Monitoring of Mediterranean marine benthic biodiversity through novel, standardized, and integrated approaches

Marine healthy ecosystems support life on Earth and human well-being thanks to their biodiversity, which is proven to decline mainly due to anthropogenic stressors. Monitoring how marine biodiversity changes trough space and time is needed to properly define and enroll effective actions towards habitat conservation and preservation. This is particularly needed in those areas that are very rich in species compared to their low surface extension and are characterized by strong anthropic pressures, such as the Mediterranean Sea. Subtidal rocky benthic Mediterranean habitats have a complex structural architecture, hosting a panoply of tiny organisms (cryptofauna) that inhabit crevices and caves, but that are still unknown. Different artificial standardized sampling structures (SSS) and methods have been developed and employed to characterize the cryptofauna, allowing for data replicability and comparability across regions. Organisms growing on these artificial structures can be identified coupling morphological taxonomy and DNA barcoding and metabarcoding. The metabarcoding allows for the identification of organisms in a bulk sample without morphological analysis, and it is based on comparing the genetic similarities of the assessed organisms with barcoding sequences present in online barcoding repositories. Nevertheless, barcoded species nowadays represent only a small portion of known species, and barcoding reference databases are not always curated and updated on a regular basis. In this Thesis I used an integrative approach to characterize benthic marine biodiversity, specifically coupling morphological and molecular techniques with the employment of SSS. Moreover, I upgraded the actual status of COI (cytochrome c oxidase subunit I) barcoding of marine metazoans, and I built a customized COI barcoding reference database for metabarcoding studies on temperate biogenic reefs. This work implemented the knowledge about diversity of Mediterranean marine communities, laying the groundworks for monitoring marine and environmental changes that will occur in the next future as consequences of anthropic and climate threats.

Relationship between phenotype and environment in marine calcifying organisms: exploring growth and shell properties of different mollusks species in past and modern scenario

Coastal ecosystems represent an inestimable source of biodiversity, being among the most productive areas on the planet. Despite the great ecological and economic value of those environments, many threats endanger the species living in this ecosystem, like the rapid warming and the sea acidification, among many other. Benthic calcifying organisms (e.g. mollusks, corals and echinoderms) in particular, are among the most exposed to those hazards. These organisms use calcium carbonate as a structural and protective material through the biomineralization process, biologically controlled by the organism, but nevertheless, strongly influenced by the environmental surroundings. Evaluating how a changing environment can influence the process of biomineralization is critical to understand how those species of great ecological and economic importance will face the ongoing climate change. This thesis investigates the mechanism of biomineralization in different mollusks’ species of the Adriatic Sea, providing detailed descriptions of shells skeletal, biometric and growth parameters. Applying a multidisciplinary and multi-scale research approach, the influence of external environmental factors on the process of shell formation has been investigated. To achieve this purpose analysis were conducted both on current populations and on fossil remain, which allows to investigate ecological responses to past climate transitions. Mollusks’ shells in fact are one of the best tools to understand climate change in the past, present and future, since they record the environmental conditions prevailed during their life, reflected on the geochemical properties, microstructure and growth of the shell. This approach allowed to overcome the time scale limit imposed by field and laboratory survey, and better understand species long term adaptive response to changing environment, a crucial issue to define proper conservation and management strategies. Furthermore, the investigation of fossil record of mollusks assemblages offered the opportunity to evaluate the long-term biotic response to anthropogenic stressors in the north Adriatic Sea.

Experiences From The Brazilian Atlantic Forest: Ecological Findings And Conservation Initiatives.

