp. elatius, wild sativum subsp. jomardii, cultivated sativum subsp. transcaucasicum, cultivated sativum subsp. elatius, wild sativum subsp. elatius, wild and P. sativum subsp. sativum, cultivated fulvum, wild sativum subsp. elatius, wild sativum subsp. sativum, cultivated sativum subsp. elatius, wild sativum subsp. elatius, wild sativum subsp. elatius, wild fulvum, wild fulvum, wild

Please cite this article as: Zaytseva, O.O., et al., Divergence and population traits in evolution of the genus Pisum L. as reconstructed using genes of two histone H1 subtypes showing different phylogenetic resolution, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.062

268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286

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Table 2 Nucleotide diversity parameters of His5 and His7 genes (after ±standard deviation values are provided).

t2:3

121 KAKAAGKPKA .....V.... .....V.... .....V.... .....V.... .....V.... .....V.... .T...V.... .......... .......... .......... .......... .......... .......... .......... .......... ..........

KAAAKPKAVA .......... .......... .......... .......V.. .......... .......... .......... .........T .......... .......... .......... .......... .......... .......... .......... --------..

Consensus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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VKSSFKLAPA .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AAKTAPAKTS .....S.... .......... .......... .......... .......... .......... .......... ...A...... .......... .......... .......... .......... .......... .......... .......--..........

AATKAPKAVT .......... ........G. .......... .......... .........A .........A .........A ........A. .......... .......... ........A. ........A. .......... ........A. .......... ..........

120 KPSAKAVTKP .......... .......... .......... .......... .......... .......... ...T...... .......... .......... .......... .......... .......... .......... ..I....... .......... ..........

KPKAKSVKAT .......... .......... .......... .......... ...S...... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

PVKKAVAKKV .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

VKKAKSVKSP .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ...V...... .......... .......... .......... .......... ..........

AKKVKSVKTP .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

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KSVASGKLVK .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

60 EEKHKDLPPT .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ...Y...... .....E.... .......... ...Y......

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61 YRKLVLLHLK .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

56 528 40 43 32 6.516 0.01234 (±0.00085) 0.035721 (±0.020748) 0.005 (±0.003513) 0.145

SSQYAITKFI .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

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Consensus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

His7

56 753 80 84 33 11.356 0.01508 (±0.00088) 0.02985 (±0.017495) 0.010666 (±0.005861) 0.355

AITSLKERTG .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

HPTFAEMITE .......... .......... .......... .......... .......... .......... .......... .......... ..X....... .......... .......... .......... .......... .......... .......... ..........

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KTAAAKKPLS .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

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1 MSTVAQTKPK .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

His5

4) WL1238 and VIR4362, with bootstrap value 75%; 5) VIR320*, VIR3249, VIR3424, VIR3439, with bootstrap value 63%. Pisum fulvum also forms a separate branch on this tree, but with a week bootstrap support (29%), although some smaller branches inside P. fulvum have better bootstrap supports. Two Pisum fulvum accession, VIR6071 and 702, are found outside the main P. fulvum branch. The Bayesian tree for His7 (Fig. S3) is slightly more informative. Branches with posterior probabilities of 1 include: 1) the P. fulvum

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Consensus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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this clade. The majority of clade 3 accessions are clustered together with 96% bootstrap support. The Bayesian His5 tree (Fig. S2) has practically the same topology with high posterior probabilities for all branches mentioned above. In contrast, the MP tree for His7 (Fig. 3), composed for the same set of accessions, resolves only some small branches, joining 1) CE1 and CE2 with bootstrap value 88%; 2) PI343993 and VIR7327 with bootstrap value 87%; 3). P. abyssinicum accessions, with bootstrap value 77%,

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Number of sequences Number of nucleotide sites in alignments without gaps (complete deletion) Polymorphic sites Total number of mutations Number of alleles Average number of nucleotide differences among all possible pairs of sequences Nucleotide diversity, average number of nucleotide differences per site among all possible pairs of sequences Synonymous sites ratio, K(s) Nonsynonymous sites ratio, K(a) ω = K(a) / K(s), arithmetical mean

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t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13

186 VKKAKK ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ......

Fig. 1. Alignment of H1-7 amino acid sequence variants reconstructed on the basis of His7 coding sequences for representatives of the genus Pisum: 1, VIR320; 2, 701 707, WL2140; 3, 702, 706, 711, 714; 4, 713; 5, JI1094, JI1095, JI1096, JI3553; 6, 703, 708, JI2724, L95, PI344008, PI344537, VIR6070; 7, VIR2523; 8, P017; 9, VIR4014; 10, 721, 722, СE1, CE2, JI254, JI2055, VIR2998, Ps008, P012, P015, PI343993, VIR7327, WL2123; 11, VIR320*, VIR3424, VIR3439; VIR3249; 12, 723, P014, Ps002, JI1794, JI1876, JI2105, VIR2524, VIR2759, VIR3567, VIR6071, WL1446; 13, VIR4362; 14, VIR1851; 15, JI1092; 16, WL1238. Globular domain sequence is filled with gray.

