Molecular Phylogenetics and Evolution 70 (2014) 244–259 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Miocene–Pliocene speciation, introgression, and migration of Patis and Ptilagrostis (Poaceae: Stipeae) Konstantin Romaschenko a,b,c, Nuria Garcia-Jacas c, Paul M. Peterson a,⇑, Robert J. Soreng a, Roser Vilatersana c, Alfonso Susanna c a b c Smithsonian Institution, Department of Botany, MRC-166, National Museum of Natural History, Washington, DC 20013-7012, USA M.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, 01601 Kiev, Ukraine Laboratory of Molecular Systematics, Botanic Institute of Barcelona, (IBB-CSIC-ICUB), Passeig del Migdia s.n., 08038 Barcelona, Spain a r t i c l e i n f o Article history: Received 11 March 2013 Revised 26 July 2013 Accepted 19 September 2013 Available online 3 October 2013 Keywords: At103 Biogeography Classification Low copy nuclear DNA sequences Patis Phylogeny a b s t r a c t Genetic interchange between American and Eurasian species is fundamental to our understanding of the biogeographical patterns, and we make a first attempt to reconstruct the evolutionary events in East Asia that lead to the origin and dispersal of two genera, Patis and Ptilagrostis. We conducted a molecular phylogenetic study of 78 species in the tribe Stipeae using four plastid DNA sequences (ndhF, rpl32-trnL, rps16-trnK, and rps16 intron) and two nuclear DNA sequences (ITS and At103). We use single copy nDNA gene At103 for the first time in the grasses to elucidate the evolutionary history among members of the Stipeae. Ampelodesmos, Hesperostipa, Oryzopsis, Pappostipa, Patis, and Stipa are found to be of multiple origins. Our phylograms reveal conflicting positions for Ptilagrostis alpina and Pt. porteri that form a clade with Patis coreana, P. obtusa, and P. racemosa in the combined plastid tree but are aligned with other members of Ptilagrostis in the ITS tree. We hypothesize that Ptilagrostis still retains the nucleotype of an extinct genus which transited the Bering land bridge from American origins in the late Miocene (minimum 7.35–6.37 mya) followed by hybridization and two plastid capture events with a Trikeraia-like taxon (7.96 mya) and para-Patis (between 5.32 and 3.76 mya). Ptilagrostis porteri and Patis racemosa then migrated to continental North America 1.7–2.9 mya and 4.3–5.3 mya, respectively. Published by Elsevier Inc. 1. Introduction The tribe Stipeae Martinov is a complex and highly specialized monophyletic lineage within the subfamily Pooideae (Davis and Soreng, 2007; Romaschenko et al., 2010, 2012). It includes between 572 and 670 species in 28 genera often with unclear generic boundaries and converging morphological traits (Tzvelev, 1977; Watson and Dallwitz, 1992; Wu and Phillips, 2006; Barkworth, 2007; Romaschenko et al., 2008, 2010, 2011, 2012; Soreng et al., 2003, 2012). Early phylogenetic studies using plastid RFLP data to locate the Stipeae among other groups of Poaceae (Soreng and Davis, 1998) was followed with more thorough sampling and the use of nrDNA ITS sequence analysis (Jacobs et al., 2000, 2007). With a different depth of sampling the Stipeae have been investigated by combining data from ITS and several plastid regions of which three noncoding regions (trnH-psbA, trnC-trnL, and trnK-rps16) were used in Barkworth et al. (2008) and two noncoding regions trnL-F and ⇑ Corresponding author. Address: Department of Botany, MRC-166, National Museum of Natural History, Smithsonian Institution, 10th and Constitution Avenue NW, Washington, DC 20013-7012, USA. Fax: +1 202 786 2563. E-mail address: peterson@si.edu (P.M. Peterson). 1055-7903/$ - see front matter Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ympev.2013.09.018 rpl16 were used in Cialdella et al. (2007). However, none of the resulting trees from these studies provided a cohesive theory concerning phylogenetic relationships within the tribe. In our previous studies (Romaschenko et al., 2010, 2011, 2012) we employed sequences of nine plastid regions (trnK-matK, matK, trnH-psbA, trnLF, rps3, ndhF, rpl32-trnL, rps16-trnK, and rps16 intron), and nrDNA ITS to recover a well-supported phylogeny using an ample set of all known Stipeae genera. We provided evidence for restricting the application of Achnatherum P. Beauv., Piptatherum P. Beauv., and Stipa to the Old World, and a monotypic Oryzopsis Michx. to the New World; resurrecting Patis Ohwi, and recognizing two new New World genera, Pappostipa (Speg.) Romasch., P.M. Peterson & Soreng and Piptatheropsis Romasch., P.M. Peterson & Soreng. We also demonstrated that the genera of subtribe Duthieinae are best placed within tribe Phaenospermateae, and that this lineage is phylogenetically isolated from Stipeae. For a sketchy view of the phylogeny of the Stipeae with poorly supported clades based on only two markers (ITS and trnK) and recognition of paraphyletic genera often portrayed as grades, see Hamasha et al. (2012). In addition, our comprehensive plastid analyses have revealed two broad phylogenetic patterns within Stipeae. First, there is a mutual exclusiveness between two groups of taxa defined on the K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 basis of lemma epidermal cells reviewed by Thomasson (1978, 1979, 1980, 1982, 1985, 1987, 2005), Barkworth and Everett (1987) and Romaschenko et al. (2008, 2010, 2011, 2012): species with long fundamental cells and lemma epidermis with sinuate sidewalls (saw-like lemma epidermal pattern [LEP]; ‘‘S’’ taxa), and species with compressed fundamental cells with straight sidewalls (maize-like LEP; ‘‘M’’ taxa). Second, there is a phytogeographic alignment of New and Old World taxa within each LEP group, with very few exceptions. This kind of pattern is of considerable biogeographic interest since it implies rarity of colonization events between the two hemispheres and allows an opportunity to determine when and how the colonization occurred, including the reconstruction of paleoclimatic events and development of novel morphological traits. Contrary to plastid analysis, the ITS data do not support an allopatric geographical pattern but suggest common origin for Asian Ptilagrostis Griseb., Patis, and American ‘‘S’’ lineages (ASL) including Piptatheropsis, Ortachne Nees ex Steud., Anatherostipa (Hack. ex Kuntze) Peñailillo, Aciachne Benth., and Piptochaetium J. Presl (Romaschenko et al., 2010, 2012). Moreover, two members of the ASL clade in the plastid analysis, Hesperostipa (M.K. Elias) Barkworth (‘‘S’’ taxon) and Pappostipa (‘‘M’’ taxon), were placed by the ITS data in different subclades in the achnatheroid clade (AC), which otherwise exclusively encompasses ‘‘M’’ taxa. This realignment leaves only two genera of Stipeae with east–west disjunctions; Patis and Ptilagrostis. Regarding the former, the North American Piptatherum racemosum (Sm.) Eaton and the Asian Pipatherum kuoi S.M. Phillips & Z.L. Wu (Oryzopsis obtusa Stapf) were shown to be closely related to the Asian, Achnatherum coreanum (Honda) Ohwi. In the course of taxonomic studies the latter taxon had been placed in different genera including Orthoraphium Nees and Patis Ohwi (as monotypic genus), and recently was returned to Achnatherum (Wu and Phillips, 2006). All three species were found in a strongly supported clade only distantly related to Achnatherum, Piptatherum, Piptatheropsis or Orthoraphium Nees (Romaschenko et al., 2011). Based on molecular evidence and shared morphological traits such as underdeveloped basal leaves and long, wide flag leaf blades (a unique combination in Stipeae), these three species were united in Patis (Romaschenko et al., 2011): as the North American Patis racemosa (Sm.) Romasch., P.M. Peterson & Soreng and Asian Patis coreanum (Honda) Ohwi and P. obtusa (Stapf) P.M. Peterson & Soreng. As for Ptilagrostis, the argument of where and how to delimit it from Stipa continues (Freitag, 1985; Tzvelev, 1977; Romaschenko et al., 2012). We showed that Stipa subsessiliflora (Rupr.) Roshev. and S. purpurea Griseb. align with Stipa s.s., rather than with other Asian Ptilagrostis s.s. Of the two species of Ptilagrostis accepted in North America (Barkworth, 2007), the morphologically isolated P. kingii (Bol.) Barkworth aligned with Piptatheropsis rather than with the Asian Ptilagrostis (Romaschenko et al., 2011, 2012). The incongruence between plastid and nuclear phylogenies is seen in many phylogeographic studies where a geographic structure in the distribution of cpDNA types (sequences of certain phylogenetic signal from the plastid genome) is detected (Humphreys et al., 2010). In such cases, phylogenies based on nuclear markers agree more often with morphological (i.e., taxonomic) boundaries (Rieseberg and Soltis, 1991; Harding et al., 2000; King and Ferris, 2000; Comes and Abbott, 2001; Grivet and Petit, 2003; Semerikov and Lascoux, 2003; Fehrer et al., 2007; Frajman and Oxelman, 2007; Ståhlberg and Hedrén, 2008). Our earlier Stipeae ITS-derived topology (Romaschenko et al., 2011, 2012) appears to represent a more ordered LEP distribution when joining Pappostipa and AC in a separate clade of ‘‘M’’ taxa, and suggests close affinities of Asian Ptilagrostis with the members of ASL, plants sometimes with similar LEP and an alpine-like growth. Such incongruence between nuclear and plastid data is usually interpreted as being an outcome of 245 hybridization among taxa with subsequent and often extensive backcrossing resulting in exchange or introgression of the nuclear genome while the plastid genome remains unchanged (Avise, 2000; Gillespie et al., 2010; Soreng et al., 2010). Fossils of Stipeae are known only from North America and the earliest stipoid spikelet was dated at 23 mya (Elias, 1942; Thomasson, 1978, 1979, 1985). Therefore, to infer a time frame for evolution of Stipeae, particularly in the Old World, we have to rely exclusively on molecular data. In our present study we focus on resolving the conflicts in the phylogenetic position of Ptilagrostis and Patis portrayed in our earlier work (Romaschenko et al., 2012). Here we employ DNA sequences of At103, a low copy nuclear gene, to assess possible reticulate evolutionary events and to compare and contrast with combined plastid (ndhF, rpl32-trnL, rps16-trnK, and rps16 intron) and nrDNA ITS-derived phylogenies. This is the first attempt to employ a low copy gene to investigate phylogenetic relationships within the Stipeae; and At103 is a new marker in phylogenetic studies of Poaceae. The application of At103 brings several advantages to the study where introgression and other kinds of genomic exchange are concerned. The At103 gene belongs to the low copy nuclear Conserved Ortholog Set (COS) and was reported to have a single copy in the rice genome (Gogarten and Olendzenski, 1999; Sonnhammer and Koonin, 2002; Li et al., 2008). It encodes Mg-protoporphyrin IX monomethyl ester cyclase, one of the key enzymes in the chlorophyll biosynthesis pathway (Hung et al., 2010). By using this new type of molecular data we were able to document past intergeneric hybridization events in the Stipeae and explain the shifting phylogenetic positions of Patis, Ptilagrostis, and a number of other related taxa in our phylogenetic trees. Genetic interchange between American and Eurasian species is fundamental to our understanding of the biogeographical patterns among the principal lineages within the Stipeae, and we make a first attempt to reconstruct the evolutionary events in East Asia that lead to the origin and dispersal of ‘‘S’’ lineages by focusing on the history of Patis and Ptilagrostis. We explore genetic interchange between lineages, investigate putative hybridization events and genome introgression, apply the molecular clock approach to recover node ages for clades of interest in plastid and nDNA phylogenies, and interpret the results in light of Miocene paleoclimatic events in East Asia. 2. Materials and methods 2.1. Taxon sampling The data consists of 78 total species, of which 70 represent the majority of phylogenetic groups in the Stipeae; seven represent the Phaenospermateae. Brachyelytrum erectum (Schreb.) P. Beauv. was included as an outgroup based on its well-documented early diverging position in subfamily Pooideae (Bouchenak-Khelladi et al., 2008; Davis and Soreng, 1993, 2007; GPWG, 2001; Hilu et al., 1999; Schneider et al., 2009; Soreng et al., 2007; Romaschenko et al., 2008, 2010, 2012). A complete list of taxa, voucher information, and GenBank numbers can be found in Appendix 1 (see Supplementary material). New sequences included in this manuscript are: 134 (all) for At103, 14 for ITS, 13 for ndhF, 12 for rpl32-trnL, 13 for rps16-trnK, and 13 for rps16 intron; others were used previously (Romaschenko et al., 2008, 2011, 2012). Our sample contains nearly a complete set of Ptilagrostis species [minus Pt. yadongensis Keng f. & J.S. Tang or Pt. schischkinii (Tzvelev) Czer.], and includes related polyploid species that had ambiguous phylogenetic positions in Romaschenko et al. (2010, 2012). We also wanted to represent the taxonomic and geographical diversity of Stipeae using primarily putative diploid species 246 K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 (2n = 20–24), confirmed by previous chromosome counts (Romaschenko et al., 2012, see Appendix 3), as well as taxa with an unknown chromosome number but which fall within clades with diploids and lacking polyploids. In the majority of cases the DNA was extracted from silica dried material collected by the authors, while a few samples were taken from other specimens in the United States National Herbarium (US). The Asian Piptatherum and Ptilagrostis samples were taken from specimens at the Komarov Botanical Institute (LE) and Austrostipa samples were provided by Surrey Jacobs from personal collections. 