Salvatore Cozzolino

Phytotoxicity to and uptake of enrofloxacin in crop plants

Phytotoxicity of enrofloxacin on crop plants Cucumis sativus, Lactuca sativa, Phaseolus vulgaris and Raphanus sativus was determined in a laboratory model: the effect of 50, 100 and 5000 microgl(-1) were evaluated after 30 days exposure by measuring post-germinative growth of primary root, hypocotyl, cotyledons and leaves. Concentrations between 50 and 5000 microgl(-1) induced both toxic effect and hormesis in plants, by significantly modifying both length of primary root, hypocotyl, cotyledons and the number/length of leaves. A toxic effect is induced by high concentration (5000 microgl(-1)), while hormesis occurs at low concentrations (50 and 100 microgl(-1)). A continuum between toxic effect and hormesis is found in the four plant species. Both toxic effect and hormesis can be related to an efficient plant drug uptake, in the order of microgg(-1). Plants are able to metabolize enrofloxacin into ciprofloxacin, as also happens in animals; Cucumis, Lactuca and Phaseolus biologically convert about one quarter of stored enrofloxacin. The ecological implication of enrofloxacin contamination in terrestrial environments is discussed.

Evolution of reproductive isolation in plants

Reproductive isolation is essential for the process of speciation and much has been learned in recent years about the ecology and underlying genetics of reproductive barriers. But plant species are typically isolated not by a single factor, but by a large number of different pre- and postzygotic barriers, and their potentially complex interactions. This phenomenon has often been ignored to date. Recent studies of the relative importance of different isolating barriers between plant species pairs concluded that prezygotic isolation is much stronger than postzygotic isolation. But studies of the patterns of reproductive isolation in plants did not find that prezygotic isolation evolves faster than postzygotic isolation, in contrast to most animals. This may be due to the multiple premating barriers that isolate most species pairs, some of which may be controlled by few genes of major effect and evolve rapidly, whereas others have a complex genetic architecture and evolve more slowly. Intrinsic postzygotic isolation in plants is correlated with genetic divergence, but some instrinsic postzygotic barriers evolve rapidly and are polymorphic within species. Extrinsic postzygotic barriers are rarely included in estimates of different components of reproductive isolation. Much remains to be learned about ecological and molecular interactions among isolating barriers. The role of reinforcement and reproductive character displacement in the evolution of premating barriers is an open topic that deserves further study. At the molecular level, chromosomal and genic isolation factors may be associated and act in concert to mediate reproductive isolation, but their interactions are only starting to be explored.

Patterns of reproductive isolation in Mediterranean deceptive orchids.

Abstract The evolution of reproductive isolation is of central interest in evolutionary biology. In plants, this is typically achieved by a combination of pre- and postpollination mechanisms that prevent, or limit, the amount of interspecific gene flow. Here, we investigated and compared two ecologically defined groups of Mediterranean orchids that differ in pollination biology and pollinator specificity: sexually deceptive orchids versus food-deceptive orchids. We used experimental crosses to assess the strength of postmating prezygotic, and postzygotic reproductive isolation, and a phylogenetic framework to determine their relative rates of evolution. We found quantitative and qualitative differences between the two groups. Food-deceptive orchids have weak premating isolation but strong postmating isolation, whereas the converse situation characterizes sexually deceptive orchids. Only postzygotic reproductive isolation among food-deceptive orchids was found to evolve in a clock-like manner. Comparison of evolutionary rates, within a common interval of genetic distance, showed that the contribution of postmating barriers was more relevant in the food-deceptive species than in the sexually deceptive species. Asymmetry in prezygotic isolation was found among food-deceptive species. Our results indicate that postmating barriers are most important for reproductive isolation in food-deceptive orchids, whereas premating barriers are most important in sexually deceptive orchids. The different rate of evolution of reproductive isolation and the relative strength of pre- and postmating barriers may have implication for speciation processes in the two orchid groups.