Please cite this article as: Zaytseva, O.O., et al., Divergence and population traits in evolution of the genus Pisum L. as reconstructed using genes of two histone H1 subtypes showing different phylogenetic resolution, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.062

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Fig. 2. Maximum parsimony phylogenetic reconstruction based on His5 sequences.

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clade, inside which all dichotomies have posterior probabilities of 1, without VIR6071 and 702; 2) all four P. abyssinicum accessions; 3) cultivated VIR3249, VIR3424, VIR3439 and wild VIR320*; 4) WL1238 and VIR4362, both sharing the same amino acid substitution that leads to a change of the globular domain charge; identical sequences of 5) CE1 and CE2; 6) PI343993, VIR7327; 7) JI1094, JI1095, JI1096, JI3553 and 8) JI1794, Ps008 and VIR1851. The Bayesian tree based on the concatenated His5 and His7 coding sequences is shown in Fig. 4. The topology of this tree is basically the same as for reconstructions based only on His5. There are three main clades: 1) P. fulvum accessions; 2) P. abyssinicum and wild P. sativum subsp. elatius from Anterior Asia and central Mediterranean; 3) all examined cultivated P. sativum subsp. sativum and some wild P. sativum subsp. elatius from Turkey, Holan Heights and Tauro-Caucasian region. The Maximum Likelihood phylogenetic reconstruction based on the concatenated His5 and His7 sequences produced a very similar tree (Fig. S4). The data on distribution of nucleotide variants between clades 1, 2, and 3 and the outgroup is shown in Supplementary Table S2.

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Fig. 3. Maximum parsimony phylogenetic reconstruction based on His7 sequences.

3.3. His5 and His7 nucleotide variation

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To explain the difference in phylogenetic resolution of two H1 subtype genes we compared parameters of their nucleotide sequences related to the mode of evolution (Table 2). Interestingly, a ω parameter related to the natural selection acting on a protein-coding sequence, calculated as the ratio of the number of nonsynonymous substitutions per nonsynonymous site to the number of synonymous substitutions per synonymous site, K(a)/K(s), appeared to be 2.5 times higher for His5 than that of His7, most probably indicating elevated selection pressure over H1-7 compared to H1-5. This is also reflected by the variation of amino acid sequences being lower for subtype 7: in the same set of accessions we have found only 15 amino acid variants of subtype 7 versus 25 variants of subtype 5. Note that none of the 12 amino acid

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Please cite this article as: Zaytseva, O.O., et al., Divergence and population traits in evolution of the genus Pisum L. as reconstructed using genes of two histone H1 subtypes showing different phylogenetic resolution, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.062

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Fig. 4. Bayesian phylogenetic tree of pea accessions reconstructed on the base of concatenated His5 and His7 gene sequences. Probability percent of node occurrence in sampled trees is indicated.

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Fig. 5. Distribution of sites with alternative (i.e. differing from the supposed ancestral state) nucleotide states along the sequences of a) His5 and b) His7 genes. On the X axis the length of alignment is plotted in base pairs, on the Y-axis — the number of sequences with alternative nucleotide states. The bold line indicates the threshold above which the position is considered highly variable.

Please cite this article as: Zaytseva, O.O., et al., Divergence and population traits in evolution of the genus Pisum L. as reconstructed using genes of two histone H1 subtypes showing different phylogenetic resolution, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.062

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3.5. His5 variation in a sample from a natural wild pea population

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The gene His5 was sequenced from 19 individual plants grown from the seeds taken from a wild pea population from North Portugal. The coding sequence, intron and short flanking 5′- and 3′-non-coding sequences (sequenced for a variable length) were found identical (GenBank IDs pending).

