Diversification of the European bladdernuts (Staphylea, Staphyleaceae) in context of the whole genus and the rich fossil record

Diversification of the European bladdernuts (Staphylea, Staphyleaceae) in context of the whole genus and the rich fossil record | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Diversification of the European bladdernuts (Staphylea, Staphyleaceae) in context of the whole genus and the rich fossil record Herlinde Vetters, Matthias Affenzeller, Andreas Tribsch This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4768147/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Staphyleaceae is a small family of shrubs and trees with its name-giving genus Staphylea having a highly disjunct distribution. Staphylea has a rich fossil record and was an important element in warm temperate Tertiary forests and is therefore regarded as a Tertiary relict. Based on DNA-sequence analyses of the nuclear marker ITS 1–2 and the chloroplast marker trnL-F as well as AFLP fingerprinting (Amplified Fragment Length Polymorphisms) we gained more insights into the evolution and diversification of the two ‶European″ bladdernut species, the widespread diploid Staphylea pinnata and the tetraploid Staphylea colchica of the Caucasus. As the Caucasus is located west of the Ural Mountains, we consider both species as European. Staphylea pinnata seems to be involved in the hybridization of the likely allo-poly-ploidization of Staphylea colchica together with an unknown, supposedly now extinct species. Ancient repeat types of ITS 1–2 in Staphylea pinnata of Central Europe and Georgia suggested possible glacial refugia in Georgia, sequence similarity (especially a characteristic gap) in ITS 1–2 sequences of Ukrainian and Central European samples indicate refugia also in Ukraine. Staphylea emodi , the only Staphylea species of Central Asia (Tibetan Plateau), was in our research more closely related to the European species than to American representatives. Staphylea Staphyleaceae disjunct distribution Tertiary relict refugia Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The small angiosperm family Staphyleaceae (Crossosomatales, APG III (Bremer et al., 2009); APG IV (Chase et al., 2016)) comprises trees and shrubs and is distributed disjunctively in Eurasia and North (South) America. Whereas deciduous species grow in temperate areas, several evergreen species reach Central America as well as tropical Asia. Takhtajan (1987) elevated the former subfamilies Staphyleoideae and Tapiscioideae to the rank of families, and Staphyleaceae now comprises three genera: Staphyle a L., Euscaphis Sieb. and Zucc., and Turpinia Vent. This taxonomic concept mainly builds on fruit morphology. Arising from a phylogenetic approach (Simmons, 2007; Simmons & Panero, 2000), however, the family has been split in two clades and genera, Staphylea L. and Dalrympelea Roxb., not congruent with the former circumscription. Harris et al. (2017) detected five major clades in Staphyleaceae. Their study included ten species of Staphylea , five of Turpinia as well as Euscaphis , evidenced with five chloroplast and two nuclear markers: (1) Old World Turpinia , (2) New World Turpinia , (3) exclusively Old World Staphylea , (4) an Asian-North American clade of Staphylea with all American species and the rest of the Old-World ones and (5) Euscaphis . There are two extant species in “Europe”: the rather widespread Staphylea pinnata L. and the Caucasian Staphylea colchica Stev. Both are elements of the Submediterranean-Nemoral Flora, i.e. of Summergreen Broad-Leaved Forests (Meusel & Jäger, 1992). In addition to regions of Central, Southern and Eastern Europe their areas incorporate parts of the neighborhood of the Black Sea (Fig. 1 a, 1 b) and therefore we rank them as European species. The bladdernuts geographically closest to Europe grow in the Himalayas ( Staphylea emodi Wall. ex Brandis), then in East Asia, e.g., Staphylea holocarpa Hemsl., Staphylea bumalda DC and Staphylea forrestii Balf.fil., and in eastern North America, e.g. Staphylea trifolia L. (Fig. 1 a). Some variation in chromosome numbers have been reported. Foster (1933) counted the chromosome numbers of Staphylea pinnata as n = 13, of Staphylea colchica as n = 26, and of Staphylea trifolia as n = 39. Staphylea pinnata ’s chromosome numbers were confirmed later on as diploid (Dobeš, Vitek, & Buttler, 2000; Peruzzi & Cesca, 2002). Thus, Staphylea colchica can be assumed as tetraploid and Staphylea trifolia as hexaploid. The largely European Staphylea pinnata and the Caucasian Staphylea colchica have been reported of being sympatric in the Western Caucasus where also natural hybrids have been postulated in spite of their different chromosome numbers (Gulisashvili, 1970; Poyarkova, 1986). Overlapping morphologic characteristics, especially of the leaves or immature fruits make the assignment to Staphylea pinnata or Staphylea colchica sometimes difficult (Table 1). Herbarium LE, scan number 2407 from Abkhasia, Poyarkova 1945 and Coll. Mus. Bot. Berol., from Adler near Sochi, Herbarium Krebs, 1987-06-10, evidence these difficulti Moreover, garden hybrids ( Staphylea pinnata x Staphylea colchica ) of unknown origin with intermediate morphology appeared in literature already in the 19th century: Staphylea x coulombieri (André, 1887), described in France, and Staphylea elegans (Zabel, 1888), documented in Germany. Weaver (1980) pointed out that both hybrids were identical according to herbarium records, and that the correct name would be Staphylea x coulombieri. European botanic gardens stick to the name Staphylea x elegans . What makes the Staphylaceae particularly interesting is the extensive fossil record of their characteristic seeds that is not only very rich but also goes far back into the mid Tertiary. There is good evidence that there was a continuous presence of bladdernuts in Europe since the Oligocene until today (Vetters, 2013). Already in Upper Oligocene fossilized seeds of Staphylea spp. were found in Western Siberia (Russia) and in Saxony (Germany) as well as Turpinia ettinghausenii seeds in the latter region. From Miocene and Pliocene numerous localities widespread across Europe, Russia and Japan with seeds of Staphylea spp. have been reported. Since the Middle Miocene also pollen discoveries have been referred. Whereas Turpinia ettinghausenii , the only Turpinia species in Europe, disappeared in Lower Pliocene (discussed in Vetters (2013)), fossil seeds of Staphylea have been found in deposits of all interglacial periods until Holsteinian (~ 450 − 370 ka before present) in northwest Europe (Willis & Niklas, 2004). Staphylea pinnata fossils have been even detected in Eemian deposits (Last interglacial period, 130 − 115 ka before present) in The Netherlands (Van der Ham et al., 2008). Recently even fossils from eastern North America of the border of Miocene to Pliocene were documented (Huang et al., 2015), and latest discoveries unearthed a Staphylea capsule of the Oligocene of Montana, USA (Zhu & Manchester, 2020). The diversification and historical biogeography of extant Staphylea species must be viewed in the light of their highly disjunct distribution in Northern Hemisphere. Geographical disjunctions of plant genera and species between North America and East Asia have been well discussed in literature since Gray (1840, 1846) and got recently even more attention (Qian, 2002; Wen, 1999). Early hypotheses for such huge disjunctions relate to migrations in former geological periods, e.g. eastwards via Bering Strait in the Tertiary (“Asa Gray disjunctions”) or westwards via the North Atlantic land bridge in Eocene, Lower Tertiary (Tiffney, 1985). Wen (1999) summarized her view as follows: “The disjunct pattern between Eastern Asia and eastern and western North America is the product of vicariance, dispersal, extinction and speciation”. Thus, it has been accepted that several different biogeographical processes did result in congruent patterns of these disjunctions. Geographical disjunctions within Eurasia, as in Staphylea spp., especially among warm temperate areas in Central and Southern Europe, Caucasus, southern slopes of the Tibetan Plateau, Southern China and Japan were hypothesized to be related to late Tertiary vicariance (Mai, 1995). Several species of trees and shrubs adapted to warm temperate and wet climate had been evidently widespread along the northern coast of the Tethys in the beginning of Tertiary. Because of the cooling climate (only interrupted by a slightly warmer phase during the Mid Miocene) they migrated south, became isolated and survived, and even partly speciated as so-called Tertiary relics. Examples include the following genera: Zelkova with relics in the Caucasus region ( Z. carpinifolia ), Crete ( Z. abelicea ) and Sicily ( Z. sicula ) (Christe et al., 2014; Kozlowski & Gratzfeld, 2013). Pterocarya fraxinifolia is distributed from the Caucasus to the south-coast of the Caspian Sea (Maharramova et al., 2018), Liquidambar orientalis in southwest Turkey (Dönmez & Yerli, 2018), Styrax officinalis in eastern Mediterranean, California, Texas (Fritsch, 1996), Aesculus sect. Aesculus in the southeast of Europe (Harris, Xiang, & Thomas, 2009). Parrotia persica can be found in the southeast of Georgia and in Iran (Nakhutsrishvili et al., 2015), as well as Staphylea colchica in the Black Sea region of western Georgia and adjacent parts of Russia (Shatilova et al., 2011). A key factor causing such disjunctions is the still ongoing orogenesis of mountain ranges from the Alps all the way to the Tibetan Plateau from Oligocene onwards (Frisch & Meschede, 2013). The emergence of mountain ranges played an important role for the distribution of plants by arising steppes in connection with increasing aridity in inland areas (Harris et al., 2017; Tiffney & Manchester, 2001; Zhang et al., 2000). The latest cut in chorography was due to the Ice Ages, inhibiting migration of plants and genetic exchange from north to south and vice versa, that led to refugia for warm temperated plants in small refugial areas (Willis & Niklas, 2004). Based on a phylogenetic approach using nuclear ribosomal (nr) and chloroplast (cp) DNA sequencing data we constructed a phylogeny focusing on European taxa of Staphyleaceae. Using AFLP fingerprinting additionally to DNA sequences we evaluated the phylogeography and relationships of Staphylea colchica and Staphylea pinnata . At first, we wanted to test if the dated phylogeny of Staphyleaceae was in line with the Tertiary vicariance hypothesis, secondly, we tested if the two taxa of Europe and the Black Sea region were closest relatives as suggested by morphology. Another question was, if Staphylea pinnata has been involved in the origin of the tetraploid Caucasian Staphylea colchica . Finally, we discuss our results in the light of possible glacial refugia and post-glacial colonization of Europe. Material and methods Taxon Sampling For DNA sequencing, we sampled Staphylea pinnata from Central Europe, Poland, Ukraine and Turkey as well as from Georgia, Staphylea colchica from Georgia and Southern Russia. The presumed garden hybrid Staphylea x elegans , a cross between Staphylea pinnata and Staphylea colchica (Zabel, 1888) from the Botanical Garden of Madrid is also included. Staphylea species from the Tibet Plateau, East Asia and eastern North America are added, predominantly newly sequenced, partly taken from NCBI ( NCBI, National Center for Biotechnology Information , 1988-) as well as the vouchers of the outgroup ( Crossosoma bigelovii , Stachyurus praecox , Stachyurus reticulata , Stachyurus chinensis , Stachyurus himalaicus ). Table S1 (Supplementary informations, SI) summarizes voucher information for all samples and NCBI accessions used for sequencing. For AFLP analysis, we surveyed 94 samples of three Georgian populations each of Staphylea pinnata and Staphylea colchica and two Central European populations of Staphylea pinnata (Tab. S2, SI). Two of the Georgian Staphylea pinnata populations included individuals from Adzharia and Bordžomi. Dmitriewa (1990) and Gulisashvili (1970) only described Staphylea colchica populations there, but we only found Staphylea pinnata. Geographic distributions as well as locations of taxa and populations used in the present study are shown in Fig. 1 . Data for the distribution maps (Fig. 1 ) were taken from the herbaria B, LE, TBI, MNHN-P, MW, W and from personal collections (Vetters 2003, 2004, 2005 in Georgia and the vicinity of Sochi, Russia), as well as from more than 30 references (“Chorology of the genus Staphylea – Methods and References”, SI). In the Black Sea region overlapping areas of Staphylea pinnata and Staphylea colchica are not yet resolved. That might be a problem of identification because of the genetic influence (see AFLP results) of Staphylea colchica seen in populations of Staphylea pinnata in that region (in contrast to the Central European bladdernuts) and its effects on morphology. Areas of the other Staphylea species used in Fig. 1 a ( Staphylea bumalda DC, Staphylea emodi Wall., Staphylea forrestii Balf.f., Staphylea holocarpa Hemsl., Staphylea trifolia L.) were taken from GBIF, Global Biodiversity Information Facility (1999 onwards) . DNA extraction and molecular techniques Genomic DNA was extracted according to a standard CTAB protocol (Doyle & Doyle, 1987; Murray & Thompson, 1980). For amplification of the ITS region, ITS1-5.8S-ITS2, primers AB 101 (5’-ACG AAT TCA TGG TCC GGT GAA GTG TTC G-3’) and AB 102 (5’-TAG AAT TCC CCG GTT CGC TCG CCG TTA C-3’), both were used (Douzery et al., 1999). Due to some ambiguous ITS sequences ten samples (Sp_A1, Sp_PO, Sp_TR1, Sp_U1, Sp_U2, Sp_U3, Sp_G1, Sc_K, Sc_M, Sel, see Tab. S1) were cloned using TA Cloning Kit (Life Technologies, California, USA). Following Harpke and Peterson (2008) we compared the three conserved motifs that should indicate functionality with our sequences. Amplification of trnL-F region was conducted using primers Tab C (5’-CGA AAT CGG TAG ACG CTA CG-3’) and Tab F (5’-ATT TGA ACT GGT GAC ACG AG-3’; (Taberlet et al., 1991; Zhao et al., 2011). PCR products were cleaned using illustra ExoProStar (GE Healthcare, Freiburg, Germany)) and sequenced by Macrogen (Seoul, S-Korea) or eurofins (Ebersberg, Germany) using primers ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’; White et al. (1990)) for ITS and Tab F for trnL-F region respectively. For Amplified fragment length polymorphisms (AFLP) we followed the protocol of Vos et al. (1995) with modifications according to Rebernig (2009) for 94 samples (eight populations in total) of Staphylea pinnata and Staphylea colchica (see also Tab. S2). For the preselective PCR we chose the pair of primers Eco RI-A (5’-GAC TGC GTA CCA ATT + A-3’) and Mse I-C (5’-GAT GAG TCC TGA GTA + C-3’). For selective PCR primers with three additional selective bases were used, three primer combinations in total. The Eco RI primers were fluorescently labeled: Eco RI-ACA / Mse l-CTG; Eco RI (6-FAM); Eco RI-AAC / Mse l-CTG; Eco RI (NED) and Eco RI-AGG / Mse l-CAT; Eco RI (HEX). The selective amplification program ran in a thermal cycler (90% ramp time). Separation and visualization of fragments was done on two polyamide gels using an automated sequencer (ABI 377, Perkin Elmer). GeneScan™ ROX TM -500 dye Size Standard was added to the fragments. Alignment of the raw data was done using ABI Prism GeneScan® Analysis Software v. 3.2.1 ( GeneScan Analysis Software 2001) Macintosh version). Subsequently the GeneScan files were imported into Genographer v. 1.1.0 (Benham et al., 1999) for scoring the AFLP fragments. Using the ‘thumbnail’ option for comparison of the signals of each fragment, we scored them as present or absent. Peaks of low intensity were included into the analysis when unambiguous scoring was possible. Computational Analyses For phylogenetic reconstruction DNA-sequences were edited and aligned using Geneious Pro 5.5.6 software © Biomatters Ltd. (Drummond et al., 2011) under