Allopolyploidy, diversification, and the Miocene grassland expansion.

The role of polyploidy, particularly allopolyploidy, in plant diversification is a subject of debate. Whole-genome duplications precede the origins of many major clades (e.g., angiosperms, Brassicaceae, Poaceae), suggesting that polyploidy drives diversification. However, theoretical arguments and empirical studies suggest that polyploid lineages may actually have lower speciation rates and higher extinction rates than diploid lineages. We focus here on the grass tribe Andropogoneae, an economically and ecologically important group of C4 species with a high frequency of polyploids. A phylogeny was constructed for ca. 10% of the species of the clade, based on sequences of four concatenated low-copy nuclear loci. Genetic allopolyploidy was documented using the characteristic pattern of double-labeled gene trees. At least 32% of the species sampled are the result of genetic allopolyploidy and result from 28 distinct tetraploidy events plus an additional six hexaploidy events. This number is a minimum, and the actual frequency could be considerably higher. The parental genomes of most Andropogoneae polyploids diverged in the Late Miocene coincident with the expansion of the major C4 grasslands that dominate the earth today. The well-documented whole-genome duplication in Zea mays ssp. mays occurred after the divergence of Zea and Sorghum. We find no evidence that polyploidization is followed by an increase in net diversification rate; nonetheless, allopolyploidy itself is a major mode of speciation.

Transport of boron by the tassel-less1 aquaporin is critical for vegetative and reproductive development in maize.

The element boron (B) is an essential plant micronutrient, and B deficiency results in significant crop losses worldwide. The maize (Zea mays) tassel-less1 (tls1) mutant has defects in vegetative and inflorescence development, comparable to the effects of B deficiency. Positional cloning revealed that tls1 encodes a protein in the aquaporin family co-orthologous to known B channel proteins in other species. Transport assays show that the TLS1 protein facilitates the movement of B and water into Xenopus laevis oocytes. B content is reduced in tls1 mutants, and application of B rescues the mutant phenotype, indicating that the TLS1 protein facilitates the movement of B in planta. B is required to cross-link the pectic polysaccharide rhamnogalacturonan II (RG-II) in the cell wall, and the percentage of RG-II dimers is reduced in tls1 inflorescences, indicating that the defects may result from altered cell wall properties. Plants heterozygous for both tls1 and rotten ear (rte), the proposed B efflux transporter, exhibit a dosage-dependent defect in inflorescence development under B-limited conditions, indicating that both TLS1 and RTE function in the same biological processes. Together, our data provide evidence that TLS1 is a B transport facilitator in maize, highlighting the importance of B homeostasis in meristem function.

Early inflorescence development in the grasses (Poaceae).

The shoot apical meristem of grasses produces the primary branches of the inflorescence, controlling inflorescence architecture and hence seed production. Whereas leaves are produced in a distichous pattern, with the primordia separated from each other by an angle of 180°, inflorescence branches are produced in a spiral in most species. The morphology and developmental genetics of the shift in phyllotaxis have been studied extensively in maize and rice. However, in wheat, Brachypodium, and oats, all in the grass subfamily Pooideae, the change in phyllotaxis does not occur; primary inflorescence branches are produced distichously. It is unknown whether the distichous inflorescence originated at the base of Pooideae, or whether it appeared several times independently. In this study, we show that Brachyelytrum, the genus sister to all other Pooideae has spiral phyllotaxis in the inflorescence, but that in the remaining 3000+ species of Pooideae, the phyllotaxis is two-ranked. These two-ranked inflorescences are not perfectly symmetrical, and have a clear "front" and "back;" this developmental axis has never been described in the literature and it is unclear what establishes its polarity. Strictly distichous inflorescences appear somewhat later in the evolution of the subfamily. Two-ranked inflorescences also appear in a few grass outgroups and sporadically elsewhere in the family, but unlike in Pooideae do not generally correlate with a major radiation of species. After production of branches, the inflorescence meristem may be converted to a spikelet meristem or may simply abort; this developmental decision appears to be independent of the branching pattern.

Duplication and diversification of the LEAFY HULL STERILE1 and Oryza sativa MADS5 SEPALLATA lineages in graminoid Poales.