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Bayesian Skyline coalescent model allows estimating both divergence times and effective population size changes (Ho and Shapiro, 2011). This model conventionally splits the time period considered (in our case that from the divergence of Pisum and Vavilovia divergence to the present moment) to a certain number of equal time intervals called Skyline bins. We used two model versions, with 10 and 15 Skyline bins (designated, as numbered backwards in time, as t0–t9 or t0–t14, respectively). Using these models, our quite limited data allowed making rather precise evaluations of divergence times of Pisum populations

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4. Discussion

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4.1. His5 and His7 comparative phylogenetic resolution

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So far seven non-allelic histone H1 subtypes were found in pea (Kosterin et al., 1994). Histone H1 genes in pea like in other plants are encoded by unique genes that contain one intron (Eirín-López et al., 2004), thus showing classic features of replication independent histones. However, subtype H1-7 is present only in chromatin of actively growing tissues, thus resembling replication dependent subtypes, which in animals are clustered and lack introns (Kosterin et al., 1994). Earlier we examined nucleotide diversity and obtained phylogenetic reconstructions from sequences of the gene His5, coding for pea H1 histone subtype 5 (Zaytseva et al., 2012). This reconstruction was reproduced in the present work for our set of 56 accessions. The structure of phylogenetic trees based on His5 sequences is as follows: in the base of the tree there is a clade of all P. fulvum accessions. The next clade is comprised of long and well-supported branches leading to various wild P. sativum subsp. elatius accessions from Anterior Asia and central Mediterranean, with a tight cluster of P. abyssinicum among

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Table 3 Evaluation of divergence times of Pisum main clades with relaxed clock and Bayesian Skyline coalescent model, tmax — the minimum and maximum estimation of the Pisum and Vavilovia divergence time according to Schaefer et al. (2012), T0 — clades 2 and 3 divergence time, and T1 — clade 1 divergence time.

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We did not expect to detect recombination events in self-pollinating plants, as selfers should be predominantly homozygous. Although self pollination is considered to prevail in pea, recent studies show that hybridization and recombination played a more important part in Pisum evolution than it was previously thought (Jing et al., 2007). We checked the sequences of His5 and His7 for possible recombination events with RDP5 package (Martin et al., 2010). As expected, no recombination events were registered when His5 and His7 alignments were analyzed separately. In the concatenated sequences of His5 and His7 genes, two potential recombination events in accessions 702 and VIR6071 were detected by three methods. According to RDP5 analysis both recombinant concatenated sequence were derived from His5 sequence of accessions Ps008 (P. sativum subsp. elatius) and His7 sequence of VIR3397 (P. fulvum). His5 sequences of accessions VIR6071, 702 and VIR3397 are almost identical, including intron sequence, there is only one nucleotide substitution in VIR3397. His7 sequences of VIR6071 and Ps008 are identical, including intron sequence, and differ by 6 nucleotide substitutions from 702 His7 sequence. MaxChi and SiScan methods detected both recombination events, while 3Seq detected only the first one in accession 702, and Chimaera only the second one in accession VIR6071. Both presumed recombination breakpoints were located adjacent to the joint of the concatenated sequences, with some uncertainty. Since recombination was revealed neither inside His5, nor His7 sequences when analyzed separately without concatenation, the actual breakpoints were doubtless situated between His5 and His7 coding sequences.

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(Table 3). Noteworthy, the estimated divergence times did not significantly depend on the value of tmax that is the time of divergence of Pisum and Vavilovia, which we set as its minimum and maximum evaluation by Schaefer et al. (2012). According to our estimations, the clade 2 diverged from clade 3 at T0 = 1.3 ± 0.3 MYA; (2) clade 1 diverged from the rest of Pisum just before T1 = 1.7 ± 0.4 MYA. Table 4 shows the evaluation of the evolutionary dynamics of the parameter Ne* = 4Neμ during the course of Pisum evolution, as estimated using Bayesian Skyline coalescent model. Here μ is the product of mutation rate (which cannot be evaluated precisely from our data) and the duration of one Skyline bin; it is assumed to be constant, so Ne* is proportional to the effective population size, Ne. In Table 4, the values of Ne* are shown for each Skyline bin. In general, this value increased from the divergence of Pisum from Vavilovia to the present moment, when Pisum is represented by three evolutionary clades, from 2.4 to 2.7 (depending on tmax chosen) to 10–15. The model also showed (Table 4) that Pisum could have passed through bottlenecks before the three main clades diverged, since during the first 2–3 Skyline bins, low values of Ne* (~1.9–2.8) were observed. We also attempted to analyze the population dynamics using IMa2 software (Hey, 2010). We estimated Ne* for the three main Pisum clades and for three common ancestors before two the main divergences in Pisum and the divergence of Pisum and Vavilovia (Table 5). According to these estimations, after divergence from the common ancestor with Vavilovia, the effective population size of the Pisum ancestors decreased dramatically, to Ne* values about 5 times lower than those estimated for the period after divergence of clade 2 up to the present moment. Although effective population sizes somewhat restored before recently, our estimations for three main Pisum clades are still lower than that for the common ancestor of Pisum and Vavilovia.

