Analysis of sexual phenotype and prezygotic fertility in natural populations of Fucus spiralis, F. vesiculosus (Fucaceae, Phaeophyceae) and their putative hybrids

In the genus Fucus, the character dioecy/hermaphroditism has undergone multiple state changes and hybridization is possible between taxa with contrasting mating systems, e.g. between the dioecious Fucus vesiculosus and the hermaphrodite F. spiralis. In the context of mating system evolution, we evaluated the potential consequences of hybridization by studying the variation in sexual phenotype and prezygotic fertility. Firstly, as a result of hybridization between the two sexual systems, gender variation may arise depending on the relative importance of genes with large versus small phenotypic effects. We thus qualitatively examined the extent of gender variation within and among individual hybrids in comparison with both parental species. Secondly, if hybridization breaks up co-adapted gene complexes, hybrid fertility may be reduced in comparison with both parental species. Therefore, we also quantified male and female prezygotic fertility in parental species and their hybrids in order to test for reduction in hybrid fitness. A total of 89 sexually mature individuals (20 F. spiralis, 40 F. vesiculosus, 10 hermaphrodite hybrids and 19 dioecious hybrids) were sampled in two geographically distant regions (France and Portugal) and six conceptacles per individual were observed. Within-individual variation was very restricted qualitatively – only one hybrid carried a conceptacle with a different sexual phenotype from the five others – as well as quantitatively. This suggests a simple genetic system for sex determination involving a few genes with major effects. In addition, analyses showed no significant decrease in hybrid fertility compared with parental species. Moreover, hybrids exhibited all sexual phenotypes, suggesting several generations of hybridization and backcrossing and, therefore, that hybrids are reproductively successful. Finally, the occurrence of sterile paraphyses in female and hermaphrodite individuals was interpreted as a relic of male function and suggests that, as in higher plants, evolution from hermaphroditism to dioecy may be the most parsimonious pathway

Adaptive Traits Are Maintained on Steep Selective Gradients despite Gene Flow and Hybridization in the Intertidal Zone

Gene flow among hybridizing species with incomplete reproductive barriers blurs species boundaries, while selection under heterogeneous local ecological conditions or along strong gradients may counteract this tendency. Congeneric, externally-fertilizing fucoid brown algae occur as distinct morphotypes along intertidal exposure gradients despite gene flow. Combining analyses of genetic and phenotypic traits, we investigate the potential for physiological resilience to emersion stressors to act as an isolating mechanism in the face of gene flow. Along vertical exposure gradients in the intertidal zone of Northern Portugal and Northwest France, the mid-low shore species Fucus vesiculosus, the upper shore species Fucus spiralis, and an intermediate distinctive morphotype of F. spiralis var. platycarpus were morphologically characterized. Two diagnostic microsatellite loci recovered 3 genetic clusters consistent with prior morphological assignment. Phylogenetic analysis based on single nucleotide polymorphisms in 14 protein coding regions unambiguously resolved 3 clades; sympatric F. vesiculosus, F. spiralis, and the allopatric (in southern Iberia) population of F. spiralis var. platycarpus. In contrast, the sympatric F. spiralis var. platycarpus (from Northern Portugal) was distributed across the 3 clades, strongly suggesting hybridization/introgression with both other entities. Common garden experiments showed that physiological resilience following exposure to desiccation/heat stress differed significantly between the 3 sympatric genetic taxa; consistent with their respective vertical distribution on steep environmental clines in exposure time. Phylogenetic analyses indicate that F. spiralis var. platycarpus is a distinct entity in allopatry, but that extensive gene flow occurs with both higher and lower shore species in sympatry. Experimental results suggest that strong selection on physiological traits across steep intertidal exposure gradients acts to maintain the 3 distinct genetic and morphological taxa within their preferred vertical distribution ranges. On the strength of distributional, genetic, physiological and morphological differences, we propose elevation of F. spiralis var. platycarpus from variety to species level, as F. guiryi

Invasion is a community affair: clandestine followers in the bacterial community associated to green algae, Caulerpa racemosa, track the invasion source

