Characters evolution of Encyclia (Laeliinae-Orchidaceae) reveals a complex pattern not phylogenetically determined: insights from macro- and micromorphology.

Encyclia is the second-largest genus in the neotropical subtribe Laeliinae (Orchidaceae) and has more than 150 species, which are characterized by fairly consistent flower morphology. Its taxonomy and species boundaries, however, seem to be still under debate. In the present study, we first examined the lip micromorphology of 61 species of Encyclia sensu stricto. We correlated our results with external flower morphology and phylogenetic analyses performed on a combined dataset that included both nuclear (ITS, Xdh, PhyC) and plastid markers (ycf1, rpl32, and trnL-trnF). Phylogenetic reconstruction showed that Encyclia sensu stricto species form a coherent, monophyletic group. However, it is difficult to determine the relationships between the different groups within one larger clade. The groups all form distinct lineages that evolved from a common ancestor. The UPGMA cluster analysis for the seven qualitative micromorphological features clearly divides the genus into two main groups, the larger of which is further subdivided into two subgroups. None of these, however, overlap with any of the phylogeographic units distinguished in previously published papers or in presented article. It is worth noting that the groups resulting from the UPGMA analysis cannot be defined by macromorphological features. The pattern of similarities between species, taking into account both macro- and micromorphological features, is eminently mosaic in nature, and only a multifaceted approach can explain this enigmatic group.

Evaluation of the yield, chemical composition and biological properties of essential oil from bioreactor-grown cultures of Salvia apiana microshoots.

Microshoot cultures of the North American endemic Salvia apiana were established for the first time and evaluated for essential oil production. Stationary cultures, grown on Schenk-Hildebrandt (SH) medium, supplemented with 0.22 mg/L thidiazuron (TDZ), 2.0 mg/L 6-benzylaminopurine and 3.0% (w/v) sucrose, accumulated 1.27% (v/m dry weight) essential oil, consisting mostly of 1,8-cineole, ß-pinene, α-pinene, ß-myrcene and camphor. The microshoots were adapted to agitated culture, showing biomass yields up to ca. 19 g/L. Scale-up studies demonstrated that S. spiana microshoots grow well in temporary immersion systems (TIS). In the RITA bioreactor, up to 19.27 g/L dry biomass was obtained, containing 1.1% oil with up to ca. 42% cineole content. The other systems employed, i.e. Plantform (TIS) and a custom made spray bioreactor (SGB), yielded ca. 18 and 19 g/L dry weight, respectively. The essential oil content of Plantform and SGB-grown microshoots was comparable to RITA bioreactor, however, the content of cineole was substantially higher (ca. 55%). Oil samples isolated from in vitro material proved to be active in acetylcholinesterase (up to 60.0% inhibition recorded for Plantform-grown microshoots), as well as hyaluronidase and tyrosinase-inhibitory assays (up to 45.8 and 64.5% inhibition observed in the case of the SGB culture).

The Floral Signals of the Inconspicuous Orchid Malaxis monophyllos: How to Lure Small Pollinators in an Abundant Environment.

Many orchid species have evolved complex floral signals to ensure pollination efficiency. Here, the authors combined analyses of anatomical flower structure with analyses of the volatile composition and flower-visiting insects' behaviour, as well as characterised features that can attract pollinators of the inconspicuous orchid Malaxis monophyllos. During field observations, the authors found that only small Diptera (e.g., mosquitos, drosophilids, fungus gnats) visit and are interested in the flowers of M. monophyllos, which was reflected in the characterised flower features that combine well with the pollination system, which engages dipterans. Analyses of the M. monophyllos floral scent revealed substantial concentrations of aliphatic compounds, e.g., 1-octen-3-ol and 1-octanol, which condition the mushroom-like scent and a substantial fraction of alkanes, some of which have been previously described as sex mimicry and aggregation pheromones in orchids' deceptive systems. The labellum anatomical structure exhibits a highly diverse cell cuticle surface and pronounced metabolic and secretory activity of the epidermal and subepidermal cells from all parts of the labellum. Moreover, our study provides evidence for the subsequent decoys of M. monophyllos flowers, including visual signals, such as raphides located on the labellum margin and the rewarding ones connected with lipid secretion limited to the area behind the column. Taking an integrative approach to studying M. monophyllos pollination biology, the authors provide new insight into its previously vague pollination strategies and provide evidence for complex floral signal operation in luring potential pollinators. The synergistic effect of M. monophyllos flowers' volatile and visual signals, together with additional rewarding for nectar/fungus/microbe-feeding pollinators, requires further detailed investigation that will be invaluable in explaining the evolution of Diptera-specific pollination systems in orchids.

