Phylogenetic Reconstruction and Diversification of the Triticeae

Biochemical Systematics and Ecology 50 (2013) 346–360

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Biochemical Systematics and Ecology

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Phylogenetic reconstruction and diversiﬁcation of the Triticeae (Poaceae) based on single-copy nuclear Acc1 and Pgk1 gene data

Xing Fan a,b, Li-Na Sha a, Shuang-Bin Yu a, Dan-Dan Wu a, Xiao-Hong Chen a, Xiao-Feng Zhuo a, Hai-Qin Zhang a, Hou-Yang Kang a, Yi Wang a, You-Liang Zheng a,b, Yong-Hong Zhou a,b,* a Triticeae Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China b Key Laboratory of Crop Genetic Resources and Improvement, Ministry of Education, Sichuan Agricultural University, Yaan 625014, Sichuan, China article info abstract

Article history: Two single-copy nuclear gene (Acc1 and Pgk1) sequences were used to estimate the phy- Received 23 March 2013 logeny and diversification patterns of the Triticeae. Phylogenetic analyses and diversification Accepted 18 May 2013 patterns suggested that (1) a major radiation occurring in the Triticeae about 6.1–9.2 MYA Available online might have been triggered by the late Miocene climate oscillations; (2) the Triticeae lineages from Mediterranean and Arctic-temperate regions were evolutionarily distinct; (3) the Keywords: relationship between Thinopyrum bessarabicum and Triticum/Aegilops is more closely than Triticeae that between Lophopyrum elongatum and Triticum/Aegilops;(4)Pseudoroegneria is closely Phylogeny Diversification related to Australopyrum and Lophopyrum;(5)Australopyrum might originate from coloni- Monogenomic genera zation via South-east Asia during the late Miocene; (6) Habitats, mating systems, and climate Climate oscillations oscillations may associated with the diversification rate shifts leading to the majority of Mediterranean lineage. Diversification rate shifts in Mediterranean lineage of the Triticeae not only spur the occurrence of many endemic lineages but also provide opportunity for polyploid crop origin and domestication. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Determining what processes have driven species diversification and diversity is a central goal of evolutionary and con- servation biologists. The forces acting on species richness may apply at different latitudes or biogeographic regions, indicating the importance of historical processes (e.g. dispersal events, and local speciation and extinction regimes) in mediating the environment–diversity relationship (Qian et al., 2009; Kisel et al., 2011). Theoretical and empirical investigation emphasized that historical climate fluctuations are a key factor promoting species diversification and distribution (Currie, 1991; Pearson and Dawson, 2003; Clarke and Gaston, 2006; Lavergne et al., 2010). Recent studies combining an effect of historical climatic variability with time-calibrated phylogenies suggested that variation in global climate determined the distribution, richness and diversity of plant clades, hence, indirectly influenced plant diversification (Egan and Crandall, 2008; Nyman et al., 2012).

* Corresponding author. Triticeae Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China. E-mail address: [email protected] (Y.-H. Zhou).

The wheat tribe (Poaceae: Triticeae), an economically important gene pool for genetic improvement of cereal and forage crops, includes about 450 diploid and polyploid species that distribute in a wide range of ecological habitats over the temperate and subtropical and tropic alpine regions (Dewey, 1984). In the Triticeae, 24 major basic genomes donated by diploid species have been designated and were recognized in eighteen monogenomic genera (Löve, 1984; Wang et al., 1994). Many basic genomes (e.g. A, B, D, Ns, Xm, E, St, Y, H, P, W) in various combinations (e.g. AB, ABD, NsXm, StE, StH, StY, StYH, StYP, StYW) constitute polyploid species, about 75% of the Triticeae species (Lu, 1994). Monogenomic genera have been the focus of numerous phylogenetic investigations, partly because of its economic importance (which includes barley, rye, and crested- wheat grasses), and partly because of its genome donor to the speciation of polyploid species, as well as partly because of its wide variety of species richness, morphology, ecology and distribution. It was not considered that genomic analysis can reveal a phylogeny of the monogenomic genera (Kellogg, 1989; Kellogg et al., 1996; Petersen and Seberg, 1997; Seberg and Petersen, 1998). Attempts to sort out the phylogenetic details of the monogenomic genera, phylogenetic reconstructions were carried out based on morphological characteristics (Seberg and Frederiksen, 2001), chloroplast genes (Mason-Gamer et al., 2002), internal transcribed spacers (ITS) sequences (Hsiao et al., 1995), highly repetitive nuclear DNA (Kellogg and Appels, 1995), single-copy nuclear genes (Petersen and Seberg, 1997; Helfgott and Mason-Gamer, 2004; Mason-Gamer, 2005), and multiple gene combined data (Escobar et al., 2011). No consensual definition of clades, however, has been received due to either phylogenetic tree inferred from a limited number of genes (Petersen and Seberg, 1997; Hsiao et al., 1995; Kellogg and Appels, 1995; Helfgott and Mason-Gamer, 2004; Mason-Gamer, 2005) or limited sample (Kellogg and Appels, 1995; Escobar et al., 2011) or introgression events and/or incomplete lineage sorting of ancestral polymorphisms (Mason-Gamer, 2005; Escobar et al., 2011). With the data accumulating on members of the Triticeae, the origin and diversification of the tribe have received attention. Data from natural distribution speculated that the tribe originated about in the middle of the Tertiary period (20–30 MYA), and the first step of phylogenetic diversification in the Triticeae was occurred at the diploid level (Sakamoto, 1973). Morphological analyses presumed that the general evolution of the Triticeae has been defined by divergence at the diploid level from a common diploid ancestor (West et al., 1988). Kellogg et al. (1996) hypothesized that the common ancestor of the Triticeae had a spicate inflorescence with three spikelets per node and gave rise to lineages leading to present-day Psa- thyrostachys and Hordeum. Molecular dating suggested that the divergence between Aegilops/Triticum lineage and barley range from 15 to 10 MYA (million years ago) (Gaut, 2002; Huang et al., 2002; Dvorak and Akhunov, 2005), and the diploid Triticum and Aegilops progenitors of the A, B, D, G and S genomes all radiated about 4.5 MYA (Huang et al., 2002). While these studies add to our understanding of origin of the Triticeae, little is known about the processes driving species diversification and diversity within the Triticeae. Moreover, a complete picture of evolutionary history within the tribe is needed. In this study, we carried out phylogenetic reconstructions and molecular dating using two unlinked nuclear gene (Acc1 and Pgk1) sequences for 31 species of 17 monogenomic genera within the Triticeae. Chromosome mapping indicate that a single copy of the Acc1 gene is present in each of the group 2 homoeologous chromosomes in hexaploid wheat (Gornicki et al., 1997). Analysis of the Pgk1 gene showed that it is present as a single copy per diploid chromosome in grass, and a single copy of the Pgk1 gene is present in each of the group 3 homoeologous chromosomes in hexaploid wheat (Huang et al., 2002). The objectives were: (1) to reconstruct the phylogeny of the Triticeae, especially the time-calibrated phylogeny of the tribe; (2) to document the divergence history within this tribe; (3) to estimate the diversification pattern and process of the Triticeae species.

