The Evolutionary History of Ehrhartoideae, Oryzeae, and Oryza
Rice is a member of the genus Oryza, which has a history extending back into the Miocene. Oryza is in turn a member of the tribe Oryzeae, which along with the tribe Ehrharteae is included in the subfamily Ehrhartoideae. This paper reviews current knowledge of the genus, tribe and subfamily, and places rice in the larger evolutionary context of the entire grass family. The morphological characteristics of rice are an amalgam of characters that have originated at different times in its long evolutionary history. Increasingly, genomic characteristics are also being placed in a broad evolutionary context and it is becoming possible see which are characteristic of all grasses and which are more restricted to the genus Oryza or even to rice itself.
KeywordsRice Phylogeny Evolution Genome Grasses Ehrharta Oryza
The position of Oryza sativa within the angiosperms
By placing rice in this very broad evolutionary context, it is possible to assess the level of generality of different aspects of the plant. Thus, for example, studies on megagametophyte development, or double fertilization mechanisms, might be expected to shed light on those processes elsewhere in the angiosperms. Conversely, a study of lemma epidermal development might be applicable only to the genus Oryza itself. Every organism is the product of all of its evolutionary history, both ancient and recent, with different characters having appeared at different times. This paper thus presents the time of acquisition of various aspects of the rice plant and the organisms to which it can be compared.
Rice is an angiosperm (flowering plant). With respect to the characteristics that it shares with all other angiosperms it thus can serve as a model for all flowering plants. Like all other angiosperms, rice produces its megagametophytes inside ovules, which are in turn enclosed in an ovary. It also shares with other angiosperms the process of double fertilization, whereby the microgametophyte (in the pollen) produces two sperm, one of which fertilizes the egg cell and one of which fertilizes the central cell to produce endosperm. It also shares with many other angiosperms the ability to produce vessels in the xylem, in addition to tracheids.
Within the angiosperms, rice belongs to the large group known as the monocotyledons, and is thus similar to onions, lilies, orchids, and the thousands of other monocot species. It is only distantly related to the models Arabidopsis and tomato, which are eudicotyledons. Rice thus provides a model for investigating characteristics that appear in monocots and not eudicots. Like other monocots, rice has a single cotyledon, whereas the eudicots have two. The primary root is unbranched and short-lived and is replaced early in plant development by a profusion of stem-borne (adventitious) roots (Stevens 2001). The eudicots in contrast generally have a long-lived and branched primary root. Rice and the other monocots also have characteristic vascular patterning. Vascular bundles are scattered in the stem (vs. arranged in a ring in the eudicots). The major veins of the leaf are arranged parallel to each other (vs. in a reticulate pattern). Intermediate leaf veins always connect with other veins, rather than ending blindly in the mesophyll as in eudicots.
Within the monocots, rice falls within the group known informally as the commelinid clade. Other members of this large group are palms (Arecaceae), pineapples (Bromeliaceae), gingers, and bananas (Zingiberales), as well as grasses, sedges, and rushes. Like all other commelinids and unlike all non-commelinid angiosperms (monocot or dicot), rice produces ferulic acid in its cell walls, which makes them fluoresce under ultraviolet light. Most commelinids also produce silica (SiO2) bodies in their leaves and have bracteate inflorescences. Commelinid stomata generally have two subsidiary cells with the long axis parallel to the stomate (paracytic stomata), or may have an additional two short subsidiaries at each end of the stomate, perpendicular to the long axis (tetracytic). All commelinids except the palms have abundant starch in their endosperm, an ancient characteristic that may have originated about 120 million years ago (Janssen and Bremer 2004). When early human agriculturalists exploited this character, they were thus taking advantage of a tissue that had been fixed by natural selection tens of millions of years earlier.
Limited comparative genomic data hints that the genomes of commelinids may have some similarities. A comparison of a region of almost two megabases (Mb) between two species of banana (Musa; Zingiberales) and rice found that both Musa and Oryza had relatively high GC content (39% and 43%, respectively), when compared either to onions (a non-commelinid monocot) or to Arabidopsis (a eudicot; 36%)(Lescot et al. 2008). Even though only a few BACs were sequenced from Musa, multiple regions were found with microsynteny to rice, including five to 11 syntenic genes.
Within the Poales, rice is a member of the Poaceae, the grass family, a family of flowering plants that includes over 10,000 species, including all the major cereal crops (Clayton and Renvoize 1986; Grass Phylogeny Working Group 2001; Watson and Dallwitz 1992). The grasses are distinguished from their poalean relatives by the structure of their embryo, their fruit, and their pollen. In most monocots, including the graminoid Poales, the embryo is scarcely differentiated when the seed is mature (Campbell and Kellogg 1987; Rudall et al. 2005). In the grasses, in contrast, the relative timing of seed maturation and embryo development are shifted such that the grass embryo has a clear shoot and root apical meristem, and two or more differentiated leaves. This could have occurred developmentally either by accelerating embryo development and keeping the seed maturation time constant, or by delaying seed maturation until the embryo has differentiated further (Kellogg 2000). Although clearly derived from a three-carpellary ovary, the grass ovary has a single locule with a single seed; the seed coat generally is appressed or even fused to the inside of the ovary wall. This unique fruit is called a caryopsis or grain.