Species Phylogeny versus Gene Trees: A Case Study of an Incongruent Data Matrix Based on Paphiopedilum Pfitz. (Orchidaceae).

The phylogeny of the genus Paphiopedilum based on the plastome is consistent with morphological analysis. However, to date, none of the analyzed nuclear markers has confirmed this. Topology incongruence among the trees of different nuclear markers concerns entire sections of the subgenus Paphiopedilum. The low-copy nuclear protein-coding gene PHYC was obtained for 22 species representing all sections and subgenera of Paphiopedilum. The nuclear-based phylogeny is supported by morphological characteristics and plastid data analysis. We assumed that an incongruence in nuclear gene trees is caused by ancestral homoploid hybridization. We present a model for inferring the phylogeny of the species despite the incongruence of the different tree topologies. Our analysis, based on six low-copy nuclear genes, is congruent with plastome phylogeny and has been confirmed by phylogenetic network analysis.

Effects of the entomopathogenic fungus Metarhizium flavoviride on the fat body lipid composition of Zophobas morio larvae (Coleoptera: Tenebrionidae).

Insects employ different defense strategies against fungal infections and chemicals. We aimed to identify the lipid compositions of the fat body of Zophobas morio larvae before and after fungal infection with the entomopathogenic fungus Metarhizium flavoviride. We used gas chromatography-mass spectrometry to analyze lipid extracts of the fat body isolated of Z. morio 2, 5, and 7 days after fungal infection (treatment group) and compared it with the lipid extracts in a control group injected with physiological isotonic saline. In all the samples, fatty acids were the most abundant compound found in the fat body extracts, with hexadecanoic acid/C16:0 being the most abundant lipid. However, the types and concentrations of lipids changed after fungal infection, likely as an immune response. The most considerable changes occurred in the concentrations of long-chain fatty acids, i.e., hexadecanoic acid/C16:0, octadecenoic acid/C18:1, and octadecanoic acid/C18:0. Contents of methyl ester increased significantly after infection, but that of other esters, especially octanoic acid decyl ester/OADE, decreased on the 5th day after infection. To the best of our knowledge, this is the first detailed analysis of the changes in the lipid composition of the fat body of Z. morio larvae as a result of fungal infection. Our results suggest that entomopathogenic fungal infection affects the internal lipid composition of insects, potentially as a way of adjusting to such infection. These results can help understand infection processes and defense strategies of insects against fungal infection. Ultimately, they can contribute to the creation of more effective chemicals against pest insects.

Genetic diversity and population structure of Polish Arabian horses assessed through breeding and microsatellite data.

Polish Arabian horses are one of the most important populations of this breed in the world. Their post-war history can be divided into two periods, with the dominant role of state studs until 1989, and the increasing significance of private breeding in the next years. The goal of the study was to evaluate genetic diversity and structure of the population under a new breeding policy. The analyses of breeding and microsatellite data from 1996 to 2012 provide a coherent picture of the population with constant flow of horses only in one direction from state to private studs. An increase in the number of broodmares was observed, from 396 mares in 1996 to 1021 mares in 2012. The proportion of foreign sires used in Polish studs also increased, from 7.1% to 37.0%. An increasing number of alleles and progressive differentiation in mares were observed. STRUCTURE analysis indicated that the Polish horses were clearly separated from foreign horses used in Polish breeding, although only one of the 75 alleles found can be considered as typically Polish. The high heterozygosity is an important feature of the Polish population; however, the decrease of heterozygosity in state broodmares was noted. This issue needs to be studied further.

Global warming not so harmful for all plants - response of holomycotrophic orchid species for the future climate change.

Current and expected changes in global climate are major threat for biological diversity affecting individuals, communities and ecosystems. However, there is no general trend in the plants response to the climate change. The aim of present study was to evaluate impact of the future climate changes on the distribution of holomycotrophic orchid species using ecological niche modeling approach. Three different scenarios of future climate changes were tested to obtain the most comprehensive insight in the possible habitat loss of 16 holomycotrophic orchids. The extinction of Cephalanthera austiniae was predicted in all analyses. The coverage of suitable niches of Pogoniopsis schenckii will decrease to 1-30% of its current extent. The reduction of at least 50% of climatic niche of Erythrorchis cassythoides and Limodorum abortivum will be observed. In turn, the coverage of suitable niches of Hexalectris spicata, Uleiorchis ulaei and Wullschlaegelia calcarata may be even 16-74 times larger than in the present time. The conducted niche modeling and analysis of the similarity of their climatic tolerance showed instead that the future modification of the coverage of their suitable niches will not be unified and the future climate changes may be not so harmful for holomycotrophic orchids as expected.