2.2. DNA extraction, amplification, sequencing, and cloning All procedures related to the sequencing of the combined plastid and ITS regions were performed in the Laboratory of Analytical Biology (LAB) at the Smithsonian Institution. DNA isolation, amplification, and sequencing of ndhF, rpL32-trnL, rps16-trnK, and rps16 intron were accomplished following procedures outlined in Peterson et al. (2010a,b, 2011, 2012) and Romaschenko et al. (2010, 2012). Cloning and direct sequencing of nDNA At103 region was done in the Laboratory of Molecular Systematics at the Botanic Institute of Barcelona. TOPO-TA (Invitrogen) kit for cloning Taq polymerase-amplified PCR products was used according to the protocol suggested by the manufacturer to obtain multiple copies of At103 for the following 14 species that are polyploid, of unknown ploidy or diploid (d) species of interest: Achnatherum splendens (Trin.) Nevski, Achnatherum stillmanii (Bol.) Barkworth, Ampelodesmos mauritanicus (Poir.) T. Durand & Schinz, Hesperostipa comata (Trin. & Rupr.) Barkworth, Oryzopsis asperifolia Michx., Pappostipa chrysophylla (E. Desv.) Romasch., Patis coreana (Honda) Ohwi, Patis obtusa (Stapf) Romasch., P.M. Peterson & Soreng, Patis racemosa, Psammochloa villosa (Trin.) Bor, Ptilagrostis mongholica (Turcz. ex Trin.) Griseb. (d), Ptilagrostis porteri (d), Stipa zalesskii Wilensky, and Trikeraia hookeri (Stapf) Bor. All available colonies (20–60 per plate) were chosen for PCR. Of these, from 15 to 48 PCR products of proper length were purified using the QIAquick PCR purification kit (Qiagen, Tokyo) and then used for sequencing with the same primers as in the original amplification or with universal cloning primers T7 and M13. At103 consists of five translated exons and four introns (Li et al., 2008). For At103 we used only exon III (largest portion) and all of intron III. These regions are relatively short (aligned length of the sequence is 336 bp) and can easily be obtained from old herbarium material. We screened 54 putative diploids in the Stipeae and seven from adjacent Phaenospermateae with At103 by direct sequencing. Only sequences with an unambiguous signal were used. The primers and the PCR conditions were used as described by Li et al. (2008). At103 data provide independent evidence for species relationships, and a chance to test inferences based on ITS and plastid data. 2.3. Phylogenetic analyses Sequences of ITS, ndhF, rpL32-trnL, rps16-trnK, and rps16 intron were aligned using Muscle (Edgar, 2004) implemented in Geneious 5.3.4. Likelihood parameters were estimated with jModeltest v0.1 (Posada, 2008) while choosing Akaike information criterion (AIC). The designated evolutionary models and maximum likelihood (ML) parameters are shown in Table 1. Different regions of the plastid genome are assumed to have evolved on a single tree topology despite possible hybridization among species (Graham and Olmstead, 2000; Erixon and Oxelman, 2008). Therefore, we combined the plastid data and analyzed them as a single unit. The incongruence length difference (ILD) test (Farris et al., 1994), implemented in the program WinClada ver. 1.00.08 (Nixon, 2002), was used to test for incongruence between plastid and ITS datasets. Default parameters for 1000 replicates were executed. The ILD test confirmed incongruence of plastid and ITS data; therefore, they were analyzed separately. The ML trees for combined plastid and ITS data were constructed with GARLI 0.951 (Zwickl, 2006). Bootstrap analyses (Felsenstein, 1985) were performed using GARLI with 1000 repetitions and the program PAUP v.4.0b10 (Swofford, 2000) was used to compute the majority rule consensus tree. Bootstrap (BS) values of 90–100% were interpreted as strong support. Bayesian posterior probabilities (PP) were estimated using MrBayes v3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist et al., 2005). The combined plastid data set was split into four partitions containing the ndhF, rpL32-trnL, rps16-trnK, and rps16 intron sequences. The models of sequence evolution as determined, were imposed for each sequence partition. Bayesian analysis was initiated with random starting trees and was run for four million generations for plastid data and twelve million generations for the ITS analysis with sampling frequency of trees set at 100th iteration. In analyses the variance of split sequences was less than 0.01 and potential scale reduction factor was close or equal to 1.0 which indicated convergence of the chains (Huelsenbeck and Ronquist, 2001). The search was also monitored with Tracer v1.4 (Rambaut and Drummond, 2007). The effective sample size (ESS) value was greater than 100 in both cases and 25% of the sampled values were Table 1 Summary of four plastid regions: ndhF, rpL32-trnL, rps16-trnK, rps16 intron; and nrDNA ITS and At103 regions. The parameters used in Maximum Likelihood and Bayesian searches were chosen according to Akaike Information Criterion (AIC). ndhF rpL32-trnL rps16-trnK rps16 intron Combined plastid data ITS At103 Total aligned characters Number of sequences Number of substitution types Model for among-site rate variation 783 78 6 Gamma 878 78 6 Gamma 791 78 6 Gamma 789 78 6 Gamma 3241 312 – – 621 76 6 Gamma 336 141 6 Gamma Substitution rates of GTR model 2.4740 4.3039 1.0000 2.4740 4.3039 1.3928 2.1348 1.0000 1.3928 2.1348 0.6345 2.3529 0.6345 1.0000 2.3529 1.0000 1.7806 0.6160 0.6160 1.7806 – 1.1737 3.7674 2.2375 0.4906 10.1691 0.5173 2.8259 0.5159 0.6338 3.2635 Character state frequencies 0.3151 0.1389 0.1575 0.3886 0.3690 TPM3uf + I + G 1.1970 0.3726 0.1200 0.1269 0.3805 0.0 TPM3uf + G 0.6580 0.3021 0.1508 0.1423 0.4048 0.1871 TPM2uf 1.0254 0.3132 0.1810 0.1414 0.3644 0.0 TPM1uf + G 0.1280 – 0.2278 0.3087 0.2710 0.1924 0.3741 GTR + I + G 0.7931 0.2512 0.2509 0.2125 0.2852 0.1442 GTR + I + G 0.8312 Proportion of invariable sites Substitution model Gamma shape parameter (a) – – – 247 K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 discarded. Posterior probabilities (PP) over 95% credibility interval were considered informative. cases of ancient hybridization events. We have used At103 exclusively to construct a network for locating multiple gene copies and to assess possible reticulate evolutionary events. 2.4. Network analyses 2.5. Divergence time estimation To investigate relationships among the species of Stipeae derived from different regions of the nuclear genome we constructed phylogenetic split networks for ITS and At103 datasets using the neighbor-net algorithm (Bryant and Moulton, 2004) implemented in the program SplitsTree version 4.11.3 (Huson and Bryant, 2006). Uncorrected p-distances were used to weight the splits. Prior to network analyses, sequences were corrected by imposing corresponding models of DNA evolution. Within the Stipeae we have well-resolved combined plastid and ITS phylogenies with high support for the nodes. In order to reconstruct the evolutionary history of Patis, Ptilagrostis, and relatives that have multiple gene copies we used At103 to detect putative We rejected clock-like mutation rates for all plastid regions at the P < 0.01 level using v2 tests of best ML topologies derived from PAUP analyses. The v2 statistics were obtained by comparing likelihood scores of unrooted and unconstrained topologies against rooted topologies where the molecular clock was enforced. The ages of the major lineages in Stipeae were calculated using the uncorrelated lognormal relaxed clock method using Bayesian inference implemented in BEAST v. 1.5.1 (Drummond and Rambaut, 2007; Table 2). Tree priors were set using the Yule process. Convergence of runs and effective sample sizes (ESS) for priors (to be above 100) were verified using program Tracer v1.4. Table 2 Age estimates for the major nodes of Stipeae derived from Bayesian relaxed clock analysis conducted in BEAST v1.4.1 for concatenated sequences from four plastid regions (ndhF, rps16-trnK, rps16 intron, and rpl32-trnL) and for nDNA At103 region direct sequences and clones. Values are node ages (stem ages for single taxa or isolated At103 clones) given in million of years (mya). Intervals in parentheses represent 95% highest posterior density intervals (HPD).  = stem age; P- = nuclear copy corresponding to plastid clade (see colored ‘‘P’’s in circles in Fig. 3 and plastid clades in Fig. 1. Crown node derived from plastid phylogeny (see Fig. 1) Node age and 95% HPD obtained for plastid data Mean rate and 95% HPD of substitution per site per 1 myr for plastid data Stipeae Eurasian S–lineages (ESL) American S–lineages (ASL) Achnatheroid clade (AC) Piptatherum s.s. group Piptatherum s.s. excluding P. coerulescens Patis group (P-copies) Patis obtusa (P-copies) Patis obtusa (ESL copies) 28.09 (22.94–36.04) 21.29 (12.82–22.09) 18.52 (15.82–23.39) 16.24 (11.24–21.16) 10.23 (8.82–19.63) 6.21 (2.69–10.99) 0.0007 0.0004 0.0004 0.0014 0.0010 0.0010 6.91 (4.71–17.4) <6.91 – 0.0003 (0.0001–0.0006) 0.0003 (0.0001–0.0006) Patis coreana (P-copies) Patis coreana (ESL copies) Patis coreana (ASL copies)  0.0004 (0.0001–0.0009) Patis coreana (AC adjacent copy) Patis racemosa (P-copies) Patis racemosa (ESL copies) Patis racemosa (ASL copies in P2) Patis + Piptatherum + AC Piptatherum + AC Para-Patis + Ptilagrostis porteri & P. alpina Ptilagrostis group (excluding P. porteri, P. alpina, P. kingii and P. pelliotii) Ptilagrostis alpina + P. porteri Oryzopsis (ESL P-copies) Oryzopsis (ASL copy) Trikeraia (ESL P-copies) Trikeraia (ASL copy) Trikeraia (AC copy) Trikeraia (Phaenospermatae copies) Orthoraphium (ASL P-copy) Psammochloa (ESL P-copies) Psammochloa (ASL copy) Pappostipa (ASL P-copy) Pappostipa (AC copies) Hesperostipa (ASL P-copies) Hesperostipa (AC adjacent copies) Stipa (ESL P-copies) Stipa (ASL copy) Ampelodesmos (ESL P-copies) Ampelodesmos (Phaenospermataelike copies) Piptochaetium group 4.34 (0.99–7.23) – – (0.0001–0.0017) (0.0001–0.0009) (0.0001–0.0008) (0.0008–0.0025) (0.0004–0.0018) (0.0004–0.0018) Node age and 95% HPD obtained for At103 region Mean rate and 95% HPD of substitution per site per 1 myr for At103 region 28.03 (21.2–39.0) 21.66 (12.6–31.1) 17.44 (10.5–24.5) 23.02 (14.2–33.2) 7.49 (4.8–18.6) 4.65 (2.5–11.0) 0.0016 0.0013 0.0015 0.0018 0.0014 0.0014 12.21 (3.0–19.3) 6.71 (1.0–8.5) 3.0 (–) 9.46 (1.7–11.1), 14.75 (–)  6.71 (1.0–8.5) 11.5 (–) 13.48 (–) 12.52 (–), 3.92 (2.5– 10.5)  2.75 (0.0–4.2)  12.21 (2.9–19.5) 4.89 (–) 13.48 (–), 3.25 (2.5–10.5) 0.0009 (0.0001–0.0019) 0.0012 (0.0002–0.0027) 0.0011 (0.0002–0.0025)   (0.0001–0.0035) (0.0002–0.0029) (0.0002–0.0032) (0.0003–0.0036) (0.0003–0.0028) (0.0003–0.0028) 0.0012 (0.0002–0.0027) 0.0012 (0.0002–0.0026) 0.0014 (0.0003–0.0033) – 4.34 (0.99–7.23) 0.0004 (0.0001–0.0009) – 26.2 (33.1–20.5) 21.2 (26.1–16.5) 5.32 (2.5–10.2) 0.0005 (0.0001–0.0011) 0.0007 (0.0002–0.0015) 0.0004 (0.0001–0.0009) 7.35 (4.62–12.51) 0.0005 (0.0001–0.0011) 6.37 (1.5–8.9) 0.0012 (0.0002–0.0027) 5.32 (2.76–7.4)  14.06 (16.22–23.02) –  7.96 (5.7–14.24) – – – 0.0005 (0.0001–0.0011) 0.0004 (0.0001–0.0009) 3.76 (–) 14.76 (–)  13.73 (11.5–24.5) 5.07 (0.5–6.7)  3.94 (0.5–8.2)  6.77 (1.5–12.5) 4.27 (1.2–8.7) 0.0012 0.0016 0.0015 0.0019 0.0014 0.0017 0.0015  0.0007 (0.0002–0.0015) 0.0005 (0.0001–0.0011)  8.09 (–) 2.81 (0.1–4.4) 9.79 (–)  2.13 (0.4–7.9) 4.84 (0.2–5.5) 10.78 (–) 6.93 (4.5–18.1) 15.78 (6.3–26.6)  5.62 (0.0–6.5) 3.61 (2.6–14.5) 16.73 (5.7–19.2) – 0.0015 – 0.0012 0.0017 – 0.0017 0.0013 0.0012 0.0012 0.0009 2.77 (2.0–10.8) 0.0006 (0.0002–0.0012)  11.7 (7.69–16.88) 0.98 (0.01–2.28) –  12.43 (4.84–19.4) –  12.62 (12.0–21.5) –  21.2 (13.0–22.0)  0.0005 (0.0001–0.0012)  0.0003 (0.0001–0.0008) 0.0005 (0.0001–0.0010) 0.0004 (0.0001–0.0009)  5.82 (2.79–14.45) – 0.0004 (0.0001–0.0008) 9.3 (4.0–13.0) 0.0005 (0.0001–0.0010) 0.0015 0.0009 0.0017 0.0013 (0.0003–0.0036) (0.0001–0.0019) (0.0003–0.0036) (0.0002–0.0027) (0.0002–0.0027) (0.0003–0.0036) (0.0002–0.0032) (0.0004–0.0040) (0.0003–0.0033) (0.0003–0.0036) (0.0003–0.0036) (0.0003–0.0036) (0.0002–0.0028) (0.0003–0.0038) (0.0004–0.0035) (0.0003–0.0024) (0.0002–0.0028) (0.0003–0.0025) (0.0002–0.0018) 248 K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 Fossil-based divergence time estimations included an unfixed mean substitution rate, were run for 20 million generations collecting parameters every 1000 generations. Twenty-five percent of the data were discarded as ‘‘burnin’’. The lognormal-distributed prior on the age (tmrca) of the SL clade (Romaschenko et al., 2010) was set for 23 myr. This corresponded to the earliest fossils of Stipeae – Stipidium M.K. Elias (=Berriochloa M.K. Elias) which morphologically resembles contemporary Hesperostipa and Piptochaetium (Elias, 1942; Thomasson, 1978, 1979, 1980, 1985), and all members of ASL. The calibration was done using a zero offset and standard deviation of 0.05%, which resulted in 95% prior distribution of 21.25–25.05 myr. 3. Results 3.1. Amplification of At103 The At103 region was easily amplified through the entire Stipeae dataset. The length of aligned sequences is 336 nucleotide positions of which first 228 represent 76 amino-acids constituting the 30 portion of the 3rd At103 exon. The remaining 108 nucleotides represent the portion of the intron between the 3rd and 4th exons of At103 gene. A deletion of the two nucleotide position (GA) was found for two close copies of At103 in Patis coreana and one in Psammochloa villosa (Trin.) Bor. When translated these copies rendered exceptionally long amino-acids sequences. The sequence of At103 in Anisopogon avenaceus R. Br. contains a stopcodon within the putative protein coded area. This indicates that the sequence might represent a pseudogene. However, it appeared on our