Chapter 1 Introduction

Chapter 1 Introduction 1.1 DNA Sequence in Plant Systematics Although nucleic acid sequencing is a relatively new approach in plant systematics, the power of the technique and the data generated have made it become one of the most utilized of the molecular approaches for inferring phylogenetic history. DNA sequence data are the most informative tool for molecular systematics, and comparative analysis of DNA sequences is becoming increasingly important in plant systematics. There are two major reasons why nucleotide sequencing is becoming so valuable in plant systematics: 1) the characters (nucleotides) are the basic units of information encoded in organisms; 2) the potential sizes of informative data sets are immense. For example, one in 100 nucleotides is polymorphic in the human genome so that there will be about 2 10 7 polymorphisms in the human genome as a whole. Thus, for most studies, systematically informative variation is essentially inexhaustible. Furthermore, different genes or parts of the genome might evolve at different rates. Therefore, questions at different taxonomic levels can be addressed using different genes or different regions of a gene. Unlike animals, plants have an additional genome, chloroplast genome (cpdna) in addition to the nuclear (ndna) and mitochondrial (mtdna) genomes. Because of its complexity and repetitive properties, the nuclear genome is used in systematic botany less frequently. The mitochondrial genome is used at the species level due to its rapid changes in its structure, size, configuration, and gene order. On the other hand, the chloroplast genome is well suited for evolutionary and phylogenetic studies particularly above the species level, because cpdna, 1) is a relatively abundant component of plant total DNA, thus facilitating extraction and analysis; 2) contains primarily single copy genes; 3) has a conservative rate of 1

nucleotide substitution; and 3) extensive background for molecular information on the chloroplast genome is available. Therefore, most phylogenetic reconstructions in plant systematics conducted so far is based on molecular data from the cpdna genes. The most common gene used to provide sequence data for plant phylogenetic analyses is the plastid-encoded rbcl gene (Chase et al., 1993; Donoghue et al., 1993). This single copy gene is approximately 1430 base pairs in length, is free from length mutations except at the far 3' end, and has a fairly conservative rate of evolution. The function of the rbcl gene is to code for the large subunit of ribulose 1, 5 bisphosphate carboxylase/oxygenase (RUBISCO or RuBPCase). The sequence data of the rbcl gene are widely used in the reconstruction of phylogenies throughout the seed plants. However, it is apparent that the ability of rbcl to resolve phylogenetic relationships below the family level is often poor (Doebley et al., 1990). Thus, interest exists in finding other useful DNA regions that evolve faster than does rbcl to facilitate lower-level phylogenetic reconstruction. The matk gene is a promising gene in this regard. 1.2 The matk Gene 1.2.1 Overview of the matk Gene The matk gene was first identified by Sugita et al. (1985) from tobacco (Nicotiana tabacum) when they sequenced the trnk gene encoding the trna Lys (UUU) of the chloroplast. They found a 509 codon major open reading frame (ORF) in the intron of the trnk gene; no function for the ORF509 was assumed. The complete sequence of the liverwort Marchantia polymorpha (Ohyama et al., 1986) confirmed the existence of this open reading frame in the non-vascular plant. Later, Neuhaus and Link (1987) found that the anticodon loop of the trna Lys was interrupted by a 2,574 base pair intron containing a long open reading frame for 524 amino acids in mustard (Sinapis alba). They suggested a possible maturase function of the matk gene for the first time based upon a homology search result. This open reading frame was identified in the complete sequence of rice (Oryza sativa) chloroplast genome, as well (Hiratsuka et al., 1989). The trnk gene from two pine species (Pinus contorta and P. thunbergii) also contained the open reading frame (Lidholm and Gustafsson, 1991). This open 2