All but two genera of Poaceae have flowers borne in spikelets ("little spikes") (Grass Phylogeny Working Group 2001). The spikelet is subtended by two sterile bracts, the glumes, and contains one or more flowers. The flowers are sometimes called florets to distinguish them from more conventional monocot flowers. The floret is subtended by a bract-like structure (the lemma). Above the lemma on the floral axis is an adaxial structure, the palea, which is commonly interpreted as a prophyll (Arber 1934), although evidence is accumulating that it might be a highly modified outer perianth whorl (Malcomber et al. 2006; Preston and Kellogg 2007). Above the palea on the floral axis and enclosed within it are two (rarely three) flap-like structures, the lodicules, that clearly represent highly modified inner tepals (Ambrose et al. 2000; Malcomber et al. 2006; Whipple et al. 2004, 2007). Rather than serving an attractive or protective function like other inner perianth whorls, the lodicules become turgid at anthesis and force the lemma and palea apart, permitting exsertion of the stamens and stigmas. Stigma number is ancestrally three, but in most grasses (including rice) has been reduced to two (GPWG 2001). Stamen number is ancestrally six, a number that also appears in rice.
Almost all species of Poaceae fall into one of two large clades, one including the Bambusoideae, Poooideae, and Ehrhartoideae (the BEP clade), and the other including Panicoideae, Arundinoideae, Centothecoideae, Chloridoideae, Micrairoideae, Aristidoideae, and Danthonioideae (the PACCMAD clade)(Bouchenak-Khelladi et al. 2008; Clark et al. 1995; Grass Phylogeny Working Group 2001; Sánchez-Ken et al. 2007) (Fig. 2). The PACCMAD clade is certainly monophyletic. In contrast, support for the BEP clade, which includes rice, was not strong in early phylogenies (Kellogg and Linder 1995). However, the two most recent phylogenies of the grasses (Bouchenak-Khelladi et al. 2008; Vicentini et al. 2008) both find strong support for monophyly of the clade.
Because rice is in the BEP clade and maize is in the PACCMAD clade, the date of the divergence of these two clades is also the maize–rice divergence date. A recent study places the maize–rice divergence at 51.6 ± 7.9 million years ago (Vicentini et al. 2008). This estimate is quite similar to the 50-million-year date commonly attributed to the comprehensive review of grass evolution provided by Gaut (2002), who based the date in turn on a citation from Stebbins (1981).
Within the BEP clade, monophyly of the included subfamilies is unequivocal, but different studies have resolved different relationships among them. The most recent phylogenies either place Ehrhartoideae as sister to Streptogyna and the two then sister to Bambusoideae (e.g. (Grass Phylogeny Working Group 2001; Vicentini et al. 2008)), or sister to a clade comprised of Bambusoideae plus Pooideae (Bouchenak-Khelladi et al. 2008). The two studies are not comparable, however, in that Bouchenak-Khelladi et al. (2008) included only limited data for Streptogyna and thus its placement was provisional.
Vicentini et al (2008) estimated the divergence of Ehrhartoideae from Streptogyna at 43.1 ± 7.2 million years ago, during the Eocene. This was a period of earth history when global climate was considerably warmer than it is now and there were no polar ice sheets (Zachos et al. 2001).
The discrepancies among different molecular phylogenetic studies can be ascribed in part to taxon sampling. If a major group (e.g. Ehrhartoideae) is represented by only one species (e.g. rice), then DNA sequence(s) from that species will contain many mutations relative to all other species in the study. The large number of mutations effectively randomizes the sequence relative to all the other sequences, such that the rice sequence will fit about equally well (or equally badly) with multiple other sequences. Thus, the differences among phylogenies with few taxa may be simply random (see for example, Kellogg and Linder 1995). With sufficient taxon sampling and enough nucleotides, however, the BEP clade appears to be monophyletic (see for example Bouchenak-Khelladi et al. 2008).
All members of Poaceae retain the signature of a whole genome duplication (Paterson et al. 2004; Wang et al. 2005). Genomic data from a variety of cereals confirm that the duplication occurred before the common ancestor of maize and rice. Data from the early diverging grasses in subfamilies Anomochlooideae, Pharoideae, and Puelioideae are more limited, but phylogenies of many individual gene families (see e.g., Malcomber et al. 2006) indicate that the duplication occurred shortly before the common ancestor of the grass family (70.9 ± 9.2 Ma)(Vicentini et al. 2008). As expected for duplicated genes, the duplicates have generally diverged in expression pattern and presumably function (Lin et al. 2008; Malcomber et al. 2006; Preston and Kellogg 2007).
Rice belongs to subfamily Ehrhartoideae within the BEP clade. Ehrhartoideae includes the three tribes Oryzeae, Ehrharteae, and Phyllorachidae (Grass Phylogeny Working Group 2001). The placement of the latter is based solely on morphological similarity to the other two tribes; it has not been included in any phylogenetic analysis to date. Most of what we know about the subfamily is thus based on Oryzeae and Ehrharteae. The two tribes are estimated to have diverged 34.5 ± 6.8 million years ago (Vicentini et al. 2008), in the late Eocene or early Oligocene when the earth as a whole was becoming cooler and global CO2 concentration was dropping to near its pre-industrial levels. Guo and Ge (2005) cite a fossil member of Oryzeae from this time period (Litke 1968), consistent with the molecular date, but Thomasson (1987) referred this fossil simply to an unidentified grass, not specifically oryzoid.