Sympatric bromeliad species (Pitcairnia spp.) facilitate tests of mechanisms involved in species cohesion and reproductive isolation in Neotropical inselbergs

The roles of intra‐ and interspecific gene flow in speciation and species evolution are topics of great current interest in molecular ecology and evolutionary biology. Recent modelling studies call for new empirical data to test hypotheses arising from the recent shift from a ‘whole‐genome reproductive isolation’ view to a ‘genic’ view of species and speciation. Particularly scarce (and thus of particular interest) are molecular genetic data on recently radiated, naturally hybridizing species in strongly structured and species‐rich environments. Here, we studied four sympatric plant species (Pitcairnia spp.; Bromeliaceae) adapted to Neotropical inselbergs (isolated outcrops resembling habitat ‘islands’ in tropical rainforests) using nuclear and plastid DNA. Patterns of plastid DNA haplotype sharing and nuclear genomic admixture suggest the presence of both, incomplete lineage sorting and interspecific gene flow over extended periods of time. Integrity and cohesion of inselberg species of Pitcairnia are maintained despite introgression and in the face of extremely low within‐species migration rates (Nem < 1 migrant per generation). Cross‐evaluation of our genetic data against published pollination experiments indicate that species integrity is maintained by the simultaneous action of multiple prezygotic barriers, including flowering phenology, pollinator isolation and divergent mating systems. Postzygotic Bateson–Dobzhansky–Muller incompatibilities appear to contribute to isolation, as suggested by asymmetric introgression rates of single loci. Our results suggest that incomplete lineage sorting, hybridization and introgression form integral aspects of adaptive radiation in Neotropical inselberg ‘archipelagos’. Inselbergs with multiple closely related co‐occurring species should be of special interest to students of speciation in mountain systems, and to ongoing conservation programmes in the Atlantic Rainforest biodiversity hotspot.

Evolution of Postzygotic Reproductive Isolation in a Guild of Deceptive Orchids

The evolution of reproductive barriers is of central importance for speciation. Here, we investigated three components of postzygotic isolation—embryo mortality, hybrid inviability, and hybrid sterility—in a group of food‐deceptive Mediterranean orchids from the genera Anacamptis, Neotinea, and Orchis. In these orchids, pollinator‐mediated isolation is weak, which suggests that postpollination barriers exist. Based on crossing experiments and a literature survey, we found that embryo mortality caused complete reproductive isolation among 36.3% of the species pairs, and hybrid inviability affected 55.6% of the potentially hybridizing species pairs. Hybrid sterility was assessed experimentally for seven species pairs. A strong reduction of fertility in all investigated hybrids was found, together with clear differences between male and female components of hybrid sterility. Postzygotic isolation was found to evolve gradually with genetic divergence, and late postzygotic isolation (i.e., hybrid inviability and sterility) evolved faster than embryo mortality, which is an earlier postzygotic isolation stage. These results reveal that intrinsic postzygotic isolation strongly contributes to maintaining species boundaries among Mediterranean food‐deceptive orchids while establishing a prominent role for these reproductive barriers in the early stage of species isolation.

Floral isolation is the main reproductive barrier among closely related sexually deceptive orchids.

Floral isolation is an important component of pollinator‐driven speciation. However, up to now, only a few studies have quantified its strength and relative contribution to total reproductive isolation. In this study, we quantified floral isolation among three closely related, sympatric orchid species of the genus Ophrys by directly tracking pollen flow. Ophrys orchids mimic their pollinators’ mating signals, and are pollinated by male insects during mating attempts. This pollination system, called sexual deception, is usually highly specific. However, whether pollinator specialization also conveys floral isolation is currently under debate. In this study, we found strong floral isolation: among 46 tracked pollen transfers in two flowering seasons, all occurred within species. Accounting for observation error rate, we estimated a floral isolation index ≥0.98 among each pair of species. Hand pollination experiments suggested that postpollination barriers were effectively absent among our study species. Genetic analysis based on AFLP markers showed a clear species clustering and very few F1 hybrids in natural populations, providing independent evidence that strong floral isolation prevents significant interspecies gene flow. Our results provide the first direct evidence that floral isolation acts as the main reproductive barrier among closely related plant species with specialized pollination.