reading frame is flanked by two exons of the trnk gene in all land plants studied so far with only one exception. In beech drop (Epifagus virginiana), the matk gene appeared as a freestanding gene with neither the trnk exons nor the interrupting introns present (Wolfe et al., 1992). 1.2.2 Function of the matk gene: maturase MatK The first putative function of the matk gene came from a sequence homology search through the GenBank, databases for DNA sequences. Neuhaus and Link (1987) found that a segment near the carboxyl terminus of the derived Sinapsis alba matk polypeptide was structurally related to portions of the maturase-like polypeptides of introns of the mitochordrial cytochrome c oxidase subunit I gene (COXI) of yeast and Podospora anserina. In later analyses (Ems et al., 1995) it was hypothesized that the putative maturase, MatK, acts to assist the splicing of group II introns other than the one in which it is normally encoded. Two good candidates are the single group II intron in rpl2 and the second intron in rps12. The maturase MatK presumably helps fold the intron RNA into the catalytically-active structure. The 3 end of the matk was identified to contain a conserved region of about 100 amino acids and this region was named domain X (Mohr et al., 1993; Liang and Hilu, 1996). Comparison of group II introns (Mohr et al., 1993) indicated that three domains (reverse transcriptase, Zn-fingerlike, and X domain) existed in the ancestral open reading frame of all group II introns. During the evolutionary process, the reverse transcriptase and Zn-finger-like domains were lost in some cases. The retention of domain X supports the hypothesis that the matk gene plays an essential function in RNA splicing. 1.2.3 Application of the matk gene to plant systematics There have been several studies using the matk gene sequence in phylogenetic reconstruction. These studies involved six families. In Saxifragaceae, matk was found to evolve approximately three-fold faster than rbcl (Johnson and Soltis, 1994, 1995; Johnson et al., 1996). The sequences of matk in Polemoniaceae varied at an overall rate twice that of rbcl sequences (Steele and Vilgalys, 1994; Johnson and Soltis, 1995). Substitutions at the third codon position predominated in rbcl sequences, while in matk substitutions were more 3

evenly distributed across codon positions. Recently, the matk gene sequences have been also used in Orchidaceae tribe Vandeae (Jarrell and Clegg, 1995), Myrtaceae (Gadek et al. 1996), Poaceae (Liang and Hilu 1996), Apiaceae (Plunkett et al. 1996), and flowering plants (Hilu and Liang, 1997). According to the detailed analysis of the matk sequence data available in Gene Bank and preliminary studies (Liang and Hilu, 1996; Hilu and Liang, 1997), matk has higher variation than any other chloroplast genes. Although the variation is slightly higher at the 5 region than at the 3 region, approximate even distribution was observed throughout the entire gene. In addition, the high proportion of tranversion of the matk gene might provide more phylogenetic information. These factors underscore the usefulness of the matk gene in systematic studies and suggest that comparative sequencing of matk may be appropriate for phylogenetic reconstruction at subfamily and family levels. 1.3 Grass Family (Poaceae) 1.3.1 Size and Importance The grass family (Poaceae) is the fourth largest flowering plant family, with 651 genera and about 10,000 species (Clayton and Renvoize, 1986). Included in this family are many important cereal crops, such as wheat, maize, barley, rice and sorghum, and economic plants such as sugar cane, bamboo and turf grasses. There are possible energy sources such as sweet sorghum, sugar cane and maize for alcohol which could be very valuable in industrial societies. Grasslands are valuable resources for grazing, and are ecologically important as well. 1.3.2 Poaceae History Fossil evidence indicates that Poaceae may have first appeared in the Late Cretaceous, approximately 70 million years ago (Thomasson, 1987). Although there are many fossil records for the grass family, the ambiguity caused by their similarity to several related families such as Cyperaceae and Juncaceae greatly reduces their application value. The extant grass species share many unique characters in their stems, leaves, flowers and inflorescences, and caryopsis fruits, and are placed in Liliopsida (Monocotyledonae) 4

(Stebbins, 1982, 1987). The monogeneric Joinvilleaceae is thought to be the most closely related family to Poaceae, since Poaceae and Joinvilleaceae are phylogenetically allied and share a very unique inverted repeat of about 6 kilobases in their chloroplast genomes (Doyle et al., 1992). Thus, Joinvillea is the best outgroup for Poaceae in cladistic analyses. The grass family was first named by de Jussieu (1789), and the grouping of the 58 genera in his treatment was mainly based on numerical characters, such as number of styles, stamens and florets. In 1814, the grass family was divided into two tribes : Paniceae with a basal reduction of the spikelets and Poaceae with an apical reduction of the spikelets (Brown, 1814). Later, with additional evidence from leaf epidermis and anatomy (Prat, 1936; Brown, 1958), chromosome number and morphology (Avdulov, 1931), embryo structure (Reeder, 1957), and numerical taxonomy (Hilu and Wright, 1982; Watson et al., 1985), a better understanding of grass systematics was achieved and various subfamily systems were proposed. With the introduction of molecular data such as from proteins and nucleic acids, more grouping patterns and evolutionary lineages of the grass family were presented (Hamby and Zimmer, 1988; Hilu and Esen, 1988; Doebley et al., 1990; Davis and Soreng, 1993; Cummings et al., 1994; Hsiao et al., 1994; Nadot et al., 1994; Barker et al., 1995; Clark et al., 1995; Duvall and Morton, 1996). Although the number of subfamilies recognized in these publications varied from 5 to 13, the following major groups are commonly recognized: Aundinoideae, Bambusoideae, Centothecoideae, Chloridoideae, Oryzoideae, Panicoideae and Pooideae. 1.3.3 Major Subfamilies Bambusoideae Bambusoideae contains both herbaceous and woody species, with 13 tribes and 91 genera (Clayton and Renvoize, 1986). All species of this subfamily are C 3 and without Kranz structure in leaf anatomy. Most species in this subfamily also have elongated, finger-like microhairs. Bambusoideae has distinctive anatomy: arm and fusoid cells, sclerome tissue around the leaf midrib, and vertically oriented silica bodies. However, most herbaceous bamboos do not show these anatomical characters. Based upon the 5