The ancestral spikelet structure for Ehrhartoideae is a three-flowered spikelet in which the lower two flowers are sterile; this spikelet structure is uniquely derived (synapomorphic) for the subfamily (Grass Phylogeny Working Group 2001). Members of tribe Ehrharteae exhibit this structure clearly, with two glumes, two proximal sterile lemmas (remnants of the lower two flowers), and a single hermaphrodite floret. The internodes between these structures (rachilla) may be more or less elongated. Many members of the tribe Oryzeae, including O. sativa itself, retain this same ancestral structure, except that the glumes are minute ("rudimentary glumes" (Bommert et al. 2005)) (Fig. 3). In other members of Oryzeae, the glumes are reduced to a cup-like rim. In addition, the sterile lemmas may be small or missing. In some species in which glumes and/or sterile lemmas are missing, the elongated internode then appears simply as a long stalk, sometimes called a stipe, above the glumes or above the sterile lemmas (Watson and Dallwitz 1992).
Ample evidence indicates that the rice spikelet is like that of other Ehrhartoideae in having three flowers, of which only the uppermost (distal) is fertile, an interpretation first suggested by Stapf (1917). In addition to the argument from comparative morphology and evolutionary relationship, outlined in the previous paragraph and also discussed by Malcomber et al. (2006), genetic studies show that overexpression of floral genes affects the sterile lemmas but not the true glumes (Komatsu et al. 2003; Prasad et al. 2001). Nonetheless, the interpretation of the rice spikelet as having only one flower persists in the literature and some authors (e.g. Terrell et al. 2001) continue erroneously to refer to the sterile lemmas as "empty glumes".
Members of the subfamily differ in the number of stamens. The ancestral number is clearly six, a number that is retained in Oryza. However, one clade of Microlaena and Tetrarrhena species includes plants with only four stamens, Ehrharta delicatula and Ehrharta triandra have stamen number reduced to three, and another clade that includes Zotovia, two species of Microlaena and Ehrharta avenacea shares a reduction of stamen number to two (Verboom et al. 2003)(Fig. 3). Independently, stamen number is reduced to one in Chikusichloa (Watson and Dallwitz 1992), whereas an increase in stamen number is reported for some species of Luziola (Watson and Dallwitz 1992).
The leaf anatomy of Ehrhartoideae was studied in detail by Tateoka (1963) and later by Watson and Dallwitz (1992). All species of both tribes have a double bundle sheath around the veins, as is true in all C3 grasses, with the outer sheath parenchymatous and the inner with thick walls. All species investigated have microhairs somewhere on the leaf surface; these are generally bicellular and elongate, as is common in most grasses, but Watson and Dallwitz (1992) report that the microhairs in some species appear to be unicellular. In all Oryzeae, except the aquatic genus Chikusichloa, mesophyll cells have deeply invaginated cell walls (arm cells), which are otherwise found only in bamboos, some early diverging grasses, and a handful of members of the PACCMAD clade; arm cells are lacking in Ehrharteae (Fig. 3). In addition, large open cells in the mesophyll (fusoid cells) occur in Zizania, Zizaniopsis, Chikusichloa, Hygroryza, and Rhynchoryza (Tateoka 1963). Fusoid cells are also known only in bamboos and in the early-diverging grasses (Grass Phylogeny Working Group 2001). The function of these cells is unknown. The midrib of Ehrharteae species is unremarkable, but midribs in most species of Oryzeae are complex, with two or more vascular bundles placed both adaxially and abaxially (i.e. top and bottom of the leaf), and often with air spaces (Tateoka 1963) (Fig. 3). However, Luziola and Hygroryza have a simple midrib vascular structure, more similar to that in Ehrharteae. Silica bodies are present in the epidermis, particularly in the costal region (over the veins); these are mostly dumbbell shaped and elongate either perpendicular to the axis of the leaf (oryzoid type) or parallel to the axis (dumbbell type).
Most Ehrhartoideae are perennials, and this appears to be the ancestral life history for the group. The annual habit has arisen independently among the A genome species of Oryza, in Oryza brachyantha (F genome), and at least twice in Ehrharta (Fig. 3). In an F2 mapping population of O. sativa crossed with Oryza longistaminata, Hu et al. (2003) have identified multiple QTL that correlate with the presence of rhizomes, which are often correlated with the perennial habit. Two of these loci overlap with similar loci in sorghum suggesting that a few major regulators control growth form in many species of grasses. Because the switch from perennial to annual appears to occur frequently in evolutionary time, it will be interesting to learn if it reflects selection on a particular small set of loci.
