Plant Systematics and Evolution (2020) 306:84 https://doi.org/10.1007/s00606-020-01713-4

ORIGINAL ARTICLE

Orostachys spinosa (Crassulaceae) origin and diversifcation: East Asia or South Siberian Mountains? Chloroplast DNA data

Arthur Yu. Nikulin1 · Vyacheslav Yu. Nikulin1 · Andrey A. Gontcharov1

Received: 20 December 2019 / Accepted: 3 September 2020 © Springer-Verlag GmbH Austria, part of Springer Nature 2020

Abstract Limited data are available on genetic structures of the herbaceous plant species populations inhabiting mountainous areas of Siberia and Northeastern Asian (Russian Far East). Although this area was not directly impacted by the extensive ice-sheets during the Quaternary, it experienced signifcant climatic fuctuations that infuenced rich local fora. Orostachys spinosa (Crassulaceae) lacks any adaptations for long-distance dispersal, yet the species is characterized by an unusually wide range spanning from the Urals to the coast of the Pacifc Ocean. We studied O. spinosa phylogeography and genetic diversity across its range sampling 203 individuals from 21 natural populations. Using sequences from three chloroplast DNA non- coding regions, we revealed 82 haplotypes and observed high level of population diferentiation indicating presence of the phylogeographic structure (GST = 0.501 and NST = 0.822 (p < 0.01)). In concordance with the previous phylogenetic analyses based on ITS rDNA data, parsimony network revealed two distinct cpDNA haplotype lineages deferring in their structure and characteristics of genetic diversity. The split between these haplotype groups can be dated to the Pliocene (ca. 3.6 Mya). According to our estimates diversifcation in the Western group of populations took place ca. 1 Mya earlier than in the East- ern group (3.1 Mya and 2.26 Mya, respectively). Apart from the generally accepted notion about East Asian origin of O. spinosa, our results indicated that the species could have originated in mountains of Southern Siberia (Altai). This region was colonized independently from O. thyrsifora which has a largely overlapping distribution in this area.

Keywords Altai · cpDNA haplotypes · Genetic structure · Origin · Orostachys spinosa · Phylogeography

Introduction 2015; Li et al. 2012; Wroblewska 2012; Chen et al. 2014; Fu et al. 2016; Zhang et al. 2018). The patterns of genetic A number of studies have documented signifcant genetic diversity in these taxa have been shaped by the interaction diversity in plant species associated with the temperate of many factors, such as life history traits, ecological vari- mountainous areas of Europe, Northern America, and East- ables (e.g., life cycle, breeding system, pollination and dis- ern Asia (e.g., Stehlik et al. 2002; Tribsch and Schönswetter persal mechanisms, efective population size, connectivity, 2003; Ronikier et al. 2008; Cun and Wang 2010; Xu et al. etc.; Loveless and Hamrick 1984; Eckert et al. 2008; Palstra 2010; Ansell et al. 2011; Allen et al. 2012; Gussarova et al. and Ruzzante 2008), and historical events. The mountain ranges act as a barrier to species migration (Taberlet et al. 1998), but due to their complex topography and varied Handling Editor: Mike Thiv. habitat types, they also serve as a refuge during climate oscillations (Tribsch and Schönswetter 2003). The efects Electronic supplementary material The online version of this article (https​://doi.org/10.1007/s0060​6-020-01713​-4) contains of the climate changes on plant species’ geographical dis- supplementary material, which is available to authorized users. tributions and population demographies are considerable, involving latitudinal and altitudinal migrations, population * Arthur Yu. Nikulin extinction, and range fragmentation/expansion that leaves [email protected] long-lasting, detectable genetic imprints within and among 1 Federal Scientifc Center of the East Asia Terrestrial species (Abbott et al. 2000; Avise 2000; Hewitt 2000, 2004, Biodiversity of the Far Eastern Branch, Russian Academy 2011; Qiu et al. 2013; Wen et al. 2014, 2016). of Sciences, 100‑Letia Vladivostoka Prospect, 159, Vladivostok, Russia 690022