BACKGROUND: Gene duplication and the subsequent divergence in function of the resulting paralogs via subfunctionalization and/or neofunctionalization is hypothesized to have played a major role in the evolution of plant form. The LEAFY HULL STERILE1 (LHS1) SEPALLATA (SEP) genes have been linked with the origin and diversification of the grass spikelet, but it is uncertain 1) when the duplication event that produced the LHS1 clade and its paralogous lineage Oryza sativa MADS5 (OSM5) occurred, and 2) how changes in gene structure and/or expression might have contributed to subfunctionalization and/or neofunctionalization in the two lineages. METHODS: Phylogenetic relationships among 84 SEP genes were estimated using Bayesian methods. RNA expression patterns were inferred using in situ hybridization. The patterns of protein sequence and RNA expression evolution were reconstructed using maximum parsimony (MP) and maximum likelihood (ML) methods, respectively. RESULTS: Phylogenetic analyses mapped the LHS1/OSM5 duplication event to the base of the grass family. MP character reconstructions estimated a change from cytosine to thymine in the first codon position of the first amino acid after the Zea mays MADS3 (ZMM3) domain converted a glutamine to a stop codon in the OSM5 ancestor following the LHS1/OSM5 duplication event. RNA expression analyses of OSM5 co-orthologs in Avena sativa, Chasmanthium latifolium, Hordeum vulgare, Pennisetum glaucum, and Sorghum bicolor followed by ML reconstructions of these data and previously published analyses estimated a complex pattern of gain and loss of LHS1 and OSM5 expression in different floral organs and different flowers within the spikelet or inflorescence. CONCLUSIONS: Previous authors have reported that rice OSM5 and LHS1 proteins have different interaction partners indicating that the truncation of OSM5 following the LHS1/OSM5 duplication event has resulted in both partitioned and potentially novel gene functions. The complex pattern of OSM5 and LHS1 expression evolution is not consistent with a simple subfunctionalization model following the gene duplication event, but there is evidence of recent partitioning of OSM5 and LHS1 expression within different floral organs of A. sativa, C. latifolium, P. glaucum and S. bicolor, and between the upper and lower florets of the two-flowered maize spikelet.

B- and C-class gene expression during corona development of the blue passionflower (Passiflora caerulea, Passifloraceae).

PREMISE OF STUDY: The origin of the passionflower corona, a complex series of structures between the petals and stamens, has intrigued botanists for centuries, but has proven intractable using traditional approaches. Supplementing developmental data with expression analyses of three floral identity genes, we test whether the corona in Passiflora caerulea (blue passionflower) is homologous to petals or stamens or whether an alternative hypothesis of the corona as a novel structure is supported. METHODS: Corona development was investigated using scanning electron microscopy. Expression of the P. caerulea B-class genes PISTILLATA (PcPI) and TOMATO MADS6 (PcTM6), and C-class gene AGAMOUS (PcAG) was investigated using a combination of RT-PCR and mRNA in situ hybridization analyses. KEY RESULTS: Corona development starts as a ring of tissue at the base of petals. The outer radii and operculum initiate first at the periphery, followed by the inner radii and pali toward the center, and finally an annulus beneath the operculum. Late in development, a limen, the innermost component of the corona, develops from the side of the androgynophore. RT-PCR analyses indicate that the B-class genes PcPI and PcTM6 and C-class gene PcAG were all expressed in mature coronas. However, mRNA in situ hybridization analyses revealed complex temporal patterns of gene expression in the different corona elements. CONCLUSIONS: Our data support the hypothesis that the corona is a composite structure, with the radii, pali, and operculum homologous to stamens, and the limen, which only expresses PcTM6, considered to be a novel structure distinct from the androgynophore.

Phylogenomic analyses of the BARREN STALK1/LAX PANICLE1 (BA1/LAX1) genes and evidence for their roles during axillary meristem development.

The diversity of plant architectural form is largely determined by the extent and duration of axillary meristem (AM) derived lateral growth. The orthologous basic helix-loop-helix (bHLH) proteins maize BARREN STALK1 (BA1) and rice LAX PANICLE1 (LAX1) are essential for the formation of AMs during vegetative development and all lateral structures during inflorescence development, but whether BA1/LAX1 co-orthologs exist outside of the grass family is unclear. Here, we present Bayesian phylogenetic evidence of a well-supported BA1/LAX1 clade comprised monocots and eudicots, estimating an origin for the lineage at least near the base of flowering plants. Genomic analyses in Arabidopsis, papaya, medicago, rice, sorghum, and maize indicate that BA1/LAX1 genes reside in syntenic regions, although there has also been a complex pattern of gene duplication and loss during the diversification of the angiosperm clade. BA1/LAX1 mRNA expression coincided with the initiation of leaves and associated AMs in the vegetative meristems of broccoli, medicago, and papaya implicating a role for the lineage in the formation of AMs in eudicots as well as monocots. Expression on the adaxial surface of lateral inflorescence structures was conserved in all sampled flowering plants, whereas mRNA expression in leaves of Arabidopsis, broccoli, and papaya also links BA1/LAX1 co-orthologs with roles in regulating leaf development, possibly as a downstream target of auxin regulating genes. Together these data point to roles for BA1/LAX1 genes during AM formation, leaf, and inflorescence development in diverse flowering plants and lend support to the hypothesis that the same genetic mechanisms regulate the development of different AM types.