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substitutions alters the charge of H1-7 C-terminal domain, while 7 such substitutions were observed for H1-5 (Zaytseva et al., 2012). For population dynamic assessment it was important to understand the pattern of variable site distribution along the sequence. In Fig. 5, the distribution of sites with alternative nucleotide states along the sequences of a) His5 and b) His7 genes is shown. The threshold above which a position is considered highly variable was assigned according to boot R package (99% confidence interval of A, where A = M ± σ, where M is the mean variability of positions and σ is its standard deviation). The analysis of non-uniformity of polymorphic site distribution along the nucleotide sequences showed that the probability of clusterization for highly variable sites was 0.049 for His5 and 0.03 for His7 gene. Such low values indicated that the rates of substitution fixation were uneven along the sequence so that both highly variable and conserved gene regions could be found in His5 and His7 sequences.

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± ± ± ± ± ±

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Please cite this article as: Zaytseva, O.O., et al., Divergence and population traits in evolution of the genus Pisum L. as reconstructed using genes of two histone H1 subtypes showing different phylogenetic resolution, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.062

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Table 4 Dynamics of the mean Ne* = 4Neμ (where μ is an unknown mutation rate) as estimated with Bayesian Skyline coalescent model for different time periods: the interval from present to the time of divergence of Pisum and Vavilovia) was divided into 10 or 15 equal periods (Skyline bins), from t0 to t9 or t14 (depending on the number of Skyline bins) going backwards in time. tmax (MYA) is variable time of Pisum and Vavilovia divergence within its estimations by Schaefer et al. (2012). tmax

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Table 5 The values of Ne* (see Table 4 and the text) estimated using IMa2 software for three main clades of Pisum observed in phylogenetic reconstructions and for their common ancestors, depending on the tmax value assumed, which is Pisum and Vavilovia divergence time (Schaefer et al., 2012).

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which are mostly self-pollinators, as well as between populations, which are rather small and local. Nucleotide diversity as such, being the average number of nucleotide differences per site among all possible pairs of sequences, was only slightly lower in His7 than in His5. However, comparison of nucleotide diversity parameters revealed a parameter differing greatly between His5 and His7, the ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site (ω), which appears to be 2.5 times higher for His5 gene. This might indicate that His7 evolved under stricter evolutionary constraints than His5, so that most of variability was limited to synonymous substitutions. This is supported by the fact that no substitutions altering the charge of C-terminal domain were found in H1-7 derived amino acid sequences, while such substitutions are common in the derived amino acid sequence of subtype H1-5. The C-terminal domain binds to linker DNA, thus its positive charge is very important for stability of this interaction. Subtype H1-7 is the only one being expressed only in growing tissues and disappearing rapidly with aging, while H1-5 is a minor subtype sharing its dynamic during ontogenesis with at least three other subtypes (Kosterin et al., 1994). Therefore it is possible that His7 amino acid structure is more conserved, as its interaction with DNA is crucial for cells undergoing rapid division and it is not secured by any paralogous subtype. We may conclude that applicability of histone H1 genes for phylogenetic reconstructions at species and intra-species levels, claimed by Zaytseva et al. (2012), in fact depends on the H1 gene chosen. His7, unlike His5, failed to resolve phylogeny for the same set of accessions, although its genetic variation does not differ significantly from that of His5 gene. His5 codes for a minor subtype H1-5 which is most probably interchangeable with other subtypes in chromatin. His7 is unique in its mode of expression only in actively growing tissues where its product, subtype H1-7, comprises a considerable proportion of the total H1 (Kosterin et al., 1994); probably its function has some relation to maintenance of transcriptionally active state of chromatin. Dispensability of H1-5 and indispensability of H1-7 is indirectly supported by our extensive study of histone H1 electrophoretic spectrum among 883 pea landraces: we found a case where H1.5 was missing from the spectrum (Berdnikov et al., 1993) while H1-7 was present invariably (Kosterin et al., 1994). It would not be surprising that a minor and probably dispensable subtype is scarcely conserved in evolution and hence turned