recent ndhf sequence data, two new subfamilies were proposed that include the herbaceous bamboos: Anomochloa and Pharus (Clark and Judziewcz, 1996). Bambusoideae has been suggested to be the basal group of the grass family according to its morphological, anatomical, cytogenetic and molecular characters (Stebbins, 1956; Hilu and Wright, 1982; Kellogg and Campbell, 1987; Kellogg and Watson, 1993). Oryzoideae Oryzoideae has been treated as tribe Oryzeae in the Bambusoideae because it has arm cells and finger-like microhairs, which are shared with bambusoid species. However, fusoid cells are not always present. According to numerical and immunological studies, Oryzeae should be treated as a separate subfamily Oryzoideae (Hilu and Wright, 1982; Esen and Hilu, 1989). All species of Oryzoideae are C 3. Pooideae Pooideae has been called Festucoideae and is one of the largest subfamilies of Poaceae with about 160 genera and 3000 species (Clayton and Renvoize, 1986). Pooideae is characterized by the absence of microhairs, C 3 pathway, and a unique stoma with parallel-sided subsidiary and overlapped guard cells. Panicoideae Most panicoid species have C 4 pathway, and a unique intermediate C 3 -C 4 type exists in this subfamily in some Panicum species (Morgan and Brown, 1979). Occasionally, a single species such as Alloteropsis semialate has both C 3 and C 4 pathways (Ellis, 1974). These species are of particular interest both functionally and taxonomically and could help to understand the evolution of the C 4 photosynthetic pathway. Panicoideae includes 7 tribes and 207 genera and is mostly characterized by its spikelet characters (Clayton and Renvoize, 1986). Anatomically, it is a diverse group (Ellis, 1987). Chloridoideae Chloridoideae includes about 5 tribes and 145 genera and all are C 4 except for one species of Eragrostis (Clayton and Renvoize, 1986). Chloridoid species are anatomically distinct with inflated, spherical microhairs and Kranz structure. This subfamily was considered to be a homogeneous group but the homogeneity has been questioned by numerical and molecular studies (Hilu and Wright, 1982; Esen and Hilu, 1989). 6

Arundinoideae Arundinoideae is a diverse assemblage without reliable diagnostic features. Most arundinoid species are C 3 with Kranz structure and rarely C 4 without Kranz structure. There are no arm cells or fusoid cells in this subfamily, but elongated, finger-like microhairs exist throughout arundinoid species. The subfamily has 4 tribes and 45 genera and is a taxonomically problematic group (Clayton and Renvoize, 1986). Centothecoideae Centothecoideae is a small monotribal subfamily with 10 genera (Clayton and Renvoize, 1986). The subfamily is mainly characterized by its distinctive embryo and was previously treated as a tribe Centotheceae in subfamily Arundinoideae (Stebbins, 1956; Tsvelev, 1983). 1.3.4 Difficulties in Grass Systematics Compared to other plant families, systematics and evolutionary studeis in Poaceae have the following particular difficulties: 1) large numbers of taxa to cover; 2) the simplicity of floral and vegetative morphology; 3) coexistance of advancement and reduction for their character evolution, or bidirectional character evolution, which makes it difficult to determine the polarity of a character during phylogenetic analyses (Stebbins, 1987); 4) widespread hybridization and polyploidy. About 80% of the taxa studied for chromosome numbers have undergone polyploidy sometime during their evolutionary histories (de Wet, 1987); and 5) frequent parallel evolution caused by adaptation to similar environments and mosaic evolution of different characters at different rates along the same line (Stebbins, 1956, 1987; Hilu and Wright, 1982; Pohl, 1987). Because of these difficulties, the systematics and evolution of the Poaceae has been studied extensively in the past few decades using morphological, anatomical, cytological, numerical, physiological, biochemical, and molecular approaches. Among these methods, DNA sequencing appears to be very promising. 7