MADS-box gene expression and implications for developmental origins of the grass spikelet.

Basic questions regarding the origin and evolution of grass (Poaceae) inflorescence morphology remain unresolved, including the developmental genetic basis for evolution of the highly derived outer spikelet organs. To evaluate homologies between the outer sterile organs of grass spikelets and inflorescence structures of nongrass monocot flowers, we describe expression patterns of APETALA1/FRUITFULL-like (AP1/FUL) and LEAFY HULL STERILE-like (LHS1) MADS-box genes in an early-diverging grass (Streptochaeta angustifolia) and a nongrass outgroup (Joinvillea ascendens). AP1/FUL-like genes are expressed only in floral organs of J. ascendens, supporting the hypothesis that they mark the floral boundary in nongrass monocots, and JaLHS1/OsMADS5 is expressed in the inner and outer tepals, stamen filaments and pistil. In S. angustifolia, SaFUL2 is expressed in all 11 (or 12) bracts of the primary inflorescence branch, but not in the suppressed floral bract below the abscission zone. In contrast, SaLHS1 is only expressed in bracts 6-11 (or 12). Together, these data are consistent with the hypotheses that (1) bracts 1-5 of S. angustifolia primary inflorescence branches and glumes of grass spikelets are homologous and that (2) the outer tepals of immediate grass relatives, bracts 6-8 of S. angustifolia, and the lemma/palea are homologous, although other explanations are possible.

Evidence for distinct roles of the SEPALLATA gene LEAFY HULL STERILE1 in Eleusine indica and Megathyrsus maximus (Poaceae).

LEAFY HULL STERILE1 (LHS1) is an MIKC-type MADS-box gene in the SEPALLATA class. Expression patterns of LHS1 homologs vary among species of grasses, and may be involved in determining palea and lemma morphology, specifying the terminal floret of the spikelet, and sex determination. Here we present LHS1 expression data from Eleusine indica (subfamily Chloridoideae) and Megathyrsus maximus (subfamily Panicoideae) to provide further insights into the hypothesized roles of the gene. E. indica has spikelets with three to eight florets that mature acropetally; E. indica LHS1 (EiLHS1) is expressed in the palea and lemma of all florets. In contrast, M. maximus has spikelets with two florets that mature basipetally; M. maximus LHS1 (MmLHS1) is expressed in the palea and lemma of the distal floret only. These data are consistent with the hypothesis that LHS1 plays a role in determining palea and lemma morphology and specifies the terminal floret of basipetally maturing grass spikelets. However, LHS1 expression does not correlate with floret sex expression; MmLHS1 is restricted to the bisexual distal floret, whereas EiLHS1 is expressed in both sterile and bisexual floret meristems. Phylogenetic analyses reconstruct a complex pattern of LHS1 expression evolution in grasses. LHS1 expression within the gynoecium has apparently been lost twice, once before diversification of a major clade within tribe Paniceae, and once in subfamily Chloridoideae. These data suggest that LHS1 has multiple roles during spikelet development and may have played a role in the diversification of spikelet morphology.

Evolution of unisexual flowers in grasses (Poaceae) and the putative sex-determination gene, TASSELSEED2 (TS2).

Unisexuality has evolved repeatedly in flowering plants, but its genetic control is not understood in most cases. In maize (Zea mays), unisexual flower development is regulated by a short-chain dehydrogenase/reductase protein, TASSELSEED2 (TS2), but its role in other grass lineages is unknown. TS2 was cloned and sequenced from a broad range of grasses and compared to available sequences from other flowering plants using phylogenetic analysis and tests for selection. Gene expression was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. TS2 orthologs appear to be restricted to monocots. The TS2 protein sequence was found to be generally under purifying selection in bisexual and unisexual lineages alike. Only one site, in unisexual herbaceous bamboos, is potentially under positive selection. TS2 was expressed broadly in all sampled tissues of unisexual and bisexual grasses, and was also expressed in rice flowers in floral organs that do not abort. TS2 may have a more general developmental role in most grasses than programmed cell death of the developing gynoecium, but has been co-opted to this role within a subset of Poaceae, probably as a result of alterations in the activity or regulation of other genes in the gynoecial pathway.