Effect on plants of sulphadimethoxine used in intensive farming (Panicum miliaceum, Pisum sativum and Zea mays)

Abstract Animal wastes from intensive farming are generally collected for field fertilisation. They contain drugs that pollute soil. To evaluate the effect of sulphonamide contamination in terrestrial systems, sulphadimethoxine has been subjected to standardisable laboratory tests using seeds of Panicum miliaceum, Pisum sativum, and Zea mays. The drug alters normal post-germinative development and growth of roots, hypocotyls and leaves in the three species; this toxic effect depends on the bioaccumulation rate, and is higher for C4 plants (Panicum and Zea) than for a C3 plant (Pisum). The environmental risk of sludge application on soils and the possible contamination of food chains are discussed.

Evidence for pollinator sharing in Mediterranean nectar-mimic orchids: absence of premating barriers?

Pollinator specificity has traditionally been considered the main reproductive isolation mechanism in orchids. Among Mediterranean orchids, however, many species attract and deceive pollinators by mimicking nectar-rewarding plants. To test the extent to which deceptive orchid species share pollinators, we collected and identified hemipollinaria-carrying insects, and used ribosomal sequences to identify the orchid species from which hemipollinaria were removed. We found that social and solitary bees, and also flies, carried hemipollinaria belonging to nine orchid species with different degrees of specialization. In particular, Anacamptis morio, Dactylorhiza romana and Orchis mascula used a large set of pollinator species, whereas others such as Orchis quadripunctata seemed to be pollinated by one pollinator species only. Out of the insects with hemipollinaria, 19% were found to carry hemipollinaria from more than one orchid species, indicating that sympatric food-deceptive orchids can share pollinators. This sharing was apparent even among orchid sister-species, thus revealing an effective overlap in pollinator sets among closely related species. These results suggest varying degrees of pollinator specificity in these orchids, and indicate that pollinator specificity cannot always act as the main isolation mechanism in food-deceptive temperate orchids.

Effect of sulphadimethoxine contamination on barley (Hordeum distichum L, Poaceae, Liliopsida)

Animal wastes from intensive farming are generally collected for field fertilisation. They may contain drugs that can become soil pollutants. To evaluate the possible effects of such contamination in terrestrial systems, sulphadimethoxine has been subjected to laboratory tests (in vitro, synthetic medium, and soil) using seeds of barley (Hordeum distichum L.). The drug suppressed normal post-germinative development and growth of roots and leaves in both test conditions; this effect was dependent on the bioaccumulation rate, which was higher on synthetic medium than in soil. Bioaccumulation was higher in roots than foliage and this was markedly evident in soil and, in particular, in soils with a low humus content. The environmental risk of sludge application on soils and the possible contamination of food chains are discussed.

Evidence for reproductive isolate selection in Mediterranean orchids: karyotype differences compensate for the lack of pollinator specificity

Mediterranean orchids of the subtribe Orchidinae are highly diverse and display a range of fascinating pollination strategies. Based on observations that orchid–pollinator relationships are often highly specialized and species specific, Darwin and others have argued that selection for different pollinators has been the driving force behind the evolutionary diversification of orchids. This may be true for orchids that attract different, specialized pollinators that act as prezygotic reproductive barriers. It is, however, not clear how closely related co–flowering Mediterranean orchids that share pollinators survive the challenge of sympatry. We show that species pairs with a generalized pool of pollinators have significantly more divergent karyotypes compared with species pairs with different pollinators. These results show that karyotype differences that act as postzygotic reproductive barriers may have played an important role in the evolution of Mediterranean orchid diversity.