MAFFT algorithm (Katoh et al., 2005; Katoh et al., 2002). We employed JModelTest 0.1.1 (Posada, 2008) to determine models of molecular evolution, ranked according to the “Akaike Information Criterion”, AIC (Akaike, 1973). For the trnL-F region the model TPM1uf + G best fitted the data. As this model is not implemented in MrBayes (Ronquist, Huelsenbeck, & Teslenko, 2011), the next best fitting model GTR + G (general time reversible model with a gamma distribution of site-specific rates), discussed in Hood et al. (2010), was chosen for both matrices. RAxML vs 1.3 (Stamatakis, 2014) was applied to conduct a Maximum Likelihood (ML) tree search analyses for both markers separately as well as concatenated under the GTR + G substitution model and 1000 rapid bootstrap replicates for statistical node support. ML bootstrap values ≥ 75 were considered as a threshold for good support (Pattengale et al., 2010). To create a time-calibrated phylogeny BEAST v1.7.5. (Drummond et al., 2012) was employed on ITS and trnL-F marker separately as well as on the combined ITS and trnL-F matrices. ITS repeats exist in many copies in the genome. Some of them may degenerate through evolution and lose their functionality but may be kept in the genome. Their existence may however lead to incorrect time data of the phylogenetic trees (Álvarez & Wendel, 2003). Therefore, the 5.8S regions of all ITS sequences were checked according to the three conserved motifs of Harpke and Peterson (2008). Then we compared phylogenies including and excluding sequences without presumed functionality. For all analyses a GTR + G substitution model, an uncorrelated log-normal relaxed clock and a birth-death process of speciation served as tree priors. Three runs each with 30 Mio generations for the trnL-F and ITS files or 100 Mio ones for the combined matrix with a sampling frequency of every 1000th, respectively every 10000th generation. We combined the tree files after a burnin of 25% in LogCombiner and constructed maximum clade credibility trees with mean node heights using TreeAnnotator (both programs are part of BEAST package, Drummond et al. (2012); Huson and Bryant (2006)). Trees were visualized using FigTree v1.3.1 (Rambaut & Drummond, 2009). For calibrating the trees, we took following considerations into account. Wikström, Savolainen and Chase (2001) involved Crossosomataceae, Stachyuraceae, Staphyleaceae - representing our dataset - as well as Aphloiaceae and Strasburgeriaceae into their molecular clock calculations. The crown age of Crossosomatales, systematically integrated according to APG III, Angiosperm Phylogeny Group classification III (Bremer et al., 2009) as well as in APG IV (Angiosperm et al., 2016; Chase et al., 2016) resulted in 95 ± 4 Mya. The stem age of Staphyleaceae accouted for 62 ± 6 Mya, the split between Crossosomataceae and Stachyuraceae for 44 ± 5 Mya (estimated by maximum parsimony with ACCTRAN optimisations). Wikström, Savolainen and Chase (2001) used the split between Fagales and Cucurbitales for calibrating their tree. Since Wikström, Savolainen and Chase (2001) many further approaches were done to calibrate the “tree of life” (Magallón & Castillo, 2009; Magallón et al., 2015; Palazzesi et al., 2012; Wang et al., 2009). In most publications, samplings of Crossosomatales’ sequences were not as complete as in Wikstroem, except for Magallón et al., who focused primarily on the rise of the angiosperms. They calibrated Crossosomatales by means of the fossil age of Turpinia , Staphyleaceae (Tiffney, 1979), with a minimum age of 28.45 Mya (beginning of Upper Oligocene). Using Bayesian relaxed clocks for calculation the result was a young age of 31.3 Mya - compared to Wikstroem - for the node of Staphyleaceae / Crossosomataceae, Stachyuraceae and Guamatelaceae, albeit with a 95% highest posterior density (HPD) interval of 30.62 Myr from 28.45 to 59.02 Mya. We considered such a large confidence interval not useful for our dating approach. Magallón et al. (2015) admitted that estimates for the crown ages of several families could be probably too young because of three reasons. The taxonomic sample may not represent the crown node of the targeted family, fossils used may not be its oldest members, and the assignment of calibrations to nodes was mostly based on apomorphies that could have already appeared in a more inclusive clade. On the other hand, Penalized Likelihood in Magallón et al. (2015) resulted in 66.5 Mya for the stem group of Staphyleaceae with a confidence interval of 14.52 Myr between 59.96 and 74.48 Mya, quite similar but even older than Wikstroem’s results. Therefore, we calibrated our phylogenetic tree according to Wikström, Savolainen and Chase (2001), corresponding also to Forest and Chase (2009). We used two calibration points for our analyses. First, the root (stem group of Staphyleaceae) was set to 62 ± 6 Mya and second, the outgroup (stem group of Stachyuraceae and Crossosomataceae) was set to 44 ± 5 Mya. We assumed a normal distribution prior for both nodes. Additionally, the uncorrected log-normal relaxed clock mean (ucld.mean) prior was set to 0.00164 substitutions per site per Myr, and standard deviation to 0.001 for ITS (Dick et al., 2013). For the trnL-F matrix the ucld.mean was set to 0.000929 substitutions per site per Myr, calculating the mean of the mutation rates from Inga (1.3x10 − 3 /Mya), Phylica (4.87x10 − 4 /Mya) and the “general mutation rates” of chloroplast markers (1.00x10 − 3 /Mya) in equal shares (Values taken from Richardson et al. (2001)) with a standard deviation of 0.0002255. Phylogenetic network estimation was executed by the program TCS 1.21 (Clement et al., 2000–2005; Clement, Posada, & Crandall, 2000). It calculated parsimony networks for each marker separately. For the calculation, the ITS file was reduced to sequences from Staphylea pinnata , Staphylea colchica , Staphylea elegans and Staphylea emodi without the three extremely aberrant ribotypes Sp_G2_1, Sp_G2_9 and Sp_A1_8. The trnL-F-file contained all members of the ingroup, i.e., of the family Staphyleaceae. Both files were manually gapcoded (“simple indel coding” according to Bena et al. (1998) and Simmons and Ochoterena (2000) with the gaps serving as fifth state in TCS. The probability levels were 95%. To gain network information within Staphylea pinnata and Staphylea colchica we used the program TCS (Clement et al., 2000–2005; Clement, Posada, & Crandall, 2000). For AFLP data analysis the presence / absence matrix of all samples was imported to SplitsTree V4.19.0 (Huson & Bryant, 2006; SplitsTree4 , 2010) for computing a Neighbor-Net tree according to Nei and Li (1979). AMOVA (Excoffier, Smouse, & Quattro, 1992; Weir, 1996; Weir & Cockerham, 1984) conducted hierarchical analysis of variance. Arlequin Ver 3.1 (Excoffier, Laval, & Schneider, 2006) estimated molecular diversity indices (Ewens, 1972; Nei, 1987; Tajima, 1993; Zouros, 1979), using the general settings (weight = 1, epsilon value = 1e − 07 , significant digits for output = 5, use of original haplotype definition for AMOVA and allowed level of missing data = 0.05. Structure v. 2.2 (Pritchard, Xiaoquan, & Falush, 2007) delimited the amount of admixture of populations in a Bayesian analysis of population structure using the models of recessive alleles and admixture, assuming one group and correlated allele frequencies. The burn-in period was set to 200000 and 500000 replicates followed as recommended by Pritchard, Xiaoquan and Falush (2007). Fossils The rich material of fossil seeds plus one fruit and one uncertain flower (Table 2) was plotted according to the periods of the fossil’s occurences into the gained phylogeny to associate them with the appearance of extant lineages. (Dorofeev, 1963, p. 210 f; Engelhardt & Kinkelin, 1908, p. 265; Geissert, Gregor, & Mai, 1990, pp. 50–51; Gregor, 1978, pp.79–84; Hernández-Damián, Cevallos-Ferriz, & Huerta-Vergara, 2019; Huang et al., 2015; Kolakovsky, 1964; Kovar-Eder & Krainer, 1988, pp. 41–42; Kovar-Eder & Meller, 2001, p. 86; Mai, 1997, p. 63; 2001, p. 115; Mai & Walther, 1988, p. 171; Mc Clennen, Jenkins, & Uhen, 2017; Negru, 1972, pp. 123–127; Ozaki, 1991; Palamarev, Ivanov, & Bozukov, 1999, p. 16; Palamarev & Petkova, 1987, p. 121; Shatilova et al., 2011; Szafer, 1946; Szafer, 1947, pp. 298–301; 1954, p. 47; Teodoridis, Kvaček, & Uhl, 2009; Van der Burgh, 1983, pp. 60–61; 1987, p. 325; Van der Ham et al., 2008, p. 133; Zhu & Manchester, 2020) Results ITS marker Functionality of the 5.8S gene Eight ITS sequences were evidenced as (presumably) non-functional copies. Three ribotypes were extremely aberrant in motif one/two/three (Harpke & Peterson, 2008): Sp_A1_8 (3/0/1), Sp_G2_1 (3/3/1) and Sp_G2_9 (4/3/0). Five differed only in one base in motif one (Turp_tern_2 (KR532697) and Sp_U1_5), in motif two (Sp_TR1_6 and Sp_U3_9) or in motif three (Sp_G2_7). Therefore, we gained two Beast trees of ITS: including all ribotypes (90 ribotypes and isolates, 722 characters, Fig. S1 , SI) to test the roughly estimated chronological relationships of the European Staphylea species to Asian and American ones, and excluding the presumably non-functioning ribotypes (82 ribotypes, 715 characters, Fig. S2 , SI), leading to correct times (Álvarez & Wendel, 2003). Diversities within the whole ITS data set and of the extremely aberrant ribotypes The diversity within the ribotypes, focusing on Staphylea pinnata and Staphylea colchica with functional ITS copies can be seen in Tab. S3 (SI). The different clusters in Tab. S3 arose from informative insertions and deletions gained by TCS Analysis (see: Phylogenetic network estimation). Because of striking similarities of the extremely aberrant ITS ribotypes of Staphylea pinnata with those of the Asian and American Staphylea species we did not skip them, but we compared them separately with the non-European species (Table 3). To ensure reproducibility nucleotid positions in Tab. S3 (SI) and Table 3 were taken from the same file, the file inclusive of the ribotypes with (presumably) non-functional 5.8S gene (Fig. S1 , SI). Staphylea emodi and Staphylea bumalda emerged as the nearest relatives to the European bladdernuts. Staphylea emodi ’s ITS sequences comprised 12 substitutions and a gap of three bases (position 88–90) compared to the 59 sequences of Staphylea pinnata and Staphylea colchica with a functional 5.8S gene. These changes were typical for all other Staphyleaceae of our research as well as for the outgroup. Staphylea emodi contained less substitutions than Staphylea bumalda (39 plus the gap of three bases mentioned above) or any other sequence. Phylogenetic network estimation (TCS) Characteristic insertions and deletions within the ITS haplotypes of Staphylea pinnata , Staphylea colchica and Staphylea elegans lead to more insights into geographic patterns on a European scale by the program TCS (Fig. 2 , but also Tab. S3 (SI)). All samples of Staphylea colchica and Staphylea elegans contained unique ribotypes with the insertion of CAA (three CAA repeats) in position 547–549 of the total alignment. The deletion of nine bases (AGTGTGGTT in position 153–161) in some ribotypes of Staphylea pinnata was also remarkable. We neither could find this deletion in ribotypes from Turkey and Georgia and Sp_Mi from Serbia nor in any other taxon analyzed. A gap of three bases (position 88–90) was characteristic for all Asian and American Staphylea species and Turpinia occidentalis as well as for the outgroup, but also found sporadically in Staphylea pinnata and Staphylea colchica (Sp_A1_8, Sp_G2_9 and Sc_K_6), seen in Table 3 and forming clades in Tab. S3 (SI) respectively. A 0 (Fig. 2 and Tab. S3) included ribotypes of Staphylea pinnata without the characteristic deletion of nine bases, but also of Staphylea colchica without the typical insertion of three bases. Haplotypes B 0 , C 0 and D 0 included only Staphylea pinnata ribotypes without this deletion. E 0 comprised all ribotypes of Staphylea pinnata with the deletion of the nine bases. F 0 contained only Staphylea colchica- and Staphylea elegans -ribotypes with the insertion of CAA for the third time. The three extremely aberrant ribotypes, and even Sc_K_6 were excluded by the TCS program (Tab. S3) trnL-F marker The alignment of 35 trnL-F - sequences comprised of 950 characters including the outgroup. Regarding this chloroplast region, only a few variable nucleotide positions existed within the ingroup (BEAST tree seen in Fig. S3 , SI). Due to the small amount of variation (Tab. S4, SI) and the chosen mutation rates from Richardson et al. (2001) young divergence times were gained. The TCS analysis of the chloroplast trnL-F dataset contained all Staphyleaceae, shown in Fig. S4 (SI). The program excluded only Turpinia occidentalis . Staphylea emodi differed in only one nucleotide from the uniform isolates of Staphylea pinnata , all but one Staphylea colchica and Staphylea elegans and was networked with Staphylea trifolia , Staphylea forrestii and Staphylea holocarpa. The two Staphylea bumalda isolates differed in one or two characters respectively from the uniform isolates and were networked with Sc_M, the only differing Staphylea colchica isolate from Russia. Concatenation The Beast trees of ITS (Fig. S1 – whole alignment, Fig. S2 - only haplotypes with functional 5.8S gene, Supplements) and trnL-F (Fig. S3 ) were incongruent concerning the different position of Staphylea bumalda , but the nodes in question of the ITS tree (Fig. S2 ) were supported with 87% (RAxML), and of the trnL-F tree (Fig. S3 ) only with 62% (RAxML). So, the trees of both molecular markers could be concatenated (Norup et al., 2006). The concatenated trees are shown in Fig. 3 (whole alignment) and in Fig. S5 (reduced alignment, see Supplements). The ages of important divergence times of all BEAST analyses are summarized in Tab. S5 (SI), including their confidence intervals (95%). Fossil data Based on the concatenated tree of Fig. 3 and the rich fossil data (Table 2) we could compare the results and evaluate the age of divergence of aberrant ITS ribotypes as well. The clade NE America plus SE Asia 2 separated from all other Staphylea species at 27.46 ± 12 Mya, where already Staphylea woodworthensis in USA and three Siberian Staphylea species had occurred. The two Georgian Staphylea pinnata ITS ribotypes (Sp_G2_1 and Sp_G2_9) separated from the sister clade of Staphylea bumalda , Staphylea emodi and the mixed group of Staphylea pinnata and Staphylea colchica at 19.15 ± 8.8 Mya. At that time Staphylea microsperma and Staphylea bessarabica lived in Central Europe, and at the end of the confidence interval also Staphylea pliocaenica , which is regarded as the ancestor or at least as a near relative of Staphylea pinnata (Mai, 2001). The aberrant ITS ribotype of Staphylea pinnata from Austria (Sp_A1_8) separated at about 11.06 ± 5.5 Mya from the clade of Staphylea emodi plus the mixed group of Staphylea pinnata and Staphylea colchica. AFLP data of Staphylea colchica and Staphylea pinnata Analysis of the genome structures of populations enabled insights in the relationship of both Staphylea species as well as in differences of Staphylea pinnata populations separated by geographical distances (Fig. 4 a, see also Fig. S6 , SI). In total we scored analyzed 113 fragments, 53 (6-FAM), 28 (NED) and 32 (HEX), 24 fragments were monomorphic in all 94 individuals. The Neighbor Joining tree of AFLP analysis shown in Fig. 4 a evidenced two lineages corresponding to the two species, but Staphylea pinnata of Central Europe also form a highly supported lineage separated from Georgia`s Staphylea pinnata populations. To show the distribution of shared AFLP markers of geographical regions a Venn diagram was drawn (Fig. 4 b). All three regions had 51, accordingly 45.1% of the markers, in common. Staphylea colchica shared markers mainly with Georgian Staphylea pinnata (16.9%) and had a high number of specific markers absent in Staphylea pinnata (31.0%). Only five markers (7% of the