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these accessions. The third clade unites some wild P. sativum subsp. elatius accessions from Crimea, Caucasus, Turkey and Holan Heights along with cultivated P. sativum accessions. This is consistent with our earlier phylogeographical results suggesting that wild pea arose in the Middle East, spread westwards to inhabit the entire Mediterranean and then some group, corresponding to our clade 3, returned through Tauro-Caucasian region to Turkey and even reached North Israel (Kosterin and Bogdanova, 2008; Kosterin et al., 2010). In contrast, the analysis based on His7, the gene for subtype H1-7, failed to resolve phylogeny as offering few small well-supported branches. Differences in the topologies of His5 and His7 trees could be explained by recombination between these two genes, while the lack of nucleotide diversity in His7 sequences could account for insufficient phylogenetic resolution. Recombination events between His5 and His7 genes are possible, as those two genes are located on the same chromosome 40 cM apart from each other (Kosterin et al., 1994). However, we detected only two possible recombination events by three out of seven applied methods. In both cases representatives of P. fulvum were involved in the recombination events detected as one of the parents and as a resulting recombinant. Both events seem to have occurred somewhere between His5 and His7 genes, so that His7 sequence in the recombinant P. fulvum originated from P. sativum. P. fulvum is confined to Levant and the adjacent regions of Turkey, so most likely the other accession involved in these recombination events should originate from the same region. Accession Ps008 was identified to have a His7 sequence which was a potential participant in both recombination events. This accession is a wild pea P. sativum subsp. elatius from Siirt Province in the north-east of Turkey, so recombination could in fact have occurred between it and some P. fulvum. The sequence resulting from recombination is shared by accessions VIR6071 (also P. fulvum), VIR2524 (P. sativum subsp. elatius) from Israel and P014 (P. sativum subsp. sativum from Tokat Province in northern Turkey). All they originated from generally the same region, although represent a variety of taxa. Therefore, despite reproductive isolation, introgression might be possible between P. fulvum and P. sativum. However, these two recombination events alone could hardly account for the differences in tree topology beyond positions of VIR6071, 702 and P014. In general, recombination should be infrequent in wild peas because of very limited gene exchange between plants,

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Please cite this article as: Zaytseva, O.O., et al., Divergence and population traits in evolution of the genus Pisum L. as reconstructed using genes of two histone H1 subtypes showing different phylogenetic resolution, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.062

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We thank Dr. Petr Smykał for providing dried leaves of V. formosa. This work was supported by the project 14-04-32008 mol_a by the Russian Fund for Fundamental Research and the project VI.53.1.3. “Genetic control of mechanisms of incompatibility between plant taxa and their adaptation to unfavorable environmental conditions”. Sanger sequencing was performed at SB RAS Genomics Core Facility. Plants were grown at ICG Artificial Plant Growing Facility.

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Populations of wild peas are known to occupy small areas and often to include very small number of individuals (Abbo et al., 2008, 2013). The last author's personal experience concerned three populations of wild P. sativum subsp. elatius: two in Crimea (in 1991) and one in Portugal (in 2010). In Crimea, a thorough search in two localities resulted in just two and about a dozen plants, respectively; in a population in North Portugal about hundred plants were found. Wild peas are thought to propagate predominantly by self-pollination (but the actual degree of cross pollination is unknown). Combination of small populations (which imply frequent bottlenecks) and selfing would result in high intrapopulation homogeneity (and intra-population heterogeneity). However, no actual population data has been published for wild peas at all. To somewhat fill this gap, we sequenced the more variable of the two here studied gene, His5, in a limited sample of 19 plants grown from seeds taken from a natural population in North Portugal, which were the only populational material at our disposal. The revealed identity of the sequences, including synonymous positions and intron, was in line with the expectation that natural population of wild peas are nearly (or completely) clones. This further motivated us to apply algorithms used for assessment of population dynamics to our set of His5 and His7 sequence data where each accession (that is population) was represented with a single plant only. These algorithms were worked out for population analysis, however, under the assumption that the present-day population are nearly clones, they are somewhat analogous to individuals representing populations of their common ancestors, and in this sense these data do reflect the processes used to take place in ancestor populations. Hence, applying populational algorithms we may attempt to infer some approximate information concerning populations of the common ancestors of main groups of recent peas, e.g. the contemporary P. fulvum, or the two revealed branches of wild P. sativum ‘ssp. elatius’, or the common ancestor of Pisum and Vavilovia. Hence application of those algorithms could be informative. These algorithms exclude from analysis certain types of substitutions, as they cannot be used for assessing population dynamics in the evolutionary history of Pisum L. The first type of non-informative substitutions are those specific for the outgroup (7.7%) and absent in Pisum L. The second type corresponds to substitutions specific for only one of the examined sequences (5.1% of all observed substitutions), as those unique substitutions could be fixed in local pea populations recently. However, the rate of substitution fixation is inversely related to the population size, so, frequency of such substitutions in the entire data set may be indicative for the small effective population size of ancient Pisum populations. The third type is represented by positions in the alignment where an alternative nucleotide variant is observed with higher frequencies than the presumed ancestor variant (Supplementary Table S3). The latter situation could be explained either as a result of genetic drift or selection for specific variants (as indicated in supplementary Table S2: 16 positions for His5 and 7 positions for His7, 1.7% of all substitutions). Hence, only 7% of all nucleotide sites (96 sites out of 1383) provide information for simultaneous estimation of divergence

