Molecular Phylogenetics and Evolution 135 (2019) 45–61

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Molecular Phylogenetics and Evolution

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Evolutionary history of the Pasque-flowers (Pulsatilla, Ranunculaceae): Molecular phylogenetics, systematics and rDNA evolution T ⁎ Gábor Sramkóa,b, Levente Laczkób, Polina A. Volkovac,d, Richard M. Batemane, Jelena Mlinarecf, a MTA-DE “Lendület” Evolutionary Phylogenomics Research Group, Egyetem tér 1., H-4032 Debrecen, Hungary b Department of Botany, University of Debrecen, Egyetem tér 1., H-4032 Debrecen, Hungary c Papanin Institute for Biology of Inland Waters RAS, 152742, Borok, Nekouz District, Yaroslavl Region, Russia d Moscow Grammar School in the South-West No. 1543, 26 Bakinskikh Komissarov Street, 3–5, 119571 Moscow, Russia e Jodrell Laboratory, Royal Botanic Gardens Kew, Richmond, Surrey TW9 3NJ, UK f Division of Molecular Biology, Department of Biology, Faculty of Science, Horvatovac 102a, HR-10000 Zagreb, Croatia

ARTICLE INFO ABSTRACT

Keywords: Pulsatilla (Anemoneae, Ranunculaceae) is sister to Anemone s.s. and contains ca 40 perennial species of con- Aridification siderable horticultural and medical importance. We sequenced 31 of those species, plus nine subspecies, two Central Asian origin cultivars and six outgroups, for two nuclear regions (high-copy nrITS and low-copy MLH1) and three plastid fi Classi cation regions (rbcL, accD–psaI, trnL intron) in order to generate the first comprehensive species-level phylogeny of the Dated phylogeny genus. Phylogenetic trees were constructed using both concatenation-based (maximum likelihood and Bayesian rDNA inference) and coalescence methods. The better supported among the internal nodes were subjected to molecular Steppe development clock dating and ancestral area reconstruction, and karyotypic characters identified by us using Fluorescence In Situ Hybridization were mapped across the tree. The preferred species tree from the coalescence analysis formed the basis of a new infrageneric classification based on monophyly plus degree of divergence. The earliest di- vergent of the three subgenera, Kostyczewianae, is represented by only a single species that is morphologically intermediate between Anemone s.s. and ‘core’ Pulsatilla. Subgenus Pulsatilla is considerably richer in species than its sister subgenus Preonanthus and contains three monophyletic sections. Species possessing nodding flowers and pectinately dissected leaves are phylogenetically derived compared with groups possessing erect flowers and palmately lobed leaves. Pulsatilla separated from Anemone s.s. at ca 25 Ma. Our results indicate a central Asian mountain origin of the genus and an initial diversification correlated with late Tertiary global cooling plus regional mountain uplift, aridification and consequent expansion of grasslands. The more rapid and extensive diversification within subgenus Pulsatilla began at ca 3 Ma and continued throughout the Quaternary, driven not only by major perturbations in global climate but also by well-documented polyploidy.

1. Introduction 1.1. Phylogenetics and classification of tribe Anemoneae

We have reconstructed the phylogeny of Pulsatilla Mill. (Pasque- Monophyly of tribe Anemoneae has been demonstrated by several flowers), a genus now classified alongside other horticulturally im- molecular phylogenetic studies that differed considerably in the portant genera such as Anemone, Hepatica and Clematis in tribe number of species included and the number and nature of genic regions Anemoneae, subfamily Ranunculoideae, family Ranunculaceae sequenced (Hoot and Palmer, 1994; Hoot, 1995; Ro et al., 1997; Cai (Tamura, 1993). Its ca 40 species of perennial herbs span the north- et al., 2010; Hoot et al., 2012; Cossard et al., 2016; Lehtonen et al., temperate, subarctic and montane zones from Europe (nine species) to 2016; Wang et al., 2016; Jiang et al., 2017; Liu et al., 2018). Critical North America (two species), but are concentrated in Asia (29 species: comparison of the resulting topologies suggests the existence of three Aichele and Schwegleb, 1957). Most of the species prefer grassland main clades: (i) Clematis s.l. plus Anemonclema, (ii) Anemone s.s. plus habitats. Pulsatilla plus three further small monophyletic genera (Barneoudia, Knowltonia, Oreithales) that are better aggregated as a single genus Knowltonia, and (iii) Hepatica plus Anemonastrum (=Anemone subgenus

⁎ Corresponding author. E-mail address: [email protected] (J. Mlinarec). https://doi.org/10.1016/j.ympev.2019.02.015 Received 7 November 2018; Received in revised form 25 January 2019; Accepted 17 February 2019 Available online 01 March 2019 1055-7903/ © 2019 Elsevier Inc. All rights reserved. G. Sramkó, et al. Molecular Phylogenetics and Evolution 135 (2019) 45–61

Anemonidium). Cytogenetically, groups (i) and (ii) have x = 7 whereas Table 1 group (iii) has the apparently derived number of x = 8. However, no Overview of previous comprehensive taxonomic treatments of the genus consensus has yet been achieved regarding the relative phylogenetic Pulsatilla. positions of these three groups. Aichele and Schwegleb Tamura (1995) Grey-Wilson (2014) This phylogenetic ambiguity has major implications for classifica- (1957) tion within tribe Anemoneae, as it has become a battle-ground between ‘lumpers’ favouring a broad, inclusive genus Anemone (Hoot et al., P. sect. Pulsatilla P. subg. Pulsatilla P. subg. Pulsatilla P. subsect. Pratenses P. sect. Pulsatilla P. sect. Pulsatilla 2012) and the majority of taxonomic users, including horticulturalists P. pratensis P. subsect. P. ser. Pratenses and medicinal herbalists, who understandably wish to retain such Pulsatilla prominent genera as Clematis, Hepatica and Pulsatilla. In fact, a utili- P. montana P. ser. Pratenses P. pratensis s.l. tarian classification consistent with monophyly can be achieved simply P. rubra P. pratensis P. montana P. montana P. rubra by treating the x = 7 clade formerly attributed to Anemone s.l. as the P. subsect. Vulgares P. rubra P. ser. Pulsatilla genus Anemonastrum, as advocated by Mosyakin (2016). Within group P. vulgaris P. nigricans P. vulgaris (ii), Pulsatilla has been placed as sister to either Anemone s.s. (Hoot P. grandis P. ser. Pulsatilla P. grandis et al., 2012)orKnowltonia (Jiang et al., 2017), but in neither case with P. taurica P. vulgaris P. halleri strong statistical support. Fortunately, all previous molecular phyloge- P. slavica P. grandis P. turczaninovii P. velezensis P. halleri P. ser. Bungeanae netic studies agreed regarding the unequivocal monophyly of genus P. styriaca P. turczaninovii P. bungeana Pulsatilla. P. halleri P. ajanensis P. magadenensis P. ser. Vernales P. millefolium 1.2. Phylogenetics and classification of genus Pulsatilla P. vernalis P. sukaczewii P. subsect. Patentes P. ser. Patentes P. tenuiloba P. patens P. patens P. ser. Albanae In spite of its horticultural (Grey-Wilson, 2014) and medical im- P. flavescens P. flavescens P. albana portance (Cheng et al., 2008; Xu et al., 2012; Ling et al., 2016), and the P. nuttalliana P. nuttalliana P. ambigua conservation interest expressed in many of its species, Pulsatilla has P. multifida P. andina received only limited attention from molecular phylogeneticists. P. subsect. Albanae P. ser. Albanae P. armena P. albana P. albana P. campanella Zetzsche (2004) studied 12 species of the P. alpina complex by se- P. campanella P. campanella P. wallichiana quencing nrITS, a genic region also used in a study of hybridization P. wallichiana P. wallichiana P. georgica between six Far East members of the genus by Sun et al. (2014). P. millefolium P. bungeana P. violacea Ronikier et al. (2008) assessed plastid sequence variation among ten P. regeliana P. millefolium P. sect. Semicampanaria European Pulsatilla species to explore their phylogeography, and Wang P. turczaninovii P. regeliana P. ser. et al. (2016) sequenced 12 Pulsatilla species but for a fundamentally Semicampanaria ecological study. More recently, Jiang et al. (2017) included 17 Pulsa- P. bungeana P. ser. P. cernua tilla species in a broader study intended to reconstruct phylogenetic Tatewakianae relationships within tribe Anemoneae. Despite using nrITS plus six P. ajanensis P. tatewakii P. chinensis P. sugawarae P. dahurica plastid markers, they found only limited phylogenetic resolution within P. subsect. Vernales P. ser. Cernuae P. tongkangensis Pulsatilla. P. vernalis P. cernua P. ser. Vernales Although several infrageneric classi fications have been proposed for P. dahurica P. vernalis Pulsatilla, even the three most recent (Aichele and Schwegleb, 1957; P. sect. Semicampanaria P. ser. Chinenses P. sect. Patentes P. subsect. Cernuae P. chinensis P. ser. Patentes Tamura, 1995; Grey-Wilson, 2014) were based on traditional her- P. cernua P. patens s.l. barium-based taxonomy with some support from studies of anatomy P. dahurica P. ser. and chromosomes (Table 1). The only common element is the division Tatekawianae of the genus – at various taxonomic ranks – into three main groups: (i) P. subsect. Chinenses P. ajanensis Kostyczewianae (containing only the central Asian P. kostyczewii, which P. chinensis P. sugawarai P. sect. Iostemon P. tatewakii resembles Anemone s.s. in more morphological characters than do other P. subsect. P. subsect. P. subg. Pulsatilla species), (ii) Preonanthus (arctic-alpine species of Eurasia and Kostyczewianae Kostyczewianae Kostyczewianae North America), and (iii) “core Pulsatilla” (most of the widely re- P. kostyczewii P. kostyczewii P. kostyczewii cognized species). None of the traditional infrageneric classifications of P. subg. Preonanthus the genus Pulsatilla has previously been evaluated explicitly against a P. sect. Preonanthus P. sect. P. sect. molecular phylogenetic topology. Preonanthus Preonanthus P. subsect. Alpinae P. alpina P. alpina 1.3. Karyotypic background P. alpina P. alba P. aurea P. alba P. aurea P. occidentalis P. aurea P. occidentalis P. scherfelii Karyotypic changes such as polyploidy, dysploidy and chromosomal P. occidentalis P. apiifolia rearrangements are widely acknowledged as important evolutionary P. sect. Preonanthopsis P. sect. P. sect. forces shaping plant genomes and underpinning angiosperm diversity Preonanthopsis Preonanthopsis (Cai et al., 2006; Weiss-Schneeweiss et al., 2007; Leitch and Leitch, P. subsect. Taraoianae P. taraoi P. taraoi P. taraoi P. nipponica P. nipponica 2008). Polyploidy can play a particularly significant role in plant spe- P. subg. P. subg. Miyakea ciation due to evolutionary advantages attributed to the plasticity of Miyakea plant genomes and to increased genetic variability, generating in- P. integrifolia P. integrifolia dividuals capable of exploiting new niches (Soltis et al., 2015). Poly- ploidy is linked to numerous epigenetic/genomic changes such as chromosome rearrangements, transposable element mobilization, gene proven an excellent tool for tracing karyotypic changes in many plant silencing and genome downsizing, a set of processes that are collec- groups, including Anemone (Mlinarec et al., 2012a). The rDNA pheno- fi tively termed ‘diploidization’ (e.g., Schnable et al., 2011; Ainouche types are often species-speci c, thereby providing valuable cytotaxo- et al., 2012). Mapping of tandemly-arrayed repetitive sequences such as nomic markers (Lan and Albert, 2011; Mlinarec et al., 2012b). the 5S and 18S-5.8S-26S (=35S) ribosomal RNA genes (rDNA) has Although chromosome numbers have played an important role in