Like rice, most members of Ehrhartoideae have a chromosome base number of x = 12. The only exceptions are in the genera Zizania and possibly Microlaena. Members of Zizania have 2n = 30 or 34, numbers that appear to reflect expansion of the genome and some segmental duplications. A genome map of Zizania palustris shows that the base number of x = 15 reflects a duplication of portions of rice chromosomes 1, 4, and 9 (Kennard et al. 2000). As in most other groups of grasses, polyploidy is common. Although Watson and Dallwitz (1992) report a chromosome base number of 10 for some species of Microlaena, recent data find that Microlaena avenacea, Microlaena carsei, Microlaena polynoda, and Microlaena stipoides have 2n = 48 chromosomes, so are tetraploids with a base number of x = 12 (Murray et al. 2005).
The common ancestor of Oryza and Leersia occurred in tropical Africa, and species in other parts of the world arrived there by independent dispersals. For example, the ancestor of Oryza granulata dispersed to India and China from Africa. A separate west-to-east dispersal resulted in the arrival of Oryza eichingeri and Oryza rhizomatis in Sri Lanka, and Oryza officinalis in India and eastern Asia. A third such event led to dispersal of Oryza rufipogon to India and eastern Asia. Oryza meridionalis and Oryza australiensis represent two independent dispersal events to Australia. Among other members of the tribe, Prosphytochloa and Potamophila represent another African-Australian pair. The phylogeny does not permit assessment of the direction of dispersal, but west to east is certainly plausible. Wide disjunctions have been documented for many aquatic plants (Les et al. 2003), so the broad distribution of many Oryzeae is not a surprise.
The phylogeny suggests dispersal from Australia to southern Africa in Ehrharteae. However, this could be an analytical artifact. The tree presented here is a grafted tree, created by simply pasting the phylogeny of Verboom et al. (2003) on to that of Guo and Ge (2005). If the Ehrharteae subclade were in fact rooted along the internode leading to the Cape Province Ehrharta species, then the ambiguity of the ancestral node would be removed and an African origin would be inferred. In addition, the parsimony optimization used here does not include any estimation of branch lengths, and thus does not take in to account the time available for long distance dispersal. Finally, all biogeographic analyses are painfully sensitive to taxon sampling and tree topology; inclusion of more species and/or minor changes in the phylogeny could easily affect the inference. A more rigorous analysis of the entire subfamily will be required to develop a better estimate of biogeographic history.
The phylogeny indicates at least three independent colonizations of North America: one each in Leersia, Zizania, and Luziola. It is likely that each occurred via a different route. For example, Chikusichloa occurs as far north as Japan, as does Zizania, suggesting that dispersal between Asia and North America may have occurred via a northern route. Conversely, Luziola is suggested to be the result of northward migration of South American species and Leersia represents a connection between Africa and North America. Taxon sampling in all three genera is poor outside of North America and many species have yet to be included in molecular phylogenetic analyses. Fortunately, a morphological phylogeny and monograph of the nine species of Luziola have been published recently, setting the stage for molecular phylogenetic work (Martínez-y-Pérez et al. 2005, 2008). A full understanding of the history of Oryzeae will require investigation of all species within these widespread genera.
All evidence points to the ancestor of Ehrhartoideae being a plant of moist habitats. All members of Oryzeae grow in damp areas, many occupy flooded sites, and still others (e.g. Hygroryza) are truly aquatic. The ancestors of Ehrharteae are reconstructed as being plants of high rainfall areas (689–1,400 mm per annum) (Verboom et al. 2003). Ehrharta species later radiated in the much drier habitats of the fynbos of South Africa, probably during the late Miocene. This represents a shift from habitats with year-round rainfall to those with winter rainfall only. Many grasses are plants of moist shade (e.g. all members of Anomochlooideae, Pharoideae, and Puelioideae, many Bambusoideae) and many species grow at least partially submerged in water (e.g. Glyceria in subfamily Pooideae, Phragmites in Arundindoideae, Spartina in Chloridoideae, to name just a few), so the Ehrhartoideae are not particularly unusual in this aspect of their biology.
The tribe Oryzeae
Oryzeae includes nine or ten genera and 60 to 70 species, and is very likely monophyletic (Ge et al. 2002; Guo and Ge 2005; Kellogg and Watson 1993). In all studies to date, the number of outgroup taxa included is not large enough to rule out completely the possibility of paraphyly, but available morphological and molecular data suggest that this is unlikely.
The phylogeny of Oryzeae has been addressed by Ge et al. (2002) and by Guo and Ge (2005). Both nuclear and chloroplast genes find that Oryza and Leersia are sister genera (Fig. 3). Sister to the Oryza/Leersia clade is a clade composed of the genera Prosphytochloa, Potamophila, Chikusichloa, Rhynchoryza, Zizania, Zizaniopsis, and Luziola. The position of Hygroryza is uncertain; it may be sister to Oryza/Leersia, or sister to the clade comprised of Rhynchoryza, Zizania, Zizaniopsis, and Luziola. (Somewhat unhelpfully, species of Zizania are known in North America as wild rice, whereas in most other regions of the world the name "wild rice" generally refers to O. rufipogon.) The two major clades are recognized and named as subtribes (Oryzinae and Zizaniinae) by some authors (e.g. (Guo and Ge 2005)). However, the rank of subtribe is not required by the International Code of Botanical Nomenclature (Vienna Code) (2006), and formal subdivision seems unnecessary for a tribe with only a dozen genera.