2. Materials and methods

2.1. Taxon sampling

A total of 31 diploid taxa representing 18 basic genomes are used in this study. While generic definitions within the Triticeae have been extremely variable (Barkworth, 2000), this sample represents nearly all of the monogenomic genera accepted in genome-based classifications of the tribe. Acc1 and Pgk1 sequences of Aegilops/Triticum group, Secale, and Hor- deum vulgare were obtained from the data reported by Huang et al. (2002). The remaining sequences of the Triticeae, along with Bromus inermis were obtained from our published data (Fan et al., 2009, 2012). Plant material with sample information and GenBank identification numbers are listed in Table S1.

2.2. Data analysis

The Acc1 and Pgk1 sequences were edited using SeqMan (DNASTAR package, Inc. Madison, Wisconsin) and adjusted manually where necessary. The basic sequence statistics, including nucleotides frequencies, transition/transversion ratio and variability in different regions of the sequences were examined by MEGA3 (Kumar et al., 2004). The partition homogeneity test, implemented in the program PAUP*4.0b10 (Swofford D L, Sinauer Associates, http://www.sinauer.com) was used to determine whether the Acc1 and Pgk1 genes contained significantly different signals. The partition homogeneity test can generate significant results if one gene has experienced many multiple substitutions or contains random information. The result of the partition homogeneity test showed that the sequence data sets for the Acc1 and Pgk1 loci were congruent (P ¼ 0.1000) and could therefore be combined for purposes of estimating the phylogeny and diversification pattern of the Triticeae. 348 X. Fan et al. / Biochemical Systematics and Ecology 50 (2013) 346–360

Three data matrixes, including the Acc1 data, Pgk1 data and combined dataset (Acc1 þ Pgk1), were used separately to carry out phylogenetic analyses. Phylogenetic analyses were conducted using maximum-likelihood (ML) method with PAUP* 4.0b10 and Bayesian inference (BI) method with MrBayes version v3.1.2 (Huelsenbeck and Ronquist, 2001). ModelTest 3.0 (Posada and Crandall, 1998) was used to determine the appropriate DNA substitution model and gamma rate heterogeneity using the Akaike information criterion (AIC). The best-fit model identified was GTR þ G for the Acc1 data, GTR þ G þ I for the Pgk1 data and GTR þ G þ I for the combined dataset. In ML analysis, Heuristic searches were employed (ACCTRAN; starting tree based on neighbor joining reconstruction; TBR branch swapping; STEEPEST DESCENT). Statistical support for nodes in ML analysis was estimated by using 1000 fast bootstrap replicates. BI analysis was performed using MrBayes v3.0 (Huelsenbeck and Ronquist, 2001). BI analyses of the Acc1 data, Pgk1 data and combined dataset were carried out under the same evolutionary model as ML analysis. Four MCMC (Markov Chain Monte Carlo) chains (one cold and three heated) were run for 170, 000 generations for the Acc1 data, 300,000 generations for the Pgk1 data, and 800,000 generations for the combined dataset, each sampling every 100 generations. The first 250, 600, and 2000 trees were stationary discarded as “brun-in” for the Acc1 data, Pgk1 data and combined dataset and the remaining trees were used to construct a 50% majority rule consensus tree (BI tree), respectively. The statistical confidence in nodes was evaluated by posterior probabilities (PP). To visualize phylogenetic structure and possible reticulating relationships within the Triticeae, the NeighborNet algorithm (Bryant and Moulton, 2004), implemented in the program SplitsTree version 4.10 (Huson, 1998), was used to generate phylogenetic networks for the Acc1 data, Pgk1 data and combined dataset. To assess the support for the observed structure, a bootstrap analysis was conducted with 1000 replicates. An effective approach of examining character evolution is through ancestral state reconstruction in a given phylogeny using statistical approaches (Pagel, 1999). To examine the evolution of geographical origin, mating systems, and growing habitats, their ancestral states were traced on our ML phylogenetic tree inferred from the combined dataset using weighted squared-change parsimony in the software Mesquite v2.5 (Maddison and Maddison, 2008). Weighted squared-change parsimony minimizes the sum of squared change along all branches of the tree, weighting branches by their length, and is equivalent to the ML estimate assuming a Brownian motion model of evolution (Finarelli and Flynn, 2006). Three geographic areas, the Arctic-temperate region, the Mediterranean and adjacent region, and Australia, were reconstructed for the ancestor of geographical origins. Ancestral character state reconstruction for mating systems employed three states including cross-pollination, moderate self- pollination, and self-pollination. Annual and perennial states were used to reconstruct the ancestral state of growing habitats. The hypothesis of rate constancy of the combined dataset was evaluated with a likelihood ratio test comparing the likelihood scores from the unconstrained and clock-constrained analyses. Substitution rates were significantly heterogenous (c2 ¼ 160.57, df ¼ 27, P < 0.0001), implying a very poor fit to the molecular clock. Therefore, divergence times with 95% confidence intervals were estimated using Bayesian relaxed molecular clock method, implemented in BEAST v1.4.6 (Drummond and Rambaut, 2007). The lack of fossils for the Triticeae precluded a direct calibration of tree topologies. Instead, molecular dating was based on the divergence age of 12 MYA between Hordeum and Aegilops/Triticum lineage because the assumption of a split between Hordeum and Aegilops/Triticum lineage ranged from 13 to 11 MYA (Gaut, 2002; Huang et al., 2002; Blattner, 2006). Calibration points were performed using a relaxed uncorrelated lognormal molecular clock. The Yule process was used to describe speciation. MCMC searches were run for 10,000,000 generations under GTR þ G þ I model (with the associated parameters specified by ModelTest v3.0 as the priors). Tracer v1.4 (Rambaut and Drummond, 2007)was used to ensure the convergence of the mixing in terms of the effective sample size (ESS) values and the coefficient rate. Resulting trees were analyzed using TreeAnnotator available in BEAST where the “burn-in” (2000 trees) was removed and a maximum credibility tree was constructed. Trees were then viewed in FigTree v. 1.3.1 (http://tree.bio.ed.ac.uk/). Analysis of diversification pattern, including the temporal dynamics and rate-variation of diversification, were estimated based on the combined dataset. The temporal dynamics of diversification in the Triticeae were visualized with lineage- through-time (LTT) plots, implemented in the software Mesquite v2.5 (Maddison and Maddison, 2008) and Genie v3.0 (Pybus and Rambaut, 2002). The topological program SymmeTREE v1.1 (Chan and Moore, 2005) was employed to conduct whole-tree tests for diversification rate-variation. The M-statistics (Mr, MS, MS*, MP and MP*) were used to test the presence of significant rate shifts (Chan and Moore, 2002). Locate shifts were estimated using the Slowinski-Guyer (SG) statistic (Slowinski and Guyer, 1989) and D1/D2 likelihood rate shift statistics, and the BI and Ic statistics were used to assess tree imbalance (Heard, 1992).

