Pierluigi Cortis

Pollinator convergence and the nature of species' boundaries in sympatric Sardinian Ophrys (Orchidaceae).

BACKGROUND AND AIMS In the sexually deceptive Ophrys genus, species isolation is generally considered ethological and occurs via different, specific pollinators, but there are cases in which Ophrys species can share a common pollinator and differ in pollen placement on the body of the insect. In that condition, species are expected to be reproductively isolated through a pre-mating mechanical barrier. Here, the relative contribution of pre- vs. post-mating barriers to gene flow among two Ophrys species that share a common pollinator and can occur in sympatry is studied. METHODS A natural hybrid zone on Sardinia between O. iricolor and O. incubacea, sharing Andrena morio as pollinator, was investigated by analysing floral traits involved in pollinator attraction as odour extracts both for non-active and active compounds and for labellum morphology. The genetic architecture of the hybrid zone was also estimated with amplified fragment length polymorphism (AFLP) markers, and pollination fitness and seed set of both parental species and their hybrids in the sympatric zone were estimated by controlled crosses. KEY RESULTS Although hybrids were intermediate between parental species in labellum morphology and non-active odour compounds, both parental species and hybrids produced a similar odour bouquet for active compounds. However, hybrids produced significantly lower fruit and seed set than parental species, and the genetic architecture of the hybrid zone suggests that they were mostly first-generation hybrids. CONCLUSIONS The two parental species hybridize in sympatry as a consequence of pollinator overlap and weak mechanical isolation, but post-zygotic barriers reduce hybrid frequency and fitness, and prevent extensive introgression. These results highlight a significant contribution of late post-mating barriers, such as chromosomal divergence, for maintaining reproductive isolation, in an orchid group for which pre-mating barriers are often considered predominant.

Ménage À Trois—Two Endemic Species of Deceptive Orchids and One Pollinator Species

In the sexually deceptive orchid genus Ophrys, reproductive isolation is based on the specific attraction of males of a single pollinator species by mimicking the female species-specific sex pheromone. Changes in the odor composition can lead to hybridization and speciation by the attraction of a new pollinator that acts as an isolation barrier toward other sympatrically occurring Ophrys species. On Sardinia, we investigated the evolutionary origin of two sympatrically occurring endemic species, Ophrys chestermanii and O. normanii, which are both pollinated by males of the cuckoo bumblebee Bombus vestalis. Chemical and electrophysiological analyses of floral scent and genetic analyses with amplified fragment length polymorphisms and plastid-markers clearly showed that O. normanii is neither a hybrid nor a hybrid species. The two species evolved from different ancestors, viz. O. normanii from O. tenthredinifera and O. chestermanii from O. annae, and converged to the same pollinator attracted by the same bouquet of polar compounds. In spite of sympatry, pollinator sharing and overlapping blooming periods, no evidence has been obtained for gene flow between O. chestermanii and O. normanii indicating an unusual case among sexually deceptive orchids in which postmating rather than premating reproductive isolation mechanisms strongly prevent interspecific gene flow.

Evaluating the efficacy of restoration plantings through DNA barcoding of frugivorous bird diets.

Frugivores are critical components of restoration programs because they are seed dispersers. Thus, knowledge about bird-plant trophic relationships is essential in the evaluation of the efficacy of restoration processes. Traditionally, the diet of frugivores is characterized by microscopically identifying plant residues in droppings, which is time-consuming, requires botanical knowledge, and cannot be used for fragments lacking detectable morphological characteristics (e.g., fragmented seeds and skins). We examined whether DNA barcoding can be used as a universal tool to rapidly characterize the diet of a frugivorous bird, Eurasian blackcap (Sylvia atricapilla). We used the DNA barcoding results to assess restoration efforts and monitor the diversity of potentially dispersed plants in a protected area in northern Italy. We collected 642 Eurasian Blackcap droppings at the restored site during the autumn migration over 3 years. Intact seeds and fragmented plant material were analyzed at 2 plastidial barcode loci (rbcL and trnH-psbA), and the resulting plant identifications were validated by comparison with a reference molecular data set of local flora. At least 17 plant species, including 7 of the 11 newly transplanted taxa, were found. Our results demonstrate the potential for DNA barcoding to be used to monitor the effectiveness of restoration plantings and to obtain information about fruit consumption and dispersal of invasive or unexpected plant species. Such an approach provides valuable information that could be used to study local plant biodiversity and to survey its evolution over time.