Herbarium-based studies on taxonomy, biogeography and ecology of Psilochilus (Orchidaceae).

BACKGROUND: Psilochilus is a poorly studied orchid genus distributed from southern Mexico to south-eastern Brazil. A taxonomic revision of this Neotropical endemic based on morphological data is presented. MATERIAL AND METHODS: Over 170 dried herbarium specimens and flowers preserved in liquid of Psilochilus were analyzed. Morphological variation among examined taxa was described based on multivariate analysis. To evaluate the similarity between niches occupied by various Psilochilus species ecological niche modeling (ENM) was applied. Species richness and the distribution patterns of Psilochilus representatives were analyzed based on squares of 5° latitude and longitude while similarities among floras between biogeographical units were measured using the Bray-Curtis index for presence/absence data. RESULTS AND DISCUSSION: A new species of the P. physurifolius-complex is described based on Central American material. Psilochilus crenatifolius is reduced to the rank of variety as P. macrophyllus var. crenatifolius. A key to 18 accepted Psilochilus species is provided. The illustrations of perianth segments of all recognized taxa are presented. The climatic niches preferred by the particular Psilochilus representatives are well separated based on ecological niche modeling analysis. Their distribution is limited mainly by the isothermality and temperature seasonality. The highest Psilochilus species richness is observed in the North Andean, Panamanian, Brazilian Planalto and Central American biogeographical provinces. A high level of endemism is observed in all those regions as well as Yungas biogeographical province. Most Psilochilus species occur in areas above 800 m of elevation. The populations were most often reported from the tropical rain forest and tropical moist deciduous forest.

Effect of Conidiobolus coronatus on the Cuticular and Internal Lipid Composition of Tettigonia viridissima Males.

Conidiobolus coronatus is an entomopathogenic fungus which has a potential as a biological control agent of insects. The cuticular and internal lipid composition of infected and noninfected Tettigonia viridissima males were analyzed by GC/MS. A total of 49 compounds were identified in the infected and noninfected males, including fatty acids, fatty acid methyl esters (FAMEs), n-alkanes, alcohols, sterols, and other organic compounds. The most abundant components of the cuticular and internal lipids of the insects were fatty acids. After exposure to C. coronatus, the cuticular lipids of the T. viridissima males contained 17 free fatty acids from C(8) to C(22), while the cuticular lipids of the noninfected insects contained only 15 fatty acids from C(12) to C(24). The cuticular and internal lipids of both the infected and the noninfected males also contained five FAMEs from C(15) to C(19), seven n-alkanes from C(25) to C(34), five alcohols from C(16) to C(25), five sterols, and the following six other organic compounds: azelaic acid, phenylacetic acid, glutaric acid, benzoic acid, sebacic acid, and glycerol. The compounds which were present only in the cuticular lipids of the infected males could be due to fungal infection.

Glacial Refugia and Future Habitat Coverage of Selected Dactylorhiza Representatives (Orchidaceae).

The intensively discussed taxonomic complexity of the Dactylorhiza genus is probably correlated with its migration history during glaciations and interglacial periods. Previous studies on past processes affecting the current distribution of Dactylorhiza species as well as the history of the polyploid complex formation were based only on molecular data. In the present study the ecological niche modeling (ENM) technique was applied in order to describe the distribution of potential refugia for the selected Dactylorhiza representatives during the Last Glacial Maximum. Additionally, future changes in their potential habitat coverage were measured with regard to three various climatic change scenarios. The maximum entropy method was used to create models of suitable niche distribution. A database of Dactylorhiza localities was prepared on the grounds of information collected from literature and data gathered during field works. Our research indicated that the habitats of majority of the studied taxa will decrease by 2080, except for D. incarnata var. incarnata, for which suitable habitats will increase almost two-fold in the global scale. Moreover, the potential habitats of some taxa are located outside their currently known geographical ranges, e.g. the Aleutian Islands, the western slopes of the Rocky Mountains, Newfoundland, southern Greenland and Iceland. ENM analysis did not confirm that the Balkans, central Europe or central Russia served as the most important refugia for individual representatives of the Dactylorhiza incarnata/maculata complex. Our study rather indicated that the Black Sea coast, southern Apennines and Corsica were the main areas characterized by habitats suitable for most of the taxa.