3. Results

3.1. Phylogenetic analyses

3.1.1. Acc1 gene data The aligned Acc1 sequences yielded a total of 1662 characters, of which 439 were variable characters, and 220 were informative. ML analysis yielded a single phylogenetic tree (Ln likelihood ¼ 6134.3935), with the following estimated ML parameters: the assumed nucleotide frequencies A: 0.2484, C: 0.1826, G: 0.2118, T: 0.3572, gamma shape parameter ¼ 0.5146. ML and Bayesian analyses recovered the same topology. The tree illustrated in Fig. 1 was the ML tree with posterior probabilities (PP) above and bootstrap support (BS) below branches. In ML tree, ﬁve clades (Clade I–V) with consistent statistic support (>70% BS; >90% PP) were found (Fig. 1A). The Clade I included Aegilops/Triticum complex, Thinopyrum (Eb genome), X. Fan et al. / Biochemical Systematics and Ecology 50 (2013) 346–360 349 350 X. Fan et al. / Biochemical Systematics and Ecology 50 (2013) 346–360

Pseudoroegneria (St genome), Lophopyrum (Ee genome), Australopyrum (W genome), Secale (R genome), Henrardia (O genome), and Taeniatherum (Ta genome) (100% PP and 97% BS). In this clade, Aegilops/Triticum complex formed one group, and Thinopyrum was placed outside this group. Pseudoroegneria was grouped with Lophopyrum and Australopyrum. Henrardia was sister to Secale with moderate values (100% PP and 75% BS). The Clade II contained Dasypyrum (V genome), Peridictyon (Xp genome), Crithopsis (K genome), and Herteranthelium (Q genome) (93% PP). The Clade III consisted of Hordeum (H/I genome) (100% PP and 100% BS). The Clade IV comprised Psathyrostachys (Ns genome) and Eremopyrum distans (F genome) (97% PP). The Clade V included Agropyron (P genome) and Eremopyrum triticeum (F genome) (100% PP and 100% BS). Phylogenetic network analysis yielded a high-resolution network, and ﬁve major splits (Split I–V) were recovered (Fig. 1B), which is consistent with the groupings generated from ML analysis. The Split I included Aegilops/Triticum complex, Thino- pyrum, Pseudoroegneria, Lophopyrum, Australopyrum, Henrardia, and Taeniatherum (100% BS). The Split II contained Dasypy- rum, Peridictyon, Crithopsis, and Herteranthelium (100% BS). The Split III consisted of Hordeum (100% BS). The Split IV comprised Psathyrostachys and E. distans (100% BS). The Split V included Agropyron and E. triticeum (100% BS).

3.1.2. Pgk1 gene data Of 1554 total characters of the Pgk1 data, 438 characters were variable, and 181 characters were informative. ML analysis yielded a single phylogenetic tree (Ln likelihood ¼ 6093.5423), with the following estimated ML parameters: the assumed nucleotide frequencies A: 0.2696, C: 0.1936, G: 0.2339, T: 0.3029, the proportion of invariable sites ¼ 0.3495, gamma shape parameter ¼ 0.7996. ML and Bayesian analyses recovered the same topology. The tree illustrated in Fig. 2 was the ML tree with PP above and BS below branches. In ML tree, six clades (Clade I–VI) with consistent statistic support (>70% BS; >90% PP) were recognized (Fig. 2A). The Clade I included Aegilops/Triticum complex, Taeniatherum, Thinopyrum, Crithopsis, Henrardia, Her- teranthelium, and Secale (100% PP and 96% BS). In this clade, Aegilops/Triticum complex, Taeniatherum, and Thinopyrum formed one group (100% PP and 74% BS), and Crithopsis was placed outside this group (98% PP and 55% BS). Henrardia was sister to Herteranthelium (91% PP and 59% BS). Secale was basal to the rest of the Clade I. The Clade II contained Eremopyrum, Agropyron, and Dasypyrum (91% PP). The Clade III comprised Lophopyrum, Pseudoroegneria, and Australopyrum (100% PP and 84% BS). The Clade IV included Peridictyon. The Clade V was comprised of Psathyrostachys (95% PP). The Clade VI contained Hordeum (100% PP and 100% BS). Consistent with the groupings generated from ML analysis, phylogenetic network analysis yielded a high-resolution network, and six major splits (Split I–VI) were recovered (Fig. 2B).