1.4 Previous Studies on Poaceae Using DNA Sequences There are only ten publications focusing on phylogenetic reconstruction using DNA sequence data in the grass family; earliest being that of Hamby and Zimmer (1988). Three studies utilized the non-coding intergenic transcription region (ITS) and phytochrome gene (Hamby and Zimmer 1988; Hsiao et al. 1994; Mathews and Sharrock, 1996). Most used the chloroplast genome, especially the rbcl gene (Doebley et al., 1990; Barker et al., 1995; Duvall and Morton, 1996). The species number included in these studies ranged from 9 to 45 and the subfamilies involved were 3 to 7. Different lengths of DNA were used in these studies and the informative sites ranged from 85 of 18S rrna and 26S rrna to 487 of ndhf (Clark et al., 1995). The most common outgroup is Joinvillea in the Joinvilleaceae, but some studies used very distant outgroups such as Nicotiana and Spinacia (Doebley et al., 1990; Cummings et al., 1994). Some of the previous nucleic acid studies were not comprehensive because of their small sample sizes that did not include the major groups of the grass family, or they had insufficient information to resolve the major lineages (Hamby and Zimmer, 1988; Doebley et al., 1990; Hsiao et al., 1994; Nadot et al., 1994). As to the phylogenies of Poaceae generated from the previous studies using DNA sequence data, there are some consistencies and disagreements. The following points and questions remain to be resolved. 1) Which group is the root of the grass family? Bambusoideae appears most likely to be the most primitive subfamily of Poaceae. The DNA sequence studies involving bambusoid species done so far supported the primitive postion of Bambusoideae and it appeared as the basal group of the grass family phylogeny. The results agree with its trimerous flowering parts and bracteate indeterminate inflorescence, which are considered to be primitive characters. However, other subfamilies that were also suggested to be the basal group of Poaceae are Oryzoideae (Hsiao et al., 1994), Arundinoideae (Cummings et al., 1994) or Panicoideae (Doebley et al., 1990). While the Clark et al. (1995) study 8

indicated a polyphyletic origin of the Bambusoideae, all other studies supported its monophyletic position. 2) Is Aundinoideae a polyphyletic or monophyletic group? Most nucleic acid studies supported the polyphyletic origin of Aruninoideae. Among the four studies including species from Arundinoideae, only one supported the monophyletic orgin of this group; the study included only two species of the Arundinoideae (Duvall and Morton, 1996). These results confirmed that Arundinoideae might be a diverse assemblage without reliable diagnostic features in their morphology. The relationships between Arundinoideae and other groups varied from one study to another. Arundinoideae was grouped with Pooideae (Cummings et al., 1994), with Panicoideae (Clark et al., 1995), or with Chloridoideae (Barker et al., 1995). 3) What is the systematic position of oryzoid species? In most cases, it appears as a clade near to the basal lineage Bambusoideae. However, it was also grouped with Panicoideae and in one study it appeared as a polyphyletic group (Cummings et al., 1994). 4) What is the circumscription of Pooideae? Most studies supported the monophyly of the Pooideae, with one exception (Cummings et al., 1994). In the cladogram of that study, Pooideae was separated by a species of Arundinoideae (Lygenum spartum). This result indicates that Pooideae is a group of very closely related taxa and agrees with its obvious diagnostic morphological characters of C 3 pathway, a unique stoma and absence of microhairs throughout the subfamily (Ellis, 1987). 5) Is Panicoideae a monophyletic or polyphyletic group? Based on the rbcl gene result, Pancoideae is a monophyletic group (Barker et al. 1995). However, ndhf and rpoc2 data indicated that Panicoideae was a polyphyletic group (Cummings et al., 1994; Clark et al., 1995). While only one study showed that 9

Panicoideae was the basal lineage of the grass family (Doebley et al., 1990), all other studies placed the Panicodeae near the bases of the cladograms. 6) Chloridoideae is a poorly studied group. Morphologically, Chloridoideae is not a well defined group despite its large number of species. The representatives of Chloridoideae were included in only three studies (Barker et al., 1995; Clark et al. 1995; Duvall and Morton 1996). The chloridoid species always were grouped together as a single group and appeared to be related to Arundinoideae. The monophyletic origin of Chloridoideae based on DNA sequences is not supported by the previous numerical and immunological studies (Hilu and Wright, 1982; Hilu and Esen, 1988). 7) Centhothecoideae is a small group with questionable phylogenetic status. Based on its morphology and anatomy, it was placed within the Arundinoideae. The current nucleic acid results suggested that it was related either to Panicoideae or Arundinoideae (Barker et al., 1995; Clark et al., 1995). Thus, it remains to be seen if this group of grasses deserves a subfamily status and where it should be in the grass phylogeny. 8) There are some problematic tribes or genera, such as Aristedeae, Stipeae, Bromus, Brachpodium and Ehrharta. These tribes or genera often share morphological and anatomical characters from different groups and their taxonomic positions are not well resolved. There are about 40 tribes in the grass family according to common classification systems. Most studies so far only covered few tribes and used one or two representative species from each tribe. Table 1.1 summarizes the sample representatives for these previous DNA sequence studies. 10