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Still relatively little is known about the genetic structures among species of the genus Hylotelephium H. Ohba (Mayu- of the plant species that are restricted to the mountains and zumi and Ohba 2004; Gontcharova et al. 2006; Nikulin et al. adjacent Siberia and Northeastern Asian (Russian Far East) 2015b). The clade Appendiculatae was resolved as a sister areas which are characterized by rich local foras (Krasnob- to the Hylotelephium/Orostachys lineage and harbored the orov 1976; Malyshev and Peshkova 1984; Revushkin 1988; monotypic genus Meterostachys. These patterns of phylo- Stepanov et al. 2003; Malyshev et al. 2012). Genetic data genetic relationships require a number of taxonomic adjust- are very scarce for herbaceous species having wide distribu- ments (e.g., classifcation of Hylotelephium species under tion areas spanning Circumboreal and Eastern Asiatic phy- the name Orostachys and Orostachys subsection Appen- togeographic zones (Takhtajan 1986). Such contemporary diculatae members under the name Meterostachys; Nikulin distribution patterns could be remnants of an ancient range et al. 2015a, b). The divergence time estimation showed that or a result of the recent range expansion. Although south- O. spinosa diverged from its closest relatives circa 6.5 Mya ern Siberian Mountains were not directly impacted by the (Late Miocene). Analyses of multiple O. spinosa ITS rDNA unifed ice-sheets during the Quaternary, they experienced sequences revealed a split between specimens from west- a much cooler and dryer climate during the glacial periods ern and eastern parts of the species range dated to 3.5 Mya (Sun and Chen 1991) and likely served as refugia for many (Nikulin et al. 2015b). Diversifcation in the Western lineage organisms (Schmitt and Varga 2012). These climatic fuctua- (includes ribotypes found in Siberia and Northeastern Asia) tions should have signifcant efects on the genetic structure also took place in the Late Pliocene circa 3 Mya, while in of the local biota, but the responses of local plants to the the Eastern lineage (ribotypes from the northeast of China glaciation are still poorly understood. High levels of genetic and south of the Russian Far East), it started much later in diversity are expected for areas that are thought to be refu- the Early Pleistocene, circa 1.2 Mya, but still long before the gial (such as Altai, Sayan, Sikhote-Aline Mountains). These last glaciation period. refugia were mostly confned to the mountainous regions Although the O. spinosa concept has never been ques- because they enjoyed relative stability during the Quaternary tioned, it was noted that populations in the eastern part of climatic cycles due to moisture availability (orographic rain- the range are represented by individual monocarpic rosettes, fall), and their diverse topographies have provided sheltered whereas in the more continental western areas, the same spe- habitats from the cold winds (Fjeldsaå and Lovett 1997; cies shows diferent growth habits with numerous densely Tzedakis et al. 2002; Kaltenrieder et al. 2009; Médail and crowded vegetative rosettes (Bezdeleva 1995; Gontcharova Diadema 2009; Muellner-Riehl 2019). Importantly, an altitu- 2006). It remains unclear whether these morphotypes refect dinal retreat of only 10 m might be equivalent to an approxi- distinct genotypes or resulted from adaptations to more xeric mately 10 km latitudinal retreat (Jump et al. 2009); therefore, environments. mountains allow plant species to follow warm interglacial/ This study provides molecular data for a better under- cold glacial trends by means of relatively narrow altitudinal standing of the evolutionary history of the Asian crassu- shifts instead of larger latitudinal migrations. lacean fora, specifcally the relationships between O. spi- Orostachys spinosa (L.) Sweet is one of the species nosa populations. We characterized wide-range patterns of characterized by an unusually wider range than most other genetic diversity in the species focusing on the contrasts in crassulacean taxa. It covers a signifcant part of Northern genetic diferentiation between western and eastern popula- Asia from the coast of the Pacifc Ocean to the Urals. This tions and examining the species’ phylogeography based on species grows on rock crevices and dry slopes at sea level intergenic plastid DNA sequences. up to 3000 m altitude. The seeds lack any specifc adap- tations for long-distance dispersal thus falling close to the maternal plant. They are small and light, produced in high Materials and methods quantities, and can be dispersed a limited distance by wind. The species’ broad altitudinal and latitudinal ranges imply Plant materials substantial thermal tolerance that along with adaptation to xeric conditions, may have enhanced survival during the Leaves or inforescences were sampled from 21 O. spinosa Late Pleistocene. (203 individuals) and one O. japonica (Maxim.) A.Berger Orostachys spinosa is a member of the East Asian cras- (10 individuals) natural populations (Table 1; Fig. 1) cover- sulacean genus Orostachys Fisch., and along with fve more ing most of the distribution range of this species. species, is part of subsection Appendiculatae (Borissova) H. The sample size was from eight to ffteen individuals per Ohba (Eggli 2005). Taxomonic relationships in the genus are population, except for fve populations (P3, P4, P16–P18; complex, and the genus itself is polyphyletic. It was shown Table 1), of which one to four individuals were sampled. that subsection Appendiculatae only has a distant relation- To minimize sampling from within the progeny of a single ship to the Orostachys-type subsection that was nested maternal plant, the individual samples were collected from

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Table 1 Sampling sites locations, codes and sample sizes of Orostachys spinosa populations Pop. # Sample size Haplotype Latitude/longitude Origin