3.1.3. The combined dataset The combined two-gene data matrix contains 2947 characters, of which 835 were variable characters, and 388 were parsimony informative. ML analysis yielded a single phylogenetic tree (Ln likelihood ¼ 12458.7972), with the following estimated ML parameters: the assumed nucleotide frequencies A: 0.2567, C: 0.1889, G: 0.2217, T: 0.3327, the proportion of invariable sites ¼ 0.3169, gamma shape parameter ¼ 0.8173. ML and Bayesian analyses recovered the same topology. The tree illustrated in Fig. 3 was the ML tree with PP above and BS below branches. A well-supported phylogenetic framework (70% of ingroup internodes received >70% BS and 95% PP) was generated using the combined dataset, and six clades (Clade I–VI) were distinguished depending on BS and PP values (Fig. 3A). The Clade I included Aegilops/Triticum complex, Thinopyrum, Tae- niatherum, Henrardia, Secale, Crithopsis, and Heteranthelium (100% PP and 73% BS). In this clade, Thinopyrum and Aegilops/ Triticum complex formed one group (100% PP and 86% BS), and Taeniatherum was placed outside this group. Henrardia was grouped with Secale with well-supported values (100% PP and 93% BS), and Crithopsis was sister to Heteranthelium with moderate support (100% PP and 89% BS). The Clade II contained Psedoroegneria, Lophopyrum, and Australopyrum (100% PP and 99% BS). The Clade III was comprised of Peridictyon and Dasypyrum (96% PP and 69% BS). The Clade IV consisted of Hordeum (100% PP and 100% BS). The Clade V contained Agropyron and Eremopyrum (100% PP and 83% BS). The Clade VI comprised Psathyrostachys (100% PP and 100% BS). Phylogenetic network analysis yielded a high-resolution network, and six major splits were recovered (Fig. 3B). Phylo- genetic network and tree-based phylogeny reconstruction revealed highly similar groupings.

3.1.4. Ancestral state reconstruction Ancestral states including the geographical origin, mating systems, and growing habitats of the Triticeae were traced on the ML tree inferred from the combined dataset using weighted squared-change parsimony (Fig. 4). The reconstruction of character evolution revealed a single origin of the character states of geographical range and growing habitats. The ancestral distributions for the Triticeae was most likely the Arctic-temperate region, with one dispersal event into the Mediterranean region from the Arctic-temperate region (Fig. 4A). The ancestral character of growing habitat may be perennial plant, and six transformations in growing habitat occurred between perennial and annual plants (Fig. 4B). Ancestral state reconstructions of mating systems showed a two origin, with one being cross-pollination and the other being self-pollination (Fig. 4C).

Fig. 1. Phylogeny of the Triticeae based on the Acc1 gene data. Maximum-likelihood tree was generated under GTR þ G model (A). Numbers with bold above nodes are Bayesian posterior probability values 90% numbers, and numbers below nodes are bootstrap values 50%. The capital letters in bracket indicate the genome type of the species. Phylogenetic network was generated under the NeighborNet algorithm (B). Numbers above branches are bootstrap values 90%. Abbreviations of species names are listed in Table S1. Different color labeled the geographic information of monogenomic genera. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) X. Fan et al. / Biochemical Systematics and Ecology 50 (2013) 346–360 351 352 X. Fan et al. / Biochemical Systematics and Ecology 50 (2013) 346–360

3.1.5. Divergence dating Based on the combined dataset, divergence times with 95% confidence intervals using BEAST analyses generated a time- calibrated tree (Fig. 5). Time-calibrated and tree-based phylogeny reconstruction revealed the same topology. Under a lognormal relaxed clock, the coefficient of rate variation was estimated to be 0.267 (95% C.I., 0.159–0.371), indicating that relaxed clock was appropriate. The birth rate estimated by the Yule prior is 0.124 (95% C.I., 0.082–0.170). The mean ages with 95% confidence intervals were indicated in the chronogram (Fig. 5). Time calibration analysis demonstrated that the time to the most recent common ancestor (tMRCA) of the Triticeae was dated to 18.2 MYA, and a major radiation of the Triticeae took place about 6.1–9.2 MYA. Hordeum originated about 10.1 MYA (95% C.I., 8.1–12.3), and the divergence time of Pseudoroegneria, Lophopyrum, and Australopyrum was dated to 8.0 MYA (95% C.I., 5.6–10.3). The tMRCA of Aegilops/Triticum, Thinopyrum, Taeniatherum, Henrardia, Secale, Crithopsis, Heteranthelium was 9.2 MYA (95% C.I., 7.4–10.4). The diploid Triticum and Aegilops progenitors of the A and B genomes may have diverged about 4.3 MYA (95% C.I., 3.0–4.6).

3.1.6. Variation in diversification rates and rate shift Based on the combined dataset, variation in diversification rates and rate shift were estimated. A lineage-through-time (LTT) plot, which reflected the number of lineages in each split point of the tree, revealed a more or less continuous lineage increase, and if a geological time scale were superimposed onto the plot, one might notice an implication of an increase of lineages during 6.1–9.2 MYA (bottom of Fig. 5). Results from the topological method implemented in SymmeTREE using whole-tree tests for rate-variation (M-statistics: Mr, MS, MS*, MP and MP*- presence of significant rate shifts; BI and Ic statistics to assess tree imbalance) confirmed the occurrences of both tree imbalance and significant variation in diversification rates among the Triticeae lineages (Table 1). The Slowinski-Guyer (SG) and delta statistic help to localize the diversification rate shift to a certain area of the phylogeny. Using the SG and delta likelihood rate shift statistics, marginally significant P-values for a rate shift deep in the tree were obtained (D1 ¼ 0.0522, D2 ¼ 0.0556; SG ¼ 0.0556) (Fig. 5), indicating the presence of a single shift point of diversification rate.