phylogram in the same clade with the other representatives of Phaenospermateae. Direct sequencing of species with known diploid status yielded a single copy of At103 without ambiguous nucleotide positions. For all of the species with unknown chromosome number from Ptilagrostis, Piptatherum, Anatherostipa and Orthoraphium the analysis also yielded unambiguous sequences. The sampling within the other genera was complicated by the general rarity of chromosome counts for Stipeae. The only known count for the Austrostipa was 2n = 44 for A. stipoides (Hook. f.) S.W.L. Jacobs & J. Everett (Murray et al., 2005). However, despite of the polyploid status of the species it yielded unambiguous sequence of At103 along with five other species of the genus all representing Austrostipa subgen. Tuberculatae S.W.L. Jacobs & J. Everett and subgen. Lobatae S.W.L. Jacobs & J. Everett. Thus, these species were tentatively included in final analysis for a complete survey of the phylogenetic splits within achnatheroid grasses. Among the available species of American genera Jarava, Amelichloa and the representative of former New World branch of the genus Achnatherum, – A. multinode, the chromosome number is known only for Jarava ichu (2n = 40; Davidse and Pohl, 1994). Direct sequencing of this species along with J. castellanosii (F.A. Roig) Peñailillo, J. pseudoichu (Caro) F. Rojas, Amelichloa clandestina (Hack.) Arriaga & Barkworth, and Achnatherum multinode (Scribn. ex Beal) Valdés-Reyna & Barkworth returned unambiguous sequences, and thus, they were preserved in our analysis. For all polyploid species in question we obtained several cloned copies of At103 falling into two to four groups (nDNA types) which were displayed in different parts of the network. We preserved all divergent copies in the analysis. In addition to allelic variation, Taq DNA polymerase can sometimes introduce errors. The number of copies for the tested taxa was: Ampelodesmos mauritanicus – six copies falling in two groups, Oryzopsis asperifolia – six copies in two groups, Patis obtusa – eight copies in two groups, Patis racemosa – seven copies in three groups, Patis coreana – 12 copies in four groups, Hesperostipa comata – five copies in two groups, Pappostipa chrysophilla – three copies in two groups, Stipa zalesskii – three copies in two groups, Psammochloa villosa – three copies in two groups, Trikeraia hookerii – three copies in two groups, and Achnatherum stillmanii – three copies in two groups. Among polyploid species Achnatherum splendens (2n = 46, Tzvelev, 1976) yielded one copy of At103, while Ptilagrostis porteri yielded three copies which were found in two groups and Ptilagrostis mongholica (2n = 22, Tzvelev, 1976) yielded three close copies found in single group. 3.2. Plastid and ITS phylogenies The ML tree from the combined analysis of four plastid regions (ndhF, rpL32-trnL, rps16-trnK, and rps16 intron) has high support for many of the nodes (Fig. 1, tree on left). Support for the monophyly of the Stipeae is strong (BS = 100, PP = 1.00) but resolution of the Phaenospermateae clade is weak (BS = 68, PP = 1.00). Within the Phaenospermateae the only strong support was found for the clade containing Stephanachne pappophorea (Hack.) Keng, S. nigrescens Keng, and Sinochasea trigyna Keng (BS = 100, PP = 1.00). The Stipeae is divided into five major lineages: Eurasian ‘‘S’’ lineage (ESL; BS = 66, PP = 1.00), American ‘‘S’’ lineage (ASL; PP = 0.83), Patis group (BS = 100, PP = 1.00), Piptatherum (BS = 100, PP = 1.00), and achnatheroid clade (AC; BS = 100, PP = 1.00). The ESL and ASL are sister (BS = 69, PP = 1.00) and together they are sister to Patis-Piptatherum-AC clade (BS = 76, PP = 0.95). The Patis group is sister to Piptatherum-AC (BS = 99, PP = 1.00) and the sister relationship between Piptatherum and AC is strongly supported (BS = 100, PP = 1.00). The ESL contains many monotypic (mon.) genera which, with the exception of Oryzopsis asperifolia (mon.) from North America, are of Eurasian origin and include: Stipa, Ampelodesmos (mon.), Achnatherum splendens (mon. as ‘‘Neotrinia’’), Psammochloa (mon.), Trikeraia, Orthoraphium (mon.), and Ptilagrostis. There are two strongly supported clades in ESL: Ampelodesmos-Achnatherum splendens-Psammochloa (BS = 93, PP = 1.00) and Trikeraia–Orthoraphium–Ptilagrostis (BS = 91, PP = 1.00). The ASL contains the following six strongly supported clades all of North American origin: two species of Ortachne (BS = 100, PP = 1.00), five species of Piptatheropsis (BS = 99, PP = 1.00), four species of Piptochaetium (BS = 100, PP = 1.00), two separate pairs of Anatherostipa species that are not sister, and two species of Aciachne (BS = 100, PP = 1.00). The Patis group includes East Asian Patis obtusa sister to a well supported clade (BS = 97, PP = 1.00) with two separate pairs: East Asian Patis coreana-North American P. racemosa (BS = 68, PP = 1.00) and East Asian Ptilagrostis alpina and North American P. porteri (BS = 95, PP = 1.00. The Piptatherum clade includes P. coerulescens of Circum-Mediterranean origin as sister to eight other species of Piptatherum of Asian origin (BS = 100, PP = 1.00). Sister to Piptatherum is North American Achnatherum stillmanii (BS = 72, PP = 0.71). The internal structure of the AC is well developed and it includes a major American clade (MAC; BS = 100, PP = 1.00) with Amelichloa clandestina, Achnatherum multinode, and three species of Jarava; two Asian clades, one we refer to as the Timouria group with Achnatherum chinense, A. caragana, and Ptilagrostis pelliotii (Achnatherum pelliotii (Danguy) Röser & H.R. Hamasha) [BS = 93, PP = 1.00] and the other Asian clade with Piptatherum miliaceum (Oloptum miliaceum (L.) Röser & H.R. Hamasha), P. thomasii, P. paradoxum, and P. virescens, Stipa parviflora (Stipellula parviflora (Desf.) Röser & H.R. Hamasha), Achnatherum confusum, and A. jacquemontii (BS = 0.51, PP = 0.75). The other strongly supported clade within the AC includes a monophyletic Austrostipa (BS = 100, PP = 1.00) represented by six species. The ITS ML tree (Fig. 1, tree on right) is considerably less supported than the plastid tree especially along the backbone. Incongruence between the ITS and plastid tree, and partial or complete rearrangements among the taxa is easily observed in Fig. 1. Most 249 K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 Plastid 0.66 68 100 1.00 78 0.95 1.00 0.92 66 93 1.00 1.00 1.00 * 0.82 0.86 91 93 96 1.00 1.00 1.00 0.91 1.00 69 0.92 69 1.00 90 0.95 1.00 57 1.00 100 1.00 0.75 99 99 1.00 0.83 1.00 100 1.00 1.00 100 1.00 100 62 1.00 100 1.00 ITS Brachyelytrum erectum (Outgr.) Phaenosperma globosa Danthoniastrum compactum Stephanachne pappophorea Phaenosp. Sinochasea trigyna Phaenosp. Stephanachne nigrescens Anisopogon avenaceus Duthiea brachypodium Stipa zalesskii Ampelodesmos mauritanicus Achnatherum splendens Psammochloa villosa ESL ESL Oryzopsis asperifolia Trikeraia hookeri Orthoraphium roylei Ptilagrostis luquensis Ptilagrostis junatovii Ptilagrostis concinna Ptilagrostis dichotoma Ptilagrostis mongholica Ptilagrostis malyschevii 1.00 97 1.00 98 73 1.00 1.00 100 78 1.00 1.00 100 1.00 95 97 1.00 1.00 68 100 1.00 1.00 Pappostipa chrysophylla Ortachne breviseta Ortachne rariflora Ptilagrostis kingii Piptatheropsis exigua Piptatheropsis shoshoneana Piptatheropsis canadense Piptatheropsis micrantha ASL Piptatheropsis pungens Hesperostipa comata Piptochaetium avenaceum Piptochaetium brachyspermum Piptochaetium montevidense Piptochaetium panicoides Anatherostipa hans-meyeri Anatherostipa rosea Aciachne acicularis Aciachne flagellifera Anatherostipa obtusa Anatherostipa rigidiseta Ptilagrostis alpina Ptilagrostis porteri Patis racemosa Patis coreana Patis obtusa Achnatherum stillmanii Piptatherum coerulescens Piptatherum munroi Piptatherum laterale Piptatherum hilariae Piptatherum aequiglume Piptatherum ferganense Piptatherum holciforme Piptatherum kokanicum Piptatherum songaricum Piptatherum miliaceum Piptatherum thomasii Stipa parviflora Achnatherum confusum Piptatherum paradoxum Piptatherum virescens Achnatherum jacquemontii Austrostipa stipoides Austrostipa pubinodis Austrostipa rudis nervosa Austrostipa geoffrey Austrostipa pubescent Austrostipa rudis rudis Achnatherum chinense Timouria saposhnikovii Achnatherum caragana Ptilagrostis pelliotii Amelichloa clandestina Achnatherum multinode Jarava ichu Jarava castellanosii Jarava pseudoichu Patis Patis * 72 0.71 100 76 1.00 100 0.95 1.00 99 1.00 72 1.00 62 0.82 70 1.00 0.50 99 100 1.00 1.00 91 1.00 51 0.75 96 1.00 0.65 89 1.00 0.54 100 1.00 100 1.00 1.00 93 1.00 72 0.99 57 1.00 100 MAC 1.00 99 1.00 92 1.00 Pipt. Pipt. AC Brachyelytrum erectum Phaenosperma globosa Stephanachne nigrescens Sinochasea trigyna Stephanachne pappophorea Danthoniastrum compactum Anisopogon avenaceus Duthiea brachypodium Ampelodesmos mauritanicus Achnatherum splendens Psammochloa villosa Oryzopsis asperifolia Trikeraia hookeri Achnatherum stillmanii Piptatherum coerulescens Piptatherum laterale Piptatherum hilariae Piptatherum aequiglume Piptatherum munroi Piptatherum holciforme Piptatherum kokanicum Piptatherum ferganense Piptatherum songaricum 61 99 71 1.00 0.70 0.94 56 0.83 1.00 86 99 1.00 88 1.00 55 1.00 0.67 60 0.67 0.83 53 70 73 0.66 1.00 100 0.67 1.00 1.00 0.99 69 1.00 0.93 Stipa zalesskii 0.96 Ptilagrostis junatovii Ptilagrostis luquensis Ptilagrostis dichotoma Ptilagrostis mongholica Ptilagrostis malyschevii Ptilagrostis alpina Ptilagrostis porteri Orthoraphium roylei Patis obtusa Patis racemosa Piptochaetium avenaceum Piptochaetium brachyspermum Piptochaetium montevidense Piptochaetium panicoides Ortachne breviseta Ortachne rariflora Anatherostipa hans-meyeri Aciachne acicularis Aciachne flagellifera Anatherostipa rosea Anatherostipa obtusa Anatherostipa rigidiseta Ptilagrostis kingii Piptatheropsis exigua Piptatheropsis shoshoneana Piptatheropsis canadense Piptatheropsis micrantha Piptatheropsis pungens Hesperostipa comata Pappostipa chrysophylla Stipa parviflora Achnatherum jacquemontii Piptatherum paradoxum Piptatherum virescens Achnatherum chinense Achnatherum confusum Ptilagrostis pelliotii Achnatherum caragana Timouria saposhnikovii Austrostipa stipoides Austrostipa pubinodis Austrostipa rudis nervosa Austrostipa geoffrey Austrostipa pubescent Austrostipa rudis rudis Amelichloa clandestina Piptatherum miliaceum Piptatherum thomasii Achnatherum multinode Jarava ichu Jarava castellanosii Jarava pseudoichu 91 1.00 0.99 53 0.95 91 0.57 85 1.00 93 0.95 1.00 95 1.00 71 84 0.91 1.00 88 1.00 95 0.93 68 80 0.62 0.95 58 0.89 93 0.53 0.99 57 1.00 100 1.00 100 54 52 0.98 75 1.00 86 0.99 0.97 0.99 51 100 1.00 0.81 96 100 1.00 1.00 Fig. 1. Cladograms of the maximum likelihood trees from analysis of combined plastid data (left) and nuclear ITS data (right). Numbers above branches are bootstrap values; numbers below branches are posterior probability values; AC = Achnatheroid clade; ASL = American Stipeae lineage; ESL = Eurasian Stipeae lineage; MAC = Major American clade; Phaenosp. = Phaenospermateae; Pipt. = Piptatherum s.s.;  indicates that Oryzopsis asperifolia is a New World taxon included in the ESL. 250 K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 notable differences in the ITS tree include: (1) the placement of the Piptatherum as sister to Ampelodesmos, Achnatherum splendens, Psammochloa, Oryzopsis and Trikeraia of the ESL (BS = 60, PP = 0.83); (2) a Ptilagrostis that includes all species (BS = 91, PP = 1.00); (3) the expansion of ASL to include Ptilagrosits, Patis and Orthoraphium; (4) the placement of Hesperostipa as sister to the AC; and (5) the novel placement of Stipa zalesskii in a grade removed from the ESL. Similarities between the ITS and plastid trees include: retention of the AC with addition of Pappostipa chryosohylla (BS = 100, PP = 0.99), retention of Piptatherum (BS = 100, PP = 1.00), retention of Piptochaetium (BS = 91, PP = 1.00), retention of four species of Anatherostipa and two species of Aciachne in a clade (BS = 84, PP = 1.00), retention of Ortachne (BS = 95, PP = 1.00), and retention of Austrostipa (BS = 54, PP = 0.98). 