Table 1.1 Tribes and genera in the previous studies with DNA sequence Subfamilies and tribes Bambusoideae Anomochloeae Bambuseae Brachyelytreae Diarrheneae Ehrharteae Olyreae Oryzeae Phaenospermatae Phareae Streptochaeteae Pooideae Aveneae Poeae Stpeae Triticeae Centostecoideae Centotheceae Arundinoideae Aristideae Arundineae Micrairaeae Thysanolaeneae Chloridoideae Chlorideae Eragrosteae Pappophoreae Zoysieae Panicoideae Andropogoneae Arundinelleae Paniceae Species 13 tribes and 91 Genera Anomochloa Arundinaria, Bambusa, Cephalostachyum, Chesquea, Guadua, Phyllostachys Brachyelytrum Diarrhena Ehrharta Lithachne, Olyra, Raddia Leersia, Lithachne, Oryza, Zizania Phaenosperma Pharus Streptochaeta, 10 tribes and 152 Genera Avena Poa, Puccinellia Stipa Aegilops, Hordeum, Triticum 1 tribe and 10 genera Zeugites, Chasmanthium 4 tribes and 45 genera Aristida, Stipagrostis Arundo, Centropodia, Danthonia, Gynerium, Karroochloa, Merxmuellera, Moliniopsis, Monachather, Rytidosperma, Plinthanthesis, Molinia, Pharagmites Micraira Thysanolaena 5 tribes and 145 genera Eustachys Eragrostis, Erioneuron, Muhlenbergia, Sporobolus Enneapogon Zoysia 7 tribes and 207 genera Hyparrhenia, Saccharum, Sorghum, Tripsacum, Zea Danthoniopsis, tristachya Neurachne, Panicum, Pennisetum, Setaria 11

1.5 Objectives of the Research There are three major objectives for this research. The first one is to study the utility of the matk gene in plant evolution. For this goal, the following questions will be answered: 1. How much variation does the matk gene have? What proportion of the variation is phylogenetically informative? Are there any differences in this variation for different plant groups? 2. What is the distribution of the variation throughout the different regions of the matk gene? Which part of the matk gene is variable and which part is more conservative? 3. What is the phylogeny of the representative species from various plant groups generated from the matk gene sequences? Which part of the matk gene provides more reliable phylogenetic information? And at which taxonomic level could these regions be used to reconstruct a phylogeny? 4. How many nucleotides of the matk gene are sufficient to generate a robust phylogeny and from which part of the matk gene? The second objective is to characterize the matk gene in the grass family. The following questions will be addressed: 1. What is the rate and pattern of nucleotide variation of the matk gene in Poaceae? 2. What are the transition and transversion ratios (tr/tv) among the major groups of Poaceae? Is the (tr/tv) related to G+C content or region specific in the gene?. Transition and transversion are important characters in phylogenetic reconstruction since transition tend to be weighted heavily in some studies. Is there any relationships between the (tr/tv) and the phylogenetic hierarchy of Poaceae? 3 At which taxonomic levels is the matk gene useful in Poaceae? Which parts of the matk gene have more phylogenetic signal for the Poaceae? The last major goal is to address the phylogenetic questions in the Poaceae using the matk sequences from representatives of different grass groups. 12

