Consequences of plant population size for pollinator visitation and plant reproductive success

Habitat loss and fragmentation have a prominent role in determining the size of plant populations, and can affect plant-pollinator interactions. It is hypothesized that in small plant populations the ability to set seeds can be reduced due to limited pollination services, since individuals in small populations can receive less quantity or quality of visits. In this study, I investigated the effect of population size on plant reproductive success and insect visitation in 8 populations of two common species in the island of Lesvos, Greece (Mediterranean Sea), Echium plantagineum and Ballota acetabulosa, and of a rare perennial shrub endemic to north-central Italy, Ononis masquillierii. All the three species depended on insect pollinators for sexual reproduction. For each species, pollen limitation was present in all or nearly all populations, but the relationship between pollen limitation and population size was only present in Ononis masquillierii. However, in Echium plantagineum, significant relationships between both open-pollinated and handcrossed-pollinated seed sets and population size were found, being small populations comparatively less productive than large ones. Additionally, for this species, livestock grazing intensity was greater for small populations and for sparse patches, and had a negative influence on productivity of the remnant plants. Both Echium plantagineum and Ballota acetabulosa attracted a great number of insects, representing a wide spectrum of pollinators, thereby can be considered as generalist species. For Ballota acetabulosa, the most important pollinators were megachilid female bees, and insect diversity didn’t decrease with decreasing plant population size. By contrast, Ononis masquillierii plants generally received few visits, with flowers specialized on small bees (Lasioglossum spp.), representing the most important insect guild. In Echium plantagineum and Ballota acetabulosa, plants in small and large populations received the same amount of visits per flower, and no differences in the number of intraplant visited flowers were detected. On the contrary, large Ononis populations supported higher amounts of pollinators than small ones. At patch level, high Echium flower density was associated with more and higher quality pollinators. My results indicate that small populations were not subject to reduced pollination services than large ones in Echium plantagineum and Ballota acetabulosa, and suggest that grazing and resource limitation could have a major impact on population fitness in Echium plantagineum. The absence of any size effects in these two species can be explained in the light of their high local abundance, wide habitat specificity, and ability to compete with other co-flowering species for pollinators. By contrast, size represents a key characteristic for both pollination and reproduction in Ononis masquillierii populations, as an increase in size could mitigate the negative effects coming from the disadvantageous reproductive traits of the species. Finally, the widespread occurrence of pollen limitation in the three species may be the result of 1) an ongoing weakening or disruption of plantpollinator interactions derived from ecological perturbations, 2) an adaptive equilibrium in response to stochastic processes, and 3) the presence of unfavourable reproductive traits (for Ononis masquillierii).

Image analysis in the morphological and morphometric study of teeth

The subject of this doctoral dissertation concerns the definition of a new methodology for the morphological and morphometric study of fossilized human teeth, and therefore strives to provide a contribution to the reconstruction of human evolutionary history that proposes to extend to the different species of hominid fossils. Standardized investigative methodologies are lacking both regarding the orientation of teeth subject to study and in the analysis that can be carried out on these teeth once they are oriented. The opportunity to standardize a primary analysis methodology is furnished by the study of certain early Neanderthal and preneanderthal molars recovered in two caves in southern Italy [Grotta Taddeo (Taddeo Cave) and Grotta del Poggio (Poggio Cave), near Marina di Camerata, Campania]. To these we can add other molars of Neanderthal and modern man of the upper Paleolithic era, specifically scanned in the paleoanthropology laboratory of the University of Arkansas (Fayetteville, Arkansas, USA), in order to increase the paleoanthropological sample data and thereby make the final results of the analyses more significant. The new analysis methodology is rendered as follows: 1. Standardization of an orientation system for primary molars (superior and inferior), starting from a scan of a sample of 30 molars belonging to modern man (15 M1 inferior and 15 M1 superior), the definition of landmarks, the comparison of various systems and the choice of a system of orientation for each of the two dental typologies. 2. The definition of an analysis procedure that considers only the first 4 millimeters of the dental crown starting from the collar: 5 sections parallel to the plane according to which the tooth has been oriented are carried out, spaced 1 millimeter between them. The intention is to determine a method that allows for the differentiation of fossilized species even in the presence of worn teeth. 3. Results and Conclusions. The new approach to the study of teeth provides a considerable quantity of information that can better be evaluated by increasing the fossil sample data. It has been demonstrated to be a valid tool in evolutionary classification that has allowed (us) to differentiate the Neanderthal sample from that of modern man. In a particular sense the molars of Grotta Taddeo, which up until this point it has not been possible to determine with exactness their species of origin, through the present research they are classified as Neanderthal.