3.3. Network analyses A split network of 78 species of Stipeae, Phaenospermateae, and Brachyelytrum using nrDNA ITS is given in Fig. 2. The network is semi tree-like easily separating the major lineages of Stipeae despite low support values of the deep relationships shown in the ITS phylogenetic tree (Fig. 1). The fit index for the ITS split network comparing the least squares fit between the pairwise distances in the graph and matrix is high (LSfit = 98.3), indicating only 1.7% of the data was discarded. A split network for 145 nDNA At103 sequences is given in Fig. 3. The network is less tree-like than for ITS and represents a radiative pattern. The LSfit = 95.2, only slightly lower than reported for the ITS split network. Again the major lineages (AC, ASL, ESL, and Piptatherum) of the Stipeae were well separated (see four nearly congruent clusters of the same color in Figs. 2 and 3). The analysis was successful in retrieving putative orthologues (P-sequences) for all species in question as seen by their phylogenetic position being similar to that in combined plastid tree (Fig. 1). We anchored such positions of At103 copies by letter ‘‘P’’ on the graph, which stands for ‘‘plastid’’. In addition, the non-P copies of At103 (i.e., not sharing the recent common ancestor with anchored sequences) were found for all species in question with the exception of the Ptilagrostis mongholica and Achnatherum splendens. These alternative copies were found for Oryzopsis (H sequence located in ESL) in ASL stemming from the same branch with two copies of Patis coreana; for Patis coreana (P1, P2; Patis) first – in ESL stemming from the same branch with Patis racemosa, P. obtusa, Psammochloa villosa (P), Oryzopsis (H), Achnatherum splendens (the only copy), and Trikeraia hookeri (P); and second – in two sets of copies within ASL, one aligned with Oryzopsis and another aligned with two sequences of Patis racemosa; and third – a single copy positioned at the base of AC; for Patis racemosa (P; Patis) – one copy was found in ESL and another in ASL closely aligned only with Patis coreana; for Patis obtusa (P; Patis) – several close copies were found but located only in ESL; for Stipa zalesskii (P; ESL) – a single copy was found in ASL; for Hesperostipa (P; ASL) – three copies were found at the base of AC; for Psammochloa (P; ESL) – a single copy was found in ASL; for Trikeraia (P; ESL) – one copy was found in ASL and another two copies aligned with Sinochasea and Stephanachne in Phaenospermatae; for Ampelodesmos (P; ESL) – three copies were found aligned with Phaenosperma; and for Ptilagrostis porteri (P; ASL) – a single copy was found at the base of AC aligned with ASL copies of Hesperostipa and Patis coreana. There is one Achnatherum stillmanii copy of At103 found at the base of Piptatherum clade replicating the plastid position (Fig. 1) while another At103 copy was found at the base of ESL between ESL and one of the two anchored sequences of Patis. Both anchored positions of Patis (P1 and P2) are equally plausible since they are nested between ASL and Piptatherum clade which corresponds to its position in plastid tree (Fig. 1). There are four areas of incompatible splits (network boxes) in Fig. 3: (1) between sequences of Patis (P) and ESL; (2) between Achnatherum stillmanii (P-copy) and Piptatherum; (3) between second copy of A. stillmanii, Stipa zalesskii and Ampelodesmos (H); and (4) between ESL copies of Ampelodesmos, Hesperostipa, Ptilagrostis porteri, Patis coreana, and Phaenospermateae including Brachyelytrum. All homologous copies of At103 for Ptilagrostis porteri and P. alpina are separated in a single group with P. mongholica. The common branch for these three species of Ptilagrostis produces no parallel splits with other representatives of the Patis. 3.4. Divergence time estimation The age estimates for the major nodes in the Stipeae (see Fig. 1, plastid tree), posterior density intervals, and main rates of substitution per site are given in the Table 2. We also include At103 marker age estimates (see Fig. 3) for comparison and for interpretation of conflicting positions of several taxa in plastid and ITS phylogenies. Based on the combined plastid data the age of the crown node for Stipeae is estimated at 28.09 mya (with 95% posterior density interval of 22.94–36.04 mya). The age of earliest diverging lineages within the Stipeae, the ESL and the ASL, are estimated to be 21.29 mya and 18.52 mya, respectively. The age for the crown node of AC is estimated to be 16.24 mya and the separation of the major clades containing Patis and Piptatherum s.l. phylogenetic groups with AC are estimated to be 26.2 mya and 21.4 mya, respectively. However, the ages for the crown nodes for the clades including extant taxa are estimated at 6.91 mya for the Patis group and 10.23 mya for Piptatherum s.s. The age for the Asian core of Piptatherum group (i.e., excluding basal Mediterranean P. coerulescens; Fig. 1) is estimated to be 6.21 mya (with 95% posterior density interval 2.69–10.99). Within Patis group, Patis obtusa is estimated to diverge first at 6.91 mya. Two independent groups are estimated to have arisen soon after separation of P. obtusa; first group includes Patis coreana and Patis racemosa (4.34 mya) and the second includes Ptilagrostis alpina and P. porteri (2.9 mya). Within ESL Stipa is the earliest diverging lineage at 21.2 mya. The age for the crown node of the clade including Oryzopsis, Ampelodesmos, Psammochloa, and Achnatherum splendens is estimated at 14.06 mya where Oryzopsis represents the single basal lineage within the group. The separation of the other three taxa is dated at 5.8 mya for Ampelodesmos and 0.98 mya for Psammochloa and Achnatherum splendens. The age for the clade including Orthoraphium (earliest split), Trikeraia, and Ptilagrostis group is estimated at 11.7 mya. The age for the crown node of Ptilagrostis group (excluding Pt. alpina and Pt. porteri) is estimated to be 7.35 mya. The stem age for Pappostipa and Hesperostipa within the ASL is date to about 12 mya and the age of Piptochaetium clade is estimated to be 9.8 mya. Despite different rates of evolution and number of variable sites between the combined plastid and nDNA At103 data sets (Table 2), the ages obtained in our relaxed clock analyses are generally in agreement. However, At103 region returned older ages for AC (23.02 versus 16.24 mya in the plastid data) and for Patis group (12.21 versus 6.91 mya). The ages for the ESL copies for Patis range between 11–15 mya, while the ASL copies of Patis racemosa and Patis coreana (Patis obtusa has no ASL copy) are estimated at much later age of 3–4 mya. The largest disparity between At103 and plastid data was found for Pappostipa. The P-copy of At103 yielded an origin of 2.13 mya whereas the combined plastid data estimated it to be 12.43 mya. The age of the non-P copy (AC) for Pappostipa is estimated at 4.84 mya. For Stipa and Hesperostipa the P copies of At103 region are nearly two to three times older than alternative sequences for ASL and AC, respectively. The opposite scenario is detected for Ampelodesmos where the age of the At103 P-copy is estimated at 3.61 mya (versus 5.82 mya for plastid data), and the 251 ium on te v hy iden sp se er m ae um tiu m av en ac eu ac pto ch He er m br Pt ila gr os tis ng m os tip a co ki ii e m lu oi ig nr e qu mu rm e a ifo m c c l u r ho um the e r um ia r he ta erum hilar he at ip at pt P tath rum le pt e p Pi tera Pi la Pi tath um Pip tather ip P ense fergan erum Piptath ia os a et Pi ns Ps am mo Ac chlo hn av a illo sa Or theru m Tr yzo sp ike len ps de ra is ns ia as ho pe ok rif er ol i m p po ina rte ri iu is et et ha ha ac em ha oc oc a Pa tis r oc pt pt pt sp ellifera Aciachne flag cularis ne aci ta Aciach obtusa idise a ig stip ar thero rostip nsis e Ana ade tha ath can ran ens gua An psis mic ng xi ero sis pu sis e na a tath rop sis Pip tathe erop erop one Pip tath tath osh i p sh Pip P is ps ro he at pt es Pi id co ni pa m iu Pi Pi Pi Anatherostipa rosea Anatherosti Ortachne rariflora pa hans-mayeri Ortachne breviseta Orthorap hium ro Ptila ylei Ptilaggrostis juna tovii ro Pti s P lagros tis dich Ptitilagros tis luqueontoma Pa sis tis Ptilalagro tis mo ng ob g s tus ros tis a olica t l K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 os s cu ni ita r au m sm e od l pe ce es ul r oe Piptatherum kokanikum Piptatherum songoricum Am m at a 0.01 Achnatherum stillmanii Brachyelytrum erectum (OUTgr.) Anisopogon avenaceum Pa pp Duthiea brachypodium Danthoni os astrum tip ac hry so ph Step Achn atheru St ipa eno sperm a glo bosa a va ich u ii tin is rudis rudis a pubinod ultin nd cla mm tipa Austrostip es ns tros yi oid Aus fre eru “S” of tip sce nath ge as rvosa Ach a loa ip ch “M” st tip ube a ridus ne ap eli ro os stip str stro Au st Au ode es ra Pha sk Au Ja za les Austrostip Am um Stephana Sinoch chne pa asea ppophore hana trigyn a chne a nigre scen s ylla Stip a pa rviflo m jac qu ra Piptathe rum vires emontii cens Piptatherum paradoxum magrostis Achnatherum cala tii tis pellio nikovii Ptilagros a posh gan uria sa cara Timo rum e th na Ach s oide um brom fus erum con nath m u Ach r the a hn Ac m eu iac mil ii m as u r hu om he t h t a oic t m ud Pip u er se h p t sii ta va no ra Pip lla Ja te s ca va ra Ja compact Fig. 2. Split network of nuclear ITS data. AC = Achnatheroid clade; ASL = American Stipeae lineage; ESL = Eurasian Stipeae lineage; red-dashed line is the separation between the ‘‘M’’ = maize-like lemma epidermal pattern and ‘‘S’’ = saw-like lemma epidermal pattern; scale bar = 1% sequence divergence. Color corresponds to common clades recovered in At103 and ITS networks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) non-P copy is dated at much older age of 16.73 mya, located among the Phaenospermateae (Fig. 3). 4. Discussion 4.1. Phylogenetic relationships Our phylogenetic analyses reveal conflicting positions for several taxa between the combined plastid and ITS trees (Fig. 1). In this paper, we focus on the peculiar placements of Ptilagrostis and Patis as these are the only two genera of Stipeae (as we understand them at present, although the taxonomy has not entirely caught up) shared between the New and Old World. These two disjunctions are all the more interesting because Ptilagrostis (as we circumscribe it, i.e., excluding Pt. kingii and Pt. pelliotii; see Romaschenko et al., 2012; Hamasha et al., 2012), although coherent in morphology and nDNA types, has two very different cpDNA types, one shared with the ESL clade, and one shared with Patis. We show that the core species of Ptilagrostis (Pt. concinna, Pt. dichotoma, Pt. junatovi, Pt. luquensis, Pt. malyschevii, and Pt. mongholica) resolve in the plastid tree within the ESL clade, while Pt. alpina and Pt. porteri are nested within the isolated Patis clade. This contrasts with the analyses of nuclear data where Ptilagrostis is well resolved as monophyletic, and the species are all contained within the ASL cluster within the At103-derived network. The Patis-Ptilagrostis grouping dissolved in the ITS tree, and Patis contains various At103 copies aligning in the independent ASL and ESL clusters and in between them. A pattern of strict intercontinental separation between lineages is seen across our plastid tree with only three individual exceptions: Oryzopsis asperifolia from North America (NA) placed in ESL; Patis racemosa and Pt. porteri known only from NA placed in the Patis group with the other three species from Asia, and Achnatherum stillmanii (Romaschenko et al., 2012 indicate this species is unrelated to the Asian genus in which it still resides) from NA is sister to the Asian genus Piptatherum. We interpret the placement of Pt. alpina, known only from Eastern Asia, and Pt. porteri, from the Rocky Mts., within the Patis group as an artifact of past hybridization events. The former two species strongly resemble Pt. mongholica from Asia and are small, alpine-like, tussock forming species with intravaginal shoots which bare no resemblance to the stout, broad-leaved species of Patis with extravagial shoots and cataphyllous basal leaves. The placement of Pt. porteri is consistent with previous molecular analyses (Barkworth et al., 2008), and a similar result has been confirmed for six other samples of this species as well as for three Siberian samples of Pt. alpina (Romaschenko et al., unpublished). Our ITS phylogeny disrupts the strict (allopatric) geographic pattern yielded by plastid analysis by mixing taxa primarily from the Old and New World S lineages (Figs. 2 and 3). This suggests there was incomplete geographic isolation of the lineages and some gene flow did occur among Old and New World lineages after their primary segregation. 252 K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 An loa ath 4 m es lod m os pe s en Am l sp m ru he a 5 t a s 6 hn tu sa 7 Ac s ob btu sa 8 6 ri 1 ti o btu a a us ke Pa atis is o btus hoo eri 2 bos anic glo P at is o ia urit s5 ok o P at a rma ma icu P riker aia h an spe os o it m r n u e T iker des ma Pha Tr pelo os Am sm de elo p Am um e nac ave gon opo dium Anis o p chy bra iea Duth eri 3 ook h eri 4 eraia Trik ia hook era Trik a trigyna 1 se a Sinoch na 2 Sinochasea trigy Stephanachne nigrescens Stepha nachne pappop horea Brachyelytrum erectum (OUTgr.) Danthoniastrum compactum tii Achnatherum Amelichloa multinodis clandestin J a J arava Au Pa Jaraarava castellan str osii Pa pp va ichu A o Au ust stipa os pse p p s ro g os tipa udo Autros stip eoff ich tip u st tip a s rey a chry ro a tip i ch s st pu oi ry op ip b de h so a es s ph ylla pu ce 2 yl bi ns la no 3 di s Ptilag rostis pellio comata 3 Hesperostipa ata 4 com tipa Hesperos ata 5 PSEUDO Hesperostipa com Ptilagrostis por teri 3 PSEUDO Patis corea na 12 PSEU DO dis ru a dis os ru rv a ne lora O tip is UD if os rud arv PSE str pa a p 6 Auosti Stip okeri m ho str xu ia do Au era ara Trik um p s ovii r cen posnik he ires ria sa tat mv Timou Pip theru ta Pip Achnatherum stillmanii 1 hilariae Piptatherum Piptatherum coerulescens Pa Pa tis r Pa tis ace c Pa tis c ore mos tis or an a Pi Pipt Pa corean a 7 7 p a P tis ea a Pip ipta tath the c r e tat Pati oreana 89 t he heru rum um s c na rum m ore 10 f so ana Pipta a ko erg ng 11 theru equigkan ane aric iku n u lu m m m se m h Piptath erum olciforme munro e Piptatherum late i rale P An A An ath na th e a ero ther rost ero s tipa ostip ipa sti villo sa 3 han a r ob pa o PSE s Acia me seatusarigid U Hes chn yeri ise pero Stip DO e fl s a ta Hesp tipa com zaless Aciaagellif erost a k ii 3 chn era ipa co ta 1 e acic Ptilagros mata 2 ular tis mon is go Ptilagrostis mon lica 1 golica 2 Ptilagrostis mongolic a3 Ptilagrostis porteri 1 Ptilagrostis kingii teri 2 se Ptilagrostis por alpina viden s stis monte icoide Ptilagro aetiumtium panpermum ch to e s ip P cha chy na Pipto um bra DO ea ti on SEU chae sh ns Pipto ceum P a 6 a5 ho nge an li a n o e s e r if v . u er co ma ,P .p 6 asp atis etiu ua P sa P sis cha 5 xig is, mo zop Pipto eri e ns e Ory ok psis ade rac o a h ero can atis 6 h rai P na ike ptat , P. rea Tr Pi tha co is t an Pa icr m is ps ro he at t p Pi och us nic rita au d en Achn Piptatherum miliaceum atheru m ca ragan Piptatherum thomasii jacquemontii a Achnatherum confusum Achnatherum chinensis mm P ana 1 Patis core tusa 