Diversification of inflorescence development in the PCK clade (Poaceae: Panicoideae: Paniceae)

In grasses, inflorescence diversification and its correlation with species evolution are intriguing and not well understood. Part of this problem lies in our lack of comprehension about the inflorescence morphological complexity of grasses. We focused our study on the PCK clade (named for phosphoenol pyruvate carboxykinase), a well-supported monophyletic group for which the relationships among its taxa are not well resolved. Interestingly, the PCK clade has an extensive diversity of adult inflorescence forms. A comparative developmental approach can help us to understand the basis of such morphological differences as well as provide characters that can be used in phylogenetic studies of the group. Using SEM studies, we demonstrate that inflorescence morphology in this clade is even more complex than what is typically observed in adult forms. We describe a number of new characters, and some classical features previously used for taxonomic purposes are redefined on the basis of development. We also define four morphological groups combining adult inflorescence form and development, and we discuss some of the evolutionary aspects of inflorescence diversification in the PCK clade. Taxonomic delimitation among genera in the PCK clade remains confusing and unclear where molecular and morphological studies support different classifications.     

Inflorescence diversity and evolution in the PCK Clade (Poaceae: Panicoideae: Paniceae)

The PCK Clade, represented by six to nine genera, is a monophyletic group situated within the Paniceae tribe. The highly diverse inflorescences within the PCK Clade provide an interesting system for the study of morphological evolution and also may aid in better understanding its unclear systematics. The inflorescence structure of 110 members of the PCK Clade has been investigated. Inflorescences are polytelic showing different levels of truncation. At least 21 different inflorescence subtypes were identified. Fourteen variable inflorescence characters were found, among which some have suprageneric or infrageneric value and others are polymorphic. A key for the identification of inflorescence types is presented. Nine processes have been identified as responsible for inflorescence diversification. Highly branched inflorescences with different internode lengths are present in the basal genus whereas truncated inflorescence morphologies appear late in the history of the clade. The precise timing of morphological changes is impossible to assess until we have a well supported phylogeny for the PCK Clade.     

A molecular phylogeny of Panicum (Poaceae: Paniceae): tests of monophyly and phylogenetic placement within the Panicoideae

Panicum L. is a cosmopolitan genus with approximately 450 species. Although the genus has been considerably reduced in species number with the segregation of many taxa to independent genera in the last two centuries, Panicum remains a heterogeneous assemblage, as has been demonstrated in recent years. The genus is remarkably uniform in its floral characters but exhibits considerable variation in anatomical, physiological, and cytological features. As a result, several classifications, and criteria of what the genus should really include, have been postulated in modern literature. The purpose of this research, based on molecular data of the chloroplast ndhF gene, is to test the monophyly of Panicum, to evaluate infrageneric classifications, and to propose a robust phylogenetic hypothesis. Based on the present results, previous morphological and molecular phylogenetic studies, and inferred diagnostic morphological characters, we restrict Panicum sensu stricto (s.s.) to the former subgenus Panicum and support recognition of Dichanthelium, Phanopyrum, and Steinchisma as distinct genera. We have transfered other species of Panicum to other genera of the Paniceae. Most of the necessary combinations have been made previously, so few nomenclatural changes have been required. The remaining species of Panicum sensu lato (s.l.) are included within Panicum incertae sedis representing isolated species or species grouped within monophyletic clades. Additionally, we explore the performance of the three codon position characters in producing the supported phylogeny.     

Abnormal cytokinesis in microsporogenesis of Brachiaria humidicola (Poaceae: Paniceae)

Microsporogenesis was evaluated in the Brachiaria humidicola collection of the Embrapa Beef Cattle Center, represented by 60 accessions. One accession (H121) presented an abnormal pattern of cytokinesis that had never been reported in this genus. Among 900 meiocytes analyzed in the first division, 10.7% underwent precocious and multiple cytokinesis in metaphase I, fractionating the genome and the cytoplasm into two or more parts. The expected cytokinesis after telophase I did not occur. The abnormal meiocytes from the first division entered the second division but the second cytokinesis after telophase II was also abnormal. Among the 857 meiocytes analyzed in the second division, 10.9% presented abnormal, incomplete or total absence of cytokinesis. Dyads and binucleated microspores were recorded among the meiotic products. The use of this accession in the Embrapa breeding program is compromised.     

Understanding spikelet orientation in Paniceae (Poaceae)

Spikelet structure and grouping are key characters to identify grasses. Here we tested the possibility that spikelet pairs, a distinctive morphological structure of many Andropogoneae and Paniceae, are the starting point for a secondary single spikelet condition that can also explain the change of spikelet orientation among Paniceae genera. As a first approach, we studied the inflorescence development of Paspalum simplex, P. stellatum, and Axonopus sufultus to clarify the origin of the spikelet orientation and other basic homologies. The results support that solitary spikelets of A. suffultus are homologous to the subsessile spikelets of P. simplex and that solitary spikelets of P. stellatum are homologous to the pedicellate spikelet of P. simplex. This last homology supports that spikelet orientation results from a differential reduction/abortion of either the pedicellate or the subsessile spikelet primordia. We also discuss the possibility that the RAMOSA and polar auxin pathways could play a role in the abortion of the lateral subsessile spikelets in P. stellatum. However, the apical meristem inhibition observed in A. suffultus and P. stellatum seems to depend on a very different genetic control, suggesting that the single spikelet condition is homoplasic within Paniceae and derived from at least two different genetic mechanisms.     