The crown node of Orzyeae (divergence of Oryza–Leersia from the other genera) was placed at 20.5 mya by Guo and Ge (2005) and the divergence of Oryza from Leersia at 14.2 (Fig. 4). (Note that the methods used by Guo and Ge were different from those applied by Vicentini et al. (2008), and did not permit assessment of confidence intervals on dates.)
Several species in Oryzeae are so morphologically distinctive that previous taxonomists have given each its own genus. Molecular phylogenetic data, however, have placed these odd species in other genera. For example, Hydrochloa caroliniensis is the sole representative of the genus Hydrochloa, which is unusual in being a floating aquatic plant, with unisexual flowers (Fig. 3). The staminate and pistillate flowers are in separate inflorescences, with the staminate ones terminal on the plant and the pistillate ones axillary; the inflorescences are often reduced to a single spikelet (Watson and Dallwitz 1992). The inflorescence morphology of the genus Luziola is similar; the flowers are also unisexual and in separate inflorescences, which led Terrell and Robinson (1974) to suggest that H. caroliniensis is simply an odd species of Luziola. Accordingly, they moved H. caroliniensis to Luziola as L. fluitans. This classification is supported by molecular data (Guo and Ge 2005).
The taxonomic history of Porteresia is analogous to that of Hydrochloa, in that it is a name given to a slightly odd member of a genus. The species Oryza coarctata was originally described by Roxburgh in 1832 (Roxburgh 1832) (p. 206) based on a specimen collected in India in the Ganges Delta. Tateoka (1965) later concluded that O. coarctata was different from all other species of Oryza because it has leathery (coriaceous) leaf blades that are strongly ribbed. Curiously, each rib has a major vascular bundle near the abaxial side and a minor bundle just above (adaxial) to it. Tateoka (1964) also reported that the species has an unusually large embryo with a large free epiblast and a distinct cleft between the coleorhiza and the scutellum. (Embryo characters were relied on heavily in the pre-DNA era as evidence of phylogenetic relationship (see for example, Reeder 1957), and often do correlate with current views of phylogeny (Grass Phylogeny Working Group 2001), but not always.) Accordingly, he created the new genus Porteresia to accommodate the species and to recognize the differences formally.
Molecular phylogenies based on the chloroplast genes maturaseK (matK) and the transfer RNA Leucine (trnL) intron, the mitochondrial gene NADH dehydrogenase (nad1), and the nuclear genes alcohol dehydrogenase1 (Adh1), alcohol dehydrogenase2 (Adh2), and G protein alpha subunit1 (GPA1) (Ge et al. 1999, 2002; Guo and Ge 2005) show that P. coarctata is derived from within Oryza. Therefore, the characteristics that Tateoka observed do not indicate a separate evolutionary origin for O. coarctata. The species was returned to its original position as a member of Oryza by Lu and Ge (2004).
Placing O. coarctata back in Oryza leaves open the question of the origin of its unusual morphology, habitat (wet saline sites), and distribution (coastal India). The species is tetraploid. It may share the H genome with other tetraploids such as Oryza schlechteri and Oryza ridleyi (Fig. 3). One molecular phylogenetic study suggests a close relationship with O. schlechteri, but the latter is native to New Guinea and has leaf morphology more similar to that of other Oryza species. The composition of repeat sequences in the genome of O. coarctata is also unusual (see below). The leaf morphology of O. coarctata is reminiscent of that of some species of Ehrharta, which have radiated into dry environments. Because saline environments also create physiological challenges similar to drought, it is perhaps not surprising that the leaf morphology of O. coarctata might have converged with that of more distantly related species.
The polyploid event that led to the formation of O. coarctata may provide a clue to its odd morphology and distribution. Polyploidy may have led to extensive genomic change that affected many aspects of the genome, including expression of genes that could be selected for a novel environment (Hegarty and Hiscock 2008). Polyploid species often have geographic ranges that are broader than or quite distinct from those of their diploid ancestors, so this would not be an unexpected outcome. A second more radical hypothesis is that O. coarctata is formed from a wide cross between an ancestor similar to rice and another ancestor more similar to another member of the tribe or subfamily. Such very wide crosses have been documented in wheat relatives (e.g. (Mason-Gamer 2004)) and are unusual but not unprecedented. Deep sequencing of the genome of O. coarctata, as well as broader phylogenetic studies in other Oryzeae, could help address this question.
Taxonomists have disagreed on the number of genera that should be recognized to accommodate the five species variously assigned to Potamophila, Maltebrunia, and Prosphytochloa. Potamophila was originally described by Robert Brown (1810), Maltebrunia by Kunth (1829), and Prosphytochloa by Schweickerdt (1961). However, Duistermaat (1987) thought that this division was a mistake, given the morphological similarities among them. The molecular phylogeny of Guo and Ge (2005) supports the close relationship, at least between Potamophila and Prosphytochloa, and suggests that the two could be combined.