P1 12 H1–H4 45° 14′ 27′′/131° 59′00′′ Russia, Primorsky Terr., Khankayski Distr., Khanka Lake, sandy dunes P2 12 H5–H7 43° 54′ 44′′/131° 59′ 36′′ Russia, Primorsky Terr., Mikhailovsky Distr., hill P3 2 H8 63° 69′ 41′′/152° 24′ 08′′ Russia, Magadan Terr., Susumansky Distr., mountain slope P4 3 H9, H10 54° 54′ 20′′/137° 40′ 37′′ Russia, Khabarovsky Terr., Shantar Islands P5 12 H7, H11–H13 43° 52′ 00′′/132° 55′ 43′′ Russia, Primorsky Terr., Anuchinsky Distr., clifs P6 12 H14–H16 54° 50′ 03′′/56° 11′ 25′′ Russia, Rep. of Bashkortostan, Ufa P7 13 H17, H25–H26 43° 24′ 40′′/133° 53′ 31′′ Russia, Primorsky Terr., Lazovsky Distr., clifs P8 12 H18–H22 52° 01′ 32′′/113° 27′ 34′′ Russia, Zabaikalski Terr., Chita, rocks P9 10 H23–H24 54° 05′ 00′′/57° 36′ 30′′ Russia, Rep. of Bashkortostan, Abelilovsky Distr., Karatas Mt. P10 15 H27–H30 50° 22′ 11′′/127° 39′ 20′′ Russia, Amur Terr., Blagoveshchensk, sandy hill P11 12 H31–H32 51° 52′ 28′′/104° 47′ 46′′ Russia, Irkutsk Reg., L. Baikal, clif P12 12 H31, H33–H38 51° 46′ 33′′/104° 10′ 43′′ Russia, Irkutsk Reg., L. Baikal, clif P13 12 H39–H48 51° 09′ 58′′/86° 09′ 15′′ Russia, Altai Rep., Katun Riv. P14 12 H49–H60 51° 07′ 57′′/86° 10′ 57′′ Russia, Altai Rep., Katun Riv. P15 12 H61–H62 49° 13′ 38′′/120° 53′ 11′′ China, Inner Mongolia, Yákèshí Shì, clif P16 4 H63 47° 42′ 59′′/136° 35′ 26′′ Russia, Khabarovsky Terr., Khor River, clif P17 1 H64 54° 6′ 55′′/124° 36′ 53′′ Russia, Amur Terr., Skovorodinsky Distr., Solovyovsk, limestone clif P18 3 H65 50° 21′ 33′′/127° 4′ 4″ China, Heilongjiang, Heihe, stony slope P19 8 H66–H70 49° 15′ 17′′/140° 20′ 18′′ Russia, Khabarovsky Terr., Vaninskii Distr., sea shore P20 12 H71–H80 50° 40′ 17′′/87° 49′ 39′′ Russia, Altai Rep., Bashkaus Riv. P21 12 H81, H82 54° 58′ 54′′/83° 02′ 37′′ Russia, Novosibirsky Reg., Novosibirsk, Kluch–Kamyshenskoe plateau Outgroup 10 H83, H84 44° 04′ 09′′/131° 24′ 10′′ Russia, Primorsky Terr., Oktyabr’ski Distr., Fadeevka, Razdolnaya Riv., clif

Fig. 1 Map of sample sites for natural populations of Orostachys spinosa (21 populations, white circles). Location details and population codes (P) correspond to those in Table 1. The map was based on the image retrieved from: https​://maps-for-free.com/

1 3 84 Page 4 of 14 A. Yu. Nikulin et al. plants at least 10 m apart. Plant material was transferred to of genetic variation among populations, groups and indi- the laboratory and stored at − 80° C until extraction. Speci- viduals using Arlequin v.3.11 (Excofer et al. 2005). The mens were collected from public land; therefore, feld per- signifcance of the results was tested using a nonparametric mits were not required. permutation procedure with 1000 permutations. Mismatch distribution analysis was used to test the DNA extraction, polymerase chain reaction (PCR) hypothesis of demographic expansion of O. spinosa (Rogers and sequencing and Harpending 1992). The observed number of diferences between pairs of haplotypes was compared to the theoretical Total genomic DNA was extracted using DNeasy Plant distribution using a sudden (stepwise) expansion model with Mini Kit (Qiagen, Maryland, USA), in accordance with the the DnaSP5 v.5.10.1. A mismatch distribution test was per- manufacturer’s instructions. Amplifcation of three cpDNA formed for 16 populations of O. spinosa represented by ≥ 8 regions (trnH–psbA, trnQ–rps16, and rpl32–trnL) was per- accessions each. formed by PCR using previously reported universal primers To infer the divergence time between Orostachys line- (Shaw et al. 2005, 2007). ages, we used the cpDNA data set to conduct a dating anal- The PCR products were sequenced in both directions ysis with the BEAST software (Drummond et al. 2012). at the Instrumental Center of Biotechnology and Gene The analyses of the constancy of molecular evolution rate Engineering of FSCEATB FEB RAS using an ABI 3130 among lineages, estimation the divergence times and conf- genetic analyzer (Applied Biosystems, USA) with a Big- dence intervals were according Zhang et al. (2014a) and Li Dye terminator v. 3.1 sequencing kit (Applied Biosystems, et al. (2018). Chloroplast DNA substitution rates for most Maryland, USA) and the same primers used for PCR. All angiosperm species have been estimated to be in the range newly obtained sequences have been submitted to GenBank 1–3 × 10−9 substitutions per site per year (s/s/year) (Wolfe under the accession numbers LT222500–LT223120 for O. et al. 1987). As the fragments we used are non-coding spinosa and MT673848–MT673877 for O. japonica (Online regions of the cpDNA genome, we assumed an evolution- Resource 1). ary rate of 1.52 × 10−9 s/s/year for the plastid dataset and 10 years were used as an approximation for g (Yamane Data analysis et al. 2006; Li et al. 2018). We used GTR + G + I substitu- tion model, lognormal relaxed and strict molecular clocks Sequences were assembled with the Staden Package v.1.4 for the cpDNA data set, a Yule process for tree prior and (Bonfeld et al. 1995), aligned manually in the SeaView a UPGMA starting tree. We sampled all parameters once program (Galtier et al. 1996) and concatenated into a single every 10,000 generations from 100,000,000 MCMC gen- data matrix (Online Resource 2). The cpDNA haplotypes erations with a burn-in of 1000 generations. We then used were determined by nucleotide substitutions and indels of Tracer v.1.6 (Rambaut and Drummond 2007) to examine the aligned sequences with DnaSP5 v.5.10.1 package (Lib- convergence of chains to the stationary distribution, and the rado and Rozas 2009). A statistical parsimony haplotype net- analysis was repeated before combining the two independent work was obtained with TCS v. 1.21 (Clement et al. 2000) runs. FigTree v. 1.3.1 (Rambaut 2009) was used to display a by using the 95% connection probability limit and treating tree with ages for each node and their 95% highest posterior indels as single evolutionary events (Nagy et al. 2012). density (HPD). Inversions were manually reversed and retained in the data set. Orostachys japonica, representing a sister lineage of O. spinosa (Nikulin et al. 2015b) was used as an outgroup. Results Number of polymorphic sites (pS); haplotypes (nH); pri- vate haplotypes (nHu); haplotype diversity (h), nucleotide cpDNA sequences variation diversity per population (π) (Nei and Tajima 1983), and pairwise diferences were estimated using DnaSP5 v.5.10.1 A total of 609 sequences of three cpDNA intergenic spacers package. This software was also used to assess GST and NST were obtained from 203 plants representing 21 populations. values. GST only considers haplotype frequencies, whereas Sequence characteristics are given in Table 2. NST considers both haplotype frequencies and their genetic The sequences contained six homopolymeric stretches divergence. NST > GST usually indicates the presence of (fve in trnQ–rps16 and one in rpl32–trnL) ranging from phylogeographic structure, that is, the more frequent occur- 2 to 6 nucleotides that difered in length between popula- rence of closely related haplotypes in the same area than less tions and accessions. Due to the uncertain homology of the closely related haplotypes (Pons and Petit 1996). repeated nucleotides, these positions were removed from Analysis of molecular variance (AMOVA; Excofer et al. the dataset (Kelchner 2000). Also, a number of structural 1992) was applied to evaluate the hierarchical partitioning autapomorphies and synapomorphies were revealed in the