4 Patis obobtusa 3 a 1 Patis racemos 2 Patis a s 1 obtu tusa Patis i s ob oyle Pati mr ii phiu lla 1 ov a ora phy viset nat Orth o z hry bre a ju a c ne lor P. stip rtach rarif is, ppo O e ns Pa hn ue a t a c u q om O r t i s l hot o s ic gr s d ila ti P t ros g ila Pt Psa P Achnatherum P Stipa zalesskii 2 1 Stipa zalesskii 3 Achnatherum stillmaniianii 2 rum stillm Achnathe P Ampelodesmos mauritanicus 1 Ampelodesmos mauritanicus 2 Ampelodesmos mau ritanicus 3 Patis coreana 2 Patis corean a3 Patis ra c Patis emos Patis co a reana 4 Pati racemo 2 s s Patiracem a 3 s ra osa 4 Or cem yz op osa sis 5 as p Ps erifo Or Ory yz am lia z O ryz opsis opsis Ps m 5 op a am och sis aspe sper l o m a Or asp rifoli ifolia oc v 1 yz erif a 2 hl illo op oa sa o sis lia vil 1 as 3 lo pe sa rifo 2 lia 4 P 0.01 Fig. 3. Split network of nuclear At103 data. Colored ‘‘P’’s indicate At103 alignment with the cpDNA type sequence for each group, where there is more than one At103 sequence per sample, the taxon name is followed by a number found in Appendix 1; numbers greater than 1 are cloned copies. AC = Achnatheroid clade; ASL = American Stipeae lineage; ESL = Eurasian Stipeae lineage; P = cpDNA type; scale bar = 1% sequence divergence. Color corresponds to common clades recovered in At103 and ITS networks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 4.2. Evolution and introgression The At103-derived network reveals a complex evolutionary history leading to the formation of the Eurasian and American lineages of the Stipeae, and it has elucidated the importance of Patis in these events. Patis includes two East Asian species, P. coreana and P. obtusa, and one North American species, P. racemosa (Romaschenko et al., 2011). Three main nDNA types of At103 (Fig. 3) were discovered and these are found in ASL, Patis, and ESL. Known chromosome counts for Patis are 2n = 46, 48 (P. racemosa; Johnson, 1945; Barkworth, 2007), and 2n = 46 (P. coreana; Tateoka, 1986). Considering Patis obtusa has two distinct At103 types found in Patis and ESL, we hypothesize it to be polyploid. Assuming 2n = 20–24 is a common diploid number in the Stipeae (Romaschenko et al., 2012), ancestral Patis might have formed via hybridization among diploids followed by allopolyploidization in order to account for three main nDNA types. However, the possibility that this pattern represents retention of ancestral polymorphisms (orthologs or paralogs in this case) cannot be ruled out. Evidence against ancestral polymorphisms includes: the very different ages and rates inferred for each lineage (Table 2), the substantial genetic distance between them (gauged from the splits and branch length on the network (Bryant and Moulton, 2004; Huson and Bryant, 2006), and the high level of diversification among the ESL and ASL nDNA type copies (Fig. 3). The ages of the nodes inferred for Patis from plastid and nDNA analyses are different. For Patis, the plastid analysis suggests an age of 6.91 mya (see Table 2) and the At103 analysis suggests 12.21 mya. In the latter analysis, the mean rate of nucleotide substitution per site found for P. racemosa (Patis nDNA type) is among the lowest for all the Stipeae sampled (0.0009). The plastid phylogeny strongly suggests primary separation of P. obtusa followed by a split between P. racemosa and P. coreana (Fig. 1). Based on the At103 data the age for the split giving rise to P. obtusa and P. coreana is estimated at 6.71 mya. If the rate of nucleotide substitution for P. racemosa is somehow underestimated (thus overestimating the date of divergence) in the At103 analysis and the primary split of P. obtusa is accurate, then the age of 6.71 mya is congruent with the date yielded by the plastid analysis. The ESL At103 copies stem dates for Patis range between 13.48 and 14.75 mya and thus are twice as old as the crown node dates (4.89–11.5 mya, average = 8.61 mya). The ASL copies of Patis are dated at 3.25 and 3.92 (with the exception of P. obtusa which has no ASL copies of K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 At103) and are twice as young as the P-nDNA type sequences. The most parsimonious solution is that the separate ESL and ASL nDNA types for Patis were introduced by different progenitors. Oryzopsis or proto-Oryzopsis (assuming a former diploid status), could be a plausible candidate for the ESL nDNA type progenitor. Evidence favoring these interpretations are: ESL copies of Oryzopsis are putative nDNA types and their position corresponds to the phylogenetic position of Oryzopsis according to the plastid analysis (Figs. 1 and 3); they share the same branch with the Patis ESL copies in the At103 network (Fig. 3); and the age inferred for the Oryzopsis plastid group of 14.06 mya (Table 2) is within the range of dates found for the Patis ESL copies (13.48 and 14.75 mya). The origin of Patis ASL nDNA type is rather ambiguous. Possession of the Patis cpDNA type by two species of Ptilagrostis, Pt. alpina, and Pt. porteri, suggests there was a genetic interchange between Patis and Ptilagrostis. Indeed, in At103 analysis, the crown node age for these two species of Ptilagrostis is estimated at 3.76 mya which falls within the range of dates estimated for ASL sequences of Patis racemosa and P. coreana (3.25–3.92 mya). This correlation renders Pt. alpina and Pt. porteri as the most logical progenitors of the ASL nDNA type in Patis. In our ITS tree all species of the Ptilagrostis (excluding Pt. kingii and Pt. pelliotii) constitute a single, strongly supported clade which is sister to the core ASL (Fig. 1) plus Patis and Orthoraphium. In the plastid analysis Ptilagrostis (as narrowly defined here) is a morphologically homogeneous genus comprised of two sets of taxa with distantly related cpDNA types since Pt. alpina and Pt. porteri share the Patis cpDNA type (Fig. 1) while the remaining species share the Trikeraia-like cpDNA type from the ESL. No alternative copy of At103 has yet been recovered from the genomes of Ptilagrostis (2n = 22, Tzvelev, 1976; Probatova and Sokolovskaya, 1980; Freitag, 1985; Tateoka, 1986) which could be derived from any of the two progenitors (Trikeraia or Patis) indicated by the plastid analysis. Despite a nearly complete set of Ptilagrostis species in the analysis, no corresponding ASL cpDNA types were found in the genus. Statistical analysis indicated hard incongruence between plastid and ITS trees in relation to placement of Ptilagrostis; rejecting the possibility of Ptilagrostis placement in ASL of the plastid tree or in ESL of the ITS tree (Romaschenko et al., 2012). In the current analysis, both alternative hypotheses are equally supported by Bayesian analysis (Fig. 1). This evidence supports the exchange of nuclear genomes between Ptilagrostis or protoPtilagrostis (assuming former possession of correspondent cpDNA type) and Patis, as well as between Ptilagrostis and Trikeraia (or its close relative/ancestor) mediated by intergeneric hybridization and extensive backcrossing. Assuming plastids are maternally inherited as is typical of grasses (Palmer, 1987), this exchange resulted in the complete (or nearly so) substitution of the maternal nuclear genome in the contact zone populations of Patis and Trikeraia by the paternal nuclear genome of Ptilagrostis, but retention of the foreign cpDNA types. This hybridization event favored the spreading of adaptive traits or, alternatively, the hybridization event could have been followed by a genetic bottleneck. Considering the tumultuous climatic changes and ecosystem collisions during late Miocene–Pleistocene epoch in Eastern Asia (Guo et al., 2001; Pound et al., 2011), both scenarios seem plausible. 4.3. Phytogeography of Patis and Ptilagrostis Patis is a mesophytic, temperate forest and forest glade genus of low elevations (up to 1900 m). Patis obtusa is widespread, occurring in Taiwan and Ryukyu Islands, and across the central eastern part of China including the North China Plain where it partially overlaps with P. coreana. Patis coreana is distributed mainly further north along the margins of the North China Plain, ranging from eastern China to Korea and Japan (Figs. 1 and 4). It is sister to the 253 eastern North American P. racemosa. The distribution of the Asian species of Patis is strictly confined within the borders of the Eastern Asiatic Floristic Region (Takhtajan, 1986) or Sino-Japanese Floristic Region (sensu Wu and Wu, 1996; Guan et al., 2011). As outlined by Takhtajan, the Eastern Asiatic Region is bounded by the eastern limits of the Tibetan Plateau, the steppes of northern half of the Loess Plateau, and the steppes and deserts of Inner Mongolia, all of which belong to the Irano-Turanian Region (Fig. 4). The Irano-Turanian Region is the major center of diversity of Stipeae of Asia. It stretches westward to the Mediterranean Region, and northward to the Circumboreal Region, and southward to the Sudano-Zambezian and Indian and regions, and includes all the steppes and mountains of Central Asia (Takhtajan, 1986). Eastern part of the Irano-Turanian Region is the area with the highest taxonomic diversity of genera in the Stipeae and the Phaenospermateae (Romaschenko et al., 2010, 2011, 2012), and Ptilagrostis occurs in the eastern part only. Of the Stipeae genera in the present study, Psammochloa is endemic to the region (Takhtajan, 1986; Wu and Wu, 1996). Orthoraphium and all three Trikeraia species are centered within the adjacent high lands of Eastern Asiatic Region in the Sikang-Yünnan Floristic Province, and the adjacent peninsular Eastern Himalayan Floristic Province (Takhtajan, 1986). Of the nine species, we accept in Ptilagrostis, three are endemic to the Qinghai-Tibet Plateau and adjacent floristic provinces and two are restricted to the Central Asian Mountains; one overlaps these but is restricted to these areas. Ptilagrostis mongholica is the most wide ranging species of the genus. Its distribution extends across the eastern Irano-Turanian Region, into the mountains of the East Asian Region, in the Manschuria and North China floristic provinces. In the latter provinces it apparently partially overlaps with Pt. alpina. Although overlooked by Wu and Phillips (2006), the range of Pt. alpina stretches further north in the mountains in the far east of Siberia to the Okhotsk Region (to ca. 55°N) and reaches south to Liaoning in Province in northeast China (Tzvelev, 1976, 1977; Probatova, 1985; reiterated by Peterson et al., 2005; based on Wang Sui-Kang 442, LE; our DNA voucher). In North America, the sister of Pt. alpina, Pt. porteri (Figs. 1 and 4) is known only from a few alpine areas of the southern end of the Rocky Mountain Province, in central Colorado (Barkworth, 1983, 2007; Weber, 1966) and one population in northern New Mexico (Legler, 2010). 4.4. Origin of Ptilagrostis Based on the contemporary distribution pattern of the putative contributors we present three hypotheses for the origin of Ptilagrostis. Two of these assume an American origin of Ptilagrostis supported by the similarity of nrDNA (ITS) and nDNA (At103) sequences with the ASL, and relatively young age (late Miocene–Pliocene) of the taxa. The estimated ages make improbable the early, primary separation of an ancestral Ptilagrostis nDNA type and retention of it in the Old World while the remaining lineages migrated to the New World. According to the fossil record and our age estimates, this primary separation occurred much earlier (early Miocene). In our reconstruction of the biogeographical history of Patis and Ptilagrostis (Fig. 5), we use the consensus theory. We account for: two events of genome introgression (one between Ptilagrostis and Patis, and one between Ptilagrostis and an Irano-Turanian Trikeraia-like taxon); and for evidence favoring the hypothetical American origin of the Ptilagrostis nDNA type. We also note that fewer migratory events are necessary if the second nuclear-plastid genome rearrangement of Tibetan proto-Orthoraphium occurred in concert with the interaction between Ptilagrostis and a Trikeraia-like taxon. 1. American origin with two stepwise plastid capturing events. This hypothesis includes spread of proto-Ptilagrostis through the Bering land bridge to Eastern Siberia in late 254 K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 50º 60º 1 40º 8 Irano-Turanian floristic region 50º 9 10 11 7 6 4 2 3 40º Eastern Asiatic floristic region 30º 5 30º 20º 20º 50º 70º 110º 90º 130º 100º 120º 80º Border between Irano-Turanian and Eastern Asiatic floristic regions Patis obtusa Ptilagrostis alpina 1 Achnatherum splendens 5 Duthiea brachypodium 8 Ptilagrostis junatovii Patis coreana Ptilagrostis mongolica 2 Psammochloa villosa 6 Stephanachne nigrescens 9 Ptilagrostis dichotoma Patis recemosa Ptilagrostis porteri 3 Sinochasea trigyna 7 Trikeraia hookeri 10 Ptilagrostis concinna 4 Stephanachne pappophorea 11 Ptilagrostis luquensis Fig. 4. Distriburion of Patis obtusa (tan), Patis coreana (green), Patis racemosa (olive green), Ptilagrostis (Pt.) alpina (blue), Pt. mongholica (light blue), Pt. porteri (dark blue), Achnatherum splendens (1), Psammochloa villosa (2), Sinochasea trigyna (3), Stephanachne pappophorea (4), Duthiea brachypodium (5), Stephanachne nigresans (6), Trikeraia hookeri (7), Pt. junatovii (8), Pt. dichotoma (9), Pt. concinna (10), and P. luquensis (11). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Miocene (6.37 mya as indicated by the crown node age for Ptilagrostis based on nDNA; see Table 2); followed by hybridization with and capturing a plastid from an IranoTuranian Trikeraia-like taxon; mediated by and subsequent backcrossing, south-east spreading of Ptilagrostis along the mountain ranges of Central Asia, and the rising Tibetan Plateau (Ni et al., 2010; Pound et al., 2011); with new speciation events and wide expansion of Pt. mongholica and its penetration into East Asian Region; a second round of introgression and plastid capturing from para-Patis in mid-Pliocene (3.76 mya) giving rise to the ancestor of Pt. alpina and Pt. porteri; and dispersal of the Pt. porteri lineage back to North America in the Pleistocene (theory portrayed in Fig. 5). 2. American origin with independent plastid capturing events. This hypothesis supposes a recent separation of Pt. mongholica in North America and spread of some of its populations into northern East Asia. Ptilagrostis mongholica then undergoes a plastid capturing event in the contact zone with Patis (giving rise to the ancestor of Pt. alpina and Pt. porteri) while the Irano-Turanian populations of Pt. mongholica hybridize farther west with Trikeraia-like taxa giving rise to the remaining taxa of Ptilagrostis. 3. East Asian origin with two stepwise plastid capturing events. This scenario supposes an Asian origin of Ptilagrostis where the genus is most diverse today in mountainous areas of Central Asia including the Tibetan Plateau (Fig. 4), which until the late-Quaternary did not undergo extensive glaciations (Guan et al., 2011). The putative progenitor of the Pti- lagrostis cpDNA type, the mesophytic genus Trikeraia, is abundant in the region at lower elevations and could have come in contact with alpine Ptilagrostis in the process of a shifting climate. This hypothesis suggests a northeast expansion of Pt. mongholica and a second round of introgression and plastid capture from Patis in Eastern Asia. We reject the third hypothesis as it does not account for the close affinities with the American taxa as revealed by nDNA. Moreover, the discovery of the putative ASL copies of approximately similar age (4 mya) in several Asiatic taxa (Trikeraia, Psammochloa, Patis; Fig. 3; Table 2) indicates the existence of genetic interchange between Asian species and one or more American species shortly before the opening of the Bering Strait (Marincovich and Gladenkov, 1999, 2001). At this time plant migrations were thought to be limited to cold tolerant species (Martin-Bravo et al., 2009) like Ptilagrostis (Roshevitz, 1934; Tzvelev, 1976, 1977; Barkworth, 1983; Freitag, 1985; Wu and Phillips, 2006; Wen et al., 2010). Using crown ages determined via a molecular clock model, Wen et al. (2010) demonstrated that of the 100 Eastern North American/ East Asian vascular plant disjunctions studied by her group, 56% involved migrations through Beringia rather than through the North Atlantic (21%). Although 62% of such disjunctions involved migration from the Old World, 30% originated from North America (contrary to the widely held belief that the latter pattern was rare; Donoghue and Smith, 2004). Of the mild to cool temperate plant lineages studied, most dispersal occurred between 20 and 3 mya, and were concentrated in the mid to late Miocene. If western North American/East Asian temperate groups were examined this way, K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 255 Fig. 5. Speciation, introgression, and migration hypothesis for Patis and Ptilagrostis. Mya = million years ago. we assume the rate of floristic interchange across Beringia would be much higher, and many disjunctions would be more recent. 4.5. Evolution of the nDNA type of Patis, Ptilagrostis, and relatives The node age of Stipeae is late Oligocene (28 mya). The date is in accordance with the previous evolutionary/biogeographical studies of the grasses where representatives of Stipeae were included (Bouchenak-Khelladi et al., 2010; Vicentini et al., 2008). We hypothesize the development in Asia in the time span from 23 to 15 mya of three ancient Stipeae diploid nDNA types: Meso Irano-Turanian, the Xero Irano-Turanian, and the East Asian (Fig. 5). The primary split between these developing genomes occurred with the gradual uplift of Tibetan Plateau and aridification of internal regions of the Eastern Asia beginning in middle–late Oligocene (Rea et al., 1998; Bosboom et al., 2011) and continuing in the Quaternary (Yao et al., 2010; Guan et al., 2011; Utescher et al., 2011). The East Asian nDNA type probably developed to occupy mesic sites with seasonably equitable or monsoonal precipitation near the coast. The Irano-Turanian nDNA type evolved to tolerate more variable continental, cooler and more arid conditions, eventually quite xeric habitats on one line (xero Irano-Turanian), and to moister habitats in emerging upland elevations on another line. The following extant lineages possess the meso Itano-Turanian nDNA type: proto-Oryzopsis (prior to 14 mya) and proto-Trikeraia (prior to 8, but likely between 11 and 14 mya). Based on nDNA At103 the ages of ESL- and ASL-derived separation of Oryzopsis are similar (see Table 2, 1476 and 1373 mya, respectively), implying migration to North America and hybridization with an unknown American progenitor (resulting in chromosomal duplication) happening immediately after the separation of proto-Oryzopsis and gain of an ASL nDNA type copy. Of the East Asian nDNA type only the extant species of Patis remain (Fig. 5). We hypothesize the introduction of a Meso Irano-Turanian nDNA type into para-Patis I via hybridization with proto-Oryzopsis, probably occurring about 14 mya, and ultimately giving rise to the tetraploid genome of Patis. In this scheme the origin and history of Orthoraphium are ambiguous, and require further study. In the plastid tree it is sister to Ptilagrostis, and it has an ASL At103 nDNA type (but we have not 256 K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 yet cloned for more copies), and its chromosome number is unknown. It could be Asian (Meso Irano-Turanian) or North American in origin, if the latter it could have evolved in concert with protoPtilagrostis. Independent ASL nDNA types evolved in North America sometime around the Miocene Climatic Optimum (based on fossils, and node date of divergence of 18.5 mya), giving rise to, among other new World lineages, proto-Ptilagrostis (11.7 mya) [Ptilagrostis node – 7.3 mya], proto-Hesperostipa (12.6 mya), and Piptochaetium (9.3 mya). Introduction of the proto-Ptilagrostis (an extinct ancestral taxon with the North American nDNA type and phylogenetically congruent cpDNA type) nDNA type into the three old lineages of Asian Stipeae (proto-Oryzopsis, proto-Trikeraia, and para-Patis I) occurred between 8 to 3 mya. This interchange did not result in an increase in the chromosome number of proto-Ptilagrostis.. Since no traces of the proto-Ptilagrostis cpDNA type have been found among contemporary species of Ptilagrostis or related genera, we assume the first contact of the proto-Ptilagrostis and Asian taxa happened sometime during the time the land mass of Beringia was connected to Eurasia (Wen et al., 2010). The nature of the contact zone presumably prevented close coexistence of hybridizing counterparts providing an ecological barrier much like the ecotone between mesic steppe and temperate deciduous forest vegetation (Wen et al., 2010), allowing pollen dispersal, but favoring backcrossing within the separate populations. The North American nDNA type copies found in extant Trikeraia and para-Patis II (P. coreana and P. racemosa) are of similar age (4–3 mya) which indicates the time of contact of these taxa with proto-Ptilagrostis, following the plastid capturing events. The first Ptilagrostis phylogenetic group including Pt. mongholica, Pt. dichotoma, Pt. junatovii, Pt. luquensis, Pt. malyshevii, and P. concinna received the proto-Trikeraia cpDNA type, while the second group of Pt. alpina and Pt. porteri received the para-Patis cpDNA type. The molecular clock indicates even earlier introgression of North American nDNA type into the genome of Orthoraphium (ca. 8 mya). The southern most species of Patis, P. obtusa, probably separated earlier than this since its genome does not contain the North American nDNA type. However, since two similar copies of At103 (Patis clones 1 and 6) are positioned at the base of ASL (Fig. 3) the possibility of introduction of the North American nDNA type into the yet undifferentiated genome of proto-Patis cannot be completely ruled out. Another ambiguity is the recovery of a much older ASL copy of Patis coreana (clone 5), similar in age and phylogenetic position to the ASL copy found in Oryzopsis (clone 6) (Table 2; Fig. 3). If this copy was received by proto-Patis at the same time as in Oryzopsis or was introduced by Oryzopsis along with ESL meso-Irano-Turanian nDNA type, it could have been independently altered or eliminated from the genomes of Patis obtusum or P. racemosa. Otherwise, we should assume that occasional genomic interchange occurred between Patis coreana and an unknown taxon, possibly older than proto-Ptilagrostis somewhere in Eastern Asia. The taxa with the Xero Irano-Turanian nDNA type (Ampelodesmos, Psammochloa, and Achnatherum splendens) are a strongly supported clade in ESL (Figs. 1 and 5). This nDNA type probably did not play an active part in genetic exchange between the meso and East Asian nDNA types. The altered At103 nDNA types found in Psammochloa similar to the North American nDNA type without subsequent backcrossing supports this hypothesis (Fig. 3). Additional studies are required to test for the presence of this allele among distant populations of Psammochloa. The stem age of Ampelodesmos is estimated to vary between 5.82 mya (plastid markers) and 3.61 mya (nDNA; Table 2). The divergence of Psammochloa and its continued isolation may be a consequence of the formation of the Arabian Land Bridge in the early Miocene (Jolivet and Faccenna, 2000; Kappelman et al., 2003), connecting North Africa and Central Asia. In Eurasia Ampelodesmos apparently is a result of intertribal hybridization between a member of the Stipeae and member of the Phaenospermateae (Fig. 3) resulting in a polyploid with multi-flowered spikelets aligning within the Stipeae (Soreng and Davis, 2000). 4.6. Origin and diversification of Ptilagrostis alpina and Pt. porteri Inferences based on hybrid biome reconstruction (Pound et al., 2011) suggest a contact hybridization zone existed ca. 7 mya among the cryophytic (cool-tolerant) plants (proto-Ptilagrostis in our analysis) and the mesothermal mesophytes (represented here by extant species of Trikeraia and Patis). Pound et al. (2011) suggested that temperate forests were present above 60°N and these were much wetter in the late Miocene than present conditions in North China (Liu et al., 2011). This is wholly consistent with major exchange of East Asian and Eastern North American Temperate Forest angiosperm elements across Beringia over this time period as detected by Wen et al. (2010), which was truncated by the Pleistocene (except for a few herbaceous elements). The warm-temperate forests covered much of the Eastern Asia and temperate savannah extended to the Tibetan Plateau. Areas that exhibit desert conditions today were probably grasslands and woodlands in the late Miocene (Pound et al., 2011). This might account for the presence of the mesophytic taxa ranging north into the primary contact zone with Ptilagrostis in Eastern Siberia, and the very late diversification of xero Irano-Turanian taxa (Psammochloa, Achnatherum splendens, and proto-Ampelodesmos; Fig. 4). The upper boundary of extensive PliocenePleistocene desertification of Asia coincides with distribution of the Xero Irano-Turanian taxa (Guo et al., 2001, 2002; An et al., 1999; Pope et al., 2011; Qin et al., 2011; Guan et al., 2011). In particular, the date of separation of Psammochloa (0.98 mya) corroborates the paleoclimatic data suggested in the formation of northwest arid area of China at approximately 0.85 mya (Guan et al., 2011). Aridification of the Loess Plateau and adjacent lowlands (An et al., 1999; Yao et al., 2010) was partially caused by accentuated uplift of Himalaya-Tibetan Plateau but generally was consistent with the global Miocene– Pleistocene trend of temperature (Liu et al., 2011). This perhaps pushed forest vegetation and associated mesophytic taxa like Patis further south into the East-Asian refugia (Heaney, 1991; Metcalfe et al., 2001; Bird et al., 2005; Corlett, 2009; Woodruff, 2010; López-Pujol and Ren, 2010; López-Pujol et al., 2011; Wen et al., 2010). At the same time Ptilagrostis-like plants occupied high elevation vegetation belts in the mountains of northern China and southeastern Siberia. This effectively separated the hybrid Ptilagrostis  Patis from the influx of the maternal nuclear genome and extensive backcrossing with Pt. mongholica, the putative paternal progenitor. It is possible that Ptilagrostis mongholica and Pt. alpina, which share the same nDNA type but different cpDNA types, have inhabited the same area continuously since the Pliocene, and separation and migration of Pt. porteri (counterpart of Pt. alpina) to North America (Fig. 5) under the cool conditions of the Berin land bridge (Colinvaux and West, 1984; Elias et al., 1996, 1997; Edwards et al., 2000; DeChaine, 2008; Martin-Bravo et al., 2009; Qiu et al., 2011) happened by chance through a genetic bottleneck. At present, Pt. porteri occupies somewhat more moist habitats than Pt. alpina (Tzvelev, 1976, 1977; Barkworth, 1983, 2007) which is probably an adaptation to Pleistocene cold and moist peri- and postglacial environments in North America (Andersen and Borns, 1994; Clark and Mix, 2002; Abbot and Brochmann, 2003; Brochmann et al., 2004; Gonzales et al., 2008). 