Monoecy has arisen twice in Oryzeae (Fig. 3). Zizaniopsis has staminate and pistillate flowers in separate inflorescences on the same plant, a characteristic that also appears in seven of the nine species of Luziola (including Hydrochloa). In the other two species of Luziola (L. caespitosa and L. brasiliensis), staminate and pistillate flowers are in the same inflorescence, with the staminate ones distal to the pistillate. Zizania also has staminate and pistillate flowers in the same inflorescence, but is somewhat peculiar among monoecious species in that the pistillate flowers are borne in the distal branches whereas the staminate ones are more proximal (lower).
Zaitchik et al. (2000) investigated the development of staminate and pistillate flowers in Zizania, and found that all florets initiated both an androecium and a gynoecium; in this respect, flowers of Zizania are similar to all other known unisexual flowers in the grasses (LeRoux and Kellogg 1999). Stamens in pistillate flowers ceased development soon after the outline of the anthers became visible. In staminate flowers, however, gynoecium development was arrested only after formation of the ovule and integuments and early development of the stylar arms. This is appreciably later than gynoecial abortion in maize or other panicoid grasses, and points to the possibility of a different underlying mechanism (Malcomber and Kellogg 2006).
Genera other than Oryza have received little attention from phylogeneticists, geneticists, or developmental biologists. Notably, Zizania, Zizaniopsis, Leersia, and Luziola all have broad geographic distributions and need to be investigated thoroughly throughout their ranges.
The genus Oryza
Oryza includes 20 to 24 species (the precise number depends on taxonomic preferences), distributed in tropical and subtropical regions of the world, and with a common ancestor dating between 10.2 and 8.8 mya (Guo and Ge 2005)(Fig. 4). An excellent web-based resource on the species of Oryza is provided by the International Rice Research Institute (IRRI), at http://www.knowledgebank.irri.org/wildRiceTaxonomy/default.htm. At this site are a key to the species, along with individual species descriptions, photos, and maps.
All species of Oryza share distinctive epidermal outgrowths or "tubercles," otherwise unknown among grasses, on the upper (fertile) lemma and palea (Terrell et al. 2001). The tubercles thus constitute a synapomorphy that provides evidence for monophyly of the genus. These outgrowths appear to be under the control of the SEPALLATA-like protein, LEAFY HULL STERILE1 (= OsMADS1); when LHS1 is over-expressed, not only do the sterile lemmas enlarge and become more like the fertile lemma, but they produce epidermal tubercles as well (Prasad et al. 2001).
Molecular phylogenies show that each of the genomic groups in Oryza is monophyletic, as expected from cytogenetic studies (Ge et al. 1999; Zou et al. 2008). The diploid species with the A genome—O. sativa, O. rufipogon, Oryza barthii, Oryza glaberrima, O. longistaminata, and O. meridionalis—all form a clade, with O. rufipogon as the sister taxon and presumed wild progenitor of O. sativa. Likewise, the C genome diploids, O. officinalis, O. rhizomatis, and O. eichingeri are sisters. The B, E, F, and G genomes are each represented at the diploid level by a single species, Oryza punctata, O. australiensis, O. brachyantha, and O. granulata respectively.
A recent phylogenomic study of the diploid species (Zou et al. 2008) used 124,079 bp in 142 genes to confirm that the A and B genome species are sisters and the C genome clade is sister to that (Fig. 3). Some of the genes suggested alternative relationships, which the authors ascribed to incomplete lineage sorting among some of the genes. If this is true, then future population samples of the various species should yield other alleles that produce the "correct" phylogeny.
The polyploid species are allotetraploids and combine the genomes of two disparate progenitors. In a few cases, the progenitor genomes can be identified as being quite similar to those of extant diploids (e.g. Oryza minuta and tetraploid O. officinalis with the B and C genomes) (Fig. 3). (Note that some authors consider tetraploid O. officinalis to be a separate species, Oryza malampuzhaensis (Thomas et al. 2001).) In other cases, however, one or both of the progenitor genomes is extinct, or enough genomic rearrangement has occurred since polyploidization that similarities to extant diploids are lost. Thus, Oryza alta, Oryza grandiglumis, and Oryza latifolia contain the C and D genomes and are likely derived from an ancestor similar to one of the C genome diploids, but chromosome pairing data indicate that the D genome is distinct from known diploids, a result supported by genomic in situ hybridization (Li et al. 2001). However, phylogenetic analyses suggest that the D genome may be derived from an ancestor similar to the E genome of O. australiensis (Bao and Ge 2004).
Chloroplast phylogenies have helped to place the maternal parent of some of the polyploids. The chloroplasts of the polyploids O. alta, O. grandiglumis, and O. latifolia are closely related to those of the C genome diploids (O. officinalis, O. rhizomatis, and O. eichingeri), indicating that the maternal parent of the CD polyploids had a C genome (Bao and Ge 2004). The chloroplasts in the polyploids O. coarctata and O. schlecteri (the latter with the H and K genomes) are closely related to each other, and share a recent common ancestor with the ancestral chloroplast of the A, B, and C genomes (Ge et al. 2002) (Fig. 3, dotted line). Likewise the chloroplast of Oryza longiglumis (HJ) appears closely related to that of O. brachyantha (F genome) (Ge et al. 2002) (Fig. 3, dotted line). Figure 3 places the other HJ polyploid, O. ridleyi, in this position as well, but this is by inference from O. longiglumis, and needs to be checked by DNA sequence data.