46 G. Sramkó, et al. Molecular Phylogenetics and Evolution 135 (2019) 45–61 the systematics of tribe Anemoneae, the base chromosome number contrasting rates of molecular evolution: (iii) the plastid gene encoding within Pulsatilla is uniformly x =8(Baumberger, 1971; Tamura, 1995; the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit

Mlinarec et al., 2012b). More detailed molecular cytogenetic studies of (rbcL) (Palmer et al., 1988), (iv) the trnLUAA (type I) intron of the trnT- Pulsatilla are limited to two works (Lee et al., 2005; Mlinarec et al., trnF region (Taberlet et al., 1991), and (v) the intergenic spacer se- 2012b) that together report data for only eight species. The typical parating the genes acetyl-CoA carboxylase beta subunit and the pho- karyotype consists of four pairs of metacentric, one pair of submeta- tosystem I subunit VIII (accD–psaI). centric or metacentric, and three pairs of acrocentric chromosomes, where the second pair carries a satellite. Although the majority of 2.3. DNA extraction, PCR conditions and sequencing Pulsatilla taxa are diploid (2n =2x = 16), some clades are reliably tetraploid and multiple ploidies have occasionally been observed within Genomic DNA was isolated from leaves using a modified CTAB species; for example, P. montana contains diploid, tetraploid and hex- protocol (Doyle and Doyle, 1987). In brief, the volumes were scaled aploid races (Baumberger, 1971; Tamura, 1995). down to microliters, the CTAB-lysed, grounded tissue was treated with chloroform: isoamyl alcohol twice, and then the DNA was precipitated 1.4. Present study with equal volume of isopropanol and 0.08 V ammonium acetate. The DNA pellet was washed twice with 70% ethanol, air-dried and re-dis- Here, we provide a comprehensive molecular phylogenetic frame- solved in 10 mM Tris (for a detailed protocol see Sramkó et al., 2014). work for 31 of the 38 Pulsatilla species listed in the monograph of Grey- The nrITS region was amplified using the primers ITS1A (Gulyás Wilson (2014) plus nine subspecies (four subspecies of P. pratensis and et al., 2005) and ITS4 (White et al., 1990), whereas the plastid regions four subspecies of P. patens plus one of P. halleri), based on two nuclear were amplified using universal plant plastid primers (Taberlet et al., regions (one low copy) and three plastid regions. We also add con- 1991; Sang et al., 1997; Small et al., 1998; Shaw et al., 2005; Shaw siderably to the range of karyotypes available for the genus, and map et al., 2007). All PCRs were run under the conditions as specified for those karyotypes across the preferred phylogeny. We use that phylo- nrITS and plastid regions by Sramkó et al. (2014). For the MLH1 region genetic hypothesis to: (i) develop a revised supraspecific classification we devised new primers based on the available whole-genome sequence of the genus using the modern principles of monophyly and relative of Aquilegia coerulea v.3.1 (available from the “Aquilegia coerulea branch length (Table 1); (ii) estimate divergence times of the better- Genome Sequencing Project, http://phytozome.jgi.doe.gov/”), which supported clades; (iii) infer ancestral areas of those major clades; and we compared with transcripts of this gene in Ranunculus muricatus (iv) interpret karyotype evolution within the genus. (GenBank accession number KM823030) and Anemone flaccida In order to maximize intelligibility, we employ our revised classi- (GEKW01014092). Our novel primers (MLH1_520fw, 5′-TTA GAG GAG fication of subgenera, sections and series (Table 1, Fig. 2) throughout AAG CAT TGG C-3′ and MLH1_900rv, 5′-GTC TTC CTT CGA GCC GTC- the paper, noting that it differs from all previous classification at the 3′) successfully amplified a ca 390 bp-long region of the MLH1 gene taxonomic levels of section and series (Table 1). located on the chromosome 6 of Aquilegia coerulea v.3.1 between bases 673,282 and 673,669. This region includes the second exon of the gene 2. Material and methods (where the forward primer anchors), the whole third exon, the fourth exon of the gene (where the reverse primer anchors), and two introns 2.1. Taxon sampling separating the above exons. PCR amplification used Phusion Green Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific Inc., Efforts were made to sample the largest possible number of species USA) as recommended by the manufacturer (including thermal cycling for the analysis (Table 2). Altogether, 51 accessions of 42 Pulsatilla taxa conditions and PCR mixture) but setting the annealing temperature of (species, subspecies or cultivars) were sampled as recognized in the the thermal cycling at 57 °C. After checking on 1% agarose gel, suc- monograph of Grey-Wilson (2014). Six species were chosen as out- cessful PCR reactions were purified and sequenced, using the original groups (Anemone multifida Poir., A. sylvestris L., A. narcissiflora L., A. primers as sequencing primers, by a commercially available service fasciculata L., Hepatica nobilis Mill. and H. transylvanica Fuss), guided by provider (Macrogen Inc., South Korea). Our original data were sup- topologies presented in previous, taxonomically broader molecular plemented with complete plastid and ribosomal array sequences for studies (Schuettpelz et al., 2002; Ehrendorfer et al., 2009). Karyotypes three species investigated by Szczecińska and Sawicki (2015). of six Pulsatilla taxa were revisited (Lee et al., 2005; Mlinarec et al., 2012b) and a further 11 taxa were examined cytogenetically for the 2.4. Phylogeny reconstruction first time (Table 2). Vouchers were preserved as herbarium/spirit spe- cimens at Herbarium Croaticum of the University of Zagreb (ZA), For each region, sequences were aligned using the online version of Herbarium of the University of Debrecen (DE) and Herbarium Uni- MultAlign v.5.4.1 (Corpet, 1988). After concatenation, phylogenetically versitatis Mosquensis of the University of Moscow (MW)(Seregin, informative regions were selected using default settings via BMGE 2018) (see Supplementary Table 1). v.1.12 (Criscuolo and Gribaldo, 2010); only the selected positions were used in downstream analyses. We generated three matrices: (i) nuclear 2.2. Choice of genic regions (nrITS + MLH1), (ii) plastid (rbcL+accD-psaI+trnL intron), and (iii) combined nuclear and plastid matrices. For the concatenation ap- The DNA regions analysed were chosen for compatibility with two proach, the combinability of both the plastid regions and the nuclear contrasting tree-building approaches: concatenation of DNA informa- and plastid datasets was checked using a ‘hard incongruence’ approach tion and subsequent phylogenetic analysis of the data (Wiens and (Wendel and Doyle, 1998); all matrices were used to build phylogenetic Cannatella, 1998) versus species-tree inference based on the multi- trees using the Maximum Likelihood (ML) method as described below, species coalescent (Heled and Drummond, 2010; Bryant et al., 2012). and the resulting trees were checked for hard incongruences that at- We therefore included two nucleus-encoded DNA-regions: (i) the multi- tracted support values exceeding 95%. Since no such incongruence was copy nuclear ribosomal internal transcribed spacer (nrITS) region detected, we combined the datasets – first the plastid regions, then the (Álvarez and Wendel, 2003; Baldwin et al., 1995; Nieto-Feliner and plastid plus the nuclear – to increase phylogenetic resolution (e.g., Rosselló, 2007) and (ii) the low-copy nuclear region MLH1 that encodes Wiens and Cannatella, 1998). a DNA mismatch repair protein and was selected because of its single- We employed two contrasting tree-building approaches. First, we copy nature documented in a wide-range of angiosperms (Zhang et al., used a Maximum Likelihood (ML) search based on an unpartitioned 2012). In addition, we sequenced three plastid regions known to show dataset as implemented in the web-based version of PhyML v.3.0

47 G. Sramkó, et al. Molecular Phylogenetics and Evolution 135 (2019) 45–61

Table 2 Details of the taxa and samples analyzed. Accessions marked by an asterisk were those only used in the cytological part of the work and omitted from the phylo- genetic analyses. More details of sampling and voucher information can be found in Supplementary Table 1.