1. How many subfamilies should the grass family be divided into? What are the relationships among these subfamilies? Which individual subfamilies are monophyletic and which are polyphyletic? 2. Are the pooids, bambusoids senso lato, or herbaceous bamboos the most basal lineages in the family? Is there an early major dichotomy in the Poaceae, such as the PACC (Panicoideae, Arundinoideae, Centeostheocoideae and Chloridoideae) and BOP (Bambusoideae, Oryzoideae, and Pooideae) clades? Do some herbaceous bamboos represent a distinct phylogenetic entity that deserves a subfamilial taxonomic treatment? 3. Do the oryzoid and bambusoid grasses represent a single monophyletic taxon? Should the oryzoids be considered as a separate family? 4. Is the PACC clade a monophyletic group? 5. Where does the Centothecoideae belong in the phylogeny? 6. What are the taxonomic position of some of the problematic tribes or genera: Stipeae, Aristedeae, Brachpodieae, Ehrharta and Bromus. This dissertation has six chapters. Chapter 1 (this chapter) introduces the grass family (Poaceae), the matk gene and the major objectives of the research. Chapter 2 deals with the general analysis of the matk gene and its application in plant systematics; it has been published in American Journal of Botany. Chapter 3 addresses the preliminary applications of the matk gene to the phylogenetic reconstruction of Poaceae; it has been published in Canadian Journal of Botany. Chapter 4 characterizes the matk genes in the Poaceae from the 13 grass species and one outgroup (Joinvillea). Chapter 5 examines the phylogeny of 48 grass species using Joinvillea as a outgroup based on 966 bps sequences at the 3 region of the matk gene. Chapter 6 will summarize the results and provide some suggestions for future work. 1.6 Literature Cited AVDULOV, N. 1931. Karyo-systematische untersuchung der familie Gramineen. Bulletin of Applied Botany, Genetics and Plant Breeding 44: 1-428. 13

BARKER, N. P., H. P LINDER, AND E. H. HARLEY. 1995. Polyphyly of Arundinoideae (Poaceae): Evidence from rbcl sequence data. Systematic Botany 20: 423-435. BROWN, R. 1814. General remarks, geographical and systematical, on the botany of Terra Australis; undertaken for the purpose of completing the discovery of that vast country, and prosecuted in the years, 1801, 1802, and 1803 2 vols. W. Bulmer & Company, London. BROWN, W. W. 1958. Leaf anatomy in grass systematics. Botanical Gazette 119: 170-178. CHASE, M. W., SOLTIS, D. E., OLMSTEAD, R. G., MORGAN, D., LES, D. H., MISHLER, B.D., DUVALL, M. R., PRICE, R. A., HILLS, H. G., QIU, Y. -L., KRON, K. A., RETTIG, J. H., CONTI, E., PALMER, J. D., MANHART, J. R., SYTSMA, K. J., MICHAELS, H. J., KRESS, W. J., KAROL, K. G., CLARK, W. D., HEDREN, M., GART, B. S., JANSEN, R. K., KIM, K. -J., WIMPEE, C. P., SMITH, J. F., FURNIER, G. R., STRAUSS, S. H., XIANG, Q. -Y., PLUNKETT, G. M., SOLTIS, P. S., SWENSEN, S. M., WILLIAMS, S. E., GRADEK, P. A., QUINN, C. J., EGUIARTE, L. E., BARRETT, S. C. H., DAYANANDAN, S., AND ALBERT, V. A. 1993. Phylogenetics of seed plants: an analysis of nucleotide sequences from the plastid gene rbcl. Annals of the Missouri Botanic Garden 80: 528-580. CLARK, L. G., W. ZHANG, AND J. F. WENDEL. 1995. A phylogeny of the grass family (Poaceae) based on ndhf sequence data. Systematic Botany 20: 436-460., AND G. JUDZIEWCZ. 1996. Two new subfamilies of Poaceae: Anomochloideae and Pharoideae. Taxon 98: 78-81. CLAYTON, W. D., AND S. A. RENVOIZE. 1986. Genera graminum. London: HMSO publications. CUMMINGS, M. P., L. M. KING, AND E. A. KELLOGG. 1994. Slipped-strand mispairing in a plastid gene: rpoc2 in grasses (Poaceae). Molecular Biology and Evolution 11: 1-8. 14

