Phylogenetics of tribe Orchideae (Orchidaceae: Orchidoideae) based on combined DNA matrices: inferences regarding timing of diversification and evolution of pollination syndromes.

BACKGROUND AND AIMS: Tribe Orchideae (Orchidaceae: Orchidoideae) comprises around 62 mostly terrestrial genera, which are well represented in the Northern Temperate Zone and less frequently in tropical areas of both the Old and New Worlds. Phylogenetic relationships within this tribe have been studied previously using only nuclear ribosomal DNA (nuclear ribosomal internal transcribed spacer, nrITS). However, different parts of the phylogenetic tree in these analyses were weakly supported, and integrating information from different plant genomes is clearly necessary in orchids, where reticulate evolution events are putatively common. The aims of this study were to: (1) obtain a well-supported and dated phylogenetic hypothesis for tribe Orchideae, (ii) assess appropriateness of recent nomenclatural changes in this tribe in the last decade, (3) detect possible examples of reticulate evolution and (4) analyse in a temporal context evolutionary trends for subtribe Orchidinae with special emphasis on pollination systems. METHODS: The analyses included 118 samples, belonging to 103 species and 25 genera, for three DNA regions (nrITS, mitochondrial cox1 intron and plastid rpl16 intron). Bayesian and maximum-parsimony methods were used to construct a well-supported and dated tree. Evolutionary trends in the subtribe were analysed using Bayesian and maximum-likelihood methods of character evolution. KEY RESULTS: The dated phylogenetic tree strongly supported the recently recircumscribed generic concepts of Bateman and collaborators. Moreover, it was found that Orchidinae have diversified in the Mediterranean basin during the last 15 million years, and one potential example of reticulate evolution in the subtribe was identified. In Orchidinae, pollination systems have shifted on numerous occasions during the last 23 million years. CONCLUSIONS: The results indicate that ancestral Orchidinae were hymenopteran-pollinated, food-deceptive plants and that these traits have been dominant throughout the evolutionary history of the subtribe in the Mediterranean. Evidence was also obtained that the onset of sexual deception might be linked to an increase in labellum size, and the possibility is discussed that diversification in Orchidinae developed in parallel with diversification of bees and wasps from the Miocene onwards.

Evolutionary Dynamics of the Repeatome Explains Contrasting Differences in Genome Sizes and Hybrid and Polyploid Origins of Grass Loliinae Lineages.

The repeatome is composed of diverse families of repetitive DNA that keep signatures on the historical events that shaped the evolution of their hosting species. The cold seasonal Loliinae subtribe includes worldwide distributed taxa, some of which are the most important forage and lawn species (fescues and ray-grasses). The Loliinae are prone to hybridization and polyploidization. It has been observed a striking two-fold difference in genome size between the broad-leaved (BL) and fine-leaved (FL) Loliinae diploids and a general trend of genome reduction of some high polyploids. We have used genome skimming data to uncover the composition, abundance, and potential phylogenetic signal of repetitive elements across 47 representatives of the main Loliinae lineages. Independent and comparative analyses of repetitive sequences and of 5S rDNA loci were performed for all taxa under study and for four evolutionary Loliinae groups [Loliinae, Broad-leaved (BL), Fine-leaved (FL), and Schedonorus lineages]. Our data showed that the proportion of the genome covered by the repeatome in the Loliinae species was relatively high (average ∼ 51.8%), ranging from high percentages in some diploids (68.7%) to low percentages in some high-polyploids (30.7%), and that changes in their genome sizes were likely caused by gains or losses in their repeat elements. Ty3-gypsy Retand and Ty1-copia Angela retrotransposons were the most frequent repeat families in the Loliinae although the relatively more conservative Angela repeats presented the highest correlation of repeat content with genome size variation and the highest phylogenetic signal of the whole repeatome. By contrast, Athila retrotransposons presented evidence of recent proliferations almost exclusively in the Lolium clade. The repeatome evolutionary networks showed an overall topological congruence with the nuclear 35S rDNA phylogeny and a geographic-based structure for some lineages. The evolution of the Loliinae repeatome suggests a plausible scenario of recurrent allopolyploidizations followed by diploidizations that generated the large genome sizes of BL diploids as well as large genomic rearrangements in highly hybridogenous lineages that caused massive repeatome and genome contractions in the Schedonorus and Aulaxyper polyploids. Our study has contributed to disentangling the impact of the repeatome dynamics on the genome diversification and evolution of the Loliinae grasses.

Phylogenomics and Systematics of Overlooked Mesoamerican and South American Polyploid Broad-Leaved Festuca Grasses Differentiate F. sects. Glabricarpae and Ruprechtia and F. subgen. Asperifolia, Erosiflorae, Mallopetalon and Coironhuecu (subgen. nov.).