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times and effective population size dynamics. Therefore such estimations cannot be precise and should be based on simple models of nucleotide substitutions. Due to insufficient data we had to use a simplified HKY substitution model, splitting codons in analyzed His5 and His7 genes into 2 partitions, corresponding to codon positions 1 & 2 and codon position 3, respectively. Such a model provided only qualitative assessment of effective population size dynamics in Pisum L. Although our data are insufficient for a detailed analysis of population dynamics, we still were able of some insights into Pisum evolution. First of all, our estimations of divergence times in the genus suggest that the earliest major divergence took place about 1.7 MYA, the latest about 1.3 MYA. Those times correspond to the early Pleistocene, when significant cooling took place in Northern hemisphere. That is in line with our previous hypothesis that further radiation of Pisum forms in the Mediterranean took place when the sea level dropped due to the climate cooling (Kosterin et al., 2010). Subsequent rise of the sea level could account for isolation of some pea population on islands and peninsulas. Another interesting observation was the dramatic reduction of the effective population size (which is proportional to the parameter Ne* estimated by the algorithms used) in the Pisum lineage after its divergence from the common ancestor with Vavilovia. Early Pisum population seems to have passed through a period of high risk of bottlenecks. These events could be connected with a shift towards self-pollination in Pisum. Note a somewhat higher Ne value reconstructed for P. fulvum than for P. sativum. This could mean P. fulvum to be either a more abundant plant in its range than P. sativum, that seems to be the case (Abbo et al., 2008) or more apt to cross-pollination. In our analysis, V. formosa was used as an outgroup. Vavilovia is a monotypic genus sister to Pisum. Unlike all Pisum representatives, V. formosa is a perennial highland plant with very large flowers and an endemic to high mountain regions of Caucasus and Asia Minor (Akopian et al., 2010). Apparently, Vavilovia is a cross-pollinator and its effective population size remained at the ancestral level.

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to be a good phylogenetic marker, while a unique subtype with a functional specificity is under string selective constraints and cannot be used for this purpose. Earlier we found a certain His5 allele to be frequently found among pea landraces from such distant areas as North Russia and the mountains of Central Asia, and registered a negative correlation of its frequency with the accumulated temperature of vegetation period in regional samples of landraces (Berdnikov et al., 1993; see also Zaytseva et al., 2012). This allele was supposed to be somehow related to cold tolerance. However, our data do not allow unequivocally recognizing pitfalls of positive selection for His5 and we suppose that its evolution mostly followed a neutral mode.

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Abbo, S., Zezak, I., Schwartz, E., Lev-Yadun, S., Gopher, A., 2008. Experimental harvesting of wild peas in Israel: implications for the origins of Near East farming. J. Archaeol. Sci. 35, 922–929. Abbo, S., Lev-Yadun, S., Heun, M., Gopher, A., 2013. On the ‘lost crops’ of the neolithic Near East. J. Exp. Bot. 64, 815–822. Akopian, J., Sarukhanyan, N., Gabrielyan, I., Vanyan, A., Mikić, A., Smýkal, P., Kenicer, G., Vishnyakova, M., Sinjushin, A., Demidenko, N., Ambrose, M., 2010. Reports on establishing an ex situ site for ‘beautiful’ Vavilovia (Vavilovia formosa) in Armenia. Genet. Resour. Crop. Evol. 57, 1127–1134. Alami, R., Fan, Y., Pack, S., Sonbuchner, T.M., Besse, A., Lin, Q., Greally, J.M., Skoultchi, A.I., Bouhassira, E.E., 2003. Mammalian linker-histone subtypes differentially affect gene expression in vivo. Proc. Natl. Acad. Sci. U. S. A. 100, 5920–5925. Berdnikov, V.A., Bogdanova, V.S., Rozov, S.M., Kosterin, O.E., 1993. Geographic patterns of histone H1 allelic frequencies found in the course of Pisum sativum L. (pea) cultivation. Heredity 71, 199–209. Berdnikov, V.A., Gorel, F.L., Bogdanova, V.S., Kosterin, O.E., Trusov, Y.A., Rozov, S.M., 1999. Effect of a substitution of a short chromosome segment carrying a histone H1 locus on expression of the homeotic gene Tl in heterozygote in the garden pea Pisum sativum. Genet. Res. 73, 93–109.