Taxon Geographic provenance Chromosome number (2n)

Pulsatilla ajanensis Regel & Tiling Russia, Magadan, Khasyn, Yablonevyi Pass P. albana Bercht. & J. Presl Azerbaijan, Xinaliq P. albana Bercht. & J. Presl* Germany, Göttingen (garden) 16 P. alpina Schrank Austria, Kalkmagerrasen, Ötscher 16 P. ambigua Turcz. ex Pritz China, Xinjiang, Yuming, Baerkelu Mts. P. andina (Rupr.) Grossh. Russia, Daghestan, Gunib P. aurea (N.Busch) Juz. Russia, Krasnodar region, Sochi distr. P. bungeana C.A.Mey. ex Ledeb. Germany, Bayreuth (garden) 32 P. campanella Fisch. ex Regel Kyrgyzstan, Thalaskiy Alatau P. cernua (Thunb.) Bercht. & J.Presl China, Liaoning, Fushun P. chinensis (Bunge) Regel China, Beijing P. dahurica (Fisch. ex DC.) Spreng. Russia, Yakutia, Kyubiute P. dahurica (Fisch. ex DC.) Spreng. Croatia, Zagreb (garden); originally collected in Russia, "Jak" 16 P. dahurica (Fisch. ex DC.) Spreng. China, Heilongjiang, Tahe P. georgica Rupr. Germany, Tübingen (garden) 16 P. grandis L.* Austria, Eichkogel 32 P. grandis L. Slovakia, Zadiel P. halleri Willd.* Germany, Potsdam (garden) P. halleri Willd. Italy, Fenestrelle, Forte Serre Marie P. halleri Willd. subsp. slavica (G.Reuss) Zämelis Slovakia, Terchová P. halleri Willd. subsp. styriaca (Pritz.) Zämelis* Germany, Potsdam (garden) 32 P. subslavica Futák ex Goliašová* Germany, Potsdam (garden) P. integrifolia (Miyabe & Tatew.) Vorosch. Russia, Sakhalin Island, Mt. Dikaya P. kostyczewii (Korsh.) Juz. Kyrgyzstan, Alay valley P. magadanensis Khokhryakov & Vorosch. Russia, Magadan, Ola left shore of River Oksa P. montana Rchb. Romania, Cluj-Napoca P. montana Rchb. Italy, Trieste, Monte Spaccato P. occidentalis (S. Watson) Freyn. U.S.A., Washington, North Cascades National Park P. patens (L.) Mill. subsp. patens Romania, Rimetea P. patens (L.) Mill. s.l. Kazakhstan, Zhaksy P. patens (L.) Mill. subsp. patens* Germany, Tübingen (garden) 16 P. patens (L.) Mill. subsp. flavescens (Zucc.) Zämelis Russia, Buryatia, Altachejsky Nature Reserve P. patens (L.) Mill. subsp. multifida (Pritz.) Zämelis Kazakhstan, Pavlodar, Sherbakty P. patens (L.) Mill. subsp. nuttalliana (DC.) Grey-Wilson Canada, Yukon, Vuntut National Park, Black Fox Creek drainage P. pratensis Mill. subsp. bohemica Skalický Czech Rep., Brno 16 P. pratensis Mill. subsp. hungarica Soó Hungary, Bagamér 16 P. pratensis Mill. subsp. nigricans (Störck) Zämelis* Hungary, Bársonyos P. pratensis Mill. subsp. nigricans (Störck) Zämelis Hungary, Kozárd P. pratensis Mill. subsp. pratensis Poland, Bocheniec P. rubra (Lam.) Delarbre Germany, München-Nymphenburg (garden); originally collected in France, Bresse 32 (garden) P. rubra (Lam.) Delarbre Germany, München-Nymphenburg (garden) P. sachalinensis Hara Russia, Sakhalin Island, Smirnykhovsky District, the mouth of the Belkina River P. sugawarai Miyabe & Tatew. Russia, Sakhalin Island, Makasovskiy district P. sukaczewii Juz. Germany, Jena (garden) 16 P. taraoi (Makino) Takeda ex Zämelis & Paegle Russia, Kuril islands, vulcano Burevestnik P. tatewakii (Kudo) Kudo Russia, Tymovsky District, near the village Zonalnoe P. tenuiloba (Hayek) Juz. Mongolia, Arbulag P. turczaninovii Krylov & Serg.* Russia, Saian Mountains (origin); seed from Göteborg 2012 16 P. turczaninovii Krylov & Serg. Russia, Buryatia, Altachejsky Nature Reserve P. turczaninovii Krylov & Serg. Kazakhstan, Sherbakty (Pavlodar region) P. turczaninovii Krylov & Serg. Mongolia, Tsetserleg P. vernalis Mill. Austria, Oberdrauburg P. vernalis Mill. Poland P. violacea Rupr. Norway, Oslo (garden) P. vulgaris Mill. Germany, Göttingen (garden) P. vulgaris Mill. ‘coccinea’ Germany, Göttingen (garden) P. vulgaris Mill. ‘coccinea’ Germany, Göttingen (garden) P. vulgaris Mill. ‘rubra’ U.K., Kew DNA Bank (ID, 30125) P. wallichiana Ulbr. Kyrgyzstan, Chuy, Panfilov district Anemone multifida Poir. Germany, Chemnitz (garden) Anemone narcissifolia L. s.d. Anemone fasciculata L. Russia, Northern Ossetia, Tsivta Anemone sylvestris L. Hungary, Budapest, Sváb Hill Hepatica nobilis Mill. Romania, Bihor, Nucet Hepatica transylvanica Fuss Romania, Ciuc, Frumoasa

(Guindon et al., 2010) with automatic model selection (Vincent Lefort, arbitrarily as ‘supported’ (aLRT ≥ 0.75) or ‘unsupported’ (aLRT < Jean-Emmanuel Longueville and Olivier Gascuel; available at http:// 0.75). www.atgc-montpellier.fr/phyml-sms/). Branch robustness was tested Our second approach used a Bayesian approach to phylogenetic tree using the Approximate Likelihood-Ratio Test (aLRT) approach building based on a partitioned dataset as implemented in MrBayes (Anisimova and Gascuel, 2006), where branch support was categorized v.3.2.6 (Ronquist et al., 2012). The sequences were partitioned by their

48 G. Sramkó, et al. Molecular Phylogenetics and Evolution 135 (2019) 45–61 function and genomic location: (i) nuclear gene exons (5.8S of nrITS Statistical Dispersal-Extinction-Cladogenesis model (S-DEC: Ree and plus exons of MLH1), (ii) nuclear spacers (ITS1 and ITS2 of nrITS) and Smith, 2008; Beaulieu et al., 2013), as implemented in RASP v.3.2 (Yu introns (of MLH1), (iii) plastid genes (entire rbcL, partial accD and psaI), et al., 2015). This approach takes account of the topological uncertainty (iv) plastid intron (trnL) and spacer (accD-psaI). We performed a priori in the phylogenetic tree. All Bayesian phylogenetic trees from the data partitioning and molecular evolution model selection using Par- StarBEAST2 analysis were imported for the analysis with 50% ‘burn-in’, titionFinder v.2.1.1 (Lanfear et al., 2017), and the recommended par- and the resulting species tree was used as a fully resolved phylogram to titions with the appropriate model settings (GTR+G for the nuclear, define the consensus tree. We took 1000 random trees and allowed the GTR+G+I for the plastid partition) were used in the Bayesian phylo- reconstruction of a maximum of two ancestral states at each node. genetic analysis. Four independent Metropolis-Coupled Markov Chain Eight biogeographical areas were defined for the Pulsatilla species, Monte Carlo (MCMCMC) analyses were run with four (three heated) based primarily on the distribution maps and information in Aichele chains for 25 million generations, sampling every 1000th generation and Schwegleb (1957): A, mountains of central Asia (Himalayas, Pamir- and using a heating parameter of 0.2. Convergence of the runs and the Alay, Tian Shan, Altai); B, the Far East; C, the eastern part of the Eur- effective sampling of the posterior space were checked using Tracer asian steppe zone (from the Altai Mountains to Manchuria); D, the v.1.6 (available from http://beast.bio.ed.ac.uk/Tracer). A maximum Caucasus Mountains; E, the central and western part of the Eurasian clade credibility tree was drawn from the four combined runs after steppe zone (from the Altai Mountains to the Pannonian basin); F, discarding 30% of the trees as ‘burn-in’ in TreeAnnotator v.2.3.2 European grasslands (outside the steppe zone); G, North America; H, (Drummond and Rambaut, 2007). Posterior probability (PP) values of European mountains. each branch were categorized as ‘supported’ (PP ≥ 0.85) or 'un- supported' (PP < 0.85). All phylogenetic trees were visualized in Fig- Tree v.1.4.2 (available at http://tree.bio.ed.ac.uk/software/figtree/). 2.7. Chromosome analyses