markers) were characteristic for both regions of Staphylea pinnata that we could not find in Staphylea colchica . We compared each Staphylea pinnata population of Georgia separately with the Central European Staphylea pinnata and Staphylea colchica of Georgia in Fig. S6 . Two populations of Georgia, collected from regions of presumed Staphylea colchica , turned out to be Staphylea pinnata (Sp Ba and Sp Bo, Fig. S6 , Tab. S2). Nevertheless, they shared AFLP fragments with Staphylea colchica which were absent in Staphylea pinnata from Central Europe, 15 and nine respectively, quantities comparable to the Georgian population of Lagodekhi (eight fragments, Sp La, Fig. S6 ) which had been declared undoubtedly as Staphylea pinnata (Tab. S2). Structure Analysis (Pritchard, Xiaoquan, & Falush, 2007) revealed K = 3 as most probable number of groups (Figs. S7, S8) congruent with the Neighborjoining results (Fig. 4 a). Admixtures of less than 13% were present within very few individuals. Two individuals from Krikhi (Sc-Kr-12 and Sc-Kr-13) as well as one from Samegrelo (Sc-Sa-7) showed most of the admixture with Staphylea pinnata from Georgia. The Staphylea pinnata populations of Batumi and Bordžomi, from regions of Staphylea colchica , contained some individuals admixed with Central European ones, only Sp_Ba_11 and 13 with Staphylea colchica . The Staphylea pinnata population of Lagodekhi (Sp_La_1–5) and the Central European populations exhibited nearly no admixture (Fig. S6 , S8). Hierarchical AMOVA (Excoffier, Smouse, & Quattro, 1992) for the three lineages (Fig. 4 ) showed significant hierarchical differentiation (see also Table 4). Most of the total AFLP variation resided among the three partitions that means about nine times of the variation among populations. Half of it can be found within populations. Staphylea colchica populations (C) featured highest percentages of gene diversity within populations and the Central European Staphylea pinnata populations (A) the lowest ones (see Fig. S8 and Table 5 ). Discussion The genus Staphylea offers the almost unique opportunity to study a highly disjunct and rather species poor genus with an exceptional fossil record during the Tertiary. Here we could show that most disjunctions are well in line with the fragmentation of warm temperate forests towards the end of the Tertiary resulting in geographical vicariance and we present additional genetic support for a continuous presence of Staphylea in Europe since the late Oligocene/Early Miocene. Europe : Staphylea colchica and Staphylea pinnata Regarding the phylogenetic tree in Fig. 3 the largely unresolved relationship of Staphylea colchica with Staphylea pinnata by the molecular markers ITS and trnL-F is striking. In contrast to that AFLP results suggested a clear genomic separation of both species (Figs. 4 a and 4 b, Fig. S6 ). The populations showed little amounts of genetic admixture in context to smaller or larger distances of Georgian Staphylea pinnata populations to Staphylea colchica (Fig. S7 , Fig. S8 ). The Georgian populations of both species showed high intra-population diversity (Table 5). Moreover, AFLP data exposed even more details within Staphylea pinnata , i.e., geographically differences between Central Europe and Black Sea region. Due to the capacity of clonal upgrowth of Staphylea ssp. the Central European populations might represent only a very few genotypes. Observations showed that Staphylea pinnata achieved dominance on isolated slopes, in diches and ravines and there were often great geographical distances between one and another population in Central, but also in Southern and Eastern Europe. Based on the present results and the presumably strong chromosomal breeding barrier between the diploid Staphylea pinnata and tetraploid Staphylea colchica , it is less likely that this sharing of markers across species-boundaries in Georgia reflects gene flow (hybridization/ introgression). Rather, it suggests genetic depletion of Staphylea pinnata near its western limit of distribution, e.g. following range expansion - “leading edge expansion” (Hewitt, 2001). The tremendous human contribution to the distribution of this plant in Europe even since Iron Age for cultural and religious reasons have also to be taken into account (Heiss et al., 2014; Vetters, 2013). Thus, Staphylea pinnata could be largely archeophytic in large parts of Europe, which also could result in genetic uniformity. An interesting observation of the seed morphology of the fossils (Heiss et al., 2014; Vetters, 2013), Table 2) shows a morphological progression from smaller to larger seeds in Europe from Late Oligocene to Pliocene. Diploid Staphylea pinnata has the largest seeds in the genus and tetraploid Staphylea colchica a seed size like the fossil Staphylea pliocaenica. The phylogenies nevertheless suggest a very close relationship of these two extant species (Fig. 3 ). Origin of the tetraploid Staphylea colchica Staphylea colchica is most likely of allotetraploid origin because of the 35 specific AFLP markers of the Staphylea colchica populations whereas Staphylea pinnata has only eight specific markers (Fig. 4 b). This suggests that Staphylea pinnata could be one of the parental species, as both extant species showed the same cpDNA haplotype (Fig. S3 ). Considering the timeframe, it is more likely that Staphylea pliocaenica , its presumptive ancestor (Teodoridis, Kvaček, & Uhl, 2009) was one parent. Its form is similar to Staphylea pinnata and size and thickness of its testa resembles Staphylea colchica (Mai, 2001). Poyarkova (1986) indicated Staphylea emodi as the nearest related species to Staphylea colchica . Indeed, their morphologies are similar including the small seeds. However, despite the similarities in the ITS sequences to Staphylea colchica and Staphylea pinnata , Staphylea emodi can be excluded as the second possible parent of Staphylea colchica because the ITS region of three samples (Sem_1, Sem_2, Sem_3) did not contain the insertion CAA (leading to threefold CAA), characteristic for Staphylea colchica (Tab. S3, clade F). The other Asian or American living species included in our study can be excluded for the same reason, therefore we presume that a now extinct European Staphylea species with small seeds might be assumed as second parent to allotetraploid Staphylea colchica . It remains speculative, as the dated ITS-phylogeny does not resolve the relationship fully (Fig. 3 ), but most likely S taphylea colchica evolved as late as the Early Pliocene where Staphylea pliocaenica and Staphylea colchica fossilis are documented from European sites (France and Poland) together with Staphylea cf. trifolia fossilis (Geissert, Gregor, & Mai, 1990; Szafer, 1947; Teodoridis, Kvaček, & Uhl, 2009). Interestingly neither Georgia nor Russia documented fossil seeds of Staphylea pinnata nor of Staphylea pliocaenica . Shatilova et al. (2011) did not even mention Staphylea pinnata up to the Holocene. Phylogeographical aspects of the European species, the Colchis refugium and cryptic refugia One of the most interesting regions is the Colchis area, which is regarded as a hot spot of Tertiary relicts, also supported by a rich fossil record. Models of late Quaternary climatic changes confirmed the existence of a deciduous forest unit in the surrounding of Sukhumi (Abkhazia) at least for 14 000 years (Connor & Kvavadze, 2009). As traditionally suggested Western Georgia can be safely viewed as refugium for Staphylea colchica although seeds of Staphylea protocolchica Kol. were mentioned only once from the border of Miocene to Pliocene of Kodori, Abkhazia (Shatilova et al., 2011), Staphylea sarmatica Krysht. from Upper Miocene of Krynka, Black Sea region (Poyarkova, 1986), Staphylea sp. from Maikopian (Mid Miocene) on the river Terek, Caucasus region (Gulisashvili, 1970). Compared to the fossil seeds, palynomorphs of Staphylea are more frequent in Eastern Europe and the Black Sea region since late Miocene. Fossil pollen of Staphylea colchica is cited in West Georgia from 7.1–0.6 Ma, and again in Holocene, of Staphylea sp. from Middle to Upper Miocene, as well as from 0.4–0.01 Ma, and missing subsequently (Shatilova et al., 2011). Fossil pollen of Staphylea sp. is documented from the Russian plain, Urals, Georgia, Ukraine, Poland, Hungary and other European countries, where Pliocene pollen of Staphylea pinnata is only referred from Siberia ( Russian paleobotanical online workshop , 2021). Usually, the taxonomic resolution of pollen is lower than that of macrofossils (Moreno-Amat et al., 2017), so it could indicate that some pollen findings of Staphylea sp. in Georgia eventually might be closer related to Staphylea pinnata than to Staphylea colchica . However, Colchis might have been not only a Tertiary refugium for Staphylea colchica but also for Staphylea pinnata that Lachashvili et al. (2021) assume at least as Tertiary relict from the region of Tbilisi. From there Staphylea pinnata could have spread along the northern and the southern Black Sea coasts in westward directions, as well as upcountry eastwards. These hypotheses are consistent with the phylogeographical patterns of Primula vulgaris (Volkova, Schanzer, & Meschersky, 2013), another species of rather warm temperate deciduous forests. Anapa, at the western border of Staphylea pinnata ‘s recent eastern part area on the NE coast of the Black Sea is also the western border of Colchis based nuclear ITS and cpDNA haplotypes of Primula vulgaris . On the south coast isolated populations of Staphylea pinnata are found westwards as far as Zonguldak. The border of Colchis haplotypes of Primula vulgaris is Trabzon, about 860 kilometers eastwards. In case of Staphylea pinnata the western borders on both coasts are also coinciding with current climatic restraints, especially with regular pronounced summer droughts (Meusel & Jäger, 1992). Staphylea ssp. were documented in Northwestern Europe in each Interglacial, but Staphylea pinnata did not reach this area during the Holocene (Van der Ham et al., 2008; Willis & Niklas, 2004). However, ice-free regions in Eastern Carpathians could have acted as refugia for Staphylea pinnata as suggested by (Derevenko, 2005) for the area between the rivers Prut and Dnister in Ukraine, an area where this species appears today. From there this species could have spread westwards in Holocene through the passageway of the gate of Przemyśl (Derevenko, 2005). This view gets support in our data by a high ribotype-diversity especially in the Ukrainian samples and is coinciding with the appearance of a characteristic gap of nine bases also found in Central Europe (Tab. S3), that was not detected in ribotypes of Georgia, Turkey and Serbia. Relictic populations of Syringa josikaea , a moisture demanding deciduous shrub endemic to the Apuseni Mountains and the Ukrainian Carpathians (Lendvay, 2014; Lendvay et al., 2016) argue also for a refugium for warm temperate deciduous forests in Eastern Europe. Diversification of contemporary Staphylea in Europe and SW-Asia and the fossil record The fossil record in Eurasia suggests a continuous presence of Staphylea spp. since the Oligocene, which is in good correspondence with the phylogeny ((Harris et al., 2017); Fig. 3 ). The European diversification probably has started in the Pliocene, but the presence of aberrant, partly non-functional ITS copies in the area might suggest that the European lineage has been attendant in the area since the Early Miocene. The dated phylogeny including such copies might be inaccurate when it comes to dating nodes, as mutation rates of non-functional copies might differ (Álvarez & Wendel, 2003; Harpke & Peterson, 2008), but the topology is nevertheless well supported. The pseudogene problem is well-known (Álvarez & Wendel, 2003) and it demands caution in the application of ITS as phylogenetic marker. In our study, we conclude, that the most aberrant sequences (Sp_G2_1, Sp_G2_9 and Sp_A1_8 with striking similarities to the Asian and American ribotypes) represent “ITS-ghosts from the past” and prove the long Tertiary history with a continuous presence of Staphylea in Europe as suggested by the fossil record (Fig. 3 ). The European - East Asian disjunction might date to the late Oligocene or Early Miocene, a period where almost continuous warm-temperate forests were present in Eurasia (Mai 1995). In SE Asia we found two clades separated from the Himalayan Staphylea emodi (Fig. 2 ) - which Browicz (1971) documented also from Iran. However, the phylogenetic position of Staphylea emodi and its sister relationship to the European species Staphylea pinnata and Staphylea colchica in our ITS phylogeny (see Supplements, Figs. S1 and S2) differs from Harris et al. (2017). Their study suggests a close relationship of Staphylea emodi with the American Staphylea species, whereas our study has evidenced unambiguously in close relationship to the European ones. This discrepancy remains contradictory and does not allow a conclusive discussion but draws attention to Staphylea emodi as a not well-studied Central Asian relictic species. Staphylea ssp. fossils in North America and Eurasia Mai (1995) already supposed that the lack of Staphylea fossils in Northern America was due to missing of appropriate embedding material compared to the numerous seed findings in coal stockyards but also in clay pits of Eurasia. New fossil discoveries, however, shed new light in this topic. Huang et al. (2015) reported on the “First fossil record of Staphylea from North America”, Staphylea levisemia sp. nova, seeds from the latest Miocene to the Earliest Pliocene from Tennessee. The second Amercian fossil recently found, a flower of Staphylea ochoterenae Hernández-Damián et al. sp. nov., bedded in amber of Miocene in South Mexico (Hernández-Damián, Cevallos-Ferriz, & Huerta-Vergara, 2019), differs from extant Staphylea flowers in lacking a flowering disc and having only one style (instead of two or three). Thus, the feature of the postgenitally united carpel tips forming a compitum is lacking, characteristic even for all recent Crossosomatales (Matthews & Endress, 2005). It grew associated with tropical elements. The most surprising discovery was Staphylea woodworthensis Zhu & Manchester sp. nov., a recently described fossil taxon from Montana (Zhu & Manchester, 2020). Its fruit (preserved pericarp) is Staphylea -like, contains two carpels, but only seed attachments scars have been conserved. The fossil has been discovered compressed in shale. The seeds may have been released from the fruit during dehiscence of the capsule. The authors report similarities to the fruits of Staphylea bumalda from East Asia. As the age of the remarkable fossil was dated to the Oligocene new questions did arise. The oldest Eurasian Staphylea fossils date from late Oligocene: Staphylea rotundata, Staphylea rugosa, Staphylea tymensis from Siberia (Dorofeev, 1963) and Staphylea microsperma from Germany (Gregor, 1978; Mai, 1997), and hence are younger (Fig. 3 ) or at least of same age as Staphylea woodworthensis . If its geological layer dated from Early or Mid-Oligocene it would be approximately as old as our diversification node for the genus Staphylea , and inside Staphyleaceae (Fig. 2 , Table 2). Harris et al. (2017) see potential of at least two phylogeographic options for the genus Staphylea , colonization from Eurasia to North America or the other way round. According to their phylogeny they suggest a rare Himalayan – North American disjunction concerning Staphylea emodi . They suppose that either the Asian-American clade could have originated in North America and colonized Asia twice or it originated in Asia, arrived in North America, and Staphylea emodi recolonized Asia. The plant sample used has been obtained from unvouchered cultivated plants (sample AA-478-78; Arnold Arboretum, see Harris et al. (2017) in Table 1 and Olmstead et al. (2000), Appendix 1). In our phylogeny Staphylea emodi is clearly the sister species of Staphylea pinnata/colchica (Fig. 3 ). As we made use of three herbarium samples from Herbarium Edinburgh obtained from wild populations (Tab. S2) we trust the phylogenetic position in our dataset. So Staphylea emodi does not seem to have been involved in migration events to North America as Harris et al. (2017) assumed. The Oligocene Staphylea woodworthensis from Northwest America may be an argument for a wide distribution of Staphylea in the Northern Hemisphere in mid Tertiary. Wen (1999) explained the general phylogeographic pattern of temperate forest elements by their continuous distribution and their fragmentation and extinction during late Tertiary and Quarternary climatic cooling and aridifications. The phylogenies presented in Harris et al. (2017; apart from Staphylea emodi ) and our dated phylogeny with Staphylea trifolia as North American representative as sister to the clade “East Asia 2”) support a more recent connection. As the deepest node in this North American – East Asian clade was dated to late Miocene/Pliocene (Fig. 3 ) this visualizes perhaps extinction of early Staphylea species and late recolonization of the American continent. The discovery of Staphylea levisemia (see above) in latest Miocene/earliest Pliocene supports this view. Declarations Authors’ contributions Vetters collected most of the silica gel dried samples in Georgia, southern Russia and Central Europe and wrote the paper. Affenzeller was responsible for sequencing in Salzburg as well as for collaboration in computational analyses with Vetters, and Tribsch contributed a lot to the interpretations, wrote part of the discussion and together with Affenzeller copy-edited the paper. Acknowledgements We want to express cordially thanks to Erich Hübl (Vienna) for accompanying and scientific support on three excursions to Georgia and Southern Russia. We like to thank Shamil Shetekauri (Tbilisi State University, Georgia) for guiding us in Georgia, as well as Carolin Anna Rebernig (University of Vienna) for doing the AFLP labwork. The study was partially supported by the HRSM-Project “Aufbau von universitären DNA-Barcoding-Pipelines für ABOL” financed by the Austrian Ministry of Science and Economy as well as of the “Stiftungs- und Förderungsgesellschaft of the University of Salzburg”. Supplementary information (SI) In SI included are annotations and the references for the chorology of the Staphylea species as well as detailed tables of the taken samples. You further can find supplementary figures of Beast Trees of the markers ITS, trnL-F and concatenated ones as well as supplementary material concerning the AFLP research. References Akaike, H. (1973). 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Supplementary Files Fig.S1.eps Fig.S2.eps Fig.S3.eps Fig.S4.eps Fig.S5.eps Fig.S6.eps Fig.S7.eps FigS8.eps Tab.S1.xlsx Tab.S2.xlsx TabS3neu.xlsx Tab.S4.xlsx Tab.S5.xlsx Tab.1.xlsx Tab.2Macrofossils.xlsx Tab.3.xlsx Tab.4.xlsx Tab.5.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 04 Feb, 2025 Reviews received at journal 03 Feb, 2025 Reviews received at journal 22 Jan, 2025 Reviewers agreed at journal 07 Jan, 2025 Reviewers agreed at journal 06 Jan, 2025 Reviewers agreed at journal 06 Jan, 2025 Reviewers agreed at journal 02 Sep, 2024 Reviewers agreed at journal 28 Aug, 2024 Reviewers invited by journal 28 Aug, 2024 Editor assigned by journal 22 Jul, 2024 Submission checks completed at journal 22 Jul, 2024 First submitted to journal 19 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4768147","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":333683298,"identity":"a040094f-aa33-4fb1-921f-1a6ba54989a3","order_by":0,"name":"Herlinde Vetters","email":"data:image/png;base64,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","orcid":"","institution":"Paris Lodron University of Salzburg","correspondingAuthor":true,"prefix":"","firstName":"Herlinde","middleName":"","lastName":"Vetters","suffix":""},{"id":333683299,"identity":"ef8d9d01-0991-49dd-b0c8-6acb710a5b27","order_by":1,"name":"Matthias Affenzeller","email":"","orcid":"","institution":"Paris Lodron University of Salzburg","correspondingAuthor":false,"prefix":"","firstName":"Matthias","middleName":"","lastName":"Affenzeller","suffix":""},{"id":333683302,"identity":"d54c2736-ade8-48db-a72e-0e6744e98510","order_by":2,"name":"Andreas Tribsch","email":"","orcid":"","institution":"Paris Lodron University of Salzburg","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Tribsch","suffix":""}],"badges":[],"createdAt":"2024-07-19 13:56:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4768147/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4768147/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62640106,"identity":"e26d0ea2-6216-49c8-b40a-6352f6029163","added_by":"auto","created_at":"2024-08-16 18:39:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11129154,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of \u003cem\u003eStaphylea \u003c/em\u003espp.,\u003c/p\u003e\n\u003cp\u003elocations of the specimens used in the analyses are given; \u003cem\u003eStaphylea trifolia\u003c/em\u003e pink, \u003cem\u003eS. pinnata\u003c/em\u003e violet, \u003cem\u003eS. colchica\u003c/em\u003e green, \u003cem\u003eS. emodi\u003c/em\u003e red, \u003cem\u003eS. forrestii\u003c/em\u003e orange, \u003cem\u003eS. holocarpa\u003c/em\u003e brown, \u003cem\u003eS. bumalda\u003c/em\u003eyellow. (See also Tabs. S1 and S2 in Supplementary Informations for details)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eGlobal overview;\u003cstrong\u003e b\u003c/strong\u003e Detail, Distribution of \u003cem\u003eStaphylea pinnata\u003c/em\u003e and \u003cem\u003eS. colchica\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e▼ Triangles on the top: samples used in the sequence studies, ▲ Triangles on the base: samples used in the AFLP study, \u003cstrong\u003eΟ\u003c/strong\u003e Dots represent isolated occurences.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4768147/v1/76c2472195fbf3a894b920b9.png"},{"id":62640107,"identity":"ab7d4ded-32e6-49f7-a435-24b4402940c6","added_by":"auto","created_at":"2024-08-16 18:39:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":389613,"visible":true,"origin":"","legend":"\u003cp\u003eHaplotype network of ITS-sequences of the European \u003cem\u003eStaphylea\u003c/em\u003e species\u003c/p\u003e\n\u003cp\u003eusing Statistical Parsimony implemented in TCS (Clement et al. 2000, 2000-2005); using simple gap coding (gaps= 5\u003csup\u003eth\u003c/sup\u003e state, statistical probability 95%); species and geographical origin are indicated by different colors; see Tab. S3 for details.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4768147/v1/16e33434c88062430eb2aa23.png"},{"id":62639896,"identity":"0a69da7c-bf76-443c-9b36-eeffd7a0dbe7","added_by":"auto","created_at":"2024-08-16 18:31:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":335805,"visible":true,"origin":"","legend":"\u003cp\u003eTime calibrated phylogeny\u003c/p\u003e\n\u003cp\u003eusing Maximum Likelihood of Staphyleaceae (ITS and trnL-F concatenated), using Beast 1.7.5. (Ronquist, Huelsenbeck, Teslenko 2011) and RAxML Vs 8 (Stamatakis 2014); ITS haplotypes with presumably non-functional 5.8S gene included (see text for details); \u003cem\u003eStaphylea\u003c/em\u003e fossils (see Tab. 2 and Tab. S3) are indicated on the timeline.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4768147/v1/0c6dd6ef2b5a3a72aa64f32e.png"},{"id":62639895,"identity":"3f222a6f-acc0-44c4-a3c3-f8f809214cad","added_by":"auto","created_at":"2024-08-16 18:31:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":419959,"visible":true,"origin":"","legend":"\u003cp\u003ePopulation analyses\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Neighbor-Net\u003c/p\u003e\n\u003cp\u003eestimated using SplitsTree V4.19.0 based on 113 AFLP-fragments scored of European \u003cem\u003eStaphylea\u003c/em\u003e species in Central Europe and Georgia. Bootstraps were calculated based on 1000 replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Venn diagram of AFLP marker distribution of European \u003cem\u003eStaphylea\u003c/em\u003especies.\u003c/p\u003e\n\u003cp\u003eGroups defined: \u003cem\u003eSp\u003c/em\u003e ME: \u003cem\u003eStaphylea pinnata\u003c/em\u003e of Central Europe, \u003cem\u003eSp\u003c/em\u003e GE: \u003cem\u003eS. pinnata \u003c/em\u003eof Georgia, \u003cem\u003eSc\u003c/em\u003eGE: \u003cem\u003eS. colchica\u003c/em\u003e of Georgia; Numbers denote the quantity of different/shared AFLP-fragments, in brackets the number of fragments present in all individuals of the unit is given.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4768147/v1/08a31bc6ac60640d24bb675a.png"},{"id":62640877,"identity":"d2fad152-4a6f-41cd-9089-3adaa31085f8","added_by":"auto","created_at":"2024-08-16 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18:39:19","extension":"xlsx","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":15200,"visible":true,"origin":"","legend":"","description":"","filename":"Tab.3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4768147/v1/380f2dae4cdef84a27cd87e5.xlsx"},{"id":62639907,"identity":"e1db512b-bb44-4f7c-bb01-4c639e035b9c","added_by":"auto","created_at":"2024-08-16 18:31:20","extension":"xlsx","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":10090,"visible":true,"origin":"","legend":"","description":"","filename":"Tab.4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4768147/v1/c57b6b86892a83f0f2d10b7c.xlsx"},{"id":62640112,"identity":"3e33b430-0cfc-4a1f-9ac8-594dac4c1d64","added_by":"auto","created_at":"2024-08-16 18:39:20","extension":"xlsx","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":11460,"visible":true,"origin":"","legend":"","description":"","filename":"Tab.5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4768147/v1/20a35b3046edc97e5b3060d2.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Diversification of the European bladdernuts (Staphylea, Staphyleaceae) in context of the whole genus and the rich fossil record","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe small angiosperm family Staphyleaceae (Crossosomatales, APG III (Bremer et al., 2009); APG IV (Chase et al., 2016)) comprises trees and shrubs and is distributed disjunctively in Eurasia and North (South) America. Whereas deciduous species grow in temperate areas, several evergreen species reach Central America as well as tropical Asia. Takhtajan (1987) elevated the former subfamilies Staphyleoideae and Tapiscioideae to the rank of families, and Staphyleaceae now comprises three genera: \u003cem\u003eStaphyle\u003c/em\u003ea L., \u003cem\u003eEuscaphis\u003c/em\u003e Sieb. and Zucc., and \u003cem\u003eTurpinia\u003c/em\u003e Vent. This taxonomic concept mainly builds on fruit morphology. Arising from a phylogenetic approach (Simmons, 2007; Simmons \u0026amp; Panero, 2000), however, the family has been split in two clades and genera, \u003cem\u003eStaphylea\u003c/em\u003e L. and \u003cem\u003eDalrympelea\u003c/em\u003e Roxb., not congruent with the former circumscription. Harris et al. (2017) detected five major clades in Staphyleaceae. Their study included ten species of \u003cem\u003eStaphylea\u003c/em\u003e, five of \u003cem\u003eTurpinia\u003c/em\u003e as well as \u003cem\u003eEuscaphis\u003c/em\u003e, evidenced with five chloroplast and two nuclear markers: (1) Old World \u003cem\u003eTurpinia\u003c/em\u003e, (2) New World \u003cem\u003eTurpinia\u003c/em\u003e, (3) exclusively Old World \u003cem\u003eStaphylea\u003c/em\u003e, (4) an Asian-North American clade of \u003cem\u003eStaphylea\u003c/em\u003e with all American species and the rest of the Old-World ones and (5) \u003cem\u003eEuscaphis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThere are two extant species in \u0026ldquo;Europe\u0026rdquo;: the rather widespread \u003cem\u003eStaphylea pinnata\u003c/em\u003e L. and the Caucasian \u003cem\u003eStaphylea colchica\u003c/em\u003e Stev. Both are elements of the Submediterranean-Nemoral Flora, i.e. of Summergreen Broad-Leaved Forests (Meusel \u0026amp; J\u0026auml;ger, 1992). In addition to regions of Central, Southern and Eastern Europe their areas incorporate parts of the neighborhood of the Black Sea (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) and therefore we rank them as European species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe bladdernuts geographically closest to Europe grow in the Himalayas (\u003cem\u003eStaphylea emodi\u003c/em\u003e Wall. ex Brandis), then in East Asia, e.g., \u003cem\u003eStaphylea holocarpa\u003c/em\u003e Hemsl., \u003cem\u003eStaphylea bumalda\u003c/em\u003e DC and \u003cem\u003eStaphylea forrestii\u003c/em\u003e Balf.fil., and in eastern North America, e.g. \u003cem\u003eStaphylea trifolia\u003c/em\u003e L. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Some variation in chromosome numbers have been reported. Foster (1933) counted the chromosome numbers of \u003cem\u003eStaphylea pinnata\u003c/em\u003e as n\u0026thinsp;=\u0026thinsp;13, of \u003cem\u003eStaphylea colchica\u003c/em\u003e as n\u0026thinsp;=\u0026thinsp;26, and of \u003cem\u003eStaphylea trifolia\u003c/em\u003e as n\u0026thinsp;=\u0026thinsp;39. \u003cem\u003eStaphylea pinnata\u003c/em\u003e\u0026rsquo;s chromosome numbers were confirmed later on as diploid (Dobeš, Vitek, \u0026amp; Buttler, 2000; Peruzzi \u0026amp; Cesca, 2002). Thus, \u003cem\u003eStaphylea colchica\u003c/em\u003e can be assumed as tetraploid and \u003cem\u003eStaphylea trifolia\u003c/em\u003e as hexaploid.\u003c/p\u003e \u003cp\u003eThe largely European \u003cem\u003eStaphylea pinnata\u003c/em\u003e and the Caucasian \u003cem\u003eStaphylea colchica\u003c/em\u003e have been reported of being sympatric in the Western Caucasus where also natural hybrids have been postulated in spite of their different chromosome numbers (Gulisashvili, 1970; Poyarkova, 1986). Overlapping morphologic characteristics, especially of the leaves or immature fruits make the assignment to \u003cem\u003eStaphylea pinnata\u003c/em\u003e or \u003cem\u003eStaphylea colchica\u003c/em\u003e sometimes difficult (Table\u0026nbsp;1). Herbarium LE, scan number 2407 from Abkhasia, Poyarkova 1945 and Coll. Mus. Bot. Berol., from Adler near Sochi, Herbarium Krebs, 1987-06-10, evidence these difficulti\u003c/p\u003e \u003cp\u003eMoreover, garden hybrids (\u003cem\u003eStaphylea pinnata\u003c/em\u003e x \u003cem\u003eStaphylea colchica\u003c/em\u003e) of unknown origin with intermediate morphology appeared in literature already in the 19th century: \u003cem\u003eStaphylea\u003c/em\u003e x \u003cem\u003ecoulombieri\u003c/em\u003e (Andr\u0026eacute;, 1887), described in France, and \u003cem\u003eStaphylea elegans\u003c/em\u003e (Zabel, 1888), documented in Germany. Weaver (1980) pointed out that both hybrids were identical according to herbarium records, and that the correct name would be \u003cem\u003eStaphylea\u003c/em\u003e x \u003cem\u003ecoulombieri.\u003c/em\u003e European botanic gardens stick to the name \u003cem\u003eStaphylea\u003c/em\u003e x \u003cem\u003eelegans\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eWhat makes the Staphylaceae particularly interesting is the extensive fossil record of their characteristic seeds that is not only very rich but also goes far back into the mid Tertiary. There is good evidence that there was a continuous presence of bladdernuts in Europe since the Oligocene until today (Vetters, 2013). Already in Upper Oligocene fossilized seeds of \u003cem\u003eStaphylea\u003c/em\u003e spp. were found in Western Siberia (Russia) and in Saxony (Germany) as well as \u003cem\u003eTurpinia ettinghausenii\u003c/em\u003e seeds in the latter region. From Miocene and Pliocene numerous localities widespread across Europe, Russia and Japan with seeds of \u003cem\u003eStaphylea\u003c/em\u003e spp. have been reported. Since the Middle Miocene also pollen discoveries have been referred.