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Please cite this article as: Zaytseva, O.O., et al., Divergence and population traits in evolution of the genus Pisum L. as reconstructed using genes of two histone H1 subtypes showing different phylogenetic resolution, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.11.062

D

P

R O

O

F

Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–1452. Lin, Q., Sirotkin, A., Skoultchi, A., 1999. Normal spermatogenesis in mice lacking the testisspecific linker histone H1t. Mol. Cell. Biol. 20, 2122–2128. Martin, D.P., Lemey, P., Lott, M., Moulton, V., Posada, D., Lefeuvre, P., 2010. RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics 26, 2462–2463. Maxted, N., Ambrose, M., 2001. Peas (Pisum L.). In: Maxted, N., Bennett, S.J. (Eds.), Plant Genetic Resources of Legumes in the Mediterranean. Springer, Netherlands, pp. 181–190. Misteli, T., Gunjan, A., Hock, R., Bustin, M., Brown, D.T., 2000. Dynamic binding of histone H1 to chromatin in living cells. Nature 408, 877–881. Ponte, I., Vidal-Taboada, J.M., Suau, P., 1998. Evolution of the vertebrate H1 histone class: evidence for the functional differentiation of the subtypes. Mol. Biol. Evol. 15, 702–708. Sancho, M., Diani, E., Beato, M., Jordan, A., 2008. Depletion of human histone H1 variants uncovers specific roles in gene expression and cell growth. PLoS Genet. 4, e1000227. Sarg, B., Gréen, A., Söderkvist, P., Helliger, W., Rundquist, I., Lindner, H.H., 2005. Characterization of sequence variations in human histone H1.2 and H1.4 subtypes. FEBS J. 272, 3673–3683. Schaefer, H., Hechenleitner, P., Santos-Guerra, A., Menezes de Sequeira, M., Pennington, R.T., Kenicer, G., Carine, M.A., 2012. Systematics, biogeography, and character evolution of the legume tribe Fabeae with special focus on the middle-Atlantic island lineages. BMC Evol. Biol. 12, 250. Song, Х., Gorovsky, M.A., 2007. Unphosphorylated H1 is enriched in a specific region of the promoter when CDC2 is down-regulated during starvation. Mol. Cel. Biol. 27, 1925–1933. Staden, R., Beal, K.F., Bonfield, J.K., 2000. The Staden package, 1998. Methods Mol. Biol. 132, 115–130. Stein, G.S., Stein, J.L., Marzluff, W.F. (Eds.), 1984. Histone Genes: Structure, Organization, and Regulation. John Willey & Sons, New York. Tamura, K., Stecher, G., Peterson, D., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Terme, J.M., Sesé, B., Millán-Ariño, L., Mayor, R., Izpisúa Belmonte, J.C., Barrero, M.J., Jordan, A., 2011. Histone H1 variants are differentially expressed and incorporated into chromatin during differentiation and reprogramming to pluripotency. J. Biol. Chem. 286, 35347–35357. Th'ng, J.P., Sung, R., Ye, M., Hendzel, M.J., 2005. H1 family histones in the nucleus. Control of binding and localization by the C-terminal domain. J. Biol. Chem. 280, 27809–27814. Trusov, Y.A., Bogdanova, V.S., Berdnikov, V.A., 2004. Evolution of regular zone of histone H1 in Fabaceae plants. J. Mol. Evol. 59, 546–555. Weiss, T., Hergeth, S., Zeissler, U., Izzo, A., Tropberger, P., Zee, B.M., Dundr, M., Garcia, B.A., Daujat, S., Schneider, R., 2010. Histone H1 variant-specific lysine methylation by G9a/ KMT1C and Glp1/KMT1D. Epigenetics Chromatin 3, 7. Wilkie, S., 1997. Plant Molecular Biology — A Laboratory Manual, Chapter I, Isolation of Total Genomic DNA. Springer, Berlin Heidelberg, p. 529. Wisniewski, J.R., Zougman, A., Krüger, S., Mann, M., 2007. Mass spectrometric mapping of linker histone H1 variants reveals multiple acetylations, methylations, and phosphorylation as well as differences between cell culture and tissue. Mol. Cell. Proteomics 6, 72–87. Zaytseva, O.O., Bogdanova, V.S., Kosterin, O.E., 2012. Phylogenetic reconstruction at the species and intraspecies levels in the genus Pisum (L.) (peas) using a histone H1 gene. Gene 504, 192–202.