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Table 2 cpDNA polymorphism Characteristics trnH–psbA trnQ–rps16 rpl32–trnL in trnH–psbA, trnQ–rps16 and rpl32–trnL intergenic spacers of Number of sites (bp) 367 ± 1.3 1244 ± 7 614 ± 1.5 Orostachys spinosa GC-content (%) 31 29 32 Variable (polymorphic) sites 29 61 25 Parsimony informative sites 19 51 12 Conserved regions 1–57 1–87; 159–224; 226–292; 310–381; 1–76; 560–626; 628–706; 1016–1080 448–518; 535–621

analyzed sequences. In trnH–psbA, a 7 bp insertion (pos. The Eastern group comprised 27 haplotypes (92 acces- 62–68) was characteristic for the population from the Shan- sions) found in populations from the southeastern part of tar Islands (P4), and a 14 bp-long insertion (pos. 308–321) the O. spinosa range (P1, P2, P5, P7, P10, P15–P19; Figs. 1, was found in one accession in the P20 population from Altai 2). Haplotypes H9 and H10 from the Shantar Islands (P4) Mts. The populations P12 (Irkutsk Reg.), P13 and P14 (Altai and H8 from the Magadan Region (P3) were not included Mts.) shared a 4-nucleotide insertion in trnQ–rps16 spacer in this group as these were expected based on their eastern (pos. 1194–1197). The former population was also distinct origin. In the Eastern group, most haplotypes showed a small in a duplication of 13 bp motif in the same locus. Insertion amount of divergence from each other (0/1–2 mutations) of seven nucleotides marked populations P6 and P9 from the even in populations characterized by a large representation/ Southern Urals. In the rpl32–trnL spacer, a notable 49 bp sample size (P1, P2, P7, P15, P16, and P19). Only two popu- inversion was revealed. It was shared by plants from popu- lations, P5 (H7, H11–H13) and P10 (H27–H30), comprised lation P19 (Khabarovsky Terr.), and some specimens from divergent haplotypes. Alternative links between the haplo- populations P6, P10, P13, and P14. The inversion was manu- types (loop structures in the network) in both groups sug- ally reverse-complemented and retained in the data matrix. gested possible homoplasy. Another inversion was observed in the trnH–psbA spacer The Western group (P3, P4, P6, P8, P9, P11–P14, (GAAA ↔ TTTC; pos. 256–259). Due to its likely homo- P20–P21) included a comparable number of accessions plastic nature, this motive was excluded from the data set. (111) but was characterized by more than a twice higher number of haplotypes (55; Fig. 2). The proportion of private haplotypes in the Western group was also high (39 vs 16 in Haplotype genealogy and distribution the Eastern group) due to exceptional haplotype diversity observed in three populations from the Altai Mountains The nucleotide substitutions and indel variations revealed described above. The Altai haplotypes H39–H48 (P13), 82 cpDNA haplotypes in 21 O. spinosa populations. The H49–H60 (P14), and H71–H80 (P20) formed several majority of these haplotypes (55; 67%) were found in a sin- “clouds” composed by representatives of diferent popula- gle specimen (private haplotypes), and almost no haplotype tions. Other populations in the Western group (P4, P6, P8, sharing between populations was observed. Only for the P9, P11, P21) had typical structures with a dominant and a geographically close (< 100 km apart) P2 and P5 popula- few allied rare/private haplotypes (Fig. 2). tions from the Primorsky Territory and P11 and P12 from In order to test for the presence of a phylogeographic the Irkutsk Region shared haplotypes were detected: H7 and structure, we calculated and compared the GST and NST indi- H31, respectively (Fig. 2). In all populations represented ces for the cpDNA dataset. It was found that GST = 0.501 and by more than four accessions, two or more haplotypes (up NST = 0.822 (p < 0.01). A signifcantly higher value for NST to 12) were observed. The highest diversity was found in than GST (p < 0.05) indicated the presence of the sampled populations from Altai Mountains (P13, P14, P20; 36 acces- haplotypes’ phylogeographic structure. sions), yielding 32 haplotypes (Fig. 2). The statistical parsimony network (Fig. 2) revealed two Genetic diversity and diferentiation of populations groups of haplotypes, called Western and Eastern in this study, separated by 11 mutation steps. Of these, two nucle- Basic parameters of genetic diversity were evaluated otide substitutions in the intergenic spacer trnQ–rps16: by Arlequin v. 3.11 are shown in Table 3. In the studied A → G (pos. 141) and A → T (pos. 1136) unambiguously populations, haplotype and nucleotide diversity (h and π, distinguished Eastern (A and A) and Western (G and T) respectively) and mean number of pairwise differences groups. Orostachys japonica was closer to the Eastern group (Pi) were unevenly partitioned and ranged 0.0000–1.0000; and was separated from the P10 by 8 mutation steps. 0.0000–0.0143; 0.0000–32.651515, respectively (Table 3).