\u003c/p\u003e \u003cp\u003eWhereas \u003cem\u003eTurpinia ettinghausenii\u003c/em\u003e, the only \u003cem\u003eTurpinia\u003c/em\u003e species in Europe, disappeared in Lower Pliocene (discussed in Vetters (2013)), fossil seeds of \u003cem\u003eStaphylea\u003c/em\u003e have been found in deposits of all interglacial periods until Holsteinian (~\u0026thinsp;450\u0026thinsp;\u0026minus;\u0026thinsp;370 ka before present) in northwest Europe (Willis \u0026amp; Niklas, 2004). \u003cem\u003eStaphylea pinnata\u003c/em\u003e fossils have been even detected in Eemian deposits (Last interglacial period, 130\u0026thinsp;\u0026minus;\u0026thinsp;115 ka before present) in The Netherlands (Van der Ham et al., 2008).\u003c/p\u003e \u003cp\u003eRecently even fossils from eastern North America of the border of Miocene to Pliocene were documented (Huang et al., 2015), and latest discoveries unearthed a \u003cem\u003eStaphylea\u003c/em\u003e capsule of the Oligocene of Montana, USA (Zhu \u0026amp; Manchester, 2020).\u003c/p\u003e \u003cp\u003eThe diversification and historical biogeography of extant \u003cem\u003eStaphylea\u003c/em\u003e species must be viewed in the light of their highly disjunct distribution in Northern Hemisphere. Geographical disjunctions of plant genera and species between North America and East Asia have been well discussed in literature since Gray (1840, 1846) and got recently even more attention (Qian, 2002; Wen, 1999). Early hypotheses for such huge disjunctions relate to migrations in former geological periods, e.g. eastwards via Bering Strait in the Tertiary (\u0026ldquo;Asa Gray disjunctions\u0026rdquo;) or westwards via the North Atlantic land bridge in Eocene, Lower Tertiary (Tiffney, 1985). Wen (1999) summarized her view as follows: \u0026ldquo;The disjunct pattern between Eastern Asia and eastern and western North America is the product of vicariance, dispersal, extinction and speciation\u0026rdquo;. Thus, it has been accepted that several different biogeographical processes did result in congruent patterns of these disjunctions.\u003c/p\u003e \u003cp\u003eGeographical disjunctions within Eurasia, as in \u003cem\u003eStaphylea\u003c/em\u003e spp., especially among warm temperate areas in Central and Southern Europe, Caucasus, southern slopes of the Tibetan Plateau, Southern China and Japan were hypothesized to be related to late Tertiary vicariance (Mai, 1995). Several species of trees and shrubs adapted to warm temperate and wet climate had been evidently widespread along the northern coast of the Tethys in the beginning of Tertiary. Because of the cooling climate (only interrupted by a slightly warmer phase during the Mid Miocene) they migrated south, became isolated and survived, and even partly speciated as so-called Tertiary relics. Examples include the following genera: \u003cem\u003eZelkova\u003c/em\u003e with relics in the Caucasus region (\u003cem\u003eZ. carpinifolia\u003c/em\u003e), Crete (\u003cem\u003eZ. abelicea\u003c/em\u003e) and Sicily (\u003cem\u003eZ. sicula\u003c/em\u003e) (Christe et al., 2014; Kozlowski \u0026amp; Gratzfeld, 2013). \u003cem\u003ePterocarya fraxinifolia\u003c/em\u003e is distributed from the Caucasus to the south-coast of the Caspian Sea (Maharramova et al., 2018), \u003cem\u003eLiquidambar orientalis\u003c/em\u003e in southwest Turkey (D\u0026ouml;nmez \u0026amp; Yerli, 2018), \u003cem\u003eStyrax officinalis\u003c/em\u003e in eastern Mediterranean, California, Texas (Fritsch, 1996), \u003cem\u003eAesculus\u003c/em\u003e sect. Aesculus in the southeast of Europe (Harris, Xiang, \u0026amp; Thomas, 2009). \u003cem\u003eParrotia persica\u003c/em\u003e can be found in the southeast of Georgia and in Iran (Nakhutsrishvili et al., 2015), as well as \u003cem\u003eStaphylea colchica\u003c/em\u003e in the Black Sea region of western Georgia and adjacent parts of Russia (Shatilova et al., 2011).\u003c/p\u003e \u003cp\u003eA key factor causing such disjunctions is the still ongoing orogenesis of mountain ranges from the Alps all the way to the Tibetan Plateau from Oligocene onwards (Frisch \u0026amp; Meschede, 2013). The emergence of mountain ranges played an important role for the distribution of plants by arising steppes in connection with increasing aridity in inland areas (Harris et al., 2017; Tiffney \u0026amp; Manchester, 2001; Zhang et al., 2000). The latest cut in chorography was due to the Ice Ages, inhibiting migration of plants and genetic exchange from north to south and vice versa, that led to refugia for warm temperated plants in small refugial areas (Willis \u0026amp; Niklas, 2004).\u003c/p\u003e \u003cp\u003eBased on a phylogenetic approach using nuclear ribosomal (nr) and chloroplast (cp) DNA sequencing data we constructed a phylogeny focusing on European taxa of Staphyleaceae. Using AFLP fingerprinting additionally to DNA sequences we evaluated the phylogeography and relationships of \u003cem\u003eStaphylea colchica\u003c/em\u003e and \u003cem\u003eStaphylea pinnata\u003c/em\u003e. At first, we wanted to test if the dated phylogeny of Staphyleaceae was in line with the Tertiary vicariance hypothesis, secondly, we tested if the two taxa of Europe and the Black Sea region were closest relatives as suggested by morphology. Another question was, if \u003cem\u003eStaphylea pinnata\u003c/em\u003e has been involved in the origin of the tetraploid Caucasian \u003cem\u003eStaphylea colchica\u003c/em\u003e. Finally, we discuss our results in the light of possible glacial refugia and post-glacial colonization of Europe.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTaxon Sampling\u003c/h2\u003e \u003cp\u003eFor DNA sequencing, we sampled \u003cem\u003eStaphylea pinnata\u003c/em\u003e from Central Europe, Poland, Ukraine and Turkey as well as from Georgia, \u003cem\u003eStaphylea colchica\u003c/em\u003e from Georgia and Southern Russia. The presumed garden hybrid \u003cem\u003eStaphylea\u003c/em\u003e x \u003cem\u003eelegans\u003c/em\u003e, a cross between \u003cem\u003eStaphylea pinnata\u003c/em\u003e and \u003cem\u003eStaphylea colchica\u003c/em\u003e (Zabel, 1888) from the Botanical Garden of Madrid is also included. \u003cem\u003eStaphylea\u003c/em\u003e species from the Tibet Plateau, East Asia and eastern North America are added, predominantly newly sequenced, partly taken from NCBI (\u003cem\u003eNCBI, National Center for Biotechnology Information\u003c/em\u003e, 1988-) as well as the vouchers of the outgroup (\u003cem\u003eCrossosoma bigelovii\u003c/em\u003e, \u003cem\u003eStachyurus praecox\u003c/em\u003e, \u003cem\u003eStachyurus reticulata\u003c/em\u003e, \u003cem\u003eStachyurus chinensis\u003c/em\u003e, \u003cem\u003eStachyurus himalaicus\u003c/em\u003e). Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (Supplementary informations, SI) summarizes voucher information for all samples and NCBI accessions used for sequencing.\u003c/p\u003e \u003cp\u003eFor AFLP analysis, we surveyed 94 samples of three Georgian populations each of \u003cem\u003eStaphylea pinnata\u003c/em\u003e and \u003cem\u003eStaphylea colchica\u003c/em\u003e and two Central European populations of \u003cem\u003eStaphylea pinnata\u003c/em\u003e (Tab. S2, SI). Two of the Georgian \u003cem\u003eStaphylea pinnata\u003c/em\u003e populations included individuals from Adzharia and Bordžomi. Dmitriewa (1990) and Gulisashvili (1970) only described \u003cem\u003eStaphylea colchica\u003c/em\u003e populations there, but we only found \u003cem\u003eStaphylea pinnata.\u003c/em\u003e Geographic distributions as well as locations of taxa and populations used in the present study are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eData for the distribution maps (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were taken from the herbaria B, LE, TBI, MNHN-P, MW, W and from personal collections (Vetters 2003, 2004, 2005 in Georgia and the vicinity of Sochi, Russia), as well as from more than 30 references (\u0026ldquo;Chorology of the genus Staphylea \u0026ndash; Methods and References\u0026rdquo;, SI).\u003c/p\u003e \u003cp\u003eIn the Black Sea region overlapping areas of \u003cem\u003eStaphylea pinnata\u003c/em\u003e and \u003cem\u003eStaphylea colchica\u003c/em\u003e are not yet resolved. That might be a problem of identification because of the genetic influence (see AFLP results) of \u003cem\u003eStaphylea colchica\u003c/em\u003e seen in populations of \u003cem\u003eStaphylea pinnata\u003c/em\u003e in that region (in contrast to the Central European bladdernuts) and its effects on morphology.\u003c/p\u003e \u003cp\u003eAreas of the other \u003cem\u003eStaphylea\u003c/em\u003e species used in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea (\u003cem\u003eStaphylea bumalda\u003c/em\u003e DC, \u003cem\u003eStaphylea emodi\u003c/em\u003e Wall., \u003cem\u003eStaphylea forrestii\u003c/em\u003e Balf.f., \u003cem\u003eStaphylea holocarpa\u003c/em\u003e Hemsl., \u003cem\u003eStaphylea trifolia\u003c/em\u003e L.) were taken from \u003cem\u003eGBIF, Global Biodiversity Information Facility\u003c/em\u003e (1999 onwards) .\u003c/p\u003e \u003cp\u003eDNA extraction and molecular techniques\u003c/p\u003e \u003cp\u003eGenomic DNA was extracted according to a standard CTAB protocol (Doyle \u0026amp; Doyle, 1987; Murray \u0026amp; Thompson, 1980). For amplification of the ITS region, ITS1-5.8S-ITS2, primers AB 101 (5\u0026rsquo;-ACG AAT TCA TGG TCC GGT GAA GTG TTC G-3\u0026rsquo;) and AB 102 (5\u0026rsquo;-TAG AAT TCC CCG GTT CGC TCG CCG TTA C-3\u0026rsquo;), both were used (Douzery et al., 1999). Due to some ambiguous ITS sequences ten samples (Sp_A1, Sp_PO, Sp_TR1, Sp_U1, Sp_U2, Sp_U3, Sp_G1, Sc_K, Sc_M, Sel, see Tab. S1) were cloned using TA Cloning Kit (Life Technologies, California, USA). Following Harpke and Peterson (2008) we compared the three conserved motifs that should indicate functionality with our sequences.\u003c/p\u003e \u003cp\u003eAmplification of trnL-F region was conducted using primers Tab C (5\u0026rsquo;-CGA AAT CGG TAG ACG CTA CG-3\u0026rsquo;) and Tab F (5\u0026rsquo;-ATT TGA ACT GGT GAC ACG AG-3\u0026rsquo;; (Taberlet et al., 1991; Zhao et al., 2011). PCR products were cleaned using illustra ExoProStar (GE Healthcare, Freiburg, Germany)) and sequenced by Macrogen (Seoul, S-Korea) or eurofins (Ebersberg, Germany) using primers ITS4 (5\u0026rsquo;-TCC TCC GCT TAT TGA TAT GC-3\u0026rsquo;; White et al. (1990)) for ITS and Tab F for \u003cem\u003etrnL-F\u003c/em\u003e region respectively.\u003c/p\u003e \u003cp\u003eFor Amplified fragment length polymorphisms (AFLP) we followed the protocol of Vos et al. (1995) with modifications according to Rebernig (2009) for 94 samples (eight populations in total) of \u003cem\u003eStaphylea pinnata\u003c/em\u003e and \u003cem\u003eStaphylea colchica\u003c/em\u003e (see also Tab. S2). For the preselective PCR we chose the pair of primers \u003cem\u003eEco\u003c/em\u003e RI-A (5\u0026rsquo;-GAC TGC GTA CCA ATT\u0026thinsp;+\u0026thinsp;A-3\u0026rsquo;) and \u003cem\u003eMse\u003c/em\u003e I-C (5\u0026rsquo;-GAT GAG TCC TGA GTA\u0026thinsp;+\u0026thinsp;C-3\u0026rsquo;). For selective PCR primers with three additional selective bases were used, three primer combinations in total. The \u003cem\u003eEco\u003c/em\u003e RI primers were fluorescently labeled: \u003cem\u003eEco\u003c/em\u003e RI-ACA / \u003cem\u003eMse\u003c/em\u003e l-CTG; \u003cem\u003eEco\u003c/em\u003e RI (6-FAM); \u003cem\u003eEco\u003c/em\u003e RI-AAC / \u003cem\u003eMse\u003c/em\u003e l-CTG; \u003cem\u003eEco\u003c/em\u003e RI (NED) and \u003cem\u003eEco\u003c/em\u003e RI-AGG / \u003cem\u003eMse\u003c/em\u003e l-CAT; \u003cem\u003eEco\u003c/em\u003e RI (HEX). The selective amplification program ran in a thermal cycler (90% ramp time).\u003c/p\u003e \u003cp\u003eSeparation and visualization of fragments was done on two polyamide gels using an automated sequencer (ABI 377, Perkin Elmer). GeneScan\u0026trade; ROX\u003csup\u003eTM\u003c/sup\u003e-500 dye Size Standard was added to the fragments. Alignment of the raw data was done using ABI Prism GeneScan\u0026reg; Analysis Software v. 3.2.1 (\u003cem\u003eGeneScan Analysis Software\u003c/em\u003e 2001) Macintosh version). Subsequently the GeneScan files were imported into Genographer v. 1.1.0 (Benham et al., 1999) for scoring the AFLP fragments. Using the \u0026lsquo;thumbnail\u0026rsquo; option for comparison of the signals of each fragment, we scored them as present or absent. Peaks of low intensity were included into the analysis when unambiguous scoring was possible.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eComputational Analyses\u003c/h2\u003e \u003cp\u003eFor phylogenetic reconstruction DNA-sequences were edited and aligned using Geneious Pro 5.5.6 software \u0026copy; Biomatters Ltd. (Drummond et al., 2011) under MAFFT algorithm (Katoh et al., 2005; Katoh et al., 2002).\u003c/p\u003e \u003cp\u003eWe employed JModelTest 0.1.1 (Posada, 2008) to determine models of molecular evolution, ranked according to the \u0026ldquo;Akaike Information Criterion\u0026rdquo;, AIC (Akaike, 1973). For the trnL-F region the model TPM1uf\u0026thinsp;+\u0026thinsp;G best fitted the data. As this model is not implemented in MrBayes (Ronquist, Huelsenbeck, \u0026amp; Teslenko, 2011), the next best fitting model GTR\u0026thinsp;+\u0026thinsp;G (general time reversible model with a gamma distribution of site-specific rates), discussed in Hood et al. (2010), was chosen for both matrices.\u003c/p\u003e \u003cp\u003eRAxML vs 1.3 (Stamatakis, 2014) was applied to conduct a Maximum Likelihood (ML) tree search analyses for both markers separately as well as concatenated under the GTR\u0026thinsp;+\u0026thinsp;G substitution model and 1000 rapid bootstrap replicates for statistical node support. ML bootstrap values\u0026thinsp;\u0026ge;\u0026thinsp;75 were considered as a threshold for good support (Pattengale et al., 2010).\u003c/p\u003e \u003cp\u003eTo create a time-calibrated phylogeny BEAST v1.7.5. (Drummond et al., 2012) was employed on ITS and trnL-F marker separately as well as on the combined ITS and\u003c/p\u003e \u003cp\u003etrnL-F matrices.\u003c/p\u003e \u003cp\u003eITS repeats exist in many copies in the genome. Some of them may degenerate through evolution and lose their functionality but may be kept in the genome. Their existence may however lead to incorrect time data of the phylogenetic trees (\u0026Aacute;lvarez \u0026amp; Wendel, 2003). Therefore, the 5.8S regions of all ITS sequences were checked according to the three conserved motifs of Harpke and Peterson (2008). Then we compared phylogenies including and excluding sequences without presumed functionality.\u003c/p\u003e \u003cp\u003eFor all analyses a GTR\u0026thinsp;+\u0026thinsp;G substitution model, an uncorrelated log-normal relaxed clock and a birth-death process of speciation served as tree priors. Three runs each with 30 Mio generations for the trnL-F and ITS files or 100 Mio ones for the combined matrix with a sampling frequency of every 1000th, respectively every 10000th generation. We combined the tree files after a burnin of 25% in LogCombiner and constructed maximum clade credibility trees with mean node heights using TreeAnnotator (both programs are part of BEAST package, Drummond et al. (2012); Huson and Bryant (2006)). Trees were visualized using FigTree v1.3.1 (Rambaut \u0026amp; Drummond, 2009).\u003c/p\u003e \u003cp\u003eFor calibrating the trees, we took following considerations into account. Wikstr\u0026ouml;m, Savolainen and Chase (2001) involved Crossosomataceae, Stachyuraceae, Staphyleaceae - representing our dataset - as well as Aphloiaceae and Strasburgeriaceae into their molecular clock calculations. The crown age of Crossosomatales, systematically integrated according to APG III, Angiosperm Phylogeny Group classification III (Bremer et al., 2009) as well as in APG IV (Angiosperm et al., 2016; Chase et al., 2016) resulted in 95\u0026thinsp;\u0026plusmn;\u0026thinsp;4 Mya. The stem age of Staphyleaceae accouted for 62\u0026thinsp;\u0026plusmn;\u0026thinsp;6 Mya, the split between Crossosomataceae and Stachyuraceae for 44\u0026thinsp;\u0026plusmn;\u0026thinsp;5 Mya (estimated by maximum parsimony with ACCTRAN optimisations). Wikstr\u0026ouml;m, Savolainen and Chase (2001) used the split between Fagales and Cucurbitales for calibrating their tree.