Unusual chromosome numbers in Paspalum L. (Poaceae: Paniceae) from Brazil

Somatic chromosome numbers were determined for 20 new germplasm accessions of Paspalum, belonging to 17 species collected in Brazil. Chromosome number is reported for the first time for P. reduncum (2n = 18), P. cinerascens (2n = 20), P. cordatum (2n = 20), P. filgueirasii (2n = 24), P. ammodes (2n = 36), P. bicilium (2n = 40), P. heterotrichon (2n = 40), and P. burmanii (2n = 48). New cytotypes were confirmed for two germplasm accessions of P. carinatum (2n = 30) and P. trachycoleon (2n = 36), one of P. clavuliferum (2n = 40) and one of P. lanciflorum (2n = 40), indicating variability in these species. The remaining chromosome numbers reported here confirm previous counts. The unexpected chromosome numbers 2n = 18, 24, 36, and 48 in Paspalum species, which are usually shown to be multiples of 10, suggest that much more collection and cytogenetic characterization are necessary to assess the whole chromosomal and genomic multiplicity present in the genus, which seems to be much more diverse than currently thought to be.     

Atypical foliar anatomy related to Kranz syndrome in Paspalum inaequivalve and Paspalum microstachyum (Poaceae: Panicoideae: Paniceae)

Paspalum L. is a large and complex genus, enclosing more than 300 species, whose boundaries and infrageneric classification are still being studied. Recent phylogenetic analyses suggest that Paspalum inaequivalve Raddi and Paspalum microstachyum J. Presl, from the Inaequivalvia informal group, should be excluded from Paspalum. Focused on the unclear taxonomic position of P. inaequivalve and P. microstachyum, their leaf anatomy was studied, and some atypical features related to C₄ photosynthesis were found. This atypical Kranz syndrome is the aim of this research. Transverse leaf blade sections from fresh and herbarium material of P. inaequivalve were studied by light, fluorescence, and transmission microscopy. Additionally, sheaths and culms of P. inaequivalve and leaf blades of P. microstachyum were observed by light microscopy. δ¹³C isotope discrimination was determinated for P. inaequivalve. We compared our results with available anatomical data from related taxa. As well as typical mesophyll cells (PCA) and mestome sheath cells (PCR), a third type of cells, here called ‘globose parenchymatous cells’, was observed in leaf blades of P. inaequivalve and P. microstachyum. These cells are placed externally to the mestome sheaths of the first and second vascular bundles, they have thin walls, with no developed suberine lamella, few chloroplasts with 1–2 starch grains, thylakoids not organized in grana, and a large central vacuole. The globose parenchymatous cells represent a novel trait in P. inaequivalve and P. microstachyum, further supporting the close relation between both species and their exclusion from the genus Paspalum. This atypical Kranz syndrome has not been described in Paspalum before, but the globose parenchymatous cells here described resemble the distinct cells considered as remnants of the outer parenchymatous sheath described for Anthaenantiopsis, some sections of Panicum L., and Chaetium Nees, providing possible taxonomic significance.     

Reproductive biology of South American Bothriochloa (Poaceae: Andropogoneae)

The reproductive biology of 13 South American taxa of Bothriochloa was studied. All surveyed species reproduce sexually and are self-compatible. Several elements also point to self-pollination as their main breeding system: (i) bud pollination that occurs in most species enhancing cleistogamy, (ii) presence of pits in the glumes that impede the opening of flowers and (iii) reduction in the number of stamens, a trend associated with outbreeding.     

Microsporogenesis in Brachiaria dictyoneura (Fig. & De Not.) Stapf (Poaceae: Paniceae)

Microsporogenesis was analyzed in five accessions of Brachiaria dictyoneura presenting x = 6 as the basic chromosome number. All accessions were tetraploid (2n = 4x = 24) with chromosome pairing in bi-, tri-, and quadrivalents. The recorded meiotic abnormalities were those typical of polyploids, including precocious chromosome migration to the poles, laggard chromosomes, and micronucleus formation. The frequency of these abnormalities, however, was lower than those reported for other polyploid accessions previously analyzed for other Brachiaria species. Cell fusion and absence of cytokinesis were also recorded in some accessions, leading to restitutional nucleus formation in some cells. Genetically unbalanced microspores, binucleate, and 2n microspores were found among normal meiotic products as results from these abnormalities. The limitation in using these accessions as pollen donor in interspecific crosses with sexual species with x = 7 or x = 9 in breeding programs is discussed.     