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Fig. 2 Statistical parsimony network of cpDNA haplotypes constructed using TCS v. 1.21. Filled circles indicate the haplotypes, the numbers in the circles identify the haplotypes (Table 1), with the size of each circle proportional to the observed frequency. The colors within the circles correspond to the diferent populations. Black dots represent missing haplo- types. E and W letters denote the Eastern and Western groups of haplotypes

The highest genetic diversity values were observed in all In order to test the hypothesis of demographic expan- populations from the Altai region (P13, P14, and P20). Four sion of O. spinosa, a mismatch distribution test was per- populations revealed no haplotype or nucleotide diversity formed for 16 populations represented by ≥ 8 accessions either because they were represented by a single speci- each (Fig. 3). The Eastern group’s plots showed mostly men only (P17) or were due to monomorphic nucleotide unimodal distribution patterns (Fig. 3a), suggesting recent sequences (P3, P16, and P18). population expansion (Harpending et al. 1998), while in

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Table 3 Within-population genetic diversity of Orostachys spinosa populations Pop.# Sample size Loci # pS nH nHu h (SD) π (SD) Pi

P1 12 2229 5 4 2 0.6970 (0.0904) 0.001040 (0.000688) 2.318182 (1.362033) P2 12 2220 2 3 1 0.4394 (0.1581) 0.000321 (0.000291) 0.712121 (0.573042) P3 2 2226 0 1 0 0.0000 (0.0000) 0.000000 (0.000000) 0.000000 (0.000000) P4 3 2232 10 2 1 0.6667 (0.3143) 0.002987 (0.002418) 6.666667 (4.327835) P5 12 2231 23 3 2 0.5606 (0.1540) 0.002615 (0.001514) 5.833333 (2.999650) P6 12 2230 7 3 1 0.5606 (0.1540) 0.000584 (0.000441) 1.303030 (0.873753) P7 13 2220 5 3 2 0.2949 (0.1558) 0.000347 (0.000304) 0.769231 (0.600139) P8 12 2222 13 5 4 0.6818 (0.1482) 0.001514 (0.000939) 3.363636 (1.853185) P9 10 2230 4 2 0 0.4667 (0.1318) 0.000837 (0.000590) 1.866667 (1.163922) P10 15 2227 27 4 3 0.3714 (0.1532) 0.002540 (0.001447) 5.657143 (2.874093) P11 12 2240 4 2 0 0.1667 (0.1343) 0.000298 (0.000276) 0.666667 (0.548651) P12 12 2247 48 6 4 0.8939 (0.0777) 0.008152 (0.004384) 18.318182 (8.749109) P13 12 2265 81 10 8 0.9848 (0.0403) 0.013914 (0.007365) 31.515152 (14.816008) P14 12 2269 78 12 12 1.0000 (0.0340) 0.014390 (0.007611) 32.651515 (15.338317) P15 12 2224 3 2 1 0.3182 (0.1637) 0.000225 (0.000231) 0.500000 (0.456254) P16 4 2221 0 1 0 0.0000 (0.0000) 0.000000 (0.000000) 0.000000 (0.000000) P17 1 2221 0 1 1 0.0000 (0.0000) 0.000000 (0.000000) 0.000000 (0.000000) P18 3 2221 0 1 0 0.0000 (0.0000) 0.000000 (0.000000) 0.000000 (0.000000) P19 12 2225 9 5 3 0.8571 (0.1083) 0.001814 (0.001153) 4.035714 (2.251321) P20 12 2250 58 10 8 0.9697 (0.0443) 0.006505 (0.003531) 14.636364 (7.055687) P21 12 2226 8 2 1 0.1667 (0.1343) 0.000599 (0.000449) 1.333333 (0.888668) pS no. of polymorphic sites, nH no. of haplotypes, nHu no. of private haplotypes in sample, h gene diversity, π nucleotide diversity, Pi mean number of pairwise diferences, SD standard deviation. Abbreviations correspond to those in Table 1