2.5. Species-tree inference with molecular dating All investigated species included in the cytogenetic analysis were grown in pots in the Botanical Garden of the University of Zagreb. For Whereas concatenation of sequences will produce a single tree that karyological studies, actively growing root-tip meristems were pre- best fits the combined sequence alignment according to the phyloge- treated with 0.05% colchicine (Sigma-Aldrich Chemie GmbH, netic criterion applied, a full Bayesian multispecies coalescent method Taufkirchen, Germany) in dark conditions for 4 h at room temperature, can in theory infer comparatively accurate species tree from even fixed in a solution of ethanol and acetic acid (3:1) for 24 h at –4 °C, and weakly informative loci (Szöllősi et al., 2015; Xu and Yang, 2016). Our stored in 70% ethanol at –20 °C until use. dated species-tree of Pulsatilla species was estimated using a multi- Chromosomes stained with acetocarmine were used for morpho- species coalescent approach as implemented in StarBEAST2 v.0.15 metric analyses. Acetocarmine staining followed a standard protocol (Ogilvie et al., 2017), an add-on package of BEAST2 v.2.5.0 (Bouckaert (Mlinarec et al., 2006). Chromosomes were identified according to et al., 2014). Two independent MCMC analyses were performed by chromosome type, total length, arm ratio and their heterochromatin running the analyses for 2 billion generations and sampling every 20kth banding pattern and Fluorescence In Situ Hybridization (FISH) signal; generation (see below). Our sample set included several accessions of they were ranked in order of decreasing chromosome length. For some species (e.g., P. turczaninovii, P. dahurica: Table 2). Accessions chromosome classification we followed the nomenclature of Levan et al. regarded a priori as different subspecies of conspecific accessions were (1964). Banding pattern was inferred based on the presence of het- assigned to different species to test for their relationships. erochromatin blocks visible after FISH. Chromosome measurements All five DNA regions were treated as unlinked for the site and clock were performed on three to four individuals per species on at least three model but were linked for the tree model of the plastid regions. The well-spread chromosome plates; each preparation was made from three model of molecular evolution most appropriate for the DNA regions was to four root-tip meristems. Slides chosen for FISH analysis were re- identified during the MCMC analysis using the Bayesian Model Test quired to show at least ten well-spread chromosome plates. v.1.1.0 (Bouckaert and Drummond, 2017), another add-on package of Chromosome preparations for FISH were performed according to the BEAST2 programme. Tajima's (1993) relative rate tests indicated Mlinarec et al. (2012b). The clone pTa794, containing the complete that a strict clock should be applied for all DNA regions. Finally, a Birth- 410 bp BamHI fragment of the 5S rRNA gene and the spacer region of Death model (Feller, 1939) was applied as prior when exploring spe- wheat (Gerlach and Dyer, 1980), was used as the 5S rDNA probe. The ciation process. To simplify computation, three monophyletic groups probe was directly labelled with Cy3-dCTP (Amersham, GE Healthcare, that received especially strong support during the concatenation ana- Little Chalfont, UK) using a nick-translation kit according to the man- lyses (Fig. 1) were topologically constrained during the coalescence ufacturer’s instructions (Roche Diagnostics GmbH, Mannheim, Ger- analysis: (i) the monophyly of the root (i.e., point of separation of many). The 2.4 kb HindIII fragment of the partial 18S rDNA and ITS1 Anemonidium from Anemone s.s.), (ii) the monophyly of genus Pulsatilla from Cucurbita pepo, cloned into UC19 (Torres-Ruiz and Hemleben, (clade I on Fig. 1), and (iii) the monophyly of section Pulsatilla (clade III 1994), was used as the 35S rDNA probe. The probe was directly labelled on Fig. 1). The root and the separation of the genus Pulsatilla were time- with Cy3-dCTP by nick-translation, as above. The hybridization mixture calibrated using a secondary dating approach, taking the estimates of (40 µl) containing 50% formamide, 10% dextran sulphate, 0.6% sodium − Wang et al. (2016) as a normally distributed prior with means de- dodecyl sulphate, 2 × SSC and 2 ng µl 1 of labelled probe was dena- termined at 27.63 Ma (sigma 1.4) and 11.64 Ma (sigma 1.2), respec- tured at 76 °C for 15 min. Chromosome preparations were denatured at tively. The two independent StarBEAST2 runs were checked for con- 73 °C for 5 min after applying the hybridization mixture. Stringent vergence in Tracer v.1.6 before being combined using a 50% burn-in. washes were performed at 42 °C in the following solutions: 2 × SSC, This combined matrix was again checked for satisfactory effective 0.1 × SSC, 2 × SSC, 4 × SSC/Tween (5 min each). sample size values in Tracer, and a maximum clade credibility tree with The preparations were mounted in antifade buffer Vectashield − mean heights was drawn using TreeAnnotator v.2.5 of the BEAST2 (Vector Laboratories, UK) containing DAPI counterstain (2 µg ml 1) package. and were stored at 4 °C. Signals were visualized and photographs cap- tured on an Olympus BX51 microscope, equipped with a highly sensi- 2.6. Ancestral area reconstruction tive Olympus DP70 digital camera. Images were merged and contrasted using Adobe Photoshop 6.0. An average of 15 well-spread metaphases The reconstructed species-tree (Fig. 2) was used as the basis of an- was analyzed for each plant. cestral area reconstruction using Bayes-Lagrange analysis following the

49 G. Sramkó, et al. Molecular Phylogenetics and Evolution 135 (2019) 45–61

Fig. 1. Phylogenetic tree of the genus Pulsatilla based on Maximum Likelihood analysis of concatenated sequences of two nuclear regions and three plastid regions. (A) Tree presented as a phylogram with branch lengths proportional to mutation changes and drawn to scale. (B) Tree presented as a cladogram, showing branch support values resulting from both ML aLRT and Bayesian posterior probability analysis (hyphens denote branches gaining less than 95% posterior probability) with unsupported branches collapsed into polytomies. Identical taxon names are distinguished by indicating their geographic provenance using ISO country codes. Taxon names follow those given in Table 2.

50 G. Sramkó, et al. Molecular Phylogenetics and Evolution 135 (2019) 45–61

Fig. 2. Species-tree constructed using a multispecies coalescent approach, derived from a StarBEAST2 analysis based on data for two nuclear and three plastid regions. The tree is drawn as a chronogram that relies upon on secondary dating of both the root and the separation of the genus Pulsatilla from Anemone s.s. Estimated dates (Ma) and error bars are shown only for nodes where statistical robustness exceeds 0.95. Below is graphed global climate change from the Oligocene to Present, as represented by estimated deep-sea mean annual temperatures (red line); also shown are the periods of aridification of central Asia and progressive uplift of central Asian mountains (adapted from Favre et al., 2015). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3. Results We have chosen to present the maximum likelihood tree as both a phylogram (Fig. 1A) and a cladogram (Fig. 1B) because 35% of the 3.1. Phylogenetic tree reconstruction internal branches yielded an aLRT of < 0.75, including the short but important branches that specify relationships among the six series and The aligned length of raw sequence reads was 3336 bp, though three sections within subgenus Pulsatilla. The phylogram helpfully BMGE subsequently removed 49 sites as phylogenetically less reliable. shows differential branch lengths, whereas in the cladogram the 'un- This matrix was submitted to PhyML-SMS as unpartitioned dataset, and supported' branches are collapsed and other short branches are artifi- Smart Model Selection identified the GTR + G model of sequence cially lengthened to allow the addition of support values for the ML and evolution as best fitting this dataset under both the AIC and BIC in- Bayesian analyses. formation criteria. For the Bayesian analysis using MrBayes, Most of the branches that receive statistical support in the ML tree PartitionFinder2 identified as optimal a tripartite partitioning of data: also occur in the species-coalescence tree (Fig. 2), and most attract (1) merged coding nuclear and plastid regions (i.e., the nuclear exons broadly similar levels of support. The topologies differ in the placement and plastid genes), (2) non-coding nuclear regions (i.e., the ITS regions of section Semicampanaria, which is nested within section Pulsatilla in and MLH1 introns) and (3) non-coding plastid regions (i.e., the plastid Fig. 1 but sister to section Pulsatilla in Fig. 2 (in both cases without intron and the spacer). For the non-coding nuclear partition it identified statistical support). Positions of the two earliest-divergent species the SYM + G model as best fitting, whereas for the non-coding plastid within section Albanae are transposed in the two trees with strong partition and for the combined coding regions the GTR + G + I model statistical support in both. Within section Pulsatilla, series Pulsatilla and was preferred. The partitions with a unique model of evolution (i.e., series Patentes can be tentatively distinguished only in Fig. 2, and nuclear exons and plastid gene with non-coding plastid regions treated precise relationships among species of series Patentes differ between the as one unit, non-coding nuclear regions as a second unit) were used as two trees, again without statistical support. Among those species that the molecular evolutionary model underlying the Bayesian MCMC participate in polytomies in Fig. 1A, P. chinensis is placed within series analysis. Semicampanaria in Fig. 2 and P. magadanensis within section

51 G. Sramkó, et al. Molecular Phylogenetics and Evolution 135 (2019) 45–61

Fig. 3. Reconstruction of ancestral areas in the genus Pulsatilla reconstructed using Bayes-Lagrange analysis. Each analyzed species is coded with its contemporary distribution (inserted key), and distribution likelihoods are shown for the consecutively numbered internal nodes. Derived states inferred by the software package RASP during the analysis are also included.

Preonanthus, though both putative relationships are unsupported. receives statistical support but series Patentes does not. Three series are We have chosen to focus our Discussion on the species-coalescence resolved within section Semicampanaria, though the relationships be- tree (Fig. 2) for three reasons, none of them wholly objective: the tween them are not. Series Pratenses and series Albanae receive statis- conceptual appeal of coalescence processes in molecular evolution (e.g., tical support, but the perceived monophyly of series Semicampanaria is Szöllősi et al., 2015), the resolved nature of the resulting tree that fa- weakened by the uncertain placements of P. cernua and especially P. cilitates supraspecific classification, and the degree of congruence of chinensis. the resulting DNA-based classification with previous morphology-based The often poorly-resolved relationships between species and sub- classifications (Table 1) – a topic discussed at greater length in section species within apparently monophyletic series are discussed in section 4 4.1. only where they are relevant to larger scale questions. Multiple, geo- Whereas the monophyly of the genus Pulsatilla is strongly supported graphically disparate accessions of five species were included in the ML in all analyses (albeit constrained in the coalescence analysis: cf. Figs. 1 tree (Fig. 1), in part to test the reliability of our analytical methods. and 2), support for most nodes along the backbone of the highly With the exception of P. patens s.l., all conspecific plants yielded near- asymmetric tree is much weaker. The division of Pulsatilla into three identical concatenated sequences. The three accessions of P. turczani- main clades (subgenera) of radically different size is strongly statisti- novii and three accessions P. dahurica resolved as statistically supported cally supported. The earliest-diverging lineage is the monospecific monophyletic groups. The one accession of P. vulgaris ‘rubra’ partici- subgenus Kostyczewianae. The less species-rich of the two remaining pated in a polytomy with other taxa of series Pulsatilla. The two ac- subgenera, Preonanthus, includes P. magadanensis with only limited cessions of P. montana associated with other named taxa that are often support, and its division into two series-level sister groups, Pre- viewed as subspecies of P. pratensis s.l. However, the last pair of sup- onanthopsis and Preonanthus, is equally tentative. posedly conspecific accessions, attributed to P. patens, were resolved as Within the species-rich subgenus Pulsatilla, section Tatewakianeae is sisters to other subspecies of P. patens s.l., albeit without statistical earliest-divergent and its circumscription is strongly supported. Both support and within the same series, Patentes. section Pulsatilla and section Semicampanaria are resolved as mono- phyletic sisters in Fig. 2, but without statistical support. Of the two apparently monophyletic series within section Pulsatilla, series Pulsatilla

52 G. Sramkó, et al. Molecular Phylogenetics and Evolution 135 (2019) 45–61

P. alpina P. patens P. georgica

A B C P. tenuiloba P. turczNaninovii P. pratensis subsp. bohemica

D E F P. halleri subsp. styriaca P. vulgaris P. vulgaris ‘coccinea’

G H I P. grandis P. bungeana

J K

Fig. 4. FISH localization of 35S rDNA (red signals) in (A) Pulsatilla alpina DE-Soo-45645, (B) P. patens ZA41504, (C) P. georgica ZA41501, (D) P. tenuiloba ZA41500, (E) P. turczaninovii ZA41498, (F) P. pratensis subsp. bohemica ZA43780, (G) P. halleri subsp. styriaca ZA41497, (H) P. vulgaris ZA41490, (I) P. vulgaris 'coccinea' ZA41491, (J) P. grandis ZA40579 and (K) P. bungeana ZA41499. Bar = 10 µm.

3.2. Molecular dating of lineage divergences The only statistically valid date obtainable within subgenus Preonanthus is the early Quaternary (1.3 Ma) separation of the Given that estimates of lineage divergence dates incur wide margins Caucasian P. aurea from the Alpine P. alpina. The onset of diversifica- for error, and that many of the more distal branches in our trees are tion within subgenus Pulsatilla coincides with the onset of Quaternary very short, we have chosen to focus our interpretation on the better climatic oscillations (3.0 Ma), marked by the separation of the newly- supported branches in the chronogram that we derived from our species promoted section Tatewakianae. Sections Pulsatilla and Semicampanaria coalescence analysis (Fig. 2). separated a little later (2.2 Ma), immediately presaging the main ra- The root of the chronogram was assessed in our analysis at 28.1 Ma, diation within genus Pulsatilla that affected both of these sections. close to the 27.6 Ma input as prior. However, our estimate for the Statistically valid dates were obtained for two further events: diversi- crown-node of genus Pulsatilla of 9.8 Ma was somewhat younger than fication within the European series Pulsatilla is estimated to have oc- the input prior of 11.6 Ma. Pulsatilla is estimated to have separated from curred at ca 310 ka and within series Albanae, the Caucasian species (P. Anemone s.s. in the late Oligocene (24.6 Ma), whereas the separation of albana, P. andina, P. georgica, P. violacea) separated from the central Hepatica from Anemonidium (the second of two distinct clades that were Asian montane species (P. campanella, P. wallichiana)atca 510 ka until recently attributed to Anemone) probably took place slightly later, (Fig. 2). in the early Miocene (21.5 Ma). Both events approximate the late Oligocene warming phase. The three subgenera diverged during the 3.3. Ancestral area reconstruction late Miocene, that of the monotypic subgenus Kostyczewianae (9.8 Ma) preceding that of the two remaining subgenera (7.2 Ma). As is often the case, our ancestral area reconstructions are