Weedy adaptation in Setaria spp. V. Effects of gaseous environment on giant foxtail (Setaria faberii) (Poaceae) seed germination

The effects that naturally occurring gases (oxygen, nitrogen, carbon monoxide) may cause in dormant giant foxtail (Setaria faberii) seed germination under favorable temperature and moisture conditions were investigated. The germination responses to gas mixtures supported the hypothesis that S. faberii germination behavior is regulated by the amount of oxygen taken into hydrated seed over time. Setaria faberii seed germination was markedly affected by O(2) concentration (in N(2)) above and below that of air (20% O(2)): the largest increase in germination (from 37 to 60%) occurred between 20-25% O(2); between 0-10% O(2), germination increased from 0-30%; and surprisingly germination at 10 and 20% O(2) was similar. These observations reveal an asymmetrical response to incremental changes in O(2) above and below that typically found in agricultural soils. Carbon monoxide had opposite effects on S. faberii germination in air depending on concentration, stimulation, and inhibition: germination increased from 37 to 56% with the addition of 1% CO, but decreased from 37 to 14% with 75% added CO. An explanation may be that there are two separate effects of CO, each occurring in different physiological systems of dormant seeds at the same time. At high concentrations (75%) in air CO inhibited seed germination, probably by inhibiting mitochondrial respiration. But low CO concentrations (0.1 or 1%) in air stimulated seed germination. It was not apparent which physiological system(s) CO and O(2) affected. It seems unlikely that CO-stimulated germination arises from effects on the respiratory apparatus, but may be a consequence of CO interactions with an as yet unknown physiological factor in the seed. We provide a model of Setaria spp. dormancy consistent with its seed morphology, the gas-germination data, and the hypothesized second physiological factor that may be involved in CO stimulated germination.     

Meiotic behaviour in three interspecific three-way hybrids between Brachiaria ruziziensis and B. brizantha (Poaceae: Paniceae)

The meiotic behaviour of three three-way interspecific promising hybrids (H17, H27, and H34) was evaluated. These hybrids resulted from the crosses between B. ruziziensis X B. brizantha and crossed to another B. brizantha. Two half-sib hybrids (H27 and H34) presented an aneuploid chromosome number (2n = 4x = 33), whereas hybrid H17 was a tetraploid (2n = 4x = 36), as expected. Chromosome paired predominantly as multivalents suggesting that genetic recombination and introgression of specific target genes from B. brizantha into B. ruziziensis can be expected. Arrangement of parental genomes in distinct metaphase plates was observed in H27 and H34, which have different male genitors. Hybrids H17 and H34 have the same male genitor, but did not display this abnormality. In H17, abnormalities were more frequent from anaphase II, when many laggard chromosomes appeared, suggesting that each genome presented a different genetic control for meiotic phase timing. Despite the phylogenetic proximity among these two species, these three hybrids presented a high frequency of meiotic abnormalities, mainly those related to irregular chromosome segregation typical of polyploids, H34, 69.1%; H27, 56.1% and H17, 44.9%. From the accumulated results obtained through cytological studies in Brachiaria hybrids, it is evident that cytogenetical analysis is of prime importance in determining which genotypes can continue in the process of cultivar development and which can be successfully used in the breeding. Hybrids with high frequency of meiotic abnormalities can seriously compromise seed production, a key trait in assuring adoption of a new apomictic cultivar of Brachiaria for pasture formation.     

A molecular phylogeny of the grass subfamily Panicoideae (Poaceae) shows multiple origins of C4 photosynthesis

DNA sequence data from the chloroplast gene ndhF were analyzed to estimate the phylogeny of the subfamily Panicoideae, with emphasis on the tribe Paniceae. Our data suggest that the subfamily is divided into three strongly supported clades, corresponding to groups with largely identical base chromosome numbers. Relationships among the three clades are unclear. In unweighted parsimony analyses, the two major clades with x = 10 (Andropogoneae and x = 10 Paniceae) are weakly supported as sister taxa. The third large clade corresponds to x = 9 Paniceae. In analyses under implied weight, the two clades of Paniceae are sisters, making the tribe monophyletic. Neither resolution is strongly supported.Our molecular phylogenies are not congruent with previous classifications of tribes or subtribes. Based on this sample of species, we infer that C(4) photosynthesis has evolved independently several times, although a single origin with multiple reversals and several reacquisitions is only slightly less parsimonious. The phosphoenol pyruvate carboxykinase (PCK) subtype of C(4) photosynthesis has evolved only once, as has the NAD-malic enzyme (ME) subtype; all other origins are NADP-ME. Inflorescence bristles are apparently homologous in the genera Setaria and Pennisetum, contrary to opinions of most previous authors. Some genera, such as Digitaria, Echinochloa, and Homolepis are supported as monophyletic. The large genus Paspalum is shown to be paraphyletic, with Thrasya derived from within it. As expected, Panicum is polyphyletic, with lineages derived from multiple ancestors across the tree. Panicum subg. Panicum is monophyletic. Panicum subg. Dichanthelium, subg. Agrostoides, and subg. Phanopyrum are unrelated to each other, and none is monophyletic. Only Panicum subg. Dichanthelium sect. Dichanthelium, represented by P. sabulorum and P. koolauense, is monophyletic. Panicum subg. Megathyrsus, a monotypic subgenus including only the species P. maximum, is better placed in Urochloa, as suggested by other authors.