the Western group, the mismatch distribution plots were Discussion generally multimodal, indicating a constant population size over the time (Fig. 3b). We revealed a clear spatial pattern of plastid DNA haplo- Analysis of molecular variance in our data set showed types in O. spinosa, a crassulacean species characterized that 76.36% of the genetic variation was due to differ- by a wide distribution range that spans from the Siberia’s ences between populations (Table 4). For the among- Pacifc coasts to the Southern Urals and grows under vari- group analysis, only 21.6% of the total genetic variance ous environmental conditions. This pattern is consistent correlated within populations of one group. The major- with the results of our previous ITS rDNA sequence com- ity of molecular variance was attributable to differences parisons that suggest a split between western and eastern among populations within groups (62.12% of the total), O. spinosa accessions (Nikulin et al. 2015a, b). and those differences were also significant (p < 0,001). Sampling three non-coding cpDNA regions from 203 The dating analysis showed (Online Resource 3) that individuals (21 populations) across the species range, we the Western and Eastern groups of O. spinosa popula- identifed two divergent haplotype groups: the Eastern and tions diverged circa 3.6 Mya (Pliocene). Further diversi- the Western. The Eastern group comprised populations fication in the Western group took place earlier then in from the southeastern part of the species distribution area the Eastern group (3.1 Mya and 2.26 Mya, respectively). only (such as the Amur River basin except its upper part These estimates are generally consistent with those based (P8), and the western coast of the Sea of Japan, while on ITS rDNA data except for a much earlier date for the populations from the Southern Urals, Southern Siberia, beginning of divergence in the Eastern group (1.1 Mya; Baikal area, and the western coast of the Sea of Okhotsk Nikulin et al. 2015b). belonged to the Western group (Figs. 1, 2). It is likely that continental areas of the Northeast Asia (Yakutia and

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a Eastern group: P1 P2 P5

P7 P10 P15

P19

b Western group: P6 P8 P9

P11 P12 P13

P14 P20 P21

Fig. 3 Mismatch distribution established for Orostachys spinosa populations of the Eastern (a) and Western (b) groups. The dashed line shows observed values; the solid line represents expected values under a model of sudden population expansion (Rogers and Harpending 1992)

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Table 4 Hierarchical AMOVA based on three cpDNA intergenic haplotype sharing, high haplotype diversity (nH), and high spacers of 21 populations of Orostachys spinosa proportion of private haplotypes (Table 3) resulted in high Source of vari- df SS VC PV Fixation index estimates of inter-populational diferentiation (NST > GST, ation 0.822 > 0.501; p < 0.05) corroborated by the fact that high All populations studied total genetic diversity in the species was due to diferences among populations (FST = 0,76364). These results suggest Among popula- 24 2509.395 12.35077 76.36 FST = 0.76364* tions the presence of a distinct phylogeographical structure (Pons Within popula- 182 695.740 3.82275 23.64 and Petit 1996; Avise 2004). Based on cpDNA data, the tions genetic structure of O. spinosa was characterized by gen- Total 206 3205.135 16.17352 erally low genetic variation within populations and high 2 groups (Western and Eastern) genetic diferentiation between populations (Table 4), imply-

Among groups 1 391.916 2.88773 16.30 FCT = 0.16302* ing a limited gene fow between populations via the seeds