DAVIS, J. I., AND R. J. SORENG. 1993. Phylogenetic structure in the grass family (Poaceae) as inferred from chloroplast DNA restriction site variation. American Journal of Botany 80: 1444-1454. DE WET, J. M. J. 1987. Hybridization and polyploidy in the Poaceae. In T. R. Soderstrom, K. W. Hilu, C. S. Campbell, and M. A. Barkworkth [eds.], Grass systematics and evolution, 188-194. Smithsonian Institution Press, Washington, DC. DOEBLEY, J., M. DURBIN, E. M. GOLENBERG, M. T. CLEGG, AND D. P. MA. 1990. Evolutionary analysis of the large subunit of carboxylase (rbcl) nucleotide sequence among the grasses (Gramineae). Evolution 44: 1097-1108. DONOGHUE, M. J., OLMSTEAD, R. G., SMITH, J. F., AND PALMER, J. D. 1993. Phylogenetic relationships of Dipsacales based on rbcl sequences. Annals of the Missouri Botanic Garden 79: 333-345. DOYLE, J. A., M. J. DONOGHUE, AND E. A. ZIMMER. 1992. Integration of morphological and ribosomal RNA data on the origin of angiosperms. Annals of the Missouri Botanic Garden 81: 419-450. DUVALL, M. R., AND B. R. MORTON. 1996. Molecular phylogenetics of Poaceae: an expanded analysis of rbcl sequence data. Molecular Phylogenetics and Evolution 5: 352-358. ELLIS, R. P. 1974. Anomalous vascular bundle sheath structure in Alloteropsis semialata leaf blades. Bothalia 11: 273-275. --------. 1987. A review of comparative leaf blade anatomy in the systematics of the Poaceae: the past twenty-five years. In T. R. Soderstrom, K. W. Hilu, C. S. Campbell, and M. A. Barkworkth [eds.], Grass systematics and evolution, 3-10. Smithsonian Institution Press, Washington, DC. EMS, S. C., C. W. MORDEN, C. K. DIXON, K. H. WOLFE, C. W. DEPAMPHILIS, AND J. D. PALMER. 1995. Transcription, splicing and editing of plastid RNAs in the nonphotosynthetic plant Epifagus virginiana. Plant Molecular Biology 29: 721-733. 15

ESEN, A., AND K. W. HILU. 1989. Immunological affinities among subfamilies of the Poaceae. American Journal of Botany 76: 196-203. GADEK, P. A., P. G. WILSON, AND C. J. QUINN. 1996. Phylogenetic reconstruction in Myrtaceae using matk, with particular reference to the position of Psiloxylon and Heteropyxis. Australian Systematic Botany. HAMBY, R. K., AND E. A. ZIMMER. 1988. Ribosomal RNA sequences for inferring phylogeny within the grass family (Poaceae). Plant Systematics and Evolution. 160: 29-37. HILU, K. W., AND A. ESEN. 1988. Prolamin size diversity in the Poaceae. Biochemical Systematics and Ecology 16: 457-465. -------, AND H. LIANG. 1997. The matk gene: sequences variation and application in plant systematics. American Journal of Botany 84: 830-839. -------, AND K. WRIGHT. 1982. Systematics of Gramineae: A cluster analysis study. Taxon 31: 9-36. HIRATSUKA. J., H. SHIRMADA, R. WHITTIER, T. ISHIBASHI, M. SAKAMOTO, M. MORI, C. KONDO, Y. YONJI, CR. SUN, BY. MENG, YQ. LI, A. KANNO, Y. NISHIZAWA, A. HIRAI, K. SHINOZAKI, AND M. SUGIURA. 1989. The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct trna genes accounts for a major plastid DNA inversion during the evolution of cereals. Molecular Gen. and Genet. 217: 185-194. HSIAO, C., N. J. CHATTERTON, K. H. ASAY, AND K. B. JENSEN. 1994. Phylogenetic relationships of 10 grass species: an assessment of phylogenetic utility of the internal transcribed spacer region in nuclear ribosomal DNA in monocots. Genome 37: 112-120. JARREL, D.C., AND M. T. CLEGG. 1995. Systematic implications of the chloroplast-encoded matk gene on the tribe Vandeae (Orchidaceae). American Journal of Botany 82: 137. 16

JOHNSON L. A., J. L. SCHULTZ, D. E. SOLTIS, AND P. S. SOLTIS. 1996 Monophyly and generic relationships of Polemoniaceae based on matk sequences. American Journal of Botany 83: 1207-1224. JOHNSON L. A., AND D. E. SOLTIS. 1994. matk DNA sequences and phylogenetic reconstruction in Saxifragaceae s. str. Systematic Botany 19: 143-156.. 1995. Phylogenetic inference in Saxifragaceae sensu stricto and Gilia (Polemoniaceae) using matk sequences. Annals of the Missouri Botanic Garden 82: 149-175. JUSSIEU, A. L., DE. 1789. Genera plantarum. Paris: Herrisant & Barrois. KELLOGG, E. A., AND C. S. CAMPBELL. 1987. Phylogenetic analysis of the Gramineae. In T. R. Soderstrom, K. W. Hilu, C. S. Campbell, and M. A. Barkworkth [eds.], Grass systematics and evolution, 310-322. Smithsonian. Institution Press, Washington, DC. -------, AND L. WATSON. 1993. Phylogenetic studies of a large data set. I. Bambusoideae, Andropogonodeae, and Pooideae (Gramineae). Botanical Review 59: 273-320. LIANG, H., AND K. W. HILU. 1996. Application of the matk gene sequences to grass systematics. Canadian Journal of Botany 74: 125-134. LIDHOLM, J., AND P. GUSTAFSSON. 1991. A three-step model for the rearrangement of the chloroplast trnk-psba region of the gymnosperm Pinus contorta. Nucleic Acids Research 19: 2881-2887. MATHEWS, S., AND R. A. SHARROCK. 1996. The phytochrome gene family in grasses (Poaceae): a phylogeny and evidence that grass have a subset of the loci found in dicot angiosperms. Molecular and Biological Evolution 13: 1141-1150. MOHR, G, P. S. PERLMAN, AND A. M. LAMBOWITZ. 1993. Evolutionary relationships among group II intron-encoded proteins and identification of a conserved domain that may be related to maturase function. Nucleic Acid Research 21: 4991-4997. 17