53 G. Sramkó, et al. Molecular Phylogenetics and Evolution 135 (2019) 45–61 disappointingly ambiguous toward the base of the chronogram (Fig. 3). rDNA signals on metacentric chromosome pair 4 (Fig. 5E), and P. pra- The S-DEC method of ancestral trait reconstruction identified the cen- tensis subsp. bohemica is unique in possessing four 5S rDNA-bearing tral Asian mountains and the Far East (AB, 17%) or the central Asian acrocentric chromosomes (Fig. 5F). mountains alone (A, 14%) as the most plausible among several poten- All species of series Pulsatilla are tetraploid (Tamura, 1995). Most tial ancestral areas for the root at the base of genus Pulsatilla (node 83). members of the group show eight major and from three to seven minor The most likely ancestral area of the common ancestor of the two re- 35S rDNA signals (Fig. 4G–J), though P. halleri subsp. styriaca has only maining subgenera is the Far East (B, 37%: node 82). Indeed, most of five major 35S rDNA signals, one being hemizygous (Fig. 4G). The the deeper nodes (78–82) resolve the Far East as the most probable major signals are telomeric and located on acrocentric chromosomes, reconstructed ancestral area, indicating the importance of this region in whereas the minor signals are centromeric and positioned on meta- the evolution of the genus. centric chromosomes (Fig. 4G–J). 5S rDNA signal number ranges from A Far Eastern origin appears most likely for subgenus Preonanthus, eight to ten, the last two being hemizygous (Fig. 5G–J). In P. coccinea, but the only statistically supported node within the subgenus (77) 5S rDNA signals are located close to the centromere on the longer arm marks the split between the Caucasian and European alpine species of of two of four metacentric chromosomes 1, close to the chromosome the section Preonanthus and suggests that their common ancestor termini on the longer arm of one of four metacentric chromosomes 2 probably occurred in both areas. and 4, on the shorter arm of two of four submetacentric chromosomes The common ancestor of section Pulsatilla is estimated to have oc- 5, and close to the chromosome termini on the longer arm of all four cupied the Far-Eastern and eastern Eurasian steppe zone or the Far East acrocentric chromosomes 7 (Fig. 5I). (node 76). Section Tatewakianae (node 44) is confined to the Far East. Series Albanae was represented only by P. bungeana, which is The common ancestor of all remaining species of subgenus Pulsatilla composed of two chromosome sets distinguishable by size, morphology (node 75) most likely lived in the eastern Eurasian steppe zone. and rDNA pattern. Eight telomeric 35S rDNA signals loci were located The ancestor of section Pulsatilla (node 74) most likely occupied on the short arm of acrocentric chromosomes 6 and 7, the signals on Eurasian steppes, though series Pulsatilla (node 67) probably originated chromosome 7 being highly heteromorphic (Fig. 4K). The seven 5S in European grasslands. Branch support is weak in section rDNA signals (one hemizygous) are located in the proximity of the Semicampanaria (node 62) but suggests an origin in the European centromere on the longer arm of one of four metacentric chromosomes mountains before spreading through the eastern Eurasian steppes be- 1, close to the chromosome termini on the longer arm of two of four fore diverging into the montane regions of the Caucasus and central metacentric chromosomes 4, and close to the chromosome termini on Asian mountains (node 51). the long arm of all four acrocentric chromosomes 7 (Fig. 5K). Most Pulsatilla taxa investigated in this study showed large hetero- 3.4. Phylogenetic mapping of ribosomal DNA patterns chromatic DAPI-positive AT-rich blocks positioned on the longer arm of an acrocentric chromosome (Fig. 6). The exceptions are P. turczaninovii, The genus Pulsatilla exhibits considerable cytogenetic diversity that which has additional DAPI bend located interstitially on longer arm of correlates well with our tree topologies. The genus contains a mixture metacentric chromosome pair, and P. georgica, which lacked DAPI of diploids (2n =2x = 16) and tetraploids (2n =4x = 32). blocks (Fig. 6). No members of subgenus Kostyczewianae or of subgenus Preonanthus section Preonanthopsis were examined. Section Preonanthus (Table 1) 4. Discussion was represented only by P. alpina, which has two terminal 35S rDNA loci (Fig. 4A) and two centromeric 5S rDNA loci (Fig. 5A). It can be 4.1. Comparison of our classification with previous classifications distinguished from other Pulsatilla species by the absence of 5S rDNA signals on acrocentric chromosomes, and is the only investigated di- Table 1 compares our DNA-based classification following mono- ploid Pulsatilla having 5S rDNA signals on a submetacentric chromo- phyly with those of three previous classifications. Here, we will focus some pair – a position otherwise observed only in polyploids of series on the two most recent classifications, presented in the morphology- Pulsatilla. based monographs of Tamura (1995) and Grey-Wilson (2014). Series Patentes was represented by P. patens subsp. patens, which The most radical contrast is that both Tamura (1995) and Grey- showed four 35S rDNA signals located terminally on the short arm of Wilson recognized P. integrifolia as separate monotypic subgenus, acrocentric chromosomes (Fig. 4B) and two 5S rDNA signals borne Miyakea, whereas our tree firmly placed this species within series Pa- terminally on longer arm of acrocentric chromosome pair (Fig. 5B). tentes of subgenus Pulsatilla, where it is the closely similar sister species Four of the eight species comprising series Albanae were in- to P. vernalis – a species that was awarded its own series within section vestigated cytogenetically: P. georgica, P. tenuiloba, P. violacea and P. Pulsatilla by Tamura (1995) and was misplaced within section Semi- albana (see also Mlinarec et al., 2012a). All investigated species are campanaria by Grey-Wilson (2014). Within section Pulsatilla, Grey- diploids (Figs. 4C, 5C) and resemble species of P. patens s.l. in the Wilson's ‘series Bungeanae’ proved to be polyphyletic, the majority of its number and position of rDNA loci. Four terminal 35S rDNA signals are species actually being distributed throughout our expanded circum- typical (Fig. 4C, D), though P. albana has only two (Mlinarec et al., scription of series Albanae (Fig. 2). The statistically significant separa- 2012a); also, heteromorphism is evident in P. georgica (Fig. 4C). 5S tion of P. bungeana and P. tenuiloba from the rest of series Albanae rDNA patterns consistently show two telomeric signals on acrocentric (Fig. 1) may indicate a more profound separation (as suggested by the chromosomes (Fig. 5C, D). earlier classifications that treat these species as a separate series Bun- Series Semicampanaria (represented by P. chinensis, P. dahurica and geanae; Table 1), but the currently applied molecular technique and P. cernua in Fig. 6) consists of diploid species (Lee et al., 2005) with four taxon sampling does not support the series-level separation of Bun- terminal 35S rDNA signals positioned on short arm of acrocentric geanae. In a more radical move, the comparatively poorly known P. chromosomes. Three or four 5S rDNA signals are located close to the magadanensis is here transferred from Grey-Wilson's series Bungeanae of centromere within the long arms of metacentric chromosome pair 4 and section Pulsatilla into section Preonanthus (a decision previously ad- in the terminal regions within long arms of acrocentric chromosome vocated by Luferov, 2004). Whereas the exact placement of P. maga- pair 7 (Lee et al., 2005). danensis within the subgenus Preonanthus is currently equivocal, its Series Pratenses consists of diploids characterized by the presence of basal position is corroborated by some important morphological char- two terminal 35S rDNA loci (Fig. 4E, F) and three 5S rDNA loci, both acters: this species shares the lack of nectariferous staminodes with terminal and centromeric (Fig. 5E, F). Pulsatilla turczaninovii can be other species in the subgenera Preonanthus and Kostyczewianae; addi- distinguished from other diploid species by the presence of telomeric 5S tional plesiomorphic characters are the greenish-yellow anthers with

54 G. Sramkó, et al. Molecular Phylogenetics and Evolution 135 (2019) 45–61

P. alpina P. patens P. georgica

A B C P. tenuiloba P. turczNaninovii P. pratensis subsp. bohemica

DD E F

P. halleri subsp. styriaca P. vulgaris P. vulgaris ‘coccinea’

G H I P. grandis P. bungeana

J K

Fig. 5. FISH localization of 5S rDNA (red signals) in (A) Pulsatilla alpina DE-Soo-45645, (B) P. patens ZA41504, (C) P. georgica ZA41501, (D) P. tenuiloba ZA41500, (E) P. turczaninovii ZA41498, (F) P. pratensis subsp. bohemica ZA43780, (G) P. halleri subsp. styriaca ZA41497, (H) P. vulgaris ZA41490, (I) P. vulgaris 'coccinea' ZA41491, (J) P. grandis ZA40579 and (K) P. bungeana ZA41499. Bar = 10 µm. grey stripes (all other species have yellow anthers) and five-sepalled considerable degree of conformity between the molecular and mor- flowers (all others have six-sepalled corolla), making this species mor- phological signals. phologically exceptional similar to the basal species, P. kostyczewii. Pulsatilla rubra was placed by both Tamura (1995) and Grey-Wilson (2014) in section Pulsatilla series Pratenses but here is placed in series 4.2. Phylogenetic relationships and evolutionary history of the genus Pulsatilla, whereas P. turczaninovii makes the converse taxonomic Pulsatilla journey from series Pulsatilla to series Pratenses. We elevate series Ta- tewakianae of Tamura (1995) and Grey-Wilson (2014) to the level of Monophyly of genus Pulsatilla, as circumscribed to include the section, and confirm that it includes P. ajanensis, a species previously Anemone-like central Asian species P. kostyczewii, is strongly supported placed in series Pulsatilla by Tamura (1995). Our results also tentatively by all aspects of our detailed study (Figs. 1 and 2) (see also Zetzsche, support Grey-Wilson's (2014) decision to place P. chinensis within sec- 2004). Possession by P. kostyczewii of a combination of morphological tion Semicampanaria series Semicampanaria, though we acknowledge character states that are plesiomorphic (absence of nectariferous sta- that a credible case remains for Tamura's (1995) recognition of a se- minodes; presence of purple stamens resembling those of Anemone s.s.) parate monotypic series Chinenses. and apomorphic (leaves divided into long-linear segments; achene awns Considered in total, and given a genus of ca 40 species, a com- hairy) for the genus accords with its status as an early-divergent, spe- paratively small number of taxonomic changes have proven necessary cies-poor lineage (e.g., Crisp and Cook, 2005); along with the com- to render the past supraspecific classifications congruent with our own paratively long branch separating P. kostyczewii from the remaining monophyly-based hierarchy, indicating (though not directly testing) a Pulsatilla species, this amply justifies its status as one of three subgenera in Pulsatilla – subgenus Kostyczewianae.

55 G. Sramkó, et al. Molecular Phylogenetics and Evolution 135 (2019) 45–61

Fig. 6. Karyotype diversification in Pulsatilla as mapped across the coalescence species-tree presented in Fig. 2. Position of 35S and 5S rDNA genes are shown as green and red dots, respectively; heterochromatin regions are shown as blue blocks. Additional data sources: *Lee et al. (2005),**Mlinarec et al. (2012b).