4. Discussion

4.1. Phylogeny of the Triticeae

Morphological and molecular analyses have increased understanding of the Triticeae phylogeny. Many previous phylogenetic reconstructions were based on either a limited number of genes, in most cases only one (Petersen and Seberg, 1997; Hsiao et al., 1995; Kellogg and Appels, 1995; Helfgott and Mason-Gamer, 2004; Mason-Gamer, 2005), or incomplete sampling of genera (Kellogg and Appels, 1995; Escobar et al., 2011). The many conﬂicts among published trees and unresolved branching details among genera and species, together with incomplete sampling of genera, prevented a clear picture of the relationships among members of the Triticeae. In this study, we carried out phylogenetic reconstruction based on combined information from two unlinked loci. Despite the absence of several genome donor (G genome, Festucopsis; M genome, Aegilops comosa; U genome, Aegilops umbellutata; T genome, Aegilops mutica; N genome, Aegilops uniaristata; Xa genome, Hordeum marinum; Xu genome, Hordeum murinum), our sample represented nearly all of the monogenomic genera accepted in genome-based classiﬁcations of the tribe. Relatively wide sampling and combined gene data increase the possibility to elucidate phylogenetic relationships within the Triticeae.

4.1.1. Subdivision of the Triticeae Data from geographic distributions (Sakamoto, 1973) and rDNA-ITS sequences (Hsiao et al., 1995) have suggested that the Triticeae should be classiﬁed as two major groups, a Mediterranean group and an Arctic-temperate group. Those genera in the Mediterranean group are restrictedly distributed in Mediterranean and adjacent regions, and those genera in the Arctic- temperate group are widely distributed in Arctic-temperate regions. In the present Acc1 gene tree, all the monogenomic genera from Mediterranean and adjacent regions were placed in the Clade I and II, and those genera from Arctic-temperate regions (except Pseudoroegneria and Australopyrum) were placed in the Clade III, IV, and V, respectively. In the Pgk1 gene tree, all the monogenomic genera from Mediterranean and adjacent regions (except Dasypyrum, Lophopyrum, and Peridictyon) formed the Clade I, and those genera from Arctic-temperate regions were placed in different clade (Clade II, III, IV, V, and VI). In the combined gene tree, all the monogenomic genera from Mediterranean and adjacent regions (except Peridictyon, Dasypyrum, and Lophopyrum) formed one clade (Clade I), and those genera from Arctic-temperate regions formed para- phyletic clade (Clade II, IV, V, and VI). Phylogenetic network analysis also presented similar patterns. These results suggest that lineages from Mediterranean and Arctic-temperate regions were evolutionarily distinct, which is in good agreement with the suggestion of Sakamoto (Sakamoto, 1973) and Hsiao et al. (1995).

Fig. 2. Phylogeny of the Triticeae based on the Pgk1 gene data. Maximum-likelihood tree was generated under GTR þ G þ I model (A). Numbers with bold above nodes are Bayesian posterior probability values 90% numbers, and numbers below nodes are bootstrap values 50%. The capital letters in bracket indicate the genome type of the species. Phylogenetic network was generated under the NeighborNet algorithm (B). Numbers above branches are bootstrap values 90%. Abbreviations of species names are listed in Table S1. Different color labeled the geographic information of monogenomic genera. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 3. Phylogeny of the Triticeae based on the combined Acc1 and Pgk1 gene data. Maximum-likelihood tree was generated under GTR þ G þ I model (A). Numbers with bold above nodes are Bayesian posterior probability values 90%, and numbers below nodes are bootstrap values 50%. The capital letters in bracket indicate the genome type of the species. Phylogenetic network was generated under the NeighborNet algorithm (B). Numbers above branches are bootstrap values 90%. Abbreviations of species names are listed in Table S1. Different color labeled the geographic information of monogenomic genera. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) 354 .Fne l iceia ytmtc n clg 0(03 346 (2013) 50 Ecology and Systematics Biochemical / al. et Fan X. – 360

Fig. 4. Ancestral state reconstructions were traced on ML tree inferred from the combined Acc1 and Pgk1 gene data using weighted squared-change parsimony. Reconstruction of ancestral state of geographic distribution (A). Reconstruction of ancestral state of growing habitat (B). Reconstruction of ancestral state of mating systems (C). Transitions indicated with asterisks above nodes. X. Fan et al. / Biochemical Systematics and Ecology 50 (2013) 346–360 355

Fig. 5. Time-calibrated phylogeny, diversiﬁcation rate shift, and lineage-through-time (LTT) plot were estimated based on the combined gene dataset. A major radiation of Triticeae diverged about 6.1–9.2 MYA is labeled with pink bar. The capital letters in bracket indicate the genome type of the species. Different color labeled the geographic information of monogenomic genera. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

4.1.2. Basal relationships within the tribe Sakamoto (1973) speculated that Psathyrostachys may be the earliest members of the Triticeae. Based on the development of spicate inﬂorescence in Poaceae, Kellogg et al. (1996) speculated that the common ancestor of the Triticeae had a spicate inﬂorescence with three spikelets per node and give rise to lineages leading to the present-day Psathyrostachys and Hordeum, but the order in which these lineages arose is uncertain. Some previous data from nuclear 5S rDNA (Kellogg and Appels, 1995), GBSSI (Mason-Gamer, 2001), DMC1 (Petersen et al., 2006), chloroplast DNA (Petersen and Seberg, 1997; Mason-Gamer et al., 2002), and 27-gene combined molecular dataset (Escobar et al., 2011) showed a hierarchical tree topology with

Table 1 The probabilities obtained in whole-tree tests for diversiﬁcation rate-variation.