Effects of habitat characteristics on cryptic fish assemblages

Habitat structure is known to influence the abundance of fishes on temperate reefs. Biotic interactions play a major role in determining the distribution and abundance of species. The significance of these forces in affecting the abundance of fishes may hinge on the presence of organisms that either create or alter habitat. On temperate reefs, for example, macroalgae are considered autogenic ecosystem engineers because they control resource availability to other species through their physical structure and provide much of the structure used by fish. On both coral and temperate reefs, small cryptic reef fishes may comprise up to half of the fish numbers and constitute a diverse community containing many specialized species. Small cryptic fishes (<100 mm total length) may be responsible for the passage of 57% of the energy flow and constitute ca. 35% of the overall reef fish biomass on coral reefs. These benthic fish exploit restricted habitats where food and shelter are obtained in, or in relation to, conditions of substrate complexity and/or restricted living space. A range of mechanisms has been proposed to account for the diversity and the abundance of small fishes: (1) lifehistory strategies that promote short generation times, (2) habitat associations and behaviour that reduce predation and (3) resource partitioning that allows small species to coexist with larger competitors. Despite their abundance and potential importance within reef systems, little is known of the community ecology of cryptic fishes. Specifically on habitat associations many theories suggested a not clear direction on this subject. My research contributes to the development of marine fish ecology by addressing the effects of habitat characteristics upon distribution of cryptobenthic fish assemblages. My focus was on the important shallow, coastal ecosystems that often serve as nursery habitat for many fish and where different type of habitat is likely to both play important roles in organism distribution and survival. My research included three related studies: (1) identification of structuring forces on cryptic fish assemblages, such as physical and biological forcing; (2) macroalgae as potential tools for cryptic fish and identification of different habitat feature that could explain cryptic fish assemblages distribution; (3) canopy formers loss: consequences on cryptic fish and relationship with benthos modifications. I found that: (1) cryptic fish assemblages differ between landward and seaward sides of coastal breakwaters in Adriatic Sea. These differences are explained by 50% of the habitat characteristics on two sides, mainly due to presence of the Codium fragile, sand and oyster assemblages. Microhabitat structure influence cryptic fish assemblages. (2) Different habitat support different cryptic fish assemblages. High heterogeneity on benthic assemblages reflect different fish assemblages. Biogenic components that explain different and diverse cryptic fish assemblages are: anemonia bed, mussel bed, macroalgal stands and Cystoseira barbata, as canopy formers. (3) Canopy forming loss is not relevant in structuring directly cryptic fish assemblages. A removal of canopy forming algae did not affect the structure of cryptic fish assemblages. Canopy formers algae on Conero cliff, does not seem to act as structuring force, probably due to its regressive status. In conclusion, cryptic fish have been shown to have species-specific associations with habitat features relating to the biological and non biological components afforded by fish. Canopy formers algae do not explain cryptic fish assemblages distribution and the results of this study and information from the literature (both from the Mediterranean Sea and elsewhere) show that there are no univocal responses of fish assemblages. Further exanimations on an non regressive status of Cystoseira canopy habitat are needed to define and evaluate the relationship between canopy formers and fish on Mediterranean sea.

Diencephalic asymmetries: morphological, immunohistochemical and ultrastructural analysis of habenular nuclei in elasmobranchs and teleosts

The habenular nuclei are diencephalic structures present in Vertebrates and they form, with the associated fiber systems, a part of the system that connects the telencephalon to the ventral mesencephalon (Concha M. L. and Wilson S. W., 2001). In representative species of almost all classes of Vertebrates the habenular nuclei are asymmetric, both in terms of size and of neuronal and neurochemical organization, although different types of asymmetry follow different evolutionary courses. Previous studies have analyzed the spread and diversity of the asymmetry in species for which data are not clear (Kemali M. et al., 1980). Notwithstanding that, it’s still not totally understood the evolution of the phenomenon, and the ontogenetic mechanisms that have led to the habenular asymmetry development are not clear (Smeets W.J. et al., 1983). For the present study 14 species of Elasmobranchs and 15 species of Teleostean have been used. Brains removed from the animals have been fixed using 4% paraformaldehyde in phosphate buffer; brains have been analyzed with different tecniques, and I used histological, immunohistochemical and ultrastructural analysis to describe this asymmetry. My results confirm data previously obtained studying other Elasmobranchs species, in which the left habenula is larger than the right one; the Teleostean show some slightly differences regarding the size of the habenular ganglia, in some species, in which the left habenular nucleus is larger than the right. In the course of studies, a correlation between the habits of life and the diencephalic asymmetry seems to emerge: among the Teleostean analyzed, the species with benthic life (like Lepidorhombus boscii, Platichthys flesus, Solea vulgaris) seem to possess a slight asymmetry, analogous to the one of the Elasmobranchs, while in the other species (like Liza aurata, Anguilla anguilla, Trisopterus minutus) the habenulae are symmetrical. However, various aspects of the neuroanatomical asymmetries of the epithalamus have not been deepened in order to obtain a complete picture of the evolution of this phenomenon, and new searches are needed to examine the species without clear asymmetry, in order to understand the spread and the diversity of the asymmetry among the habenulae between the Vertebrates.