\u003c/p\u003e \u003cp\u003eSince Wikstr\u0026ouml;m, Savolainen and Chase (2001) many further approaches were done to calibrate the \u0026ldquo;tree of life\u0026rdquo; (Magall\u0026oacute;n \u0026amp; Castillo, 2009; Magall\u0026oacute;n et al., 2015; Palazzesi et al., 2012; Wang et al., 2009). In most publications, samplings of Crossosomatales\u0026rsquo; sequences were not as complete as in Wikstroem, except for Magall\u0026oacute;n et al., who focused primarily on the rise of the angiosperms. They calibrated Crossosomatales by means of the fossil age of \u003cem\u003eTurpinia\u003c/em\u003e, Staphyleaceae (Tiffney, 1979), with a minimum age of 28.45 Mya (beginning of Upper Oligocene). Using Bayesian relaxed clocks for calculation the result was a young age of 31.3 Mya - compared to Wikstroem - for the node of Staphyleaceae / Crossosomataceae, Stachyuraceae and Guamatelaceae, albeit with a 95% highest posterior density (HPD) interval of 30.62 Myr from 28.45 to 59.02 Mya. We considered such a large confidence interval not useful for our dating approach. Magall\u0026oacute;n et al. (2015) admitted that estimates for the crown ages of several families could be probably too young because of three reasons. The taxonomic sample may not represent the crown node of the targeted family, fossils used may not be its oldest members, and the assignment of calibrations to nodes was mostly based on apomorphies that could have already appeared in a more inclusive clade.\u003c/p\u003e \u003cp\u003eOn the other hand, Penalized Likelihood in Magall\u0026oacute;n et al. (2015) resulted in 66.5 Mya for the stem group of Staphyleaceae with a confidence interval of 14.52 Myr between 59.96 and 74.48 Mya, quite similar but even older than Wikstroem\u0026rsquo;s results.\u003c/p\u003e \u003cp\u003eTherefore, we calibrated our phylogenetic tree according to Wikstr\u0026ouml;m, Savolainen and Chase (2001), corresponding also to Forest and Chase (2009).\u003c/p\u003e \u003cp\u003eWe used two calibration points for our analyses. First, the root (stem group of Staphyleaceae) was set to 62\u0026thinsp;\u0026plusmn;\u0026thinsp;6 Mya and second, the outgroup (stem group of Stachyuraceae and Crossosomataceae) was set to 44\u0026thinsp;\u0026plusmn;\u0026thinsp;5 Mya. We assumed a normal distribution prior for both nodes.\u003c/p\u003e \u003cp\u003eAdditionally, the uncorrected log-normal relaxed clock mean (ucld.mean) prior was set to 0.00164 substitutions per site per Myr, and standard deviation to 0.001 for ITS (Dick et al., 2013). For the trnL-F matrix the ucld.mean was set to 0.000929 substitutions per site per Myr, calculating the mean of the mutation rates from \u003cem\u003eInga\u003c/em\u003e (1.3x10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e/Mya), \u003cem\u003ePhylica\u003c/em\u003e (4.87x10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e/Mya) and the \u0026ldquo;general mutation rates\u0026rdquo; of chloroplast markers (1.00x10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e/Mya) in equal shares (Values taken from Richardson et al. (2001)) with a standard deviation of 0.0002255.\u003c/p\u003e \u003cp\u003ePhylogenetic network estimation was executed by the program TCS 1.21 (Clement et al., 2000\u0026ndash;2005; Clement, Posada, \u0026amp; Crandall, 2000). It calculated parsimony networks for each marker separately. For the calculation, the ITS file was reduced to sequences from \u003cem\u003eStaphylea pinnata\u003c/em\u003e, \u003cem\u003eStaphylea colchica\u003c/em\u003e, \u003cem\u003eStaphylea elegans\u003c/em\u003e and \u003cem\u003eStaphylea emodi\u003c/em\u003e without the three extremely aberrant ribotypes Sp_G2_1, Sp_G2_9 and Sp_A1_8. The trnL-F-file contained all members of the ingroup, i.e., of the family Staphyleaceae. Both files were manually gapcoded (\u0026ldquo;simple indel coding\u0026rdquo; according to Bena et al. (1998) and Simmons and Ochoterena (2000) with the gaps serving as fifth state in TCS. The probability levels were 95%.\u003c/p\u003e \u003cp\u003eTo gain network information within \u003cem\u003eStaphylea pinnata\u003c/em\u003e and \u003cem\u003eStaphylea colchica\u003c/em\u003e we used the program TCS (Clement et al., 2000\u0026ndash;2005; Clement, Posada, \u0026amp; Crandall, 2000).\u003c/p\u003e \u003cp\u003eFor AFLP data analysis the presence / absence matrix of all samples was imported to SplitsTree V4.19.0 (Huson \u0026amp; Bryant, 2006; \u003cem\u003eSplitsTree4\u003c/em\u003e, 2010) for computing a Neighbor-Net tree according to Nei and Li (1979). AMOVA (Excoffier, Smouse, \u0026amp; Quattro, 1992; Weir, 1996; Weir \u0026amp; Cockerham, 1984) conducted hierarchical analysis of variance. Arlequin Ver 3.1 (Excoffier, Laval, \u0026amp; Schneider, 2006) estimated molecular diversity indices (Ewens, 1972; Nei, 1987; Tajima, 1993; Zouros, 1979), using the general settings (weight\u0026thinsp;=\u0026thinsp;1, epsilon value\u0026thinsp;=\u0026thinsp;1e\u003csup\u003e\u0026minus;\u0026thinsp;07\u003c/sup\u003e, significant digits for output\u0026thinsp;=\u0026thinsp;5, use of original haplotype definition for AMOVA and allowed level of missing data\u0026thinsp;=\u0026thinsp;0.05. \u003cem\u003eStructure\u003c/em\u003e v. 2.2 (Pritchard, Xiaoquan, \u0026amp; Falush, 2007) delimited the amount of admixture of populations in a Bayesian analysis of population structure using the models of recessive alleles and admixture, assuming one group and correlated allele frequencies. The burn-in period was set to 200000 and 500000 replicates followed as recommended by Pritchard, Xiaoquan and Falush (2007).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFossils\u003c/h2\u003e \u003cp\u003e The rich material of fossil seeds plus one fruit and one uncertain flower (Table\u0026nbsp;2) was plotted according to the periods of the fossil\u0026rsquo;s occurences into the gained phylogeny to associate them with the appearance of extant lineages.\u003c/p\u003e \u003cp\u003e(Dorofeev, 1963, p. 210 f; Engelhardt \u0026amp; Kinkelin, 1908, p. 265; Geissert, Gregor, \u0026amp; Mai, 1990, pp. 50\u0026ndash;51; Gregor, 1978, pp.79\u0026ndash;84; Hern\u0026aacute;ndez-Dami\u0026aacute;n, Cevallos-Ferriz, \u0026amp; Huerta-Vergara, 2019; Huang et al., 2015; Kolakovsky, 1964; Kovar-Eder \u0026amp; Krainer, 1988, pp. 41\u0026ndash;42; Kovar-Eder \u0026amp; Meller, 2001, p. 86; Mai, 1997, p. 63; 2001, p. 115; Mai \u0026amp; Walther, 1988, p. 171; Mc Clennen, Jenkins, \u0026amp; Uhen, 2017; Negru, 1972, pp. 123\u0026ndash;127; Ozaki, 1991; Palamarev, Ivanov, \u0026amp; Bozukov, 1999, p. 16; Palamarev \u0026amp; Petkova, 1987, p. 121; Shatilova et al., 2011; Szafer, 1946; Szafer, 1947, pp. 298\u0026ndash;301; 1954, p. 47; Teodoridis, Kvaček, \u0026amp; Uhl, 2009; Van der Burgh, 1983, pp. 60\u0026ndash;61; 1987, p. 325; Van der Ham et al., 2008, p. 133; Zhu \u0026amp; Manchester, 2020)\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eITS marker\u003c/h2\u003e \u003cp\u003eFunctionality of the 5.8S gene\u003c/p\u003e \u003cp\u003eEight ITS sequences were evidenced as (presumably) non-functional copies. Three ribotypes were extremely aberrant in motif one/two/three (Harpke \u0026amp; Peterson, 2008): Sp_A1_8 (3/0/1), Sp_G2_1 (3/3/1) and Sp_G2_9 (4/3/0). Five differed only in one base in motif one (Turp_tern_2 (KR532697) and Sp_U1_5), in motif two (Sp_TR1_6 and Sp_U3_9) or in motif three (Sp_G2_7).\u003c/p\u003e \u003cp\u003eTherefore, we gained two Beast trees of ITS:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eincluding all ribotypes (90 ribotypes and isolates, 722 characters, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, SI) to test the roughly estimated chronological relationships of the European \u003cem\u003eStaphylea\u003c/em\u003e species to Asian and American ones, and\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eexcluding the presumably non-functioning ribotypes (82 ribotypes, 715 characters, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, SI), leading to correct times (\u0026Aacute;lvarez \u0026amp; Wendel, 2003).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eDiversities within the whole ITS data set and of the extremely aberrant ribotypes\u003c/p\u003e \u003cp\u003eThe diversity within the ribotypes, focusing on \u003cem\u003eStaphylea pinnata\u003c/em\u003e and \u003cem\u003eStaphylea colchica\u003c/em\u003e with functional ITS copies can be seen in Tab. S3 (SI). The different clusters in Tab. S3 arose from informative insertions and deletions gained by TCS Analysis (see: Phylogenetic network estimation).\u003c/p\u003e \u003cp\u003eBecause of striking similarities of the extremely aberrant ITS ribotypes of \u003cem\u003eStaphylea pinnata\u003c/em\u003e with those of the Asian and American \u003cem\u003eStaphylea\u003c/em\u003e species we did not skip them, but we compared them separately with the non-European species (Table\u0026nbsp;3). To ensure reproducibility nucleotid positions in Tab. S3 (SI) and Table\u0026nbsp;3 were taken from the same file, the file inclusive of the ribotypes with (presumably) non-functional 5.8S gene (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, SI).\u003c/p\u003e \u003cp\u003e \u003cem\u003eStaphylea emodi\u003c/em\u003e and \u003cem\u003eStaphylea bumalda\u003c/em\u003e emerged as the nearest relatives to the European bladdernuts. \u003cem\u003eStaphylea emodi\u003c/em\u003e\u0026rsquo;s ITS sequences comprised 12 substitutions and a gap of three bases (position 88\u0026ndash;90) compared to the 59 sequences of \u003cem\u003eStaphylea pinnata\u003c/em\u003e and \u003cem\u003eStaphylea colchica\u003c/em\u003e with a functional 5.8S gene. These changes were typical for all other Staphyleaceae of our research as well as for the outgroup. \u003cem\u003eStaphylea emodi\u003c/em\u003e contained less substitutions than \u003cem\u003eStaphylea bumalda\u003c/em\u003e (39 plus the gap of three bases mentioned above) or any other sequence.\u003c/p\u003e \u003cp\u003ePhylogenetic network estimation (TCS)\u003c/p\u003e \u003cp\u003eCharacteristic insertions and deletions within the ITS haplotypes of \u003cem\u003eStaphylea pinnata\u003c/em\u003e, \u003cem\u003eStaphylea colchica\u003c/em\u003e and \u003cem\u003eStaphylea elegans\u003c/em\u003e lead to more insights into geographic patterns on a European scale by the program TCS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e, but also Tab. S3 (SI)). All samples of \u003cem\u003eStaphylea colchica\u003c/em\u003e and \u003cem\u003eStaphylea elegans\u003c/em\u003e contained unique ribotypes with the insertion of CAA (three CAA repeats) in position 547\u0026ndash;549 of the total alignment. The deletion of nine bases (AGTGTGGTT in position 153\u0026ndash;161) in some ribotypes of \u003cem\u003eStaphylea pinnata\u003c/em\u003e was also remarkable. We neither could find this deletion in ribotypes from Turkey and Georgia and Sp_Mi from Serbia nor in any other taxon analyzed. A gap of three bases (position 88\u0026ndash;90) was characteristic for all Asian and American \u003cem\u003eStaphylea\u003c/em\u003e species and \u003cem\u003eTurpinia occidentalis\u003c/em\u003e as well as for the outgroup, but also found sporadically in \u003cem\u003eStaphylea pinnata\u003c/em\u003e and \u003cem\u003eStaphylea colchica\u003c/em\u003e (Sp_A1_8, Sp_G2_9 and Sc_K_6), seen in Table\u0026nbsp;3 and forming clades in Tab. S3 (SI) respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA\u003csub\u003e0\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Tab. S3) included ribotypes of \u003cem\u003eStaphylea pinnata\u003c/em\u003e without the characteristic deletion of nine bases, but also of \u003cem\u003eStaphylea colchica\u003c/em\u003e without the typical insertion of three bases. Haplotypes B\u003csub\u003e0\u003c/sub\u003e, C\u003csub\u003e0\u003c/sub\u003e and D\u003csub\u003e0\u003c/sub\u003e included only \u003cem\u003eStaphylea pinnata\u003c/em\u003e ribotypes without this deletion. E\u003csub\u003e0\u003c/sub\u003e comprised all ribotypes of \u003cem\u003eStaphylea pinnata\u003c/em\u003e with the deletion of the nine bases. F\u003csub\u003e0\u003c/sub\u003e contained only \u003cem\u003eStaphylea colchica-\u003c/em\u003e and \u003cem\u003eStaphylea elegans\u003c/em\u003e-ribotypes with the insertion of CAA for the third time.\u003c/p\u003e \u003cp\u003eThe three extremely aberrant ribotypes, and even Sc_K_6 were excluded by the TCS program (Tab. S3)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003etrnL-F marker\u003c/h2\u003e \u003cp\u003eThe alignment of 35 trnL-F - sequences comprised of 950 characters including the outgroup. Regarding this chloroplast region, only a few variable nucleotide positions existed within the ingroup (BEAST tree seen in Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, SI). Due to the small amount of variation (Tab. S4, SI) and the chosen mutation rates from Richardson et al. (2001) young divergence times were gained.\u003c/p\u003e \u003cp\u003eThe TCS analysis of the chloroplast trnL-F dataset contained all Staphyleaceae, shown in Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e (SI). The program excluded only \u003cem\u003eTurpinia occidentalis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStaphylea emodi\u003c/em\u003e differed in only one nucleotide from the uniform isolates of \u003cem\u003eStaphylea pinnata\u003c/em\u003e, all but one \u003cem\u003eStaphylea colchica\u003c/em\u003e and \u003cem\u003eStaphylea elegans\u003c/em\u003e and was networked with \u003cem\u003eStaphylea trifolia\u003c/em\u003e, \u003cem\u003eStaphylea forrestii\u003c/em\u003e and \u003cem\u003eStaphylea holocarpa.\u003c/em\u003e The two \u003cem\u003eStaphylea bumalda\u003c/em\u003e isolates differed in one or two characters respectively from the uniform isolates and were networked with Sc_M, the only differing \u003cem\u003eStaphylea colchica\u003c/em\u003e isolate from Russia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eConcatenation\u003c/h2\u003e \u003cp\u003eThe Beast trees of ITS (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u0026ndash; whole alignment, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e - only haplotypes with functional 5.8S gene, Supplements) and trnL-F (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) were incongruent concerning the different position of \u003cem\u003eStaphylea bumalda\u003c/em\u003e, but the nodes in question of the ITS tree (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) were supported with 87% (RAxML), and of the trnL-F tree (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) only with 62% (RAxML). So, the trees of both molecular markers could be concatenated (Norup et al., 2006). The concatenated trees are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e (whole alignment) and in Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e (reduced alignment, see Supplements).