Frequentiles BI Mr MS MS* MP MP* Ic 0.025 0.14286 0.02343 0.00451 0.00257 0.03465 0.02460 0.01882 0.975 0.32091 0.39673 0.09392 0.08541 0.13001 0.13709 0.13203 356 X. Fan et al. / Biochemical Systematics and Ecology 50 (2013) 346–360

Psathyrostachys and then Hordeum branching off sequentially as sister groups to all other genus in the Triticeae. Other data from b-amylase gene (Mason-Gamer, 2005), ITS sequence (Hsiao et al., 1995), EF-G (Petersen et al., 2006), and morphology (Seberg and Frederiksen, 2001) did not place the Psathyrostachys at the basal position. In this study, the basal position of the Acc1 gene tree included Agropyron and E. triticeum, and they were followed by Psathyrostachys and E. distans. In the Pgk1 gene tree, Hordeum was basal to the rest of the Triticeae, which was followed by Psathyrostachys. In the combined gene tree, the basal position of the phylogenetic tree included Psathyrostachys, Agropyron and Eremopyrum. Phylogenetic network analysis showed a reticulate pattern (the bootstrap support value is 96%) including Psathyrostachys, Agropyron, Eremopyrum and Hordeum, which is an indicative of incomplete lineage sorting of ancestral polymorphism among these four genera. Combined with previous data (Kellogg et al., 1996; Petersen and Seberg, 1997; Mason-Gamer et al., 2002; Kellogg and Appels, 1995; Sakamoto, 1973; Petersen et al., 2006), we prefer the suggestion that Psathyrostachys is basal to the rest of the tribe.

4.1.3. Relationship between Agropyron and Eremopyrum Eremopyrum is a genus with four species including two diploids (E. triticeum and E. distans), one tetraploid (Eremopyrum orientale), and one species with both diploid and tetraploid cytotypes (Eremopyrum bonaepartis)(Frederiksen, 1991). Phy- togeographically, they are distributed in the Mediterranean, Central Asia and northern China. Some speciﬁc features, such as one-keeled glumes and caryopsis morphology, are highly similar between Eremopyrum and Agropyron (Frederiksen, 1991), and Eremopyrum has even been described as an annual crested wheatgrass (Agropyron)(Barkworth, 1998). Recent studies including E. triticeum and/or E. distans based on DMC1 (Petersen et al., 2006), EF-G (Petersen et al., 2006), cpDNA (Petersen and Seberg, 1997; Mason-Gamer et al., 2002) have showed that Eremopyrum was sister to Agropyron. Data from b-amylase (Mason-Gamer, 2005) and 27-gene combined dataset (Escobar et al., 2011) showed a dual placement of Eremopyrum, with E. distans and/or E. orientale being grouped with Agropyron and E. bonaepartis being clustered with Henrardia. In the GBSSI gene tree, E. bonaepartis was placed in a group including Agropyron and Eremopyrum (Mason-Gamer, 2001). The present Acc1 gene data suggested the non-monophyletic origin of Eremopyrum, with E. distans being grouped with Psathyrostachys (97% PP and <50% BS) and E. triticeum being clustered with Agropyron (100% PP and 100% BS). In the Pgk1 gene tree, E. triticeum and E. distans were clustered with Agropyron with weak statistic support (90% PP and 60% BS). In the combined gene tree, E. triticeum and E. distans were clustered with Agropyron with well statistic support (100% PP and 96% BS). The placement of E. bonaepartis, however, is unclear because the species was not included in our study. Despite the non-monophyly of Eremopyrum indicated by the Acc1 gene data, the accumulated data indicate that Eremopyrum is closely related to Agropyron.

4.1.4. Branching position of Pseudoroegneria Previous published phylogenies inferred from morphology (Seberg and Frederiksen, 2001), the integrated repetitive nuclear loci (5S and ITS) (Kellogg et al., 1996), GBSSI (Mason-Gamer, 2001), b-amylase (Mason-Gamer, 2005), DMC1 (Petersen et al., 2006; Petersen and Seberg, 2002), ITS sequence (Hsiao et al., 1995) and cpDNA (Mason-Gamer et al., 2002) failed to lead to any consensual deﬁnition of the clade involving Pseudoroegneria. Analysis of a 27-gene combined data also suggested that the branching position of Pseudoroegneria remains uncertain (Escobar et al., 2011). Taken the published data together, the branching relationship of Pseudoroegneria was reported to be related to Lophopyrum, Dasypyrum, Australopyrum, Heter- anthelium, Peridictyon, and Taeniatherum. In this study, both the Acc1 and Pgk1 gene data showed that Pseudoroegneria, Lophopyrum, and Australopyrum formed one group. In the combined gene phylogeny, Pseudoroegneria was grouped with Lophopyrum with well statistic support (100% PP and 100% BS), and Australopyrum was placed outside the group (100% PP and 100% BS). Phylogenetic network graph showed that the branching length of node including Australopyrum, Lophopyrum and Pseudoroegneria is less than that of node including Lophopyrum and Pseudoroegneria, indicating that Australopyrum may be the earliest diverged taxa among these three genera. These results suggest that Pseudoroegneria is more closely related to Lophopyrum than to Australopyrum. Relatedness of Pseudoroegneria and Lophopyrum may reﬂect their nearly identical spike and spikelet morphology.