Among popula- 23 2117.479 11.00355 62.12 FSC = 0.74216* (Gao et al. 2009). Such genetic patterns could be due to a tions lack of long-distance dispersal (the seeds drop close to the F Within popula- 182 695.740 3.82275 21.58 ST = 0.78420* parent plant) and discontinuous distribution on scattered tions rock crevices, dry slopes, and similar xeric habitats. Gener- Total 206 3205.135 17.71402 ally, it is characteristic for many plant species having wide df degrees of freedom, SS sum of squares, VC variance components, distribution ranges (Opgenoorth et al. 2010; Zhang et al. PV F percentage of variation, ST fxation index within populations, 2010; Gao et al. 2012, 2016). F F SC fxation index among populations within groups, CT fxation Most O. spinosa populations consisted of a dominant and index among groups a few derived rare/private haplotypes. This reduced genetic Signifcance levels are based on 1000 permutations, *p < 0.001 variation could be due to their relatively recent origin and founder efects or genetic bottlenecks (e.g., Dlugosch and Magadan Territory) are also inhabited by members of this Parker 2008; Lachmuth et al. 2010; Uller and Leimu 2011; group. Long individual evolutionary histories (3.6 Mya) but see Roman and Darling 2007). Contrasting patterns shaped genetic distinctness of the Western and Eastern were reveled in three populations from Altai (P13, P14, and groups of populations. Geographic structuring within O. P20) with nearly every specimen carrying a private haplo- spinosa is further supported by the diferences between the type (Fig. 2). One population from Baikal (P12) was also groups in genetic diversity (e.g., the number of polymor- characterized by conspicuously elevated values of genetic phic sites and p-distances calculated for the Western and diversity (Table 3). Retention of high ancestral genetic vari- Eastern groups difer almost twofold—86/0.0037 ± 0.0008 ability is the most likely reason for such unusual intrapopula- vs. 42/0.0061 ± 0.0009, respectively) and their relatively tion diversity. The secondary contact zone after postglacial high FCT value (0.16302), which suggest low gene fow populations’ expansion could have also contributed to the rates. increased diferentiation observed here (Taberlet 1998; Petit Our sampling covered a wide range of environments et al. 2003). These assumptions are supported by the fact that that greatly difered in climate characteristics, landscapes, the mountains of Southern Siberia are important refuges for and edaphic conditions; therefore, genetic diferentiation many plants and animal species with only a fraction of the revealed in O. spinosa could be the result of several evo- territory covered by glaciers during the last glaciation maxi- lutionary factors, such as distance-associated isolation, mum (Ono et al. 2004; Blyakharchuk et al. 2007; Lehm- postglacial migrations, and adaptive diferentiation to local kuhl et al. 2011; Blomdin et al. 2016; Chernykh et al. 2014; environments. Strong geographic isolation and low genetic Řičánková et al. 2014; Hais et al. 2015; Chytrý et al. 2017). diversity within populations were documented in some other crassulacean taxa occurring in alpine habitats and mostly Origin area and migration routs attributed to their complex quaternary paleographic and paleoclimatic history (Rhodiola spp., DeChaine and Martin Our data on genetic structure of O. spinosa provided new 2005). insights into evolutionary history and migration routes of the species. Traditionally, it was believed that the genus Haplotype spatial pattern and evolutionary factor Orostachys has East Asian origins similar to other mem- bers of the Hylotelephium clade (tribe Telephieae (’t Hart) Comparisons of the nucleotide sequences of three cpDNA Ohba and Thiede) since most of its species are restricted intergenic spacers revealed a high level of genetic diversity to this region (Ohba 1978; Byalt 1999; Mort et al. 2001; in the species (see Results). Diferences in haplotype compo- Mayuzumi and Ohba 2004; Gontcharova 2006). However, sition among O. spinosa populations with only two cases of unlike the genus Rhodiola L., whose origin was temporally

1 3 84 Page 10 of 14 A. Yu. Nikulin et al. and spatially associated with the Himalayas and Qinghai- by introgression from co-distributed O. thyrsifora although Tibet Plateau uplift (Ohba 1987; Gao et al. 2009; Zhang no data on hybridization between these species exists. et al. 2014b), the history of Orostachys remains poorly Much earlier diversifcation in the Western lineage of O. understood. spinosa and high likely ancestral genetic diversity observed The Himalayan origin of the Hylotelephium clade is in the populations from Altai Mountains (Table 3) chal- generally supported by the molecular studies placing the lenges the East Asian origin of the species in favor of South genus Sinocrassula A. Berger, distributed in the Himala- Siberian mountains. Late Miocene origin of the species tem- yas and southwestern China, at the base of Hylotelephium porally and credibly spatially coincided with Altai orogen- clade (Mayuzumi and Ohba 2004; Nikulin et al. 2015b). It esis that could prompt species adaptation to a range of eco- was expected that Orostachys’ ancestor moved northeast- logical conditions and its distribution. There were two major ward to Northeast China, the Korean peninsula, and adja- directions of the species dispersal from the Altai: (1) west- cent regions of Russia on which the genus diversifed (Byalt ward to the Southern Urals along the Kazakh Uplands and 1999). Most of its species are confned to these territories; (2) eastward toward the coast of the Pacifc Ocean (Fig. 4). only O. spinosa occurs up to the Southern Urals, and O. In both cases, mountain ridges could serve as corridors for thyrsifora Fisch. predominantly resides in Central Asia. The the species that is a poor competitor for light and substrate and distribution pattern of the latter taxon prompted Gontch- generally restricted to rock crevices or dry stony places with arova (2006) to hypothesize an alternative migration route poor vegetation cover. As it moved eastward, the species fol- for O. thyrsifora from the Himalayas to Southern Siberia lowed the mountains of Southern Siberia (Sayan Mountains) via the Central Asian mountain corridor (Pamir-Alai Moun- to Baikal. Further dispersal was likely prompted by the Quater- tains). Yet, in her opinion, O. spinosa originated in East nary uplift of the Stanovoj Ridge that could provide the species Asia and later expanded its distribution westward. Recent with the access to the coast of the Sea of Okhotsk. Following phylogenetic analyses (Nikulin et al. 2015a,b) did not reveal the Dzhugdzhur Ridge north, O. spinosa was able to reach the close relationships between O. spinosa and O. thyrsifora, Kolyma Mountains and adjacent areas, which is its current having largely overlapping ranges in Mongolia, Altai, and northern distribution limit. But during the Late Pleistocene, westward to Urals that suggests their independent appear- the species was a part of the xerocryophilous plant communi- ance in this region. Moreover, Meterostachys sikokiana ties of the Central Taymyr Peninsula (Kienast et al. 2001). The and O. thyrsifora diverged ca. 3 Mya earlier than specia- Western group was less successful with the southward disper- tion started in O. spinosa/O. chanetii/O. japonica subclade sal at the eastern part of the range, and its representatives have (Nikulin et al. 2015b). The high genetic diversity revealed in not been encountered at the northern part of the Amur basin so the Altai populations of O. spinosa could partially be caused far (Fig. 4). We were unable to confdently locate the area of