Biological invasions rank amongst the most deleterious components of global change inducing alterations from genes to ecosystems. The genetic characteristics of introduced pools of individuals greatly influence the capacity of introduced species to establish and expand. The recently demonstrated heritability of microbial communities associated to individual genotypes of primary producers makes them a potentially essential element of the evolution and adaptability of their hosts. Here, we characterized the bacterial communities associated to native and non-native populations of the marine green macroalga Caulerpa racemosa through pyrosequencing, and explored their potential role on the strikingly invasive trajectory of their host in the Mediterranean. The similarity of endophytic bacterial communities from the native Australian range and several Mediterranean locations confirmed the origin of invasion and revealed distinct communities associated to a second Mediterranean variety of C. racemosa long reported in the Mediterranean. Comparative analysis of these two groups demonstrated the stability of the composition of bacterial communities through the successive steps of introduction and invasion and suggested the vertical transmission of some major bacterial OTUs. Indirect inferences on the taxonomic identity and associated metabolism of bacterial lineages showed a striking consistency with sediment upheaval conditions associated to the expansion of their invasive host and to the decline of native species. These results demonstrate that bacterial communities can be an effective tracer of the origin of invasion and support their potential role in their eukaryotic host’s adaptation to new environments. They put forward the critical need to consider the 'meta-organism' encompassing both the host and associated micro-organisms, to unravel the origins, causes and mechanisms underlying biological invasions

Immunity of an Alternative Host Can Be Overcome by Higher Densities of Its Parasitoids Palmistichus elaeisis and Trichospilus diatraeae

Interactions of the parasitoids Palmistichus elaeisis Delvare & LaSalle and Trichospilus diatraeae Cherian & Margabandhu (Hymenoptera: Eulophidae) with its alternative host Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) affect the success or failure of the mass production of these parasitoids for use in integrated pest management programs. The aim of this study was to evaluate changes in the cellular defense and encapsulation ability of A. gemmatalis pupae against P. elaeisis or T. diatraeae in adult parasitoid densities of 1, 3, 5, 7, 9, 11 or 13 parasitoids/pupae. We evaluated the total quantity of circulating hemocytes and the encapsulation rate versus density. Increasing parasitoid density reduced the total number of hemocytes in the hemolymph and the encapsulation rate by parasitized pupae. Furthermore, densities of P. elaeisis above 5 parasitoids/pupae caused higher reduction in total hemocyte numbers. The encapsulation rate fell with increasing parasitoid density. However, parasitic invasion by both species induced generally similar responses. The reduction in defensive capacity of A. gemmatalis is related to the adjustment of the density of these parasitoids to their development in this host. Thus, the role of the density of P. elaeisis or T. diatraeae by pupa is induced suppression of cellular defense and encapsulation of the host, even without them possesses a co-evolutionary history. Furthermore, these findings can predict the success of P. elaeisis and T. diatraeae in the control of insect pests through the use of immunology as a tool for evaluation of natural enemies

Implications of Extreme Life Span in Clonal Organisms: Millenary Clones in Meadows of the Threatened Seagrass Posidonia oceanica

The maximum size and age that clonal organisms can reach remains poorly known, although we do know that the largest natural clones can extend over hundreds or thousands of metres and potentially live for centuries. We made a review of findings to date, which reveal that the maximum clone age and size estimates reported in the literature are typically limited by the scale of sampling, and may grossly underestimate the maximum age and size of clonal organisms. A case study presented here shows the occurrence of clones of slow-growing marine angiosperm Posidonia oceanica at spatial scales ranging from metres to hundreds of kilometres, using microsatellites on 1544 sampling units from a total of 40 locations across the Mediterranean Sea. This analysis revealed the presence, with a prevalence of 3.5 to 8.9%, of very large clones spreading over one to several (up to 15) kilometres at the different locations. Using estimates from field studies and models of the clonal growth of P. oceanica, we estimated these large clones to be hundreds to thousands of years old, suggesting the evolution of general purpose genotypes with large phenotypic plasticity in this species. These results, obtained combining genetics, demography and model-based calculations, question present knowledge and understanding of the spreading capacity and life span of plant clones. These findings call for further research on these life history traits associated with clonality, considering their possible ecological and evolutionary implications

A multidisciplinary approach to the study of the brightening effects of white chalk ground layers in 15th and 16thcentury paintings

This paper employs various techniques to analyze the mixture of chalk and binder materials used, by Portuguese and Flemish painters in the 15th and 16th centuries, to enhance the reflection of light in paintings. The cases studied show evidence of the search by painters for light effects created when combining specific fillers and binders to obtain absorbent or non-absorbent ground layers in order to reflect superficial or deep light in paintings. These brightening effects are largely provided by microscopic coccoliths and calcispheres – the main constituents of chalk. The composition, size and slightly concave-convex shield-like shape of calcareous nannofossils (micrometrical dimensions) also facilitate application, thereby increasing the speed of handling. These calcareous nannofossils are crucial proof that chalk was used in the ground layers of Portuguese paintings. They have proved to be important in defining the various stages of Portuguese painting workshops, such as the Viseu Workshop (1501–1569), which used powdered chalk in the first phase and powdered limestone in the second phase in the production of a ground layer. A two-layer structure has been verified in some Flemish paintings of the period, but the use of different binders to provide different levels of light absorption and reflection in these artworks had not been previously identified. The results showing this two-layer ground structure are significant in making the connection between Flemish and Portuguese art in the context of a specific painting technique. The use of calcium carbonate ground layers was verified by SEM-EDS and confirmed by m-XRD, and m-Raman, while binders were analyzed by m-FTIR and optical microscopy, using staining test