5. Conclusion The Stipeae tribe has a complicated evolutionary history, and we used several lines of evidence to present a cohesive theory of K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 the evolutionary events which led to formation of many contemporary lineages in Stipeae. The recovered pattern of speciation of Patis and Ptilagrostis demonstrates how deep reticulation and introgressive hybridization can be examined in order to elucidate past distribution and intercontinental genomic exchange in grasses. In our assessment of the histories of these two genera, we incorporate fossil evidence, phylogenetically-supported morphological evidence, plastid and nrDNA ITS phylogenies, a network based on the single copy nuclear gene At103 (sequenced directly and cloned from species of interest to detect multiple gene copies), molecular dating, and climatological and floristic information. The genus Patis, only recently resurrected and first detected by molecular studies (Romaschenko et al., 2011, 2012), was an important early lineage in the history of the modern Stipeae. Ptilagostis is hypothesized to be of ancient hybrid origin, with its nuclear genome providing evidence of an early precursor (proto-Ptilagrostis) probably originating in North America. No cpDNA type has been found corresponding to contemporary Ptilagrostis. Instead, two different cpDNA types originating in Asia are hypothesized, one derived from a meso Irano-Turanian precursor (proto-Trikeraia) and the other derived from an east-Asian precursor (proto-Patis). In its extant state Patis is a polyploidy genus which includes three species, two in Southeast Asia (Patis coreana and P. obtusa), and one (P. racemosa) more recently entering North America; the latter two species bearing genes (markers) from proto-Ptilagrostis and proto-Trikeraia lineages. Miocene floristic interchanges resulted in American and Eurasian Stipeae plastid lingeages but traces of each other remain or were subsequently introgressed into their respective nuclear genomes. One possible contributor to the modern genome of Oryzopsis asperifolia, which is hypothesized to have originated in Asia and then migrated to North America, is the extinct fossil genus, Berriochloa (Thomasson, 1978). The Bering land bridge between two and three mya was the area or contact zone where the putative floristic interchanges, hybridizations, and speciation occurred. Acknowledgments This project was funded by the Restricted Endowment Fund, the Scholarly Studies Program, Research Opportunities, Atherton Seidell Foundation, Biodiversity Surveys and Inventories Program, National Museum of Natural History-Small Grants, and Laboratory of Analytical Biology (LAB) all part of the Smithsonian Institution. We thank the National Geographic Society Committee for Research and Exploration (Grant Number 8087-06) for field and laboratory support and the Fulbright Scholar Program to KR for a research visit to the Smithsonian Institution. We thank the following organizations and people: the Komarov Botanical Institute, Russian Academy of Sciences for the opportunity to work with herbarium collections, and Nikolai Tzvelev and Dmitry Geltman for consultation and permitting us to sample Stipeae specimens; Surrey Jacobs for providing samples from Australia, Simon Laegaard for providing samples from South America, and Jeffery M. Saarela for providing samples from North America; and three anonymous reviewers for suggesting improvements to the manuscript. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev. 2013.09. 018. References Abbot, R.J., Brochmann, C., 2003. History and evolution of the arctic flora: in the footsteps of Eric Hultén. Mol. Ecol. 12, 299–313. 257 An, Z.S., Wang, S.M., Wu, X.H., Chen, M.Y., Sun, D.H., Liu, X.M., Wang, F.B., Li, L., Sun, Y.B., Zhou, W.J., 1999. Eolian evidence from the Chinese Loess Plateau: the onset of the Late Cenozoic Great Glaciation in the Northern Hemisphere and QinghaiXizang Plateau uplift forcing. Sci. China, Ser. D Earth Sci. 42, 258–271. Andersen, B.G., Borns Jr., H.W., 1994. The Ice Age World: an Introduction to Quaternary History and Research with Emphasis on North America and Northern Europe during the Last 2.5 Million Years. Scandinavian University Press, Oslo, Norway. Avise, J.C., 2000. Molecular Markers, Natural History, and Evolution, second ed. Sinauer Associates, Sunderland, Massachusetts. Barkworth, M.E., 1983. Ptilagrostis in North America and its relationship to other Stipeae (Gramineae). Syst. Bot. 8, 395–419. Barkworth, M.E., 2007. 10. Stipeae Dumort. In: Barkworth, M.E., Capels, K.M., Long, S., Anderton, L.K., Piep, M.B. (Eds.), Magnoliophyta: Commelinidae (in Part): Poaceae, Part 1. Flora of North America north of Mexico. Oxford University Press, New York, pp. 109–186. Barkworth, M.E., Everett, J., 1987. Evolution in Stipeae: identification and relationship of its monophyletic taxa. In: Soderstrom, T.R., Hilu, K.W., Campbell, C.S., Barkworth, M.E. (Eds.), Grass Systematics and Evolution. Smithsonian Institution Press, Washington, DC, pp. 251–264. Barkworth, M.E., Arriaga, M.O., Smith, J.F., Jacobs, S.W.L., Valdes-Reyna, J., Bushman, B.S., 2008. Molecules and morphology in South American Stipeae (Poaceae). Syst. Bot. 33, 719–731. Bird, M.I., Taylor, D., Hunt, C., 2005. Environments of insular Southeast Asia during the last glacial period: a savanna corridor in Sundaland? Quatern. Sci. Rev. 24, 2228–2242. Bosboom, R.E., Dupont-Nivet, G., Houben, A.J.P., Brinkhuis, H., Villa, G., Mandic, O., Stoica, M., Zachariasse, W.J., Guo, Z., Li, C., Krijgsman, W., 2011. Late Eocene sea retreat from the Tarim Basin (west China) and concomitant Asian paleoenvironmental change. Palaeogeogr. Palaeocl. Palaeoecol. 299, 385–398. Bouchenak-Khelladi, Y., Salamin, N., Savolainen, V., Forest, F., van der Bank, M., Chase, M.W., Hodkinson, T.R., 2008. Large multi-gene phylogenetic trees of the grasses (Poaceae): progress towards complete tribal and generic level sampling. Mol. Phylogenet. Evol. 47, 488–505. Bouchenak-Khelladi, Y., Verboom, G.A., Savolainen, V., Hodkinson, T.R., 2010. Biogeography of the grasses (Poaceae): a phylogenetic approach to reveal evolutionary history in geographical space and geological time. Bot. J. Linn. Soc. 162 (4), 543–557. Brochmann, C., Brysting, A.K., Alsos, I.G., Borgen, L., Grundt, H.H., Scheen, A.C., Elven, R., 2004. Polyploidy in arctic plants. Biol. J. Linn. Soc. 82, 521–536. Bryant, D., Moulton, V., 2004. Neighbor-net: an agglomerative method for the construction of phylogenetic networks. Mol. Biol. Evol. 21, 255–265. Cialdella, A.M., Giussani, L.M., Aagesen, L., Zuloaga, F.O., Morrone, O., 2007. A phylogeny of Piptochaetium (Poaceae: Pooideae: Stipeae) and related genera based on a combined analysis including trnL-F, rpl16, and morphology. Syst. Bot. 32, 545–559. Clark, P.U., Mix, A.C., 2002. Ice sheets and sea level of the last glacial maximum. Quatern. Sci. Rev. 21, 1–7. Colinvaux, P.A., West, F.H., 1984. The Beringian ecosystem. Q. Rev. Archaeol. 5, 10– 16. Comes, H.P., Abbott, R.J., 2001. Molecular phylogeography, reticulation, and lineage sorting in Mediterranean Senecio sect. Senecio (Asteraceae). Evolution 55, 1943– 1962. Corlett, R.T., 2009. The Ecology of Tropical East Asia. Oxford University Press, Oxford. Davidse, G., Pohl, R.W., 1994. 52. Stipa L. In: Davidse, G., Sousa S., M., Chater, A.O. (Eds.), Flora Mesoamericana. Alismataceae a Cyperaceae, vol. 6. Universidad Nacional Autónoma de México, México, DF, p. 243. Davis, J.I., Soreng, R.J., 1993. Phylogenetic structure in the grass family (Poaceae) as inferred from chloroplast DNA restriction site variation. Am. J. Bot. 80, 1444– 1454. Davis, J.I., Soreng, R.J., 2007. A preliminary phylogenetic analysis of the grass subfamily Pooideae (Poaceae), with attention to structural features of the plastid and nuclear genomes, including an intron loss in GBSSI. Aliso 23, 335– 348. DeChaine, E.G., 2008. A bridge or a barrier? Beringia’s influence on the distribution and diversity of tundra plants. Plant Ecol. Div. 1, 197–207. Donoghue, M.J., Smith, S.A., 2004. Patterns in the assembly of temperate forests around the Northern Hemisphere. Philos. Trans. R. Soc. London 359, 1633–1644. Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Edwards, M.E., Anderson, P.M., Brubaker, L.B., Ager, T.A., Andreev, A.A., Bigelow, N.H., Cwynar, L.C., Eisner, W.R., Harrison, S.P., Hu, F.S., Jolly, D., Lozhkin, A.V., MacDonald, G.M., Mock, C.J., Ritchie, J.C., Sher, A.V., Spear, R.W., Williams, J.W., Yu, G., 2000. Pollen-based biomes for Beringia 18, 000, 6000 and 0 14C yr bp. J. Biogeogr. 27, 521–554. Elias, M.K., 1942. Tertiary prairie grasses and other herbs from the high plains. Spec. Pap. Geol. Soc. Am. 41, 1–176. Elias, S.A., Short, S.K., Nelson, C.H., Birks, H.H., 1996. Life and times of the Bering land bridge. Nature 382, 60–63. Elias, S.A., Short, S.K., Birks, H.H., 1997. Late Wisconsin environments of the Bering land bridge. Palaeogeogr. Palaeocl. Palaeoecol. 136, 293–308. Erixon, P., Oxelman, B., 2008. Reticulate or tree-like chloroplast DNA evolution in Sileneae (Caryophyllaceae)? Mol. Phylogenet. Evol. 48, 313–325. 258 K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 Farris, J.S., Källersjö, M., Kluge, A.G., Bult, C., 1994. Testing significance of incongruence. Cladistics 10, 315–319. Fehrer, J., Gemeinholzer, B., Chrtek Jr., J., Bräutigam, S., 2007. Incongruent plastid and nuclear DNA phylogenies reveal ancient intergeneric hybridization in Pilosella hawkweeds (Hieracium, Cichorieae, Asteraceae). Mol. Phylogenet. Evol. 42, 347–361. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Frajman, B., Oxelman, B., 2007. Reticulate phylogenetics and phytogeographical structure of Heliosperma (Sileneae, Caryophyllaceae) inferred from chloroplast and nuclear DNA sequences. Mol. Phylogenet. Evol. 43, 140–155. Freitag, H., 1985. The genus Stipa (Gramineae) in Southwest and South Asia. Notes Roy. Bot. Gard. Edinburgh 42, 355–489. Gillespie, L.J., Soreng, R.J., Paradis, L.M., Bull, R.D., 2010. Phylogeny and reticulation in Poinae subtribal complex based on nrITS, ETS, and trnTLF data. In: Seberg, O., Petersen, G., Barfod, A.S., Davis, J.I. (Eds.), Diversity, Phylogeny, and Evolution in the Monocotyledons. Aarhus University Press, Denmark, pp. 589–617. Gogarten, J.P., Olendzenski, L., 1999. Orthologs, paralogs and genome comparisons. Curr. Opin. Genet. Dev. 9, 630–636. Gonzales, A., Hamrick, J.L., Chang, S.M., 2008. Identification of glacial refugia in South-Eastern North America by phylogeographical analyses of a forest understorey plant, Trillium cuneatum. J. Biogeogr. 35, 844–852. Graham, S.W., Olmstead, R.G., 2000. Utility of 17 chloroplast genes for inferring the phylogeny of the basal angiosperms. Amer. J. Bot. 87, 1712–1730. Grass Phylogeny Working Group (GPWG), 2001. Phylogeny and subfamilial classification of the grasses (Poaceae). Ann. Missouri Bot. Gard. 88, 373–457. Grivet, D., Petit, R.J., 2003. Chloroplast DNA phylogeography of the hornbeam in Europe: evidence for a bottleneck at the outset of postglacial colonization. Conserv. Genet. 4, 47–56. Guan, Q.Y., Pan, B.T., Li, N., Zhang, J.D., Xue, L.J., 2011. Timing and significance of the initiation of present day deserts in the northeastern Hexi Corridor, China. Palaeogeogr. Palaeocl. Palaeoecol. 306, 70–74. Guo, Z.T., Peng, S.Z., Hao, Q.Z., Biscaye, P.E., Liu, T.S., 2001. Origin of the Miocene– Pliocene red-earth formation at Xifeng in Northern China and implications for paleoenvironments. Palaeogeogr. Palaeocl. Palaeoecol. 170, 11–26. Guo, Z.T., Ruddiman, W.F., Hao, Q.Z., Wu, H.B., Qiao, Y.S., Zhu, R.X., Peng, S.Z., Wei, J.J., Yuan, B.Y., Liu, T.S., 2002. Onset of Asian desertification by 22 Myr ago inferred from loess deposits in China. Nature 416, 159–163. Hamasha, H.R., Bernhard von Hagen, K., Röser, M., 2012. Stipa (Poaceae) and allies in the Old World: molecular phylogenetics realigns genus circumscription and gives evidence on the origin of American and Australian lineages. Plant Syst. Evol. 298, 351–367. Harding, T.M., Brunsfeld, S.J., Fritz, R.S., Morgan, M., Orians, C.M., 2000. Morphological and molecular evidence for hybridization and introgression in a willow (Salix) hybrid zone. Mol. Ecol. 9, 9–24. Heaney, L.R., 1991. A synopsis of climatic and vegetational change in Southeast Asia. Clim. Change 19, 53–61. Hilu, K.W., Lawrence, A.A., Liang, H., 1999. Phylogeny of Poaceae inferred from matK sequences. Ann. Missouri Bot. Gard. 86, 835–851. Huelsenbeck, J.P., Ronquist, F.R., 2001. MrBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. Humphreys, A.M., Pirie, M.D., Linder, H.P., 2010. A plastid tree can bring order to the chaotic generic taxonomy of Rytidosperma Steud. s.l. (Poaceae). Mol. Phylogenet. Evol. 55, 911–928. Hung, C.Y., Sun, Y.H., Chen, J., Darlington, D.E., Williams, A.L., Burkey, K.O., Xie, J., 2010. Identification of a Mg-protoporphyrin IX monomethyl ester cyclase homologue, EaZIP, differentially expressed in variegated Epipremnum aureum ‘Golden Pothos’ is achieved through a unique method of comparative study using tissue regenerated plants. J. Exp. Bot. 61, 1483–1493. Huson, D.H., Bryant, D., 2006. Application of phylogenetic networks in evolutionary studies. Mol. Biol. Evol. 23, 254–267. Jacobs, S.W.L., Everett, J., Barkworth, M.E., Hsiao, C., 2000. Relationships within the Stipeae (Gramineae). In: Jacobs, S.W.L., Everet, t J. (Eds.), Grasses: Systematics and Evolution. CSIRO Publishing, Collingwood, Australia, pp. 75–82. Jacobs, S.W.L., Bayer, R., Everett, J., Arriaga, M.O., Barkworth, M.E., Sabin-Badereau, A., Torres, M.A., Vázquez, F., Bagnall, N., 2007. Systematics of the tribe Stipeae using molecular data. Aliso 23, 349–361. Johnson, B.L., 1945. Cytotaxonomic studies in Oryzopsis. Bot. Gaz. 107, 1–32. Jolivet, L., Faccenna, C., 2000. Mediterranean extension and the Africa–Eurasia collision. Tectonics 19, 1095–1106. Kappelman, J., Rasmussen, D.T., Sanders, W.J., Feseha, M., Bown, T., Copeland, P., Crabaugh, J., Fleagle, J., Glantz, M., Gordon, A., Jacobs, B., Maga, M., Muldoon, K., Pan, A., Pyne, L., Richmond, B., Ryan, T., Seiffert, E.R., Sen, S., Todd, L., Wiemann, M.C., Winkler, A., 2003. Oligocene mammals from Ethiopia and faunal exchange between Afro-Arabia and Eurasia. Nature 426 (6966), 549–552. King, R.A., Ferris, C., 2000. Chloroplast DNA and nuclear DNA variation in the sympatric alder species, Alnus cordata (Lois.) Duby and A. glutinosa (L.) Gaertn. Biol. J. Linn. Soc. 70, 147–160. Legler, B.S., 2010. Additions to the vascular flora of New Mexico. Bot. Res. Inst. Texas 4, 777–784. Li, M., Wunder, J., Bissoli, G., Scarponi, E., Gazzani, S., Barbaro, E., Saedler, H., Varotto, C., 2008. Development of COS genes as universally amplifiable markers for phylogenetic reconstructions of closely related plant species. Cladistics 24, 727–745. Liu, Y.S., Utescher, T., Zhou, Z., Sun, B., 2011. The evolution of Miocene climates in North China: preliminary results of quantitative reconstructions from plant fossil records. Palaeogeogr. Palaeocl. Palaeoecol. 