Allopolyploidy is considered a driver of diversity in subtribe Loliinae. We investigate the evolution and systematics of the poorly studied Mesoamerican and South American polyploid broad-leaved Festuca L. species of uncertain origin and unclear taxonomy. A taxonomic study of seven diagnostic morphological traits was conducted on a representation of 22 species. Phylogenomic analyses were performed on a representation of these supraspecific taxa and all other Loliinae lineages using separate data from the entire plastome, nuclear rDNA 45S and 5S genes, and repetitive DNA elements. F. subgen. Mallopetalon falls within the fine-leaved (FL) Loliinae clade, whereas the remaining taxa are nested within the broad-leaved (BL) Loliinae clade forming two separate Mexico-Central-South American (MCSAI, MCSAII) lineages. MCSAI includes representatives of F. sect. Glabricarpae and F. subgen. Asperifolia plus F. superba, and MCSAII of F. subgen. Erosiflorae and F. sect. Ruprechtia plus F. argentina. MCSAII likely had a BL Leucopoa paternal ancestor, MCSAI and MCSAII a BL Meso-South American maternal ancestor, and Mallopetalon FL, American I-II ancestors. Plastome vs. nuclear topological discordances corroborated the hybrid allopolyploid origins of these taxa, some of which probably originated from Northern Hemisphere ancestors. The observed data indicate rapid reticulate radiations in the Central-South American subcontinent. Our systematic study supports the reclassification of some studied taxa in different supraspecific Festuca ranks.

Development of an ELISA approach for the determination of flavodoxin and ferredoxin as markers of iron deficiency in phytoplankton.

Quantification of the iron-nutritional status of phytoplankton is of great interest not only for the study of oceans but also for fresh waters. Flavodoxin is a small flavoprotein proposed as a marker for iron deficiency, since it is induced as a consequence of iron deprivation, replacing the iron-sulphur protein ferredoxin. Flavodoxin and ferredoxin have been frequently used as markers for determination of iron deficiency in phytoplankton. Using purified flavodoxin and ferredoxin from Scenedesmus vacuolatus and polyclonal antibodies against both proteins, individual ELISA tests have been developed. The assays have a linear response in the range of 30-600 ng/ml of protein.

Plastocyanin/cytochrome c6 interchange in Scenedesmus vacuolatus.

Plastocyanin and cytochrome c6 from the green alga Scenedesmus vacuolatus were immunoquantified in cells grown under different concentrations of copper and iron. Plastocyanin expression was constitutive, its synthesis was not significantly affected by iron availability, and increases with copper availability. On the contrary, cytochrome c6 synthesis is repressed by copper, and only residual amounts of the protein were detected at 0.1 micromol/L copper. Under copper deficiency, cytochrome c6 is slightly dependent on iron. In natural environments, plastocyanin seems to be the predominant electron donor to P700.

Dated historical biogeography of the temperate Loliinae (Poaceae, Pooideae) grasses in the northern and southern hemispheres.

Divergence times and biogeographical analyses have been conducted within the Loliinae, one of the largest subtribes of temperate grasses. New sequence data from representatives of the almost unexplored New World, New Zealand, and Eastern Asian centres were added to those of the panMediterranean region and used to reconstruct the phylogeny of the group and to calculate the times of lineage-splitting using Bayesian approaches. The traditional separation between broad-leaved and fine-leaved Festuca species was still maintained, though several new broad-leaved lineages fell within the fine-leaved clade or were placed in an unsupported intermediate position. A strong biogeographical signal was detected for several Asian-American, American, Neozeylandic, and Macaronesian clades with different affinities to both the broad and the fine-leaved Festuca. Bayesian estimates of divergence and dispersal-vicariance analyses indicate that the broad-leaved and fine-leaved Loliinae likely originated in the Miocene (13My) in the panMediterranean-SW Asian region and then expanded towards C and E Asia from where they colonized the New World. Further expansions in America (10-3.8My) showed a predominant migratory route from North to South (N America<-->the Andes<-->Patagonia). This late Tertiary scenario of successive colonizations and secondary polyploid radiations in the southern hemisphere from the northern hemisphere was accompanied by occasional transcontinental long-distance dispersal events between South America and New Zealand. Multiple Pliocene dispersal events (3.6-2.5My) from the near SW European and NW African continents gave rise to the Macaronesian Loliinae flora, while a more recent Pleistocene origin (2-1My) is hypothesized for the high polyploid lineages that successfully colonized newly deglaciated areas in both hemispheres.