Characterization of gypsum and anhydrite ground layers in 15th and 16th centuries Portuguese paintings by Raman Spectroscopy and other techniques

The purpose of this work is to characterize Portuguese painting ground layers from the 15th and 16th centuries (1450–1600), focusing particularly on the Coimbra, Lisbon, and Viseu workshops. Calcium sulfate, used regularly in these layers is common in the Iberian Peninsula as a natural resource. Its use is now confirmed, as already studied in Spanish paintings, and it was prepared generally by addition of animal glue. Besides the common esthetic language to the various painting workshops at the time, chalk/calcite (calcium carbonate), gypsum (di-hydrated calcium sulfate), and anhydrite (anhydrous calcium sulfate) ground layers have specificities that allow characterizing different compounds. The cross-sections from a set of six groups of Portuguese paintings were examined by optical microscopy and micro-confocal Raman and Raman spectroscopic mapping imaged the location of the different compounds in the cross-section. Results were integrated with those obtained by micro-X-ray diffraction and scanning electron microscopy with energy dispersive X-ray spectroscopy on the same samples. In most cases, the ground layers are made of calcium sulfate in the form of the so-called gesso grosso (mainly anhydrite with small amounts of gypsum). Differently, the Viseu paintings have calcium carbonate ground layers. These results put in evidence differences between the examined groups giving important information on the various workshop practices and also on the possible future conservative intervention on the paintings

Preserving European paintings in Asian environment. The case of Goa Cathedral former altarpiece

How to preserve a set of paintings made with European materials and taken to South Asia in the 16th century? A set of seven paintings belonging to the first altarpiece of Goa Cathedral now remain in its sacristy. Art history states that these paintings, assigned to the painter Garcia Fernandes, were produced in Lisbon circa 1530 and carried to Goa shortly after, Serrão (2011), Reis-Santos (1957). Presently, the conservation state of the paintings indicates urgent treatment. The most damaged paintings show significant loss of the original chromatic layer. This is mostly due to the high temperatures and relative humidity existing in tropical climates, such as Goa, different from the European. The Baltic oak support is covered with ground layers of calcium suphate, which present fissures and great losses due to the shrinkages of the wood support. The high reactivity of the ground materials to water is also a factor leading to specific material deterioration and degradation mechanisms. In comparison with the paintings of the same period produced in Portugal using the same materials, we verify a much higher degradation of the state of conservation. To access the procedures for preservation and restoration of this set of paintings we must take into account the possible degradation mechanisms of the constituent materials brought by specific environmental factors. This paper discusses the degradation problematic of this specific set of paintings taking into account their particular environment, while bringing results on the materials used. Concluding, this study will allow the knowledge on how to preserve and restore one of the most ancient sets of European paintings in India, kept since the 16th century in tropical climate

Material and diagnostic characterization of 17th century mural paintings by spectra-colorimetry and SEM-EDS: An insight look at José de Escovar workshop at the CONVENT of Na Sra da Saudação (Southern Portugal)

The early 17th century mural paintings set from the low-choir of the extinct Dominican Convent of Nossa Senhora da Saudac¸~ao in the town of Montemor-onovo were analyzed by a setup comprising visible spectra- colorimetry and scanning electron microscopy coupled with energy dispersive X-ray spectrometry (SEMEDS) complemented with optical microscopy (OM), micro X-ray diffraction, micro FTIR, and micro Raman spectroscopy. The main goals were material and diagnostic characterization of the paint layers and pictorial techniques used by Jose de Escovar workshop, one of the most active and controversial easel and mural painters of Evora Archiepiscopate between 1585 and 1622. The results show a differentiate use of red, blue, and green pigments in an alkaline environment. Stable mercury sulfide (HgS) was identified in almost all red areas in the latest scenes while, in the first ones, red ochre (Fe2O3) is the predominant pigment. Faded smalt (a co-potassium silicate glass) is present pure in the sky background or mixed with red ochre in the garments at the beginning of the low-choir but less at the end. Five kinds of copperbased materials, with different degrees of color alteration, were found, namely malachite and azurite, copper chlorides, copper sulfates (e.g., posjnakite), and pseudomalachite. Another curious feature is the uneven use of a limewash made with slaked calcitic lime in the carnations. Lack of material, internal organization strategies, or technical differences within the team are probably the most likely causes