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe ages of important divergence times of all BEAST analyses are summarized in Tab. S5 (SI), including their confidence intervals (95%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eFossil data\u003c/h2\u003e \u003cp\u003eBased on the concatenated tree of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e and the rich fossil data (Table\u0026nbsp;2) we could compare the results and evaluate the age of divergence of aberrant ITS ribotypes as well. The clade NE America plus SE Asia 2 separated from all other \u003cem\u003eStaphylea\u003c/em\u003e species at 27.46\u0026thinsp;\u0026plusmn;\u0026thinsp;12 Mya, where already \u003cem\u003eStaphylea woodworthensis\u003c/em\u003e in USA and three Siberian \u003cem\u003eStaphylea\u003c/em\u003e species had occurred. The two Georgian \u003cem\u003eStaphylea pinnata\u003c/em\u003e ITS ribotypes (Sp_G2_1 and Sp_G2_9) separated from the sister clade of \u003cem\u003eStaphylea bumalda\u003c/em\u003e, \u003cem\u003eStaphylea emodi\u003c/em\u003e and the mixed group of \u003cem\u003eStaphylea pinnata\u003c/em\u003e and \u003cem\u003eStaphylea colchica\u003c/em\u003e at 19.15\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8 Mya. At that time \u003cem\u003eStaphylea microsperma\u003c/em\u003e and \u003cem\u003eStaphylea bessarabica\u003c/em\u003e lived in Central Europe, and at the end of the confidence interval also \u003cem\u003eStaphylea pliocaenica\u003c/em\u003e, which is regarded as the ancestor or at least as a near relative of \u003cem\u003eStaphylea pinnata\u003c/em\u003e (Mai, 2001). The aberrant ITS ribotype of \u003cem\u003eStaphylea pinnata\u003c/em\u003e from Austria (Sp_A1_8) separated at about 11.06\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5 Mya from the clade of \u003cem\u003eStaphylea emodi\u003c/em\u003e plus the mixed group of \u003cem\u003eStaphylea pinnata\u003c/em\u003e and \u003cem\u003eStaphylea colchica.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eAFLP data of\u003c/b\u003e \u003cb\u003eStaphylea colchica\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eStaphylea pinnata\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAnalysis of the genome structures of populations enabled insights in the relationship of both \u003cem\u003eStaphylea\u003c/em\u003e species as well as in differences of \u003cem\u003eStaphylea pinnata\u003c/em\u003e populations separated by geographical distances (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, see also Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e, SI). In total we scored analyzed 113 fragments, 53 (6-FAM), 28 (NED) and 32 (HEX), 24 fragments were monomorphic in all 94 individuals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Neighbor Joining tree of AFLP analysis shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ea evidenced two lineages corresponding to the two species, but \u003cem\u003eStaphylea pinnata\u003c/em\u003e of Central Europe also form a highly supported lineage separated from Georgia`s \u003cem\u003eStaphylea pinnata\u003c/em\u003e populations. To show the distribution of shared AFLP markers of geographical regions a Venn diagram was drawn (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). All three regions had 51, accordingly 45.1% of the markers, in common. \u003cem\u003eStaphylea colchica\u003c/em\u003e shared markers mainly with Georgian \u003cem\u003eStaphylea pinnata\u003c/em\u003e (16.9%) and had a high number of specific markers absent in \u003cem\u003eStaphylea pinnata\u003c/em\u003e (31.0%). Only five markers (7% of the markers) were characteristic for both regions of \u003cem\u003eStaphylea pinnata\u003c/em\u003e that we could not find in \u003cem\u003eStaphylea colchica\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eWe compared each \u003cem\u003eStaphylea pinnata\u003c/em\u003e population of Georgia separately with the Central European \u003cem\u003eStaphylea pinnata\u003c/em\u003e and \u003cem\u003eStaphylea colchica\u003c/em\u003e of Georgia in Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e. Two populations of Georgia, collected from regions of presumed \u003cem\u003eStaphylea colchica\u003c/em\u003e, turned out to be \u003cem\u003eStaphylea pinnata\u003c/em\u003e (Sp Ba and Sp Bo, Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e, Tab. S2). Nevertheless, they shared AFLP fragments with \u003cem\u003eStaphylea colchica\u003c/em\u003e which were absent in \u003cem\u003eStaphylea pinnata\u003c/em\u003e from Central Europe, 15 and nine respectively, quantities comparable to the Georgian population of Lagodekhi (eight fragments, Sp La, Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e) which had been declared undoubtedly as \u003cem\u003eStaphylea pinnata\u003c/em\u003e (Tab. S2).\u003c/p\u003e \u003cp\u003e \u003cem\u003eStructure\u003c/em\u003e Analysis (Pritchard, Xiaoquan, \u0026amp; Falush, 2007) revealed K\u0026thinsp;=\u0026thinsp;3 as most probable number of groups (Figs. S7, S8) congruent with the Neighborjoining results (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eAdmixtures of less than 13% were present within very few individuals. Two individuals from Krikhi (Sc-Kr-12 and Sc-Kr-13) as well as one from Samegrelo (Sc-Sa-7) showed most of the admixture with \u003cem\u003eStaphylea pinnata\u003c/em\u003e from Georgia. The \u003cem\u003eStaphylea pinnata\u003c/em\u003e populations of Batumi and Bordžomi, from regions of \u003cem\u003eStaphylea colchica\u003c/em\u003e, contained some individuals admixed with Central European ones, only Sp_Ba_11 and 13 with \u003cem\u003eStaphylea colchica\u003c/em\u003e. The \u003cem\u003eStaphylea pinnata\u003c/em\u003e population of Lagodekhi (Sp_La_1\u0026ndash;5) and the Central European populations exhibited nearly no admixture (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e, S8).\u003c/p\u003e \u003cp\u003eHierarchical AMOVA (Excoffier, Smouse, \u0026amp; Quattro, 1992) for the three lineages (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e) showed significant hierarchical differentiation (see also Table\u0026nbsp;4). Most of the total AFLP variation resided among the three partitions that means about nine times of the variation among populations. Half of it can be found within populations.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStaphylea colchica\u003c/em\u003e populations (C) featured highest percentages of gene diversity within populations and the Central European \u003cem\u003eStaphylea pinnata\u003c/em\u003e populations (A) the lowest ones (see Fig. \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e and Table\u0026nbsp;5\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe genus \u003cem\u003eStaphylea\u003c/em\u003e offers the almost unique opportunity to study a highly disjunct and rather species poor genus with an exceptional fossil record during the Tertiary. Here we could show that most disjunctions are well in line with the fragmentation of warm temperate forests towards the end of the Tertiary resulting in geographical vicariance and we present additional genetic support for a continuous presence of \u003cem\u003eStaphylea\u003c/em\u003e in Europe since the late Oligocene/Early Miocene.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEurope\u003c/b\u003e: \u003cb\u003eStaphylea colchica\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eStaphylea pinnata\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRegarding the phylogenetic tree in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e the largely unresolved relationship of \u003cem\u003eStaphylea colchica\u003c/em\u003e with \u003cem\u003eStaphylea pinnata\u003c/em\u003e by the molecular markers ITS and trnL-F is striking. In contrast to that AFLP results suggested a clear genomic separation of both species (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). The populations showed little amounts of genetic admixture in context to smaller or larger distances of Georgian \u003cem\u003eStaphylea pinnata\u003c/em\u003e populations to \u003cem\u003eStaphylea colchica\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Georgian populations of both species showed high intra-population diversity (Table\u0026nbsp;5). Moreover, AFLP data exposed even more details within \u003cem\u003eStaphylea pinnata\u003c/em\u003e, i.e., geographically differences between Central Europe and Black Sea region. Due to the capacity of clonal upgrowth of \u003cem\u003eStaphylea\u003c/em\u003e ssp. the Central European populations might represent only a very few genotypes. Observations showed that \u003cem\u003eStaphylea pinnata\u003c/em\u003e achieved dominance on isolated slopes, in diches and ravines and there were often great geographical distances between one and another population in Central, but also in Southern and Eastern Europe.\u003c/p\u003e \u003cp\u003eBased on the present results and the presumably strong chromosomal breeding barrier between the diploid \u003cem\u003eStaphylea pinnata\u003c/em\u003e and tetraploid \u003cem\u003eStaphylea colchica\u003c/em\u003e, it is less likely that this sharing of markers across species-boundaries in Georgia reflects gene flow (hybridization/ introgression). Rather, it suggests genetic depletion of \u003cem\u003eStaphylea pinnata\u003c/em\u003e near its western limit of distribution, e.g. following range expansion - “leading edge expansion” (Hewitt, 2001). The tremendous human contribution to the distribution of this plant in Europe even since Iron Age for cultural and religious reasons have also to be taken into account (Heiss et al., 2014; Vetters, 2013). Thus, \u003cem\u003eStaphylea pinnata\u003c/em\u003e could be largely archeophytic in large parts of Europe, which also could result in genetic uniformity.\u003c/p\u003e \u003cp\u003eAn interesting observation of the seed morphology of the fossils (Heiss et al., 2014; Vetters, 2013), Table\u0026nbsp;2) shows a morphological progression from smaller to larger seeds in Europe from Late Oligocene to Pliocene. Diploid \u003cem\u003eStaphylea pinnata\u003c/em\u003e has the largest seeds in the genus and tetraploid \u003cem\u003eStaphylea colchica\u003c/em\u003e a seed size like the fossil \u003cem\u003eStaphylea pliocaenica.\u003c/em\u003e The phylogenies nevertheless suggest a very close relationship of these two extant species (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eOrigin of the tetraploid\u003c/b\u003e \u003cb\u003eStaphylea colchica\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eStaphylea colchica\u003c/em\u003e is most likely of allotetraploid origin because of the 35 specific AFLP markers of the \u003cem\u003eStaphylea colchica\u003c/em\u003e populations whereas \u003cem\u003eStaphylea pinnata\u003c/em\u003e has only eight specific markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). This suggests that \u003cem\u003eStaphylea pinnata\u003c/em\u003e could be one of the parental species, as both extant species showed the same cpDNA haplotype (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Considering the timeframe, it is more likely that \u003cem\u003eStaphylea pliocaenica\u003c/em\u003e, its presumptive ancestor (Teodoridis, Kvaček, \u0026amp; Uhl, 2009) was one parent. Its form is similar to \u003cem\u003eStaphylea pinnata\u003c/em\u003e and size and thickness of its testa resembles \u003cem\u003eStaphylea colchica\u003c/em\u003e (Mai, 2001).\u003c/p\u003e \u003cp\u003ePoyarkova (1986) indicated \u003cem\u003eStaphylea emodi\u003c/em\u003e as the nearest related species to \u003cem\u003eStaphylea colchica\u003c/em\u003e. Indeed, their morphologies are similar including the small seeds. However, despite the similarities in the ITS sequences to \u003cem\u003eStaphylea colchica\u003c/em\u003e and \u003cem\u003eStaphylea pinnata\u003c/em\u003e, \u003cem\u003eStaphylea emodi\u003c/em\u003e can be excluded as the second possible parent of \u003cem\u003eStaphylea colchica\u003c/em\u003e because the ITS region of three samples (Sem_1, Sem_2, Sem_3) did not contain the insertion CAA (leading to threefold CAA), characteristic for \u003cem\u003eStaphylea colchica\u003c/em\u003e (Tab. S3, clade F). The other Asian or American living species included in our study can be excluded for the same reason, therefore we presume that a now extinct European \u003cem\u003eStaphylea\u003c/em\u003e species with small seeds might be assumed as second parent to allotetraploid \u003cem\u003eStaphylea colchica\u003c/em\u003e. It remains speculative, as the dated ITS-phylogeny does not resolve the relationship fully (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e), but most likely S\u003cem\u003etaphylea colchica\u003c/em\u003e evolved as late as the Early Pliocene where \u003cem\u003eStaphylea pliocaenica\u003c/em\u003e and \u003cem\u003eStaphylea colchica fossilis\u003c/em\u003e are documented from European sites (France and Poland) together with \u003cem\u003eStaphylea\u003c/em\u003e cf. \u003cem\u003etrifolia fossilis\u003c/em\u003e (Geissert, Gregor, \u0026amp; Mai, 1990; Szafer, 1947; Teodoridis, Kvaček, \u0026amp; Uhl, 2009).\u003c/p\u003e \u003cp\u003eInterestingly neither Georgia nor Russia documented fossil seeds of \u003cem\u003eStaphylea pinnata\u003c/em\u003e nor of \u003cem\u003eStaphylea pliocaenica\u003c/em\u003e. Shatilova et al. (2011) did not even mention \u003cem\u003eStaphylea pinnata\u003c/em\u003e up to the Holocene.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhylogeographical aspects of the European species, the Colchis refugium and cryptic refugia\u003c/h2\u003e \u003cp\u003eOne of the most interesting regions is the Colchis area, which is regarded as a hot spot of Tertiary relicts, also supported by a rich fossil record. Models of late Quaternary climatic changes confirmed the existence of a deciduous forest unit in the surrounding of Sukhumi (Abkhazia) at least for 14 000 years (Connor \u0026amp; Kvavadze, 2009). As traditionally suggested Western Georgia can be safely viewed as refugium for \u003cem\u003eStaphylea colchica\u003c/em\u003e although seeds of \u003cem\u003eStaphylea protocolchica\u003c/em\u003e Kol. were mentioned only once from the border of Miocene to Pliocene of Kodori, Abkhazia (Shatilova et al., 2011), \u003cem\u003eStaphylea sarmatica\u003c/em\u003e Krysht. from Upper Miocene of Krynka, Black Sea region (Poyarkova, 1986), \u003cem\u003eStaphylea\u003c/em\u003e sp. from Maikopian (Mid Miocene) on the river Terek, Caucasus region (Gulisashvili, 1970). Compared to the fossil seeds, palynomorphs of \u003cem\u003eStaphylea\u003c/em\u003e are more frequent in Eastern Europe and the Black Sea region since late Miocene. Fossil pollen of \u003cem\u003eStaphylea colchica\u003c/em\u003e is cited in West Georgia from 7.1–0.6 Ma, and again in Holocene, of \u003cem\u003eStaphylea\u003c/em\u003e sp. from Middle to Upper Miocene, as well as from 0.4–0.01 Ma, and missing subsequently (Shatilova et al., 2011). Fossil pollen of \u003cem\u003eStaphylea\u003c/em\u003e sp. is documented from the Russian plain, Urals, Georgia, Ukraine, Poland, Hungary and other European countries, where Pliocene pollen of \u003cem\u003eStaphylea pinnata\u003c/em\u003e is only referred from Siberia (\u003cem\u003eRussian paleobotanical online workshop\u003c/em\u003e, 2021). Usually, the taxonomic resolution of pollen is lower than that of macrofossils (Moreno-Amat et al., 2017), so it could indicate that some pollen findings of \u003cem\u003eStaphylea\u003c/em\u003e sp. in Georgia eventually might be closer related to \u003cem\u003eStaphylea pinnata\u003c/em\u003e than to \u003cem\u003eStaphylea colchica\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eHowever, Colchis might have been not only a Tertiary refugium for \u003cem\u003eStaphylea colchica\u003c/em\u003e but also for \u003cem\u003eStaphylea pinnata\u003c/em\u003e that Lachashvili et al. (2021) assume at least as Tertiary relict from the region of Tbilisi. From there \u003cem\u003eStaphylea pinnata\u003c/em\u003e could have spread along the northern and the southern Black Sea coasts in westward directions, as well as upcountry eastwards. These hypotheses are consistent with the phylogeographical patterns of \u003cem\u003ePrimula vulgaris\u003c/em\u003e (Volkova, Schanzer, \u0026amp; Meschersky, 2013), another species of rather warm temperate deciduous forests. Anapa, at the western border of \u003cem\u003eStaphylea pinnata\u003c/em\u003e‘s recent eastern part area on the NE coast of the Black Sea is also the western border of Colchis based nuclear ITS and cpDNA haplotypes of \u003cem\u003ePrimula vulgaris\u003c/em\u003e. On the south coast isolated populations of \u003cem\u003eStaphylea pinnata\u003c/em\u003e are found westwards as far as Zonguldak. The border of Colchis haplotypes of \u003cem\u003ePrimula vulgaris\u003c/em\u003e is Trabzon, about 860 kilometers eastwards. In case of \u003cem\u003eStaphylea pinnata\u003c/em\u003e the western borders on both coasts are also coinciding with current climatic restraints, especially with regular pronounced summer droughts (Meusel \u0026amp; Jäger, 1992).