4.1.5. Relationship among Henrardia, Secale, Crithopsis, and Heteranthelium This subclade, though supported on the present combined gene tree, is not recovered on either the Acc1 or the Pgk1 gene tree and even any other published Triticeae trees. The placement of each of the four taxa on the GBSSI (Mason-Gamer, 2001), DMC1 (Petersen and Seberg, 2002), PEPC (Helfgott and Mason-Gamer, 2004), EF-G (Petersen et al., 2006), cpDNA (Mason- Gamer et al., 2002), and integrate highly repetitive gene (Kellogg et al., 1996) tree has not received a consensus position. In the present Acc1 and combined gene tree, Henrardia was grouped with Secale with moderate support (100% PP and >70% BS), and phylogenetic network graph based on the combined dataset also suggested a relative relationship between Henrardia and Secale (96.2% BS). This is in agreement with the result of the integrated repetitive gene data (Kellogg et al., 1996). The combined gene tree also showed that Crithopsis was sister to Heteranthelium with well statistic support, indicating a relative relationship between these two taxa. Relatedness of Crithopsis and Heteranthelium may reﬂect their similar spikelet morphology. Clayton and Renvoize (1986) regarded Heteranthelium as an advanced off-shoot of Crithopsis in morphological characteristic.

4.1.6. Relationship among Lophopyrum, Thinopyrum, and Triticum/Aegilops group Much attention has been paid on the relationship among Lophopyrum, Thinopyrum, and Triticum/Aegilops group, because Lophopyrum and Thinopyrum and their polyploid descendants are the potentially valuable source for improvement of wheat X. Fan et al. / Biochemical Systematics and Ecology 50 (2013) 346–360 357

(Triticum/Aegilops)(Wang, 2011). Löve (1984) designated J genome for Thinopyrum bessarabicum and E genome for Lopho- pyrum elongatum, respectively. Chromosome pairing analysis suggested that the genomes of Lo. elongatum and Th. bessarabicum were similar enough to be designated a single genome (E) (Wang and Hsiao, 1989). Data from the integrated repetitive nuclear loci (5S and ITS) (Kellogg et al.,1996) and b-amylase gene (Mason-Gamer, 2005) showed that Th. bessarabicum was not sister to Lo. elongatum, and they were included in one clade including Triticum/Aegilops, Crithopsis, and Taeniatherum. In the DMC1 gene tree, Triticum/Aegilops were split into two clades, one of which included Th. bessarabicum, and the other contained Lo. elongatum and Crithopsis (Petersen et al., 2006; Petersen and Seberg, 2002). Analysis of nuclear GBSSI (Mason-Gamer, 2001) and ITS sequence (Hsiao et al., 1995) showed that Th. bessarabicum was grouped with Lo. elongatum and they were clustered with Triticum/Aegilops with weak support. Data of cpDNA sequence supported that Th. bessarabicum was sister to Lo. elongatum with well support, but showed that any of each did not grouped with Triticum/Aegilops (Mason-Gamer et al., 2002). In this study, the Acc1 gene tree showed that Th. bessarabicum was placed outside the Triticum/Aegilops group but with weak support, and Lo. elongatum was included into the clade containing Pseudoroegneria and Australopyrum. Our Pgk1 gene tree also presented similar pattern of groupings. In the combined gene tree, Th. bessarabicum was sister to the Aegilops section Sitopsis and nested in Triticum/Aegilops group with moderate support (100% PP and 86% BS), while Lo. elongatum was grouped with Pseudoroegneria (100% PP and 100% BS). This suggests that Th. bessarabicum and Lo. elongatum are mostly evolutionarily distinct, and the relationship between Th. bessarabicum and Triticum/Aegilops is more closely than that between Lo. elongatum and Triticum/Aegilops.