304, 308–317. López-Pujol, J., Ren, M.X., 2010. China: a hot spot of relict plant taxa. In: Rescigno, V., Maletta, S. (Eds.), Biodiversity Hotspots. Nova Science Publishers, New York, pp. 123–137. López-Pujol, J., Zhang, F.M., Sun, H.Q., Ying, T.S., Ge, S., 2011. Centres of plant endemism in China: places for survival or for speciation? J. Biogeogr. 38, 1267– 1280. Marincovich, L., Gladenkov, A.Y., 1999. Evidence for an early opening of the Bering Strait. Nature 397 (6715), 149–151. Marincovich, L., Gladenkov, A.Y., 2001. New evidence for the age of Bering Strait. Quatern. Sci. Rev. 20, 329–335. Martin-Bravo, S., Vargas, P., Luceño, M., 2009. Is Oligomeris (Resedaceae) indigenous to North America? Molecular evidence for a natural colonization from the Old World. Am. J. Bot. 96, 507–518. Metcalfe, I., Smith, J.M.B., Morwood, M., Davidson, I., 2001. Faunal and Floral Migrations and Evolution in SE Asia-Australasia. Swets and Zeitlinger, Balkema, Lisse. Murray, B.G., De Lange, P.J., Ferguson, A.R., 2005. Nuclear DNA variation, chromosome numbers and polyploidy in the endemic and indigenous grass flora of New Zealand. Ann. Bot. 96, 1293–1305. Ni, J., Yu, G., Harrison, S.P., Prentice, I.C., 2010. Palaeovegetation in China during the late Quaternary: biome reconstructions based on a global scheme of plant functional types. Palaeogeogr. Palaeocl. Palaeoecol. 289, 44–61. Nixon, K.C., 2002. WinClada, ver.1.00.08. Ithaca, NY, Published by the Author. Palmer, J.D., 1987. Chloroplast DNA evolution and biosystematic uses of chloroplast DNA variation. Am. Nat. 130, S6–S29. Peterson, P.M., Soreng, R.J., Wu, Z.L., 2005. Ptilagrostis luquensis (Poaceae: Pooideae: Stipeae: Stipinae), a new species from China. Sida 21, 1355–1362. Peterson, P.M., Romaschenko, K., Johnson, G., 2010a. A classification of the Chloridoideae (Poaceae) based on multi-gene phylogenetics trees. Mol. Phylogenet. Evol. 55, 580–598. Peterson, P.M., Romaschenko, K., Johnson, G., 2010b. A phylogeny and classification of the Muhlenbergiinae (Poaceae: Chloridoideae: Cynodonteae) based on plastid and nuclear DNA sequences. Am. J. Bot. 97, 1532–1554. Peterson, P.M., Romaschenko, K., Barker, N.P., Linder, H.P., 2011. Centropodieae and Ellisochloa, a new tribe and genus in the Chloridoideae (Poaceae). Taxon 60, 1113–1122. Peterson, P.M., Romaschenko, K., Snow, N., Johnson, G., 2012. A molecular phylogeny and classification of Leptochloa (Poaceae: Chloridoideae: Chlorideae) sensu lato and related genera. Ann. Bot. 109, 1317–1329. Pope, J.O., Collins, M., Haywood, A.M., Dowsett, H.J., Hunter, S.J., Lunt, D.J., Pickering, S.J., Pound, M.J., 2011. Quantifying uncertainty in model predictions for the Pliocene (Plio-QUMP): initial results. Palaeogeogr. Palaeocl. Palaeoecol. 309, 128–140. Posada, D., 2008. JModelTest model averaging. Mol. Biol. Evol. 25, 1253–1256. Pound, M.J., Haywood, A.M., Salzmann, U., Riding, J.B., Lunt, D.J., Hunter, S.J., 2011. A Tortonian (Late Miocene, 11.61–7.25 Ma) global vegetation reconstruction. Palaeogeogr. Palaeocl. Palaeoecol. 300, 29–45. Probatova, N.S., 1985. Poaceae. In: Tzvelev, N.N. (Ed.), Sosudistye Rastenia Sovetskogo Dal’nego Vostoka. [updated and translated in English 2003 as Vascular Plants of the Russian Far East, vol. 1]. Science Publishers, Enfield, New Hampshire, pp. 89–488. Probatova, N.S., Sokolovskaya, A.P., 1980. A karyotaxonomic study of the grasses of the Altai Mts. J. Bot. Zhurnal 65, 509–520. Qin, F., Ferguson, D.K., Zetter, R., Wang, Y., Syabryaj, S., Li, J., Yang, J., Li, C., 2011. Late Pliocene vegetation and climate of Zhangcun region, Shanxi, North China. Glob. Change Biol. 17, 1850–1870. Qiu, Y.X., Fu, C.X., Comes, H.P., 2011. Plant molecular phylogeorgaphy in China and adjacent regions: tracing the genetic imprints of Quaternary climate and environmental change in the world’s most diverse temperate flora. Mol. Phylogenet. Evol. 59, 225–244. Rambaut, A., Drummond, A.J., 2007. Tracer v1.4. <http://beast.bio.ed.ac.uk/Tracer>. Rea, D.K., Snoeckx, H., Joseph, L.H., 1998. Late Cenozoic Eolian deposition in the North Pacific: Asian drying, Tibetan uplift, and cooling of the northern hemisphere. Paleoceanography 13, 215–224. Rieseberg, L.H., Soltis, D.E., 1991. Phylogenetic consequences of cytoplasmic gene flow in plants. Evol. Trend. Plant. 5, 65–84. Romaschenko, K., Peterson, P.M., Soreng, R.J., Garcia-Jacas, N., Futoma, O., Susanna, A., 2008. Molecular phylogenetic analysis of the American Stipeae (Poaceae) resolves Jarava sensu lato polyphyletic: evidence for a new genus, Pappostipa. J. Bot. Res. Inst. Texas 2, 165–192. Romaschenko, K., Peterson, P.M., Soreng, R.J., Garcia-Jacas, N., Susanna, A., 2010. Phylogenetics of Stipeae (Poaceae: Pooideae) based on plastid and nuclear DNA sequences. In: Seberg, O., Petersen, G., Barfod, A.S., Davis, J.I. (Eds.), Diversity, Phylogeny, and Evolution in the Monocotyledons. Aarhus University Press, Denmark, pp. 513–539. Romaschenko, K., Peterson, P.M., Soreng, R.J., Futoma, O., Susanna, A., 2011. Phylogenetics of Piptatherum s.l. (Poaceae: Stipeae): evidence for a new genus, Piptatheropsis, and resurrection of Patis. Taxon 60, 1703–1716. Romaschenko, K., Peterson, P.M., Soreng, R.J., Garcia-Jacas, N., Futoma, O., Susanna, A., 2012. Systematics and evolution of the needle grasses (Poaceae: Pooideae: Stipeae) based on analysis of multiple chloroplast loci, ITS, and lemma micromorphology. Taxon 61, 18–44. K. Romaschenko et al. / Molecular Phylogenetics and Evolution 70 (2014) 244–259 Ronquist, F., Huelsenbeck, J.P., Van der Mark, P., 2005. MrBayes 3.1 Manual, Draft 02.26.05. <http://mrbayes.csit.fsu.edu/mb3.1_manual.pdf>. Roshevitz, R.Yu., 1934. Gramineae. In: Roshevitz, R.Yu., Shishkin, B.K. (Eds.), Flora of the U.S.S.R., vol. 2. (translated in English 1963) Akademii Nauk SSSR, Leningrad, pp. 71–118. Schneider, J., Döring, E., Hilu, K.W., Röser, M., 2009. Phylogenetic structure of the grass subfamily Pooideae based on comparison of plastid matK gene-30 trnK exon and nuclear ITS sequences. Taxon 58, 405–424. Semerikov, V.L., Lascoux, M., 2003. Nuclear and cytoplasmic variation within and between Eurasian Larix (Pinaceae) species. Am. J. Bot. 90, 1113–1123. Sonnhammer, E.L.L., Koonin, E.V., 2002. Orthology, paralogy and proposed classification for paralog subtypes. Trends Genet. 18, 619–620. Soreng, R.J., Davis, J.I., 1998. Phylogenetics and character evolution in the grass family (Poaceae): simultaneous analysis of morphological and chloroplast DNA restriction site character sets. Bot. Rev. 64, 1–88. Soreng, R.J., Davis, J.I., 2000. Phylogenetic structure in Poaceae subfamily Pooideae as inferred from molecular and morphological characters: misclassification versus reticulation. In: Jacobs, S.W.L., Everett, J. (Eds.), Grasses: Systematics and Evolution. CSIRO Publishing, Collingwood, Australia, pp. 61–74. Soreng, R.J., Peterson, P.M., Davidse, G., Judziewicz, E.J., Zuloaga, F.O., Filgueiras, T.S., Morrone, O., 2003 and onwards. Catalogue of New World grasses (Poaceae): IV. Subfamily Pooideae. Contr. U.S. Natl. Herb. 48, 1–730. <http:// www.tropicos.org/Project/CNWG>. Soreng, R.J., Davis, J.I., Voionmaa, M.A., 2007. A phylogenetic analysis of Poaceae tribe Poeae sensu lato based on morphological characters and sequence data from three plastid-encoded genes: evidence for reticulation, and a new classification for the tribe. Kew. Bull. 62, 425–454. Soreng, R.J., Bull, R.D., Gillespie, L.J., 2010. Phylogeny and reticulation in Poa L. based on plastid trnTLF and nrITS sequences with attention to diploids. In: Seberg, O., Petersen, G., Barfod, A.S., Davis, J.I. (Eds.), Diversity, Phylogeny, and Evolution in the Monocotyledons. Aarhus University Press, Denmark, pp. 619–643. Soreng, R.J., Peterson, P.M., Davidse, G., Judziewicz, E.J., Zuloaga, F.O., Filgueiras, T.S., Morrone, O., Romaschenko, K., 2012. A World-wide Phylogenetic Classification of Poaceae (Gramineae): çimen, çayır, darbha, ghaas, ghas, gramas, gräser, grasses, he ben ke, hullu, kasa, kusa, pastos, pillu, pullu, zlaki, etc. <http:// www.tropicos.org/ projectwebportal.aspx?pagename=ClassificationNWG&projectid=1>. Ståhlberg, D., Hedrén, M., 2008. Systematics and phylogeography of the Dactylorhiza maculata complex (Orchidaceae) in Scandinavia: insights from cytological, morphological and molecular data. Plant Syst. Evol. 273, 107–132. Swofford, D.L., 2000. PAUP: Phylogenetic Analysis using Parsimony (and Other Methods), Version 4. Sinauer, Sunderland, Massachusetts. Takhtajan, A., 1986. Floristic Regions of the World (T.J. Crovello, A. Cronquist, Trans.). University of California Press, Berkeley. Tateoka, T., 1986. Chromosome numbers in the tribe Stipeae (Poaceae) in Japan. Bull. Natl. Sci. Mus. Tokyo 12, 151–154. Thomasson, J.R., 1978. Epidermal patterns of the lemma in some fossil and living grasses and their phylogenetic significance. Science 199, 975–977. Thomasson, J.R., 1979. Late Cenozoic grasses and other Angiosperms from Kansas, Nebraska, and Colorado: biostratigraphy and relationships to living taxa. Kan. Geol. Surv. Bull. 218, 1–68. 259 Thomasson, J.R., 1980. Paleoeriocoma (Gramineae, Stipeae) from the Miocene of Nebraska: taxonomic and phylogenetic significance. Syst. Bot. 5, 233–240. Thomasson, J.R., 1982. Fossil grass anthoecia and other plant fossils from arthropod burrows in the Miocene of Western Nebraska. J. Paleontol. 56, 1011–1017. Thomasson, J.R., 1985. Miocene fossil grasses: possible adaptation in reproductive bracts (lemma and palea). Ann. Missouri Bot. Gard. 72, 843–851. Thomasson, J.R., 1987. Fossil grasses: 1820–1986 and beyond. In: Soderstrom, T.R., Hilu, K.W., Campbell, C.S., Barkworth, M.E. (Eds.), Grass Systematics and Evolution. Smithsonian Institution Press, Washington, DC, pp. 159–167. Thomasson, J.R., 2005. Berriochloa gabeli and Berriochloa huletti (Gramineae, Stipeae), two new grass species from the late Miocene Ash Hollow Formation of Nebraska and Kansas. J. Paleontol. 79, 185–199. Tzvelev, N.N., 1976. Zlaki SSSR. Nauka Publishers, Leningrad. Tzvelev, N.N., 1977. [On the origin and evolution of Feathergrasses (Stipa L.)]. In: Lebedev, D.V., Karamysheva, Z.V. (Eds.), Problemy ekologii, geobotaniki, botanicheskoi geografii I floristiki. Academiya Nauk SSSR, Leningrad, pp. 139– 150. Utescher, T., Bruch, A.A., Micheels, A., Mosbrugger, V., Popova, S., 2011. Cenozoic climate gradients in Eurasia – a palaeo-perspective on future climate change? Palaeogeogr. Palaeocl. Palaeoecol. 304, 351–358. Vicentini, A., Barber, J.C., Aliscioni, S.S., Giussani, L.M., Kellogg, E.A., 2008. The age of the grasses and clusters of origins of C4 photosynthesis. Glob. Change Biol. 14 (12), 2963–2977. Watson, L., Dallwitz, M.J., 1992 – onwards. The Grass Genera of the World: Descriptions, Illustrations, Identification, and Information Retrieval; Including Synonyms, Morphology, Anatomy, Physiology, Phytochemistry, Cytology, Classification, Pathogens, World and Local Distribution, and References. Version: 23 Apr. 2010. <http://www.delta-ntkey.com/grass/index.htm>. Weber, W.A., 1966. Additions to the Flora of Colorado IV. Univ. Colorado Stud. Ser. Biol. 23, 2. Wen, J., Ickert-Bond, S., Nie, Z.L., Li, R., 2010. Timing and modes of evolution of eastern Asian – North American biogeographic disjunctions in seed plants. In: Long, M., Gu, H., Zhou, Z. (Eds.), Darwin’s Heritage Today – Proceedings of the Darwin 200 Beijing International Conference. Higher Education Press, Beijing, pp. 252–269. Woodruff, D., 2010. Biogeography and conservation in Southeast Asia: how 2.7 million years of repeated environmental fluctuations affect today’s patterns and the future of the remaining refugial-phase biodiversity. Biodivers. Conserv. 19, 919–941. Wu, Z., Phillips, S.M., 2006. Stipeae. In: Wu, Z., Raven, P.H., Hong, D.Y. (Eds.), Flora of China: Poaceae, vol. 22. Science Press, Beijing and Missouri Botanical Garden Press, St. Louis, pp. 188–212. Wu, Z., Wu, S., 1996. A proposal for a new floristic kingdom (realm). In: Zhang, A., Wu, S. (Eds.), Floristic Characteristics and Diversity of East Asian Plants. China Higher Education Press, Beijing, pp. 3–42. Yao, Z., Xiao, G., Wu, H., Liu, W., Chen, Y., 2010. Plio-Pleistocene vegetation changes in the North China Plain: magnetostratigraphy, oxygen and carbon isotopic composition of pedogenic carbonates. Palaeogeogr. Palaeocl. Palaeoecol. 297, 502–510. Zwickl, D.J., 2006. Genetic Algorithm Approaches for the Phylogenetic Analysis of Large Biological Sequence Datasets under the Maximum Likelihood criterion. Ph.D. Thesis, University of Texas, Austin.