The remaining Pulsatilla species are divided, with high statistical (Fig. 2) by favouring two sections. Within section Preonanthopsis, the confidence, into two main clades that are also best treated as subgenera enigmatic P. sachalinensis (currently known from only a single locality, (Fig. 2). The smaller clade encompasses approximately seven species on Sakhalin Island, and not accepted by Grey-Wilson, 2014) is tenta- that constitute subgenus Preonanthus (a taxon viewed as a genus on tively placed as sister to the pairing of P. taraoi (also a localized en- morphological grounds by Szentpéteri et al., 2008) whereas the larger demic, on the Southern Kuril Islands) and P. occidentalis, a more clade encompasses the numerous species and subspecies that constitute widespread species of North America. These relationships suggest a the third subgenus, Pulsatilla. This subgeneric distinction evident in our scenario of eastward migration of the lineage across the Far Eastern molecular data is supported by our cytogenetic data; P. alpina, re- islands and the Bering Strait into North America, thus allowing allo- presenting subgenus Preonanthus, exhibited a unique rDNA pattern that patric speciation. This hypothesis could be tested in the future by se- distinguished it from all other investigated diploid Pulsatilla species. quencing the putatively closely related Japanese endemic, P. nipponica. Moreover, P. alpina resembles Anemone s.s. in the chromosomal posi- The Far Eastern P. magadanensis is placed only tentatively in section tions of 5S rDNA loci (Mlinarec et al., 2012b). We will discuss first Preonanthus, as sister to the cold-adapted pairing of the Alpine P. alpina subgenus Preonanthus and then subgenus Pulsatilla, paying particular and Caucasian montane P. aurea (see also Zetzsche, 2004). The common attention to the hierarchical levels of section and series (Fig. 2, Table 1). ancestor of these montane species may have lived somewhere in the The ML topology would allow recognition of three sections within lowlands between the Caucasian and European mountain ranges and subgenus Preonanthus, but we follow the species coalescence topology become divided as the population migrated from the lowlands to

56 G. Sramkó, et al. Molecular Phylogenetics and Evolution 135 (2019) 45–61 mountains during the onset of interglacial periods. separate P. pratensis s.l. from P. montana (one sample of which was Within subgenus Pulsatilla, only the earliest-diverging section, taken from the locus classicus in Italy), suggesting that these taxa may be Tatewakianae, shows substantial internal molecular differentiation. All conspecific with geographic separation at the subspecies level. three species in the group possess distinctive ternate basal leaves. In Series Semicampanaria contains P. cernua, P. dahurica and, more contrast, a wide range of leaf morphologies is exhibited by species of tentatively, P. chinensis. These species share simple pinnate leaves with section Pulsatilla, including ternate and ternate-derived palmate leaves P. vernalis (Grey-Wilson, 2014), but our results suggest that this mor- (Grey-Wilson, 2014). Most notably, P. integrifolia – an endemic species phological similarity is a parallelism rather than a synapomorphy that of Sakhalin Island with entire leaves unique in the genus that was indicates shared ancestry. Jiang et al. (2017) sampled several plants of formerly awarded the status of monospecific subgenus (Grey-Wilson, each of these three species for their multigene phylogeny but were 2014) – unexpectedly nested well within series Patentes, nicely illus- unable to demonstrate the monophyly of any of the three. trating the basic principle of cladistics that monophyly has priority over The final taxonomic series of Pulsatilla to be considered is series phenetic measures based on the amount of character-state divergence Albanae of section Semicampanaria. This clade of Caucasian and central (e.g., Bateman, 2009; Stuessy, 2009). Asian high mountain species has as its earliest-divergent species first P. Series Patentes is especially morphologically polymorphic tenuiloba and then P. bungeana, both from the Eastern Eurasian steppes (Zimmermann and Miehlich-Vogel, 1962; Zimmermann, 1963). Pulsa- (see also Aichele and Schwegleb, 1957). Pulsatilla sukaczewii and P. tilla patens s.l. has palmately divided leaves resembling those of Ane- tenuiloba, typical of the northeast region of Eurasia (Siberia, Mongolia mone s.s., rather than the apomorphic pinnately divided leaf structure and China), were previously regarded as closer to P. bungeana, which is that characterizes most Pulsatilla species. However, P. patens s.l. appears the only small-bodied, semi-erect flowered species in the series. Pulsa- polyphyletic in our trees, two species (P. vernalis and P. integrifolia) tilla sukaczewii from the Baikal region of Russia was found to be nearly being nested within P. patens s.l., albeit without strong statistical sup- identical to P. georgica in our analysis (Fig. 1). Grey-Wilson (2014) ar- port. However, the significant genetic structure within P. patens s.l. gued that P. sukaczewii is commonly confused with the Caucasian P. (Fig. 1) may validate the taxonomic view of previous monographers georgica in European gardens. Given that our plant of ‘P. sukaczewii’ is (Zimmermann and Miehlich-Vogel, 1962; Zimmermann, 1963) who one of the few samples we were obliged to obtain from living collec- regarded the subspecies of P. patens s.l. as separate, allopatric species. tions (Table 2), we suspect it was mislabelled. The strongly supported This remarkable result demands a more detailed phylogeographic study species-pair of the west Himalayan P. wallichiana and the Pamir–Tian- of the P. patens ‘aggregate’. Shan–east Hindu Kush endemic P. campanella (both central Asian The inclusion of P. integrifolia and P. vernalis within series Patentes is members of the Albanae group, along with P. ambigua) clearly shows an unexpected result as no specialist of this genus has ever considered that a vicariance event induced speciation. the species included in this clade to be related. Nonetheless, the erect flowers and less-dissected leaf-blades connect the species of series 4.3. Karyotype differentiation and evolution Patentes. Species of the European series Pulsatilla are also known to readily In every investigated Pulsatilla species, whether diploid or poly- hybridize and yield a fertile F1, particularly the well-known European ploid, all of the 35S rDNA loci were separated from the 5S rDNA gene horticultural species P. vulgaris, P. halleri and P. grandis (Grey-Wilson, clusters and located terminally on the short arm of acrocentric chro- 2014). Morphological synapomorphies of the group include large, erect mosomes, whereas there was no preferential chromosomal position for or semi-erect flowers and achenes possessing long awns, and all species 5S rDNA loci; they occupy terminal, subterminal or pericentromeric are tetraploid. Although P. rubra has traditionally been classified in positions, and can be located on acrocentric, submetacentric or meta- section Semicampanaria series Pratenses, our study revealed P. rubra to centric chromosomes (Fig. 6). Clearly, 5S rDNA is far more mobile than possess sequences identical with those of P. vulgaris ‘coccinea’ (also P. 35S rDNA in Pulsatilla,reflecting a more general pattern that has re- vulgaris ‘rubra’: Fig. 1) and it was shown by Mlinarec et al. (2012b) to be cently been reported across land plants (Garcia et al., 2017). tetraploid, in contrast with series Pratensis which is uniformly diploid. Mapping of rDNA characters across a topology congruent with the Although our sample of P. rubra was a garden plant, we are confident coalescence species-tree for Pulsatilla permitted evolutionary inter- that we did not confuse it with cultivar ‘coccinea’ or ‘rubra’; the plant pretation (Fig. 6). The majority of the species examined, including the showed the required nodding flowers and finely dissected leaves, and earliest-divergent species analyzed (P. alpina of subgenus Preonanthus), its morphological identification was confirmed by expert A.N. Luferov possessed two 35S and two 5S rDNA loci, which therefore represents (I.M. Sechenov First Moscow State Medical University). Unfortunately, the most likely plesiomorphic condition. Deletion of 35S rDNA sites was the near-uniformity of DNA sequences in series Pulsatilla means that we observed only in the diploid P. albana and tetraploid P. halleri subsp. can neither confirm nor reject the hypothesis that P. halleri subsp. sla- styriaca, which had two and five 35S rDNA signals, respectively. Fre- vica is a stabilized hybrid between P. vulgaris and P. grandis (Mereďa quent duplication or deletion of 5S rDNA loci, leading to large-scale and Hodálová, 2011). polymorphism of numbers, sizes and physical positions of signals, Although the monophyly of section Semicampanaria is only weakly characterized the investigated members of series Pulsatilla. Deletion of supported by the species-tree (Fig. 2), this clade credibly unites all 5S rDNA signals was observed in P. patens and the clade consisting of P. species that produce a nodding or half-nodding flower (Grey-Wilson, albana, P. georgica and P. violacea, all of which have only one 5S rDNA 2014). Its three taxonomic series are also only tentatively circum- locus, as well as in P. cernua, which has three 5S rDNA loci. The absence scribed. of signal from one of the homologues in P. cernua may be consequence The most surprising inclusion in series Pratenses is the central and of sequence elimination due to unequal crossing-over, often related to a eastern Asian species P. turczaninovii, shown by our trees to be earliest process of concerted evolution of tandemly repeated genes or to the divergent within the series. Its semi-erect or half-nodding, brightly activities of repetitive DNA such as transposable elements. By contrast, coloured flowers with widely spreading sepals resemble those of series diversification of P. turczaninovii and P. pratensis subsp. bohemica was Pulsatilla. However, the diploid nature and three 5S rDNA loci of P. probably accompanied by the duplication of one 5S rDNA locus, which turczaninovii are consistent with the remainder of series Pratenses but was subsequently translocated to a metacentric chromosome in P. not with the uniformly tetraploid series Pulsatilla (Fig. 6). The poly- turczaninovii and to an acrocentric chromosome in P. pratensis subsp. morphic species P. pratensis s.l. has attracted much taxonomic attention bohemica. from European taxonomists (Aichele and Schwegleb, 1957; Polyploidization can precipitate numerous epigenetic and genomic Zimmermann, 1958; Szentpéteri et al., 2007). Our analysis failed to changes such as chromosome rearrangements, gene silencing and distinguish among the four subspecies of P. pratensis, nor could it genome downsizing, together with transposable element mobilization