MORGAN, J. A., AND R. H. BROWN. 1979. Photosynthesis in grass species differing in carbon dioxide fixation pathways. II. A search with intermediate gas exchange and anatomical characteristics. Plant Physiology 64: 257-262. NADOT, S. R. BAJON, AND B. LEJEUNE. 1994. The chloroplast gene rps4 as a tool for the study of Poaceae phylogeny. Plant Systematics and Evolution. 191: 27-38. NEUHAUS, H., AND G. LINK. 1987. The chloroplast trna LYS (UUU) gene from mustard (Sinapis alba) contains a class II intron potentially coding for a maturaserelated polypeptide. Current Genetics 11: 251-257. OHYAMA, K. H., FUKUZAWA, T. KOHCHI, H. SHIRAI, T. SANO, S. SANO, K. UMESONO, Y. SHIKI, M. TAKEUCHI, Z. CHANG, S. AOTA, H. INOKUCHI, AND H. OZEKI. 1986. Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322: 572-574. PLUNKETT, G. M., D. E. SOLTIS, AND P. S. SOLTIS. 1996. Evolutionary pattern in Apiaceae: inferences based on matk sequence data. Systematic Botany 21: 477-495. POHL, R. W. 1987. Man and the grasses: a history. In T. R. Soderstrom, K. W. Hilu, C. S. Campbell, and M. A. Barkworkth [eds.], Grass systematics and evolution, 355-358. Smithsonian Institution Press, Washington, DC. PRAT, H. 1936. La systematique des Graminees. Annales des Sciences Nattureles, Botanique, series 10. 18: 165-258. REEDER, J. R. 1957. The embryo in grass systematics. American Journal of Botany 44: 756-768. STEBBINS, G. L. 1956. Cytogenetics and evolution of the grass family. American Journal of Botany 43: 890-905. ------. 1982. Major trends of evolution in the Poaceae and their possible significance. In J. R. Estes, R. J. Tyrl, AND J. N. Brunken [Eds.]. Grasses and grasslands: systematics and Ecology. 3-36. University of Oklahoma Press, Norman. ---------. 1987. Grass systematics and evolution: past, present and future. In T. R. Soderstrom, K. W. Hilu, C. S. Campbell, and M. A. Barkworkth [eds.], Grass 18

systematics and evolution, 359-367. Smithsonian Institution Press, Washington, DC. STEELE, K. P., AND R. VILGALYS. 1994. Phylogenetic analyses of Polemoniaceae using nucleotide sequences of the plastid gene matk. Systematic Botany 19: 126-142. SUGITA, M., K. SHINOZAKI, AND M. SUGIURA. 1985. Tobacco chloroplast trna Lys (UUU) gene contains a 2.5-kilobase-pair intron: an open reading frame and a conserved boundary sequence in the intron. Proceeding of the National Academy of Sciences, USA 82: 3557-3561. THOMASSON, J. R. 1987. Fossil grasses: 1820-1986 and beyond. In T. R. Soderstrom, K. W. Hilu, C. S. Campbell, and M. A. Barkworkth [eds.], Grass systematics and evolution, 159-167. Smithsonian Institution Press, Washington, DC. TSVELEV, N. N. 1983. Grasses of the Soviet Union. Part I. Translated by B. R. Sharma. Amerind Publ. Co. Pvt. Ltd., New Delhi. WATSON, L., H., T. CLIFFORD, AND M. J. DALLWITZ. 1985. The classification of Poaceae: subfamilies and supertribes. American Journal of Botany 33: 433-484. WOLFE, K. H., C. W. MORDEN, AND J. D. PALMER. 1992. Function and evolution of a minimal plastid genome from a nonphotosynthetic parasitic plant. Proceedings of the National Academy of Sciences, USA 89: 10648-10652. 19