\u003c/p\u003e \u003cp\u003e \u003cem\u003eStaphylea\u003c/em\u003e ssp. were documented in Northwestern Europe in each Interglacial, but \u003cem\u003eStaphylea pinnata\u003c/em\u003e did not reach this area during the Holocene (Van der Ham et al., 2008; Willis \u0026amp; Niklas, 2004). However, ice-free regions in Eastern Carpathians could have acted as refugia for \u003cem\u003eStaphylea pinnata\u003c/em\u003e as suggested by (Derevenko, 2005) for the area between the rivers Prut and Dnister in Ukraine, an area where this species appears today. From there this species could have spread westwards in Holocene through the passageway of the gate of Przemyśl (Derevenko, 2005). This view gets support in our data by a high ribotype-diversity especially in the Ukrainian samples and is coinciding with the appearance of a characteristic gap of nine bases also found in Central Europe (Tab. S3), that was not detected in ribotypes of Georgia, Turkey and Serbia. Relictic populations of \u003cem\u003eSyringa josikaea\u003c/em\u003e, a moisture demanding deciduous shrub endemic to the Apuseni Mountains and the Ukrainian Carpathians (Lendvay, 2014; Lendvay et al., 2016) argue also for a refugium for warm temperate deciduous forests in Eastern Europe.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDiversification of contemporary\u003c/b\u003e \u003cb\u003eStaphylea\u003c/b\u003e \u003cb\u003ein Europe and SW-Asia and the fossil record\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe fossil record in Eurasia suggests a continuous presence of \u003cem\u003eStaphylea\u003c/em\u003e spp. since the Oligocene, which is in good correspondence with the phylogeny ((Harris et al., 2017); Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The European diversification probably has started in the Pliocene, but the presence of aberrant, partly non-functional ITS copies in the area might suggest that the European lineage has been attendant in the area since the Early Miocene. The dated phylogeny including such copies might be inaccurate when it comes to dating nodes, as mutation rates of non-functional copies might differ (Álvarez \u0026amp; Wendel, 2003; Harpke \u0026amp; Peterson, 2008), but the topology is nevertheless well supported. The pseudogene problem is well-known (Álvarez \u0026amp; Wendel, 2003) and it demands caution in the application of ITS as phylogenetic marker. In our study, we conclude, that the most aberrant sequences (Sp_G2_1, Sp_G2_9 and Sp_A1_8 with striking similarities to the Asian and American ribotypes) represent “ITS-ghosts from the past” and prove the long Tertiary history with a continuous presence of \u003cem\u003eStaphylea\u003c/em\u003e in Europe as suggested by the fossil record (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe European - East Asian disjunction might date to the late Oligocene or Early Miocene, a period where almost continuous warm-temperate forests were present in Eurasia (Mai 1995). In SE Asia we found two clades separated from the Himalayan \u003cem\u003eStaphylea emodi\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e) - which Browicz (1971) documented also from Iran. However, the phylogenetic position of \u003cem\u003eStaphylea emodi\u003c/em\u003e and its sister relationship to the European species \u003cem\u003eStaphylea pinnata\u003c/em\u003e and \u003cem\u003eStaphylea colchica\u003c/em\u003e in our ITS phylogeny (see Supplements, Figs. S1 and S2) differs from Harris et al. (2017). Their study suggests a close relationship of \u003cem\u003eStaphylea emodi\u003c/em\u003e with the American \u003cem\u003eStaphylea\u003c/em\u003e species, whereas our study has evidenced unambiguously in close relationship to the European ones. This discrepancy remains contradictory and does not allow a conclusive discussion but draws attention to \u003cem\u003eStaphylea emodi\u003c/em\u003e as a not well-studied Central Asian relictic species.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStaphylea\u003c/b\u003e \u003cb\u003essp. fossils in North America and Eurasia\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMai (1995) already supposed that the lack of \u003cem\u003eStaphylea\u003c/em\u003e fossils in Northern America was due to missing of appropriate embedding material compared to the numerous seed findings in coal stockyards but also in clay pits of Eurasia. New fossil discoveries, however, shed new light in this topic.\u003c/p\u003e \u003cp\u003eHuang et al. (2015) reported on the “First fossil record of \u003cem\u003eStaphylea\u003c/em\u003e from North America”, \u003cem\u003eStaphylea levisemia\u003c/em\u003e sp. nova, seeds from the latest Miocene to the Earliest Pliocene from Tennessee.\u003c/p\u003e \u003cp\u003eThe second Amercian fossil recently found, a flower of \u003cem\u003eStaphylea ochoterenae\u003c/em\u003e Hernández-Damián et al. sp. nov., bedded in amber of Miocene in South Mexico (Hernández-Damián, Cevallos-Ferriz, \u0026amp; Huerta-Vergara, 2019), differs from extant \u003cem\u003eStaphylea\u003c/em\u003e flowers in lacking a flowering disc and having only one style (instead of two or three). Thus, the feature of the postgenitally united carpel tips forming a compitum is lacking, characteristic even for all recent Crossosomatales (Matthews \u0026amp; Endress, 2005). It grew associated with tropical elements.\u003c/p\u003e \u003cp\u003eThe most surprising discovery was \u003cem\u003eStaphylea woodworthensis\u003c/em\u003e Zhu \u0026amp; Manchester sp. nov., a recently described fossil taxon from Montana (Zhu \u0026amp; Manchester, 2020). Its fruit (preserved pericarp) is \u003cem\u003eStaphylea\u003c/em\u003e-like, contains two carpels, but only seed attachments scars have been conserved. The fossil has been discovered compressed in shale. The seeds may have been released from the fruit during dehiscence of the capsule. The authors report similarities to the fruits of \u003cem\u003eStaphylea bumalda\u003c/em\u003e from East Asia. As the age of the remarkable fossil was dated to the Oligocene new questions did arise. The oldest Eurasian \u003cem\u003eStaphylea\u003c/em\u003e fossils date from late Oligocene: \u003cem\u003eStaphylea rotundata, Staphylea rugosa, Staphylea tymensis\u003c/em\u003e from Siberia (Dorofeev, 1963) and \u003cem\u003eStaphylea microsperma\u003c/em\u003e from Germany (Gregor, 1978; Mai, 1997), and hence are younger (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e) or at least of same age as \u003cem\u003eStaphylea woodworthensis\u003c/em\u003e. If its geological layer dated from Early or Mid-Oligocene it would be approximately as old as our diversification node for the genus \u003cem\u003eStaphylea\u003c/em\u003e, and inside Staphyleaceae (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eHarris et al. (2017) see potential of at least two phylogeographic options for the genus \u003cem\u003eStaphylea\u003c/em\u003e, colonization from Eurasia to North America or the other way round. According to their phylogeny they suggest a rare Himalayan – North American disjunction concerning \u003cem\u003eStaphylea emodi\u003c/em\u003e. They suppose that either the Asian-American clade could have originated in North America and colonized Asia twice or it originated in Asia, arrived in North America, and \u003cem\u003eStaphylea emodi\u003c/em\u003e recolonized Asia. The plant sample used has been obtained from unvouchered cultivated plants (sample AA-478-78; Arnold Arboretum, see Harris et al. (2017) in Table\u0026nbsp;1 and Olmstead et al. (2000), Appendix 1).\u003c/p\u003e \u003cp\u003eIn our phylogeny \u003cem\u003eStaphylea emodi\u003c/em\u003e is clearly the sister species of \u003cem\u003eStaphylea pinnata/colchica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). As we made use of three herbarium samples from Herbarium Edinburgh obtained from wild populations (Tab. S2) we trust the phylogenetic position in our dataset. So \u003cem\u003eStaphylea emodi\u003c/em\u003e does not seem to have been involved in migration events to North America as Harris et al. (2017) assumed.\u003c/p\u003e \u003cp\u003eThe Oligocene \u003cem\u003eStaphylea woodworthensis\u003c/em\u003e from Northwest America may be an argument for a wide distribution of \u003cem\u003eStaphylea\u003c/em\u003e in the Northern Hemisphere in mid Tertiary. Wen (1999) explained the general phylogeographic pattern of temperate forest elements by their continuous distribution and their fragmentation and extinction during late Tertiary and Quarternary climatic cooling and aridifications. The phylogenies presented in Harris et al. (2017; apart from \u003cem\u003eStaphylea emodi\u003c/em\u003e) and our dated phylogeny with \u003cem\u003eStaphylea trifolia\u003c/em\u003e as North American representative as sister to the clade “East Asia 2”) support a more recent connection. As the deepest node in this North American – East Asian clade was dated to late Miocene/Pliocene (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e) this visualizes perhaps extinction of early \u003cem\u003eStaphylea\u003c/em\u003e species and late recolonization of the American continent. The discovery of \u003cem\u003eStaphylea levisemia\u003c/em\u003e (see above) in latest Miocene/earliest Pliocene supports this view.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVetters collected most of the silica gel dried samples in Georgia, southern Russia and Central Europe and wrote the paper. Affenzeller was responsible for sequencing in Salzburg as well as for collaboration in computational analyses with Vetters, and Tribsch contributed a lot to the interpretations, wrote part of the discussion and together with Affenzeller copy-edited the paper.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe want to express cordially thanks to Erich H\u0026uuml;bl (Vienna) for accompanying and scientific support on three excursions to Georgia and Southern Russia. We like to thank Shamil Shetekauri (Tbilisi State University, Georgia) for guiding us in Georgia, as well as Carolin Anna Rebernig (University of Vienna) for doing the AFLP labwork. The study was partially supported by the HRSM-Project \u0026ldquo;Aufbau von universit\u0026auml;ren DNA-Barcoding-Pipelines f\u0026uuml;r ABOL\u0026rdquo; financed by the Austrian Ministry of Science and Economy as well as of the \u0026ldquo;Stiftungs- und F\u0026ouml;rderungsgesellschaft of the University of Salzburg\u0026rdquo;. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information (SI)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn SI included are annotations and the references for the chorology of the \u003cem\u003eStaphylea\u0026nbsp;\u003c/em\u003especies as well as detailed tables of the taken samples. You further can find supplementary figures of Beast Trees of the markers ITS, trnL-F and concatenated ones as well as supplementary material concerning the AFLP research.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkaike, H. 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Mutation rates, population sizes and amounts of electrophoretic variation of enzyme loci in natural populations. \u003cem\u003eGenetics\u003c/em\u003e,\u003cem\u003e 92\u003c/em\u003e(2), 623-646. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-systematics-and-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plsy","sideBox":"Learn more about [Plant Systematics and Evolution](http://link.springer.com/journal/606)","snPcode":"606","submissionUrl":"https://submission.nature.com/new-submission/606/3","title":"Plant Systematics and Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Staphylea, Staphyleaceae, disjunct distribution, Tertiary relict, refugia","lastPublishedDoi":"10.21203/rs.3.rs-4768147/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4768147/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStaphyleaceae is a small family of shrubs and trees with its name-giving genus \u003cem\u003eStaphylea\u003c/em\u003e having a highly disjunct distribution. \u003cem\u003eStaphylea\u003c/em\u003e has a rich fossil record and was an important element in warm temperate Tertiary forests and is therefore regarded as a Tertiary relict. Based on DNA-sequence analyses of the nuclear marker ITS 1\u0026ndash;2 and the chloroplast marker trnL-F as well as AFLP fingerprinting (Amplified Fragment Length Polymorphisms) we gained more insights into the evolution and diversification of the two ‶European\u0026Prime; bladdernut species, the widespread diploid \u003cem\u003eStaphylea pinnata\u003c/em\u003e and the tetraploid \u003cem\u003eStaphylea colchica\u003c/em\u003e of the Caucasus. As the Caucasus is located west of the Ural Mountains, we consider both species as European.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStaphylea pinnata\u003c/em\u003e seems to be involved in the hybridization of the likely allo-poly-ploidization of \u003cem\u003eStaphylea colchica\u003c/em\u003e together with an unknown, supposedly now extinct species.\u003c/p\u003e \u003cp\u003eAncient repeat types of ITS 1\u0026ndash;2 in \u003cem\u003eStaphylea pinnata\u003c/em\u003e of Central Europe and Georgia suggested possible glacial refugia in Georgia, sequence similarity (especially a characteristic gap) in ITS 1\u0026ndash;2 sequences of Ukrainian and Central European samples indicate refugia also in Ukraine.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStaphylea emodi\u003c/em\u003e, the only \u003cem\u003eStaphylea\u003c/em\u003e species of Central Asia (Tibetan Plateau), was in our research more closely related to the European species than to American representatives.\u003c/p\u003e","manuscriptTitle":"Diversification of the European bladdernuts (Staphylea, Staphyleaceae) in context of the whole genus and the rich fossil record","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-16 18:31:14","doi":"10.21203/rs.3.rs-4768147/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-04T12:43:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-03T20:14:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-22T08:20:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330745248978585301675046861249599529065","date":"2025-01-07T09:58:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329168677267367026990677376422853250216","date":"2025-01-06T10:27:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330066890635073671601703647209184469418","date":"2025-01-06T09:44:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"193498903356575016999871068247435661100","date":"2024-09-02T15:55:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"208212796140914796211681150491432500506","date":"2024-08-28T12:47:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-28T10:57:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-22T10:34:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-22T08:52:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Systematics and Evolution","date":"2024-07-19T13:54:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-systematics-and-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plsy","sideBox":"Learn more about [Plant Systematics and Evolution](http://link.springer.com/journal/606)","snPcode":"606","submissionUrl":"https://submission.nature.com/new-submission/606/3","title":"Plant Systematics and Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4cca5b05-80d7-419a-b483-6daaa4c7bb20","owner":[],"postedDate":"August 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-06-24T12:23:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-16 18:31:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4768147","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4768147","identity":"rs-4768147","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}