Mechanism of colonization in the Mediterranean-sea. Asperarca nodulosa: morphological and genetic analysis

Il mar Mediterraneo è un bacino acquifero peculiare per la recente colonizzazione di specie aliene, per l’evento geologico legato alla Crisi di salinità del Messiniano e per l’ampio range di salinità. L’individuazione dei meccanismi di colonizzazione si è incentrata sullo studio morfologico, istologico e molecolare delle specie Asperarca nodulosa ed Anadara demiri (Arcidae-Bivalvia-Mollusca). La ricerca si è basata sulla caratterizzazione morfologica, con utilizzo del microscopio elettronico a scansione, al fine di individuare il tipo di sviluppo larvale. Successivamente i dati rilevati al S.E.M. sono stati supportati dall’indagine istologica che ha evidenziato la presenza di gonadi a sessi distinti e la non incubazione larvale. L’ulteriore analisi filogenetica ha permesso di evidenziare la netta suddivisione tra le tre popolazioni studiate, indagine effettuata tramite marcatori arbitrari (RAPDs) e nucleari specifici (ITS). I risultati ottenuti trovano supporto da quanto noto su base morfologica. I dati, nel complesso, mostrano una perdita delle capacità di diffusione della specie tramite sviluppo larvale plantotrofico a favore di quello lecitotrofico o diretto; tale tesi è ulteriormente supportata dai dati molecolari che mostrano una netta separazione delle popolazioni prese in esame ed un conseguente isolamento tra individui appartenenti a zone di profondità del Mediterraneo (sub-bacini abissali). La ricerca ha, inoltre,esaminato i meccanismi di introduzione attuali nel bacino acquifero che è soggetto ad una nuova invasione da parte specie aliene dovuta all’apertura del canale di Suez. L’analisi si è focalizzata sullo studio per l’ individuazione dell’origine della specie aliena A. demiri , di presunta derivazione Indo-Pacifica, ma rivelatasi, nei dati preliminari, di origine Atlantica.

Molecular markers for the assessment of genetic variability in threatened plant species

La variabilità genetica è un importante strumento per lo studio e la conservazione della biodiversità in specie rare e minacciate di estinzione. Durante il mio dottorato mi sono quindi occupata di mettere a punto diverse metodologie molecolari al fine di valutare la diversità genetica in due specie rare della flora italiana che presentano problematiche diverse e specifiche. I marcatori arbitrari RAPD e i marcatori semi-arbitrari ISSR sono stati utilizzati per valutare la diversità genetica in Quercus crenata Lam. e per confermare l’ipotesi della sua origine ibridogena dalle due specie presunte parentali Quercus cerris L. e Quercus suber L., essendo Q. crenata presente in Italia settentrionale dove Q. suber è attualmente assente. I marcatori SSR o microsatelliti sono invece stati messi a punto su una specie a rischio di estinzione, endemica dell’Appennino Tosco-Emiliano, Primula apennina Widmer, applicando una metodologia specifica, basata sulla costruzione di una libreria genomica arricchita per l’isolamento di primer specifici. I marcatori RAPD e ISSR, utilizzati su un totale di 85 campioni, hanno mostrato alti livelli di diversità molecolare entro le specie studiate, eccetto per Q. suber le cui popolazioni rappresentano il margine orientale di distribuzione della specie, per questo più sottoposte ad impoverimento genetico. Oltre alla cluster analysis (UPGMA) e alla Analisi delle Componenti Principali effettuate per entrambi i marcatori, che confermano l’ipotesi dell’origine ibrida degli individui di Q. crenata diffusi in Italia Settentrionale, sono stati calcolati l’indice di ibridità basato sul maximum likelihood, che dimostra una introgressione asimmetrica di Q. crenata verso il parentale caratterizzato da superiorità demografica (Q. cerris) e il test di Mantel. Quest’ultimo ha permesso di confrontare i due marcatori RAPD e ISSR utilizzati ottenendo una bassa correlazione, a conferma del fatto che, amplificando tratti differenti del DNA nucleare, i dati non sono sovrapponibili, sebbene forniscano risultati analoghi. Per l’isolamento di loci microsatelliti ipervariabili ho utilizzato il protocolllo FIASCO (Fast isolation by AFLP of sequences containing repeats- Zane et al. 2002) che permette di costruire una libreria genomica arricchita partendo dal DNA estratto da P. apennina. Tale procedura ha previsto la digestione del DNA genomico per la produzione di una miscela di frammenti di DNA. Tramite ibridazione con opportune sonde sono stati isolati i frammenti contenenti i microsatelliti. Sequenziando i cloni ricombinanti, ho ottenuto sequenze contenenti repeats sulle cui regioni fiancheggianti sono stati costruiti 15 coppie di primer che potranno, in seguito, essere utilizzate per definire la quota di riproduzione clonale in P. apennina e per valutare la diversità genetica delle popolazioni che coprono l’areale di distribuzione della specie. Data la loro natura altamente variabile e la loro abbondanza nel DNA, gli SSR saranno, come i marcatori RAPD e gli ISSR, ugualmente validi per lo studio della variabilità genetica e per l’analisi di problematiche specifiche legate alle specie rare.