Evaluating the efficacy of restoration plantings through DNA barcoding characterization of frugivorous bird diets

Frugivores are critical components of restoration programs because they are seed dispersers. Thus, knowledge about bird-plant trophic relationships is essential in the evaluation of the efficacy of restoration processes. Traditionally, the diet of frugivores is characterized by microscopically identifying plant residues in droppings, which is time-consuming, requires botanical knowledge, and cannot be used for fragments lacking detectable morphological characteristics (e.g., fragmented seeds and skins). We examined whether DNA barcoding can be used as a universal tool to rapidly characterize the diet of a frugivorous bird, Eurasian blackcap (Sylvia atricapilla). We used the DNA barcoding results to assess restoration efforts and monitor the diversity of potentially dispersed plants in a protected area in northern Italy. We collected 642 Eurasian Blackcap droppings at the restored site during the autumn migration over 3 years. Intact seeds and fragmented plant material were analyzed at 2 plastidial barcode loci (rbcL and trnH-psbA), and the resulting plant identifications were validated by comparison with a reference molecular data set of local flora. At least 17 plant species, including 7 of the 11 newly transplanted taxa, were found. Our results demonstrate the potential for DNA barcoding to be used to monitor the effectiveness of restoration plantings and to obtain information about fruit consumption and dispersal of invasive or unexpected plant species. Such an approach provides valuable information that could be used to study local plant biodiversity and to survey its evolution over time.

A rapid diagnostic approach to identify poisonous plants using DNA barcoding data

Among plants, several groups have toxicities that are dangerous for humans. Plant exposures are indeed one of the most frequent cases reported to poison control centers. A rapid and easy screening test is required for accurate medical treatment. However, it is usually based on the morphological analysis of ingested plant portions that is expensive in terms of time and resources requiring experience in systematic botany which could also be biased by the absence of clear diagnostic traits in stomach contents. In this study, we designed specific primer pairs for the amplification of a sequence-characterized amplified region within standard barcode regions (i.e., rbcL and trnH–psbA) to detect toxic plants belonging to the genera Atropa and Colchicum. Allied edible species were also included in our data-set to exclude the cross-amplification with the toxic ones when the detection system is applied. Using real-time polymerase chain reaction, we tested the specificity of the selected primer pairs in order to develop a fast and reliable tool to be used in poison centers.

Bryophyte flora of some temporary pools in Sardinia and Corsica

Abstract A comparative study of the bryophytes in some Sardinian and Corsican wetlands of major naturalistic value, such as the “padule of Suartone” (SE Corsica) and “pauli of the Giara of Gesturi” (C Sardinia), was carried out. In Europe, these habitats (Mediterranean temporary pond) are indicated as priority natural habitats under the Habitats Directive 92/43/EEC. In the Sardinian pauli, 56 taxa (50 Bryophyta and six Marchantiophyta) were found, while in the Corsican padule, 30 taxa (21 Bryophyta and nine Marchantiophyta) are reported. Among the bryophytes collected in the padule, the discovery of Scleropodium cespitans, Riccia perennis and Campylopus introflexus is of particular phytogeographic interest. Data are here reported together with information on the phytogeography and ecology of the recorded species.