The genus Oryza has excellent genomic resources which are providing an unprecedented view into the highly dynamic nuclear genome (Kim et al. 2008). Recently genomic libraries and partial sequences have become available for many of them (the OMap project; http://www.omap.org).
Many lineages include characteristic retrotransposons; bursts of amplification of these transposons have led to considerable expansion of genome size. Members of Oryza share a group of closely related Ty3-gypsy retrotransposons, called the RWG family, whose common ancestor was at least as old as the genus itself (Ammiraju et al. 2007), although it is somewhat similar to the Grande elements of Zea mays (Ohtsubo et al. 1999). Although similar in their sequences, different lineages of the transposons have amplified independently in the various lineages of Oryza. Each of these independent amplifications has been given a separate name, such that the clade of transposons that occurs in O. sativa is known as RIRE2 (Ohtsubo et al. 1999), that in O. australiensis is Wallabi (Piegu et al. 2006), and that in O. granulata is called Gran3. In addition, a non-autonomous derivative of an RWG element, known as Dasheng, originated in the common ancestor of the A, B, and C genomes (Ammiraju et al. 2007) (Fig. 3).
Amplification of retrotransposons correlates with genome size. Among the diploid species, the largest genomes are in O. officinalis and O. granulata (Zuccolo et al. 2007). A project sequencing random sheared genomic libraries found that over 66% of the reads in O. officinalis involved repetitive sequences of some sort and the majority of these were retrotransposons. To make the data comparable among species, the authors divided the number of repeats by the total number of megabase pairs (Mbp) sequenced. With this calculation, O. granulata and O. australiensis (with large genomes) had the highest number of retrotransposons per Mbp, whereas O. brachyantha (with a small genome) had the lowest.
O. coarctata, despite being polyploid, has an unusually low percentage of repeat sequences, with few that are comparable to those in other species of Oryza (Zuccolo et al. 2007). Zuccolo et al. (2007) suggested that this might argue for recognition of the genus Porteresia. However, because genera are defined phylogenetically and because O. coarctata is clearly derived from within Oryza, it is preferable to consider O. coarctata as an unusual and intriguing member of the genus Oryza. Nonetheless, it would be of interest to know whether the unusual leaf morphology of the species were causally connected to the unusual lack of repeat elements in the genome. Because the leaf morphology of O. coarctata is similar to that of some species of Ehrharta, one might speculate that it represents a reversion to an ancestral type caused by a genome-scale change in gene expression.
All species of Oryza investigated to date have an array of genes encoding ribosomal RNA (rDNA, or nucleolar organizing region, or NOR) on chromosome 9, as observed with fluorescent in situ hybridization, although data are not available for the F and G genome species (Chung et al. 2008). Most taxa also have an rDNA array on chromosome 10. However, the chromosome 10 rDNA was not observed in O. sativa ssp. japonica cv. Nipponbare, in O. glaberrima, or in O. australiensis. Because of the phylogenetic distribution of the chromosome 10 array, the absence of the array in Nipponbare and O. glaberrima is most simply interpreted as a loss, whereas the lack of the array in O. australiensis could either be a loss peculiar to the O. australiensis lineage, or could be the ancestral condition. Oryza punctata (B genome) and its polyploid derivatives, O. punctata and O. minuta (BC) have an additional array on chromosome 4 and O. officinalis (C genome) and some of its derivatives have arrays on chromosome 5.
Early genome mapping studies in Oryza sativa identified a duplication of the distal part of chromosomes 11 and 12 (Rice Chromosomes 11 and 12 Sequencing Consortia 2005; Wu et al. 1998). The duplicated region is approximately 2 Mb in length and based on divergence between orthologous genes appears to have occurred approximately six mya (Jiang et al. 2007). If this date is correct, then the duplication is placed before the divergence of the A, B, and C genomes. The exact timing could be tested phylogenetically by assembling gene trees for genes shared in the duplicated segments. As expected with duplicate genes, the rate of molecular evolution is accelerated, particularly in the orthologues on chromosome 12 (Jiang et al. 2007). In addition, the rate of rearrangements is also elevated, again particularly on chromosome 12, such that inversions are more common than elsewhere in the genome.
Centromere evolution in Oryza is also active. Like all known centromeres, those of rice are made up of centromere-specific repeat sequences interspersed with a variety of retrotransposons (Nagaki et al. 2004). The most common centromeric repeat in Orzya is known as CentO, which is similar to the corresponding sequence in maize. However, novel repeat sequences were found in the centromeres of O. rhizomatis (C genome) and O. brachyantha (F genome). The latter, called CentO-F, is strikingly different from all other known centromeric repeat sequences in the grasses (Lee et al. 2005). Curiously CentO-F has not only replaced most of the (presumably ancestral) CentO sequences, but also the repeated retrotransposons. In addition, the centromeric region of chromosome 8 in O. sativa appears to be inverted relative to O. officinalis and O. brachyantha (Ma et al. 2007)
Combined studies of molecular and genome evolution point to an unusual history of O. brachyantha (F genome). Phylogenetic studies place O. brachyantha at the end of a longer branch (Zou et al. 2008), indicating a higher mutation rate than in other species. In addition, members of the species have the smallest genome reported in the genus, which must reflect either a marked loss of retrotransposons, or failure of retrotransposons to amplify. The novel centromere repeat and loss of centromeric retrotransposons may reflect a single underlying genomic mechanism. O. brachyantha is also an annual plant, and hence has a short life cycle. It is tempting to speculate that all these observations might be related.