57 G. Sramkó, et al. Molecular Phylogenetics and Evolution 135 (2019) 45–61

(Schnable et al., 2011; Ainouche et al., 2012). There is also significant central Asia (Miao et al., 2012). evidence in the literature of rDNA loci reduction following poly- We hypothesize that the ancestor of the genus Pulsatilla – pre- ploidization (Clarkson et al., 2005; Dvořák, 1990; Vaio et al., 2005; sumably a member of Anemone s.s. – lived in the central Asian region, in Weiss-Schneeweiss et al., 2007). Hemizygous 5S rDNA loci were ob- the generally more humid and warm Miocene evergreen or semi-ever- served in the majority of the investigated polyploid species of Pulsatilla, green forests (Spicer, 2017), but with the progressive uplift of the most likely occurring as a consequence of reduction of 5S rDNA during Pamir-Alay and Tian Shan ranges it adapted to the ensuing colder and diploidization, though this process has occurred to differing extents forest-free mountainous conditions. The subsequent global cooling among the polyploid taxa. Furthermore, Pulsatilla polyploids exhibit period (Fig. 2) is likely to have facilitated diversification of a high- additional 35S rDNA signals of smaller size and/or lower intensity that mountain lineage represented today by subgenus Preonanthus that are located in the centromeric/pericentromeric regions of the chro- subsequently spread to other high mountain ranges of the Northern mosomes, which are enriched in transposable elements (Mlinarec et al., Hemisphere, including the Rocky Mountains of North America. The Far 2016). Interestingly, dispersed 35S rDNA signals were not found in East then became a secondary centre of speciation; the gradual global Pulsatilla diploids. We postulate that transposable element mobilization cooling and aridification of central Asia allowed them to colonize the was induced by polyploidization and that the seeding of rDNA repeats grasslands of lowland Eurasia, which expanded toward the close of the to ectopic locations in the genome was transposon-mediated. A sig- Miocene (Tang et al., 2011). nificant fraction of the rDNA units in animals are interrupted by The formation of extensive grasslands during the Pliocene and – transposable elements that are highly specialized for insertion into more importantly – during the Quaternary (Guthrie, 2001) had a dra- conserved sites within the rDNA genes (Zhang et al., 2008); recent matic effect on the putative evolutionary radiation of the species; they studies suggested that they may encourage rDNA mobility (Costa et al., diversified into several lineages that migrated through grasslands from 2013). the Far East via central Asia to Europe, eventually reaching North The origin of polyploid Pulsatilla species has not previously been America on two occasions. The major climatic oscillations that char- investigated. The existence in P. grandis, P. vulgaris, P. rubra and P. acterized the subsequent Quaternary period might also have heavily halleri subsp. styriaca of two diploid chromosome sets distinguishable influenced speciation in this genus of predominantly grassland species, by size, morphology and rDNA FISH pattern indicates that series inducing repeated contraction and expansion of grasslands during the Pulsatilla had a hybrid, allopolyploid origin. Although chromosome glacial-interglacial cycles (Frenzel et al., 1992). markers such as rDNA cannot solely reveal the precise origin of poly- ploids, they can nonetheless reveal some interesting patterns (Mlinarec 5. Conclusions et al., 2012a; Jang et al., 2018). The occurrence and localization of the 5S rDNA on a particular chromosome pair can enable the identification (1) Our multi-gene phylogeny allowed reclassification within genus of markers of the two parental species involved in the formation of Pulsatilla; the resulting classification into three subgenera, five sections hybrid taxa. Pulsatilla polyploids have 5S rDNA signals positioned and eight series broadly resembles the most recent classifications ori- subterminally on the longer arm of metacentric chromosomes – a po- ginating from traditional morphological methods but differs in several sition was found only in P. turczaninovii among the investigated di- important respects. Although morphological differentiation is limited ploids. Furthermore, members of series Pulsatilla have 5S rDNA on the and an objective morphological analysis is still lacking, pinnately dis- shorter arm of submetacentric chromosomes, and a 5S rDNA-bearing sected leaves and nodding flowers are identified as apomorphic ten- submetacentric chromosome pair occurred only in P. alpina among the dencies within the genus. investigated diploids. Therefore, based on the phylogenetic distribution (2) Mapping of karyotypes across the poorly resolved sections of the rDNA FISH pattern, we hypothesize that the members of series Pulsatilla and Semicampanaria reveals phylogenetically correlated var- Pulsatilla have a single allopolyploid origin from P. turczaninovii and P. iation in the number and distribution of rDNA loci that is of value for alpina, presumably on the eastern Eurasian steppes (Fig. 3). Confirma- both circumscribing and determining the relationships of species. tion of this hypothesis would require application of more informative Particular polyploid patterns constitute apomorphies for some sections methods such as GISH (Mlinarec et al., 2012a). and series, though at least one putative species reputedly includes both diploid and tetraploid plants. 4.4. Evolutionary history (3) Reconstructing divergence dates and phylogeographic patterns of internal nodes of our coalescent species-tree suggest that species Polyploidization has frequently been implicated in both diversifi- diversity remains greatest in the region of origin of the genus in central cation and the colonization of new habitats (Fawcett and Van de Peer, Asia, potentially linked to the progressive uplift of the Pamir-Alay and 2010). Current mainstream theory states that polyploids are often Tian-Shan Mountains. abundant in harsh and unstable environments, and they often occupy (4) Subsequent diversification of this predominantly grassland new niches in which their diploid progenitors would be less likely to genus is hypothesized to have been driven by the well-documented persist (Fawcett and Van de Peer, 2010). Also, recent studies proposed global cooling of temperature and growing aridification during late that the comparative success of certain ancient polyploids might be Miocene, which is likely to have encouraged radical expansion of linked to periods of climatic change. Thus, the colonization of Europe grassland habitats in Eurasia. by the polyploid members of series Pulsatilla may have been facilitated (5) More extensive diversification, arguably constituting an evolu- by the periods of drastic climatic change that characterized the Pleis- tionary radiation, occurred during the Quaternary, presumably trig- tocene. gered by the environmental perturbations and consequent migrations According to our dated phylogeny (Fig. 2) and ancestral-state re- that characterize glacial-interglacial cycles and further encouraged by construction (Fig. 3), the ancestral area of the genus Pulsatilla most repeated polyploidization. likely lies in the central Asian mountains, a region previously identified (6) Although we have presented the most data-rich and tax- as an important area of origin for xeric grassland species in several onomically comprehensive phylogeny of the genus to date, our results other families of flowering plants (Zhang et al., 2014; Zhang et al., make clear that next-generation sequencing techniques (NGS: pre- 2015; Zhang et al., 2016; Zhang et al., 2017). Diversification of the ferably focusing on the nuclear genome) will be necessary in order to genus clearly coincided with the prolonged uplift of the northern cen- better resolve relationships at taxonomic levels up to and including tral Asian mountain ranges (Pamir-Alay, Tian Shan). Uplift became series, ideally combined with a morphological cladistic analysis rich in more pronounced in the late Miocene (Favre et al., 2015; Spicer, 2017), micromorphological characters. NGS will need to be combined with and is suspected of having contributed to the significant aridification of much more intensive sampling and morphometric surveys to achieve

58 G. Sramkó, et al. Molecular Phylogenetics and Evolution 135 (2019) 45–61 robust circumscription of species and subspecies, especially in the four # 15-29-02486], Russia and an institutional project financed by the series that contain multiple taxa that have proven molecularly near- University of Zagreb, Croatia. identical in our study (Fig. 2). Author contribution 6. Taxonomic treatment GS designed and carried out sampling, performed laboratory work, designed and performed phylogenetic analyses, wrote the manuscript; In light of our results, we provide here a revised classification for LL performed laboratory work, designed and performed phylogenetic the genus. Asterisked taxa were not included in our study so their analyses; PAV carried out sampling and wrote the manuscript; RMB classification below should be regarded as provisional; one putative performed full revision and restructuring of the manuscript; JM ob- species suffixed with a hashtag was not recognized by Grey-Wilson tained samples, designed and performed cytogenetic analyses, per- (2014): formed laboratory work, wrote the manuscript.

Genus Pulsatilla Mill. Conflict of interest

Subgenus Kostyczewianae (Aichele & Schweg.) Grey-Wilson The authors declare no conflicts of interest. species: P. kostyczewii (Korosh.) Juz. Subgenus Preonanthus (DC.) Juz. Section Preonanthus (DC.) Spach Appendix A. Supplementary material species: P. alpina Schrank, P. alba Rchb.*, P. aurea (N.Busch) Juz.,P. magadanensis Khokhryakov & Vorosch, P. scherfelii (Ullep.) Skalický Supplementary data to this article can be found online at https:// Section Preonanthopsis Zämelis & Paegle doi.org/10.1016/j.ympev.2019.02.015. species: P. taraoi (Makino) Takeda ex Zämelis & Paegle, P. nipponica (Takeda) Ohwi*, P. occidentalis Freyn, P. sachalinensis Hara# Subgenus Pulsatilla References Section Tatewakianae Tamura species: P. tatewakii Kudô, P. sugawarai Miyabe & Tatew., P. ajanensis Aichele, D., Schwegleb, H.W., 1957. Die Taxonomie der Gattung Pulsatilla. Repertorium Regel & Tiling novarum specierum regni vegetabilis 60, 1–230. https://doi.org/10.1002/fedr. Section Pulsatilla 19570600103. 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