Chemical, molecular, and proteomic analyses of moss bag biomonitoring in a petrochemical area of Sardinia (Italy)

In this study, Hypnum cupressiforme moss bags were used to examine the atmospheric deposition of trace elements in the oil refinery region of Sardinia (Italy) compared with surrounding natural zones. The concentrations of 13 elements [arsenic (As), calcium (Ca), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), sodium (Na), nickel (Ni), lead (Pb), vanadium (V), and zinc (Zn)] were determined using inductively coupled plasma optical emission spectrometry. A significant accumulation of pollutants was detected using active biomonitoring with moss bags compared with a control site. The most relevant contaminants for all of the tested sites were Cr, Cu, Ni, and Zn. Moreover, the accumulation of Cr and Zn in the refinery industrial areas, IA1 and IA2, was more than five times greater than that detected at the control site. Levels of Cd, Mg, and Pb were also higher at all of the monitored sites compared with the control site. Both genomic and proteomic methods were used to study the response of H. cupressiforme to air pollution. No DNA damage or mutations were detected using the amplified fragment length polymorphisms (AFLP) method. At the protein level, 15 gel spots exhibited differential expression profiles between the moss samples collected at the IA1 site and the control site. Furthermore, among the 14 spots that showed a decrease in protein expression, nine were associated with ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and proteins of the light-harvesting complexes of photosystem (PS) II, three were associated with protein synthesis, and three were stress-related proteins. Thus, some of these proteins may represent good moss biosensors which could be used as pre-alert markers of environmental pollution.

Onopordum illyricum L., a Mediterranean plant, as a source of anti HIV-1 compounds

Abstract Currently, many attempts have been made worldwide to isolate compounds from plants that could prevent transmission of HIV and offer new treatments. In this study, the methanolic extract of Onopordum illyricum aerial parts was evaluated for the inhibition of the HIV-1 reverse transcriptase (RT) ribonuclease H (RNase H), an attractive target for the identification of new antiretroviral inhibitors. Using an HIV-1 reverse transcriptase (RT)-associated RNase H inhibition assay, the bio-guided fractionation of the extract led to the isolation of seven compounds (luteolin, apigenin, hispidulin, arctiin, 1,5-dicaffeoylquinic acid, and two germacranes, 8α-(5-hydroxy)-angeloylsalonitenolide and onopordopicrin). Among them, luteolin was the most effective on RNase H RT-associated function (IC50 of 12.8 μM), followed by 1,5-dicaffeoylquinic acid and apigenin with IC50 values of 16.9 and 59.6 μM, respectively. Pure compounds were then assayed for their effects also on HIV-1 integrase (IN). 1,5-Dicaffeoylquinic acid, arctiin, onopordopicrin, and luteolin exhibited the most potent inhibition with IC50 values ranging from 0.50 to 22.5 μM. 1,5-Dicaffeoylquinic acid was also able to inhibit the early stages of HIV-1 replication in cell-based assays.

Chemical and biomolecular analyses to discriminate three taxa of Pistacia genus from Sardinia Island (Italy) and their antifungal activity

Abstract This work reports the results and the comparison concerning the chemical and biomolecular analyses and the antifungal activity of three wild Pistacia species (Anacardiaceae) from Sardinia. Volatile oils from leaves and twigs of Pistacia x saportae, Pistacia lentiscus and Pistacia terebinthus were characterised using GC-FID and GC-MS techniques and tested against some fungal strains. Two DNA nuclear regions (ITS and 5S-rRNA-NTS) were amplified through PCR technique and sequenced. The three **Pistacia have similar chemical profile, although there are some important quantitative differences. The analysis of ITS and 5S-rRNA-NTS regions, reveals a species-specific nucleotide variation among the three **taxa. This method could emerge as a powerful tool for the species identification, especially because the discrimination of these three **taxa appears difficult for non-expert botanists. Concerning the antifungal activity, P. lentiscus and P. x saportae show the highest activity against Cryptococcus neoformans, with a MIC value of 0.32 μL/mL.