4.2. Diversiﬁcation of the Triticeae

Grass pollen and flowers did not appear in the fossil record until between 60 and 55 Ma, and it was not until after the Miocene (5–23 MYA) that grasses became the dominant species in many ecosystems (Jacobs et al., 1999; Kellogg, 2001). Analysis of diverse grass fossils from the Miocene indicated a scenario of ongoing taxonomic diversification within Poaceae in tandem during this time (Jacobs et al., 1999). Based on geographic distribution patterns, Sakamoto (1973) speculated that the Triticeae might originate about in the middle of the Tertiary period (20–30 MYA). Molecular dating estimated that the separation of the Triticeae from Aveneae–Pooeae took place the Miocene (Inda et al., 2008). The present time calibration analysis showed that the most recent common ancestor (tMRCA) of the Triticeae was dated to 18.2 MYA (95% C.I., 15.8–19.0), and the Triticeae may have undergone ongoing evolutionary diversification during the period of 0.8–18.2 MYA since its origin. This is in good agreement with previous suggestions (Sakamoto, 1973; Jacobs et al., 1999; Inda et al., 2008). Sakamoto (1973) and Kellogg et al. (1996) hypothesized that the common ancestor of the Triticeae gave rise to lineages leading to present-day Psathyrostachys. Consistent with these suggestions, our time calibration estimate also showed that the first divergence occurred between Psathyrostachys and Agropyron/Eremopyrum (17.4 MYA), and subsequent divergence followed between Hordeum and the remaining genera of the Triticeae (16.9 MYA). The estimates of the origin of Hordeum (10.1 MYA) and the divergence (4.3 MYA) among the A genome (Triticum), B genome (Aegilops speltoides ssp. ligustica), and D genome (Aegilops tauschii) are also congruent with previous results of molecular dating (Gaut, 2002; Huang et al., 2002). Australopyrum, an Australasia’s native Triticeae genus, is restrictedly distributed in fertile semi-areas of Australia, New Zealand, and Papua New Grinea. The present distribution of Australopyrum might result from either Gondwanan origin or migration event. Geological evidence suggested that the creation of Australia and New Zealand resulted from the break-up of Gondwana, and Australia had a long history of separation from Antarctica with the formation of shallow seas at about 55 Ma and full separation in the early Oligocene (35 MYA) (Crook, 1981; Upchurch, 2008). However, grass pollen and fossil plant did not appear in the fossil record in Australia until the Miocene (Kershaw et al., 1994). Our molecular dating analysis showed a diversification occurring about 8 MYA. Combined with geological and fossil plant evidence, it can be suggested that the timing of the continental separation of Australia from the break-up of Gondwana was significant prior to that of the entry of Aus- tralopyrum into Australia. We thus prefer the explanation of migration to Australia. Previous studies based on morphological and geographic data (Sakamoto, 1973; West et al., 1988) and DNA information (Appels and Baum, 1992) implied a Eurasia origin of the Triticeae. The present ancestral state reconstructions of geographical range suggested that ancestry of the Tri- ticeae was distributed in the Arctic-temperate region (Fig. 4A), and time-calibrated phylogeny also implied that the earliest- diverging taxa included Eurasian Psathyrostachys, Agropyron, and Ermopyrum (Fig. 5). This is consistent with the suggestion of the Eurasian origin of the tribe. Evidence from geological and plate tectonic evolution have indicated that the Australian plate collided with Sundaland (South-east Asia and the Indonesian archipelago) in the Miocene (Powell et al., 1981; Pubellier et al., 2003), which led to the emergence of island and land bridge. Land bridges repeatedly connected some areas, including New Guinea and Australia, and Indochina, Sumatra, Java, and Borneo on the Sundaland shelf, allowing overland migration among the Asian mainland, Sundaland, and Australia in the late Miocene (5–10 MYA) (Nauheimer et al., 2012). Therefore, it is possible that Australopyrum might originate from colonization via South-east Asia during the late Miocene and was evolutionarily distinct from other Triticeae plants accompanied with the isolation of island. High plant-species diversification has been found to be strongly linked to climate fluctuations (Jaramillo et al., 2006; Hoorn et al., 2010). Our LTT plot reveals an increase of most Triticeae lineages during a relatively narrow period of time (6.1–9.2 MYA), indicating a major radiation of the Triticeae (Fig. 5). This diversification pattern may be related to historical climate fluctuations. During the late Miocene (5–10 MYA), atmospheric CO2 level was decreased to the bottom of the Miocene after the mid-Miocene climate optimum (14–16 MYA) (Tripati et al., 2009), resulting in climate change from greenhouse to icehouse. The rapid upliftings of the Himalayan Mountains and the Qinghai-Tibetan Plateau occurred about 8 MYA have 358 X. Fan et al. / Biochemical Systematics and Ecology 50 (2013) 346–360 enhanced aridity in the Asian interior, promoted the presence of Asian monsoons, and even affected significantly Northern Hemisphere climate change (An et al., 2001). Therefore, it is possible that the radiation of the Triticeae might have been triggered by the late Miocene climate oscillations. This possibility is further strengthened by the expansion of the habitats of its current species. The habitats preferred by most Triticeae species, especially those distributed in Mediterranean region and Central Asia, are cold and dry alpine meadow, steppe desert and dry slopes. However, several Triticeae species (e.g. Agropyron, Pseudoroegneria) can grow in shady and moist forests. Changing climatic conditions might enhance habitat isolation and well have promoted rapid speciation and radiation of the Triticeae in small, isolated populations. A recent study based on multi- locus time-calibrated phylogeny of Psoraleeae (Fabaceae) species also suggested that climate oscillations are especially influential on change in plant habitats (Egan and Crandall, 2008). It is worth pointing out that significant variation in diversification rates among Triticeae lineages was detected using the whole-tree statistics, and the delta statistics find statistic support for a single shift point of diversification rate leading to the majority of Mediterranean lineage, with shift age being dated to 12 MYA (Fig. 5). Habitats, mating systems, climate oscillations, and even a mixed process of them may associate with such diversification shifts. Movement of ancestral species from one habitat to another may spurred diversification, relegating climate-based habitat as a key innovation. Most Mediterranean lineage in the Triticeae reside in more xeric habitats characterized by hot, dry environments such as the deserts of Medi- terranean regions whereas Lo. elongatum and Th. bessarabicum reside in more mesic environments characterized by warm, moist, humid climates of Mediterranean coasts. In addition, ancestral state reconstructions for growing habitat showed one transformation occurred between clade I and II (Fig. 4B), indicating a habitat shift from perennial (Clade II) to annual (Clade I) plant. Change in mating systems could result in diversification shifts. Ancestral state reconstructions for mating system in- dicates one transformation between clade I and II (Fig. 4C), which is indicative of a shift of mating system from cross- pollination (Clade II) to self-pollination (Clade I). It has been well documented that much terrestrial biodiversity and many species’ evolutionary trajectories were drastically influenced by the climate oscillations during the late Miocene (MacFadden, 2006; Yesson and Culham, 2006). The main diversification of Mediterranean lineage in the Triticeae (most are annual plant) occurred about 9 MYA when Mediterranean climates (which is characterized by hot, dry summers alternating with cool, wet winters) are thought to have arisen. The development of the Mediterranean climate can be seen as the opening of a new and novel climatic niche, to which lineages have adapted and speciated, by accumulating morphological change in other climate zones (Yesson and Culham, 2006). It is thus likely that climate oscillations during the late Miocene, especially the estab- lishment of the Mediterranean climate, might promote Mediterranean lineage of the Triticeae rapid diversification and adaptation and have continued to diversify through the Quaternary up to the present. We can not rule out the possibility that a mixed process in habitats, mating systems, and climate oscillations could result in diversification rate shifts because uneven diversification is evident in both taxonomic and geographical realms, and no single hypothesis could complete explain the phenomena of uneven diversification (Egan and Crandall, 2008). Despite this, diversification rate shifts in Mediterranean lineage of the Triticeae not only spur the occurrence of many endemic lineages (e.g. Henrarida, Heteranthelium, Crithopsis, Taeniatherum, Secale, Triticum) but also provide opportunity for polyploid crop origin and domestication.

Acknowledgments

The authors are thankful to the National Natural Science Foundation of China (Nos. 30900087, 30901052, 31101151, 31200252, and 31270243), Special Fund for Agro-Scientiﬁc Research in the Public Interest of China (Nos. 201003021), and the Science and Technology Bureau (Nos. 2060503) and Education Bureau (Nos. 10ZA045) of Sichuan Province, China for the ﬁnancial support. We are very grateful to American National Plant Germplasm System (Pullman, Washington, USA) providing the part seed materials.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bse.2013.05.010.

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