The species O. sativa
The history of O. sativa itself has been thoroughly reviewed elsewhere (Vaughan et al. 2008a, b), and will only be summarized here. Oryza sativa is comprised of two subspecies, indica and japonica, both of which are domesticated from a wild ancestor similar to O. rufipogon. Subspecies japonica is informally divided into tropical and temperate groups, the latter presumed to be derived from the former. Additional informal groups, such as aus, aromatic, ashwina, and rayada, are recognized and apply to plants with particular characteristics of a local environment.
An unresolved question in the history of O. sativa is the number of domestication events involved. Evidence has accumulated in the literature for various evolutionary histories, summarized in tabular form by Vaughan et al. (2008b).
Methods for assessing the pattern of evolution include phylogenetic analysis of sequence data, as well as tests for selection. The latter tests compare the observed patterns of sequence variation with the patterns expected if mutation were purely random and no selection were occurring (a neutral model). If mutations occur randomly (i.e. none is favored by selection), most sequence variants (haplotypes) will each appear in only a few individual plants. Conversely, a small number of haplotypes will each appear in a large number of plants. In other words, a survey of single nucleotide polymorphisms (SNPs) in a population will find a few that are very common and many that are rare. A deficiency of rare SNPs or an excess of common ones indicates that the population is not evolving neutrally. However, there are many possible reasons why a population might depart from neutral expectation.
A recent analysis of single nucleotide polymorphisms in material of O. sativa indica, japonica, aromatic, aus, and O. rufipogon found a pattern similar to that in Fig. 5b (Caicedo et al. 2007). In addition, too many loci had SNPs that were at a high frequency in O. sativa. The authors concluded that the pattern could be explained by a sharply reduced population size (bottleneck) during domestication, combined with a selective sweep. Alternatively, the pattern might indicate that the ancestral population had been subdivided, but that subsequent gene flow had occurred among the populations.
An analogous study using microsatellite loci found a pattern similar to that in Fig. 5a, with a single domestication origin and some apparent gene flow between the two subspecies of O. sativa (Gao and Innan 2008). The microsatellite study included fewer plants and a smaller number of markers than the sequence-based study (Caicedo et al. 2007). Nonetheless the discrepancies between the two studies indicate how difficult it may be to arrive at a definitive answer to the question of rice domestication.
All studies agree that domestication reduced the population size of O. sativa relative to that of O. rufipogon, as expected. Because domestication selects only a sample of plants from the population of the wild ancestor, genetic diversity is necessarily reduced. Beyond this, however, there is little agreement among the studies. It seems unlikely that the issue of one versus two origins of O. sativa will be firmly resolved with current analytical methods and data. In the presence of gene flow and selection, both of which are occurring, the two patterns in fact cannot be distinguished. (See also discussions in Vaughan et al. (2008a, b))
For a few types of domesticated rice, the history appears easier to assess. For example, fragrant rice is a result of a mutation at the Badh2 locus (Chen et al. 2008), which encodes the gene for betain aldehyde dehydrogenase. In most rice varieties the gene product prevents formation of 2-acetyl-1pyrroline. Fragrant varieties, in contrast, contain a loss of function mutation in the gene, thus permitting synthesis of the volatile compound, which provides the aroma for which the rice is known. Because all fragrant varieties seem to contain the same mutant haplotype, fragrant rice is inferred to have arisen once.
Summary and conclusions
Like any living organism, rice carries with it the legacy of its evolutionary history. Some characteristics were acquired deep in the distant past of eukaryotic evolution, whereas other aspects were acquired more recently along with the common ancestors of other angiosperms, other monocots, or other grasses. An understanding of evolutionary history permits us to assess the generality of aspects of rice biology and conversely to understand which aspects of other plants might be comparable. For example, the pathways that permit accumulation of ferulic acid in the cell wall are likely common to all the commelinid monocots, so that data from rice could reasonably be extrapolated to cell wall structure in pineapples or bananas, and conversely. On the other hand, formation of tubercles (outgrowths) on the lemma and palea (hull) is a characteristic unique to the genus Oryza; an understanding of this aspect of hull architecture can be extrapolated to all congeneric species, but not to other Oryzeae or other cereal crops.
Use of rice as a model for other grass species or for other monocots requires extensive comparative study. Only by determining the taxonomic distribution of species that share particular characteristics will we be able to translate information from rice to other plants, both wild and cultivated. Such comparative studies can be guided by the extensive phylogenetic information now available, and will permit targeted investigation of plants likely to illuminate the generality of results from studies of rice.
I thank the editors for the opportunity to contribute this review. This work was supported in part by National Science Foundation.