R

R

E

C

T

Berdnikov, V.A., Bogdanova, V.S., Gorel, F.L., Kosterin, O.E., Trusov, Y.A., 2003. Large changes in the structure of the major histone H1 subtype result in small effects on quantitative traits in legumes. Genetica 119, 167–182. Bogdanova, V.S., Kosterin, O.E., Berdnikov, V.A., 2007. Phenotypic effect of substitution of allelic variants for a histone H1 subtype specific for growing tissues in the garden pea (Pisum sativum L.). Genetica 130, 61–72. Bogdanova, V.S., Galieva, E.R., Kosterin, O.E., 2009. Genetic analysis of nuclear-cytoplasmic incompatibility in pea associated with cytoplasm of an accession of wild subspecies Pisum sativum subsp. elatius (Bieb.) Schmahl. Theor. Appl. Genet. 118, 801–809. Brown, D.T., 2001. Histone variants: are they functionally heterogeneous? Genome Biol. 2 (REVIEWS0006). Corpet, F., 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 16, 10881–10890. De, S., Brown, D.T., Lu, Z.H., Leno, G.H., Wellman, S.E., Sittman, D.B., 2002. Histone H1 variants differentially inhibit DNA replication through an affinity for chromatin mediated by their carboxyl-terminal domains. Gene 292, 173–181. Drummond, A.J., Suchard, M.A., Xie, D., Rambaut, A., 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29, 1969–1973. Eirín-López, J.M., González-Tizón, A.M., Martínez, A., Méndez, J., 2004. Birth-and-death evolution with strong purifying selection in the histone H1 multigene family and the origin of orphon H1 genes. Mol. Biol. Evol. 21, 1992–2003. Han, M.V., Zmasek, C.M., 2009. PhyloXML: XML for evolutionary biology and comparative genomics. BMC Bioinformatics 10, 356. Happel, N., Doenecke, D., 2009. Histone H1 and its isoforms: contribution to chromatin structure and function. Gene 431, 1–12. Hey, J., 2010. Isolation with migration models for more than two populations. Mol. Biol. Evol. 27, 905–920. Ho, S.Y., Shapiro, B., 2011. Skyline-plot methods for estimating demographic history from nucleotide sequences. Mol. Ecol. Resour. 11, 423–434. Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. Izzo, A., Kamieniarz-Gdula, K., Ramírez, F., Noureen, N., Kind, J., Manke, T., van Steensel, B., Schneider, R., 2013. The genomic landscape of the somatic linker histone subtypes H1.1 to H1.5 in human cells. Cell Rep. 3, 2142–2154. Jedrusik, M.A., Schulze, E., 2001. A single histone H1 isoform (H1.1) is essential for chromatin silencing and germline development in Caenorhabditis elegans. Development 128, 1069–1080. Jing, R., Johnson, R., Seres, A., Kiss, G., Ambrose, M.J., Knox, M.R., Ellis, T.H., Flavell, A.J., 2007. Gene-based sequence diversity analysis of field pea (Pisum). Genetics 177, 2263–2275. Kosterin, O.E., Bogdanova, V.S., 2008. Relationship of wild and cultivated forms of Pisum L. as inferred from an analysis of three markers, of the plastid, mitochondrial and nuclear genomes. Genet. Resour. Crop. Evol. 55, 735–755. Kosterin, O.E., Bogdanova, V.S., Gorel, F.L., Rozov, S.M., Trusov, Y.A., Berdnikov, V.A., 1994. Histone H1 of the garden pea (Pisum sativum L.): composition, developmental changes, allelic polymorphism and inheritance. Plant Sci. 101, 189–202. Kosterin, O.E., Zaytseva, O.O., Bogdanova, V.S., Ambrose, M., 2010. New data on three molecular markers from different cellular genomes in Mediterranean accessions reveal new insights into phylogeography of Pisum sativum L. subsp. elatuis (Beib.) Schmahl. Genet. Resour. Crop. Evol. 57, 733–739. Kosterin, O.E., Bogdanova, V.S., Kechin, A.A., Zaytseva, O.O., Yadrikhinskiy, A.K., 2012. Polymorphism in a histone H1 subtype with a short N-terminal domain in three legume species (Fabaceae, Fabaeae). Mol. Biol. Rep. 39, 10681–10695. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948. Lee, H., Habas, R., Abate-Shen, C., 2004. MSX1 cooperates with histone H1b for inhibition of transcription and myogenesis. Science 304, 1675–1678.

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