SEPALLATA gene diversification: brave new whorls.

SEPALLATA (SEP) genes form an integral part of models that outline the molecular basis of floral organ determination and are hypothesized to act as co-factors with ABCD floral homeotic genes in specifying different floral whorls. The four SEP genes in Arabidopsis function redundantly, but the extent to which SEP genes in other flowering plants function similarly is unknown. Using a recent 113-gene SEP phylogeny as a framework, we find surprising heterogeneity among SEP gene C-terminal motifs, mRNA expression patterns, protein-protein interactions and inferred function. Although some SEP genes appear to function redundantly, others have novel roles in fruit maturation, floral organ specification and plant architecture, and have played a major role in floral evolution of diverse plants.

Primaclade--a flexible tool to find conserved PCR primers across multiple species.

Primaclade is a web-based application that accepts a multiple species nucleotide alignment file as input and identifies a set of polymerase chain reaction (PCR) primers that will bind across the alignment. Primaclade iteratively runs the Primer3 application for each alignment sequence and collates the results. Primaclade creates an HTML results page that recaps the original alignment, provides a consensus sequence and lists primers for each alignment area, with primers color-coded to reflect the level of degeneracy in the primer.

Heterogeneous expression patterns and separate roles of the SEPALLATA gene LEAFY HULL STERILE1 in grasses.

SEPALLATA (SEP) genes exhibit distinct patterns of expression and function in the grass species rice (Oryza sativa) and maize (Zea mays), suggesting that the role of the genes has changed during the evolution of the family. Here, we examine expression of the SEP-like gene LEAFY HULL STERILE1 (LHS1) in phylogenetically disparate grasses, reconstruct the pattern of gene expression evolution within the family, and then use the expression patterns to test hypotheses of gene function. Our data support a general role for LHS1 in specifying determinacy of the spikelet meristem and also in determining the identity of lemmas and paleas; these two functions are separable, as is the role of the gene in specifying floret meristems. We find no evidence that LHS1 determines flower number; it is strongly expressed in all spikelet meristems even as they are producing flowers, and expression is not correlated with eventual flower number. LHS1 expression in only the upper flowers of the spikelet appears to be the ancestral state; expression in all flowers is derived in subfamily Pooideae. LHS1 expression in pistils, stamens, and lodicules varies among the cereals. We hypothesize that LHS1 may have affected morphological diversification of grass inflorescences by mediating the expression of different floral identity genes in different regions of the floret and spikelet.

Phylogeny of Gaertnera Lam. (Rubiaceae) based on multiple DNA markers: evidence of a rapid radiation in a widespread, morphologically diverse genus.

Phylogenetic relationships among 28 of the 68 species of the paleotropical genus Gaertnera (Rubiaceae) and two related genera were inferred from nucleotide sequence variation in four nuclear DNA (nDNA) markers: the internal transcribed spacers of nuclear rDNA (ITS), the large and small copies of phosphoenolpyruvate carboxylase (PepC-large and PepC-small), and triose phosphate isomerase (Tpi). Phylogenetic analysis of the combined nDNA dataset suggested that Gaertnera is monophyletic, but genetic variation among species was insufficient to reconstruct well-supported relationships within the genus. This was counter to expectations based on the very distinct morphologies and widespread distribution of the genus (West Africa to Sulawesi). Molecular clock analyses suggested variable dates of origin for Gaertnera depending upon the calibration method used. The most plausible calibration implies that Gaertnera migrated to Africa during the early Tertiary, possibly via a boreotropical land bridge and suggests that Gaertnera started to radiate 5.21 +/- 0.14 million years ago. This implies that range expansion in the group has occurred via a number of long-distance dispersal events rather than vicariance. The molecular clock estimate in turn estimated an unusually rapid lineage diversification rate within the radiation of 0.717-0.832 species/million years, comparable to those estimated for radiations on oceanic islands. Although low interspecific competition levels may have contributed to the diversification of Gaertnera on Mauritius, the mechanisms driving the rapid radiation of the group in other parts of its range remain elusive.