– supposed center of O. spinosa origin and ancestral diversity (Altai Mts.) – hypothetical distribution routes of O. spinosa – estimated boundary between the Western and Eastern groups of O. spinosa populations

E

500 km

Fig. 4 Hypothetical scheme of Orostachys spinosa range expansion based on the cpDNA data. The map was based on the image retrieved from: https​://www.googl​e.ru/maps

1 3 Orostachys spinosa (Crassulaceae) origin and diversifcation Page 11 of 14 84 the Western and Eastern groups’ divergence. Currently, their about its East Asian origin, our results suggested that the representatives could be in contact at the upper part of Amur species could initially diversify in mountains of South- basin (Argun and Shilka basins) and likely Eastern Mongo- ern Siberia (Altai). Unusually, high genetic diversity was lia in which O. spinosa is frequently found. However, in the revealed in populations from the Altai Mountains and some haplotype network, the Eastern and the Western groups are adjacent areas, and this diversity was attributed to ancestral connected through the most eastern and the most diverse popu- genetic variability. In the majority of populations sampled lation P19 and a pool of haplotypes from Altai Mountains, across the species distribution range, restricted genetic vari- respectively (Fig. 3). ation was observed likely due to their relatively recent origin According to our feld observations in the population sam- and founder efects or genetic bottlenecks. We revealed that pled, analysis of a large image gallery (http://www.plantarium​ ​ populations in the southeastern part of the species range .ru/page/view/item/26045​.html) and molecular data, we can (middle and lower basin of Amur River and the Sea of conclude that the distinct morphotypes observed in O. spinosa Japan cost) comprise a divergent (Eastern) group that was (individual monocarpic rosettes versus numerous crowded unambiguously characterized by two marker substitutions in vegetative rosettes forming a cushion or crust) are not explic- trnQ–rps16 IGS. Molecular dating suggests a more recent itly linked to a specifc genotype/haplotype although generally diversifcation in the Eastern group that also supports west- associated with the Eastern and Western groups. Plants having ern origins of O. spinosa. cushion habit (thought to be a result of adaptation to a xeric condition) were observed in the coastal populations P4 (West- Acknowledgements We thank Shamil Abdullin, Vadim Bakalin, Vik- tor Bogatov, Roman Dudkin, Konstantin Kiselev, Alexandra Dubrovina, ern group) and P19 (Eastern group) that are neither moisture Yuri Ovchinnikov, Valery Shokhrin, Valentina Shokhrina, Dmitry limited nor afected by low winter temperatures typical for Sidorov and Valentin Yakubov for sampling natural populations of O. alpine and arctic habitats. In the Eastern group agglomera- spinosa; Marko Dobos for sharing his personal Orostachys collection tions of vegetative rosettes connected by stolons are not limited and Sun Yan for assistance during sampling in North-Eastern China. to the Tatar Strait coast but also typical for populations from Authors’ contribution AN and AG conceived the paper with VN. AN the Sikhote-Aline Mountains and lower Amur River current, performed the analyses and wrote the manuscript under the guidance inhabiting gravel slopes and rock crevices as well as at the of AG and with critical reviews, editing and contributions from VN. western edge of the group distribution area (P15, Inner Mon- golia). Contrasting environmental conditions at these localities Funding The study was supported by Russian Foundation for Basic suggest that diferent ecological factors can lead to a conver- Research according to the research Project No. 18-34-00436. gent cushion-like habit appearance in O. spinosa. Morphologi- cal convergence toward such a habit was also observed in some Compliance with ethical standards populations of closely related O. japonica having overlapping Conflict of interest The authors declare that they have no confict of with the Eastern group distribution (Gontcharova 2006). interest. Distinctive genetic structures and geographical ranges of the Western and Eastern groups of populations revealed in Information on Electronic Supplementary Mate- O. spinosa (Nikulin et al. 2015a; present study) may call for rial their taxonomic recognition either as species or intraspecifc taxa. There is no any evidence for the existence of repro- Online Resource 1. GenBank accession numbers of the cpDNA se- ductive barriers between these lineages and their diver- quences. Online Resource 2. Alignment used in analyses. gence time (3.6 Mya) falls within the range typical for most Online Resource 3. Bayesian chronogram of cladogenesis of Orostach- intraspecifc taxa of herbaceous plants (Levin and Samuel ys spinosa populations. 2017). Thus, most likely we deal with intraspecifc diver- gence. At the moment, we refrain from making a formal taxonomic proposal about this, pending more molecular and References particularly morphological data that allow precise discrimi- nation of O. spinosa intraspecifc taxa based on phenotypic Abbott RJ, Smith LC, Milne RI, Crawford RM, Wolf K, Balfour J characters. (2000) Molecular analysis of plant migration and refugia in the Arctic. Science 289:1343–1346. https​://doi.org/10.1126/scien​ ce.289.5483.1343 Allen GA, Marr KL, McCormick LJ, Hebda RJ (2012) The impact Conclusion of Pleistocene climate change on an ancient arctic–alpine plant: multiple lineages of disparate history in Oxyria digyna. 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