The phylogeny of the BEP clade in grasses revisited: Evidence from the whole-genome sequences of chloroplasts

Abstract

Despite the considerable efforts to reconstruct the phylogeny of grasses, the relationships among the subfamilies Bambusoideae, Pooideae and Ehrhartoideae in the BEP clade remain unresolved. Here we completely sequenced three chloroplast genomes of representative species from Bambusoideae and Ehrhartoideae and obtained 19 additional chloroplast genome sequences of other grasses from GenBank. Using sequences of 76 chloroplast protein-coding genes from the 22 grass species, we fully resolved the phylogeny of the BEP clade. Our results strongly supported the (B,P)E hypothesis, i.e., Bambusoideae and Pooideae are more closely related than Ehrhartoideae. This result was not biased by systematic or sampling errors and was impervious to phylogenetic methods or model specification. The divergence time estimate suggests that the initial diversification of the BEP clade into three subfamilies happened within a short time period (∼4 MY). The presence of these short internal branches may explain the inability of previous studies to achieve a confident resolution of the BEP clade. The combination of the sequences of the entire chloroplast genomes provided sufficient phylogenetic information to resolve the BEP phylogeny fully. These results provide a valuable evolutionary framework for comparative and functional genomic studies using the grass family as a model system.

Introduction

The grass family (Poaceae) is one of the most diverse angiosperm families and consists of approximately 700 genera and more than 10,000 species (Clayton and Renvoize, 1986, GPWG (Grass Phylogeny Working Group), 2001). The grasses not only include many economically important crops, such as rice (Oryza sativa), corn (Zea mays), wheat (Triticum aestivum) and sorghum (Sorghum bicolor), but also dominate various natural and agricultural landscapes of the world (Clayton and Renvoize, 1986). With the accomplished and ongoing genome sequencing of many cereal crops, grass species have become a model system for functional and comparative genomic research.

In the past decades, the phylogenetic relationships of Poaceae have been the focus of extensive studies. An evolutionary framework has been thoroughly established at the family level (Clark et al., 1995, GPWG (Grass Phylogeny Working Group), 2001, Duvall et al., 2007, Bouchenak-Khelladi et al., 2008, Vicentini et al., 2008, Kellogg, 2009). To date, two major clades within the family have been identified, with one comprising the three subfamilies Bambusoideae, Ehrhartoideae, and Pooideae (BEP clade) and the other consisting of the seven subfamilies Panicoideae, Arundinoideae, Chloridoideae, Centothecoideae, Micrairoideae, Aristidoideae, and Danthonioideae (PACCMAD clade) (GPWG (Grass Phylogeny Working Group), 2001, Duvall et al., 2007, Bouchenak-Khelladi et al., 2008, Kellogg, 2009). However, the phylogenetic relationships within these clades have not been fully resolved (GPWG (Grass Phylogeny Working Group), 2001, Bouchenak-Khelladi et al., 2008, Kellogg, 2009). Using the plastid ndhF sequences, Clark et al. (1995) first recovered a tree with two major groups (the PACC and BOP clades) and found that bambusoid (i.e., Bambusoideae) and oryzoid (i.e., Ehrhartoideae) clustered together and were sister to pooid (i.e., Pooideae). The (B,E)P relationship, with Bambusoideae and Ehrhartoideae being more closely related than Pooideae, was confirmed by subsequent studies (GPWG (Grass Phylogeny Working Group), 2001, Vicentini et al., 2008, Sungkaew et al., 2009). Nevertheless, many other studies have revealed either a sister relationship of Bambusoideae and Pooideae, (B,P)E (Zhang, 2000, Bouchenak-Khelladi et al., 2008, Leseberg and Duvall, 2009, Peng et al., 2010) or a sister relationship of Ehrhartoideae and Pooideae, (E,P)B (Mason-Gamer et al., 1998, Mathews et al., 2000). The problem of the relationship among the BEP lineages is similar to the well-known “trichotomy problem” involving chimpanzee, gorilla, and human (Satta et al., 2000) in which all three possible relationships have been recovered in various studies.

Apart from the inconsistent phylogenies within the BEP clade, it is noteworthy that the different topologies were generated if different sampling strategies (gene and taxon) were employed and if different methods of analysis were used. For instance, based on sequences of 43 orthologous loci selected from 10,608 putative full-length cDNA sequences of bamboos, Peng et al. (2010) obtained different phylogenetic relationships within the BEP clade using different phylogenetic methods. Ehrhartoideae was the basal lineage on the maximum likelihood and Bayesian trees, i.e., (B,P)E. In contrast, Pooideae was the basal lineage on the Neighbor-Joining tree, i.e., (B,E)P. Therefore, despite considerable efforts to reconstruct the phylogeny of the grasses, the BEP relationship remains unclear.

The cpDNA sequences are increasingly used for resolving the deep phylogeny of plants because of their low rates of nucleotide substitutions and structural changes (Soltis et al., 2004, Jansen et al., 2007, Moore et al., 2010). Concatenating sequences from many genes may overcome the problem of multiple substitutions that results in a loss of phylogenetic information between chloroplast lineages and can reduce sampling errors due to substitutional noise (Delsuc et al., 2005). Here, we sequenced the complete chloroplast genomes of three grass species and obtained 19 additional chloroplast genomes of grasses from GenBank. Using sequences of 76 plastid protein-coding genes (70,700 bp in total) that are derived from the complete plastid genomes of the 22 grass species, we were able to resolve the phylogenetic relationships of the BEP clade fully. Based on the divergence time estimates, we found that two rapid speciation events within the BEP clade occurred in a small time interval (roughly 4 million years). These events might be responsible for the inconsistent phylogenetic reconstructions that have been reported in previous studies.