The Brazilian Atlantic Forest hosts one of the world's most diverse and threatened tropical forest biota. In many ways, its history of degradation describes the fate experienced by tropical forests around the world. After five centuries of human expansion, most Atlantic Forest landscapes are archipelagos of small forest fragments surrounded by open-habitat matrices. This 'natural laboratory' has contributed to a better understanding of the evolutionary history and ecology of tropical forests and to determining the extent to which this irreplaceable biota is susceptible to major human disturbances. We share some of the major findings with respect to the responses of tropical forests to human disturbances across multiple biological levels and spatial scales and discuss some of the conservation initiatives adopted in the past decade. First, we provide a short description of the Atlantic Forest biota and its historical degradation. Secondly, we offer conceptual models describing major shifts experienced by tree assemblages at local scales and discuss landscape ecological processes that can help to maintain this biota at larger scales. We also examine potential plant responses to climate change. Finally, we propose a research agenda to improve the conservation value of human-modified landscapes and safeguard the biological heritage of tropical forests.

Molecular Genetic Variability Of Commercial And Wild Accessions Of Passion Fruit (passiflora Spp.) Targeting Ex Situ Conservation And Breeding.

Passiflora species are distributed throughout Latin America, and Brazil and Colombia serve as the centers of diversity for this genus. We performed cross-species amplification to evaluate 109 microsatellite loci in 14 Passiflora species and estimated the diversity and genetic structure of Passiflora cincinnata, Passiflora setaceae and Passiflora edulis. A total of 127 accessions, including 85 accessions of P. edulis, a commercial species, and 42 accessions of 13 wild species, were examined. The cross-species amplification was effective for obtaining microsatellite loci (average cross-amplification of 70%). The average number of alleles per locus (five) was relatively low, and the average diversity ranged from 0.52 in P. cincinnata to 0.32 in P. setacea. The Bayesian analyses indicated that the P. cincinnata and P. setacea accessions were distributed into two groups, and the P. edulis accessions were distributed into five groups. Private alleles were identified, and suggestions for core collections are presented. Further collections are necessary, and the information generated may be useful for breeding and conservation.

Fast And Direct Na And K Determination In Table, Marine, And Low-sodium Salts By X-ray Fluorescence And Chemometrics.

X-ray fluorescence (XRF) is a fast, low-cost, nondestructive, and truly multielement analytical technique. The objectives of this study are to quantify the amount of Na(+) and K(+) in samples of table salt (refined, marine, and light) and to compare three different methodologies of quantification using XRF. A fundamental parameter method revealed difficulties in quantifying accurately lighter elements (Z < 22). A univariate methodology based on peak area calibration is an attractive alternative, even though additional steps of data manipulation might consume some time. Quantifications were performed with good correlations for both Na (r = 0.974) and K (r = 0.992). A partial least-squares (PLS) regression method with five latent variables was very fast. Na(+) quantifications provided calibration errors lower than 16% and a correlation of 0.995. Of great concern was the observation of high Na(+) levels in low-sodium salts. The presented application may be performed in a fast and multielement fashion, in accordance with Green Chemistry specifications.

Molecular Diversity Of Fungal And Bacterial Communities In The Marine Sponge Dragmacidon Reticulatum.

The present work aimed to investigate the diversity of bacteria and filamentous fungi of southern Atlantic Ocean marine sponge Dragmacidon reticulatum using cultivation-independent approaches. Fungal ITS rDNA and 18S gene analyses (DGGE and direct sequencing approaches) showed the presence of representatives of three order (Polyporales, Malasseziales, and Agaricales) from the phylum Basidiomycota and seven orders belonging to the phylum Ascomycota (Arthoniales, Capnodiales, Dothideales, Eurotiales, Hypocreales, Pleosporales, and Saccharomycetales). On the other hand, bacterial 16S rDNA gene analyses by direct sequencing approach revealed the presence of representatives of seven bacterial phyla (Cyanobacteria, Proteobacteria, Actinobacteria, Bacteroidetes, Lentisphaerae, Chloroflexi, and Planctomycetes). Results from statistical analyses (rarefaction curves) suggested that the sampled clones covered the fungal diversity in the sponge samples studied, while for the bacterial community additional sampling would be necessary for saturation. This is the first report related to the molecular analyses of fungal and bacterial communities by cultivation-independent approaches in the marine sponges D. reticulatum. Additionally, the present work broadening the knowledge of microbial diversity associated to marine sponges and reports innovative data on the presence of some fungal genera in marine samples.

The syntheses of the marine metabolites 3-bromoverongiaquinol and 5-monobromocavernicolin

An efficient synthesis of the marine metabolite 3-bromoverongiaquinol (1) and the first total synthesis of 5-monobromocavernicolin (2), both isolated from the marine sponge Aplysina cavernicola, have been described based on the 1,2 addition of the lithium enolate of N,O-bistrimethylsilylacetamide (BSA, 4) to 1,4-benzoquinone (3). Bromination and purification of the crude product on silica gel chromatography provided 3-bromoverongiaquinol (1) in 50% overall yield. Under alkaline conditions, the crude product of the bromination reaction was converted to 5-monobromocavernicolin (2) in 20% yield which was also obtained in 13% yield (25% yield based on recovered starting material) from 3-bromoverongiaquinol (1).

Critical applications of SW 846 US EPA methods to evaluation of marine samples quality

Technical evaluation of analytical data is of extreme relevance considering it can be used for comparisons with environmental quality standards and decision-making as related to the management of disposal of dredged sediments and the evaluation of salt and brackish water quality in accordance with CONAMA 357/05 Resolution. It is, therefore, essential that the project manager discusses the environmental agency's technical requirements with the laboratory contracted for the follow-up of the analysis underway and even with a view to possible re-analysis when anomalous data are identified. The main technical requirements are: (1) method quantitation limits (QLs) should fall below environmental standards; (2) analyses should be carried out in laboratories whose analytical scope is accredited by the National Institute of Metrology (INMETRO) or qualified or accepted by a licensing agency; (3) chain of custody should be provided in order to ensure sample traceability; (4) control charts should be provided to prove method performance; (5) certified reference material analysis or, if that is not available, matrix spike analysis, should be undertaken and (6) chromatograms should be included in the analytical report. Within this context and with a view to helping environmental managers in analytical report evaluation, this work has as objectives the discussion of the limitations of the application of SW 846 US EPA methods to marine samples, the consequences of having data based on method detection limits (MDL) and not sample quantitation limits (SQL), and present possible modifications of the principal method applied by laboratories in order to comply with environmental quality standards.

Influence of spawning procedure on gametes fertilization success in Salminus hilarii Valenciennes, 1850 (Teleostei: Characidae): Implications for the conservation of this species

Artificial reproduction and gamete fertilization were evaluated in Salminus hilarii wild and domesticated broodstocks. Wild and domesticated broodstocks were artificially induced to reproduction using a carp pituitary treatment. Four groups were considered: Group 1 (G1), fish caught in the wild maintained for three years in the same conditions as the domesticated broodstocks and spawned naturally; Group 2 (G2), broodstock born and raised in captivity and spawned naturally; Group 3 (G3), wild broodstocks, which were manually stripped for gamete collection and dry fertilization; and Group 4 (G4), domesticated males and females, also manually stripped. Oocytes, eggs, and larvae were sampled at different time intervals throughout embryonic development. Yolk sac absorption occurred approximately 24-29 h after hatching. Twenty-six h after hatching, the larvae mouths opened. Cannibalism was identified just 28-30 h after hatching. There was no morphological difference in embryonic development among all groups. The number of released eggs per gram of female was: G1: 83.3 ± 24.5 and G2: 103.8 ± 37.4; however, the fertilization success was lower in G2 (42.0 ± 6.37 %) compared with G1 (54.7 ± 3.02%) (P = 0.011). Hand-stripping of oocytes was not successful and the fertilization rate was zero. The reproduction of this species in captivity is viable, but it is necessary to improve broodstock management to enhance fertilization rates and obtain better fingerling production for restocking programs.

Localization of ribosomal genes in three Pimelodus species (Siluriformes, Pimelodidae) of the São Francisco River: 5S genes as species markers and conservation of the 18S rDNA sites

Pimelodidae is one of the most representative of Neotropical catfish families. However, these fish are still poorly studied in terms of cytogenetics, especially regarding the application of more accurate techniques such as the chromosomal localization of ribosomal genes. In the present work, fluorescent in situ hybridization with 5S and 18S rDNA probes was employed for rDNA site mapping in Pimelodus sp., P. fur and P. maculatus from the São Francisco River in the Três Marias municipality - MG. The results from the application of the 18S probe confirmed the previous data obtained by silver nitrate staining, identifying a simple nucleolar organizing region system for these species. However, the labeling results from the 5S rDNA probe demonstrated a difference in the number and localization of these sites between the analyzed species. The obtained data allowed inferences on the possible processes involved in the karyotypic evolution of this genus.

A multi-screening approach for marine-derived fungal metabolites and the isolation of cyclodepsipeptides from Beauveria felina

Extracts obtained from 57 marine-derived fungal strains were analyzed by HPLC-PDA, TLC and ¹H NMR. The analyses showed that the growth conditions affected the chemical profile of crude extracts. Furthermore, the majority of fungal strains which produced either bioactive of chemically distinctive crude extracts have been isolated from sediments or marine algae. The chemical investigation of the antimycobacterial and cytotoxic crude extract obtained from two strains of the fungus Beauveria felina have yielded cyclodepsipeptides related to destruxins. The present approach constitutes a valuable tool for the selection of fungal strains that produce chemically interesting or biologically active secondary metabolites.

Comparative analysis of microsatellite variability in five macaw species (Psittaciformes, Psittacidae): application for conservation

Cross-amplification was tested and variability in microsatellite primers (designed for Neotropical parrots) compared, in five macaw species, viz., three endangered blue macaws (Cyanopsitta spixii [extinct in the wild], Anodorhynchus leari [endangered] and Anodorhynchus hyacinthinus [vulnerable]), and two unthreatened red macaws (Ara chloropterus and Ara macao). Among the primers tested, 84.6% successfully amplified products in C. spixii, 83.3% in A. leari, 76.4% in A. hyacinthinus, 78.6% in A. chloropterus and 71.4% in A. macao. The mean expected heterozygosity estimated for each species, and based on loci analyzed in all the five, ranged from 0.33 (A. hyacinthinus) to 0.85 (A. macao). As expected, the results revealed lower levels of genetic variability in threatened macaw species than in unthreatened. The low combined probability of genetic identity and the moderate to high potential for paternity exclusion, indicate the utility of the microsatellite loci set selected for each macaw species in kinship and population studies, thus constituting an aid in planning in-situ and ex-situ conservation.

Marine influence in the Barreiras Formation, State of Alagoas, northeastern Brazil

Tidal processes were important for deposition of the Barreiras Formation located in northern Brazil, while correlatable deposits in northeastern Brazil have been traditionally related to continental environments. Facies analysis in southern Alagoas revealed that the Barreiras Formation consists of cross-stratified conglomerates and sandstones (facies Sx and Cgx), compound cross-stratified sandstones (facies Cx), and heterolithic beddings (facies H). A significant portion of these deposits occurs within channel morphologies displaying fining and thinning upward successions. An abundance of sedimentary features is comparable to those from the northern Brazilian counterpart. These include: tidal bundles; herringbone cross-stratification; heterolithic beddings with sandstone and mudstone beds in sharp contacts; and ichnofossils mostly consisting of Ophiomorpha nodosa, Skolithos and Planolites. Altogether, these features point to a marginal marine depositional setting dominated by tidal processes, which are related to an estuarine system, an interpretation also provided for the Barreiras Formation in northern Brazil. The widespread occurrence of deposits with unambiguous evidence of tidal processes in the Barreiras Formation of northern Brazil, and now in the State of Alagoas, leads to argue that the early/middle Miocene worldwide marine transgression might have left a much more widespread sedimentary record along the Brazilian coast than currently regarded.

Ichthyofauna of Rio Jurubatuba, Santos, São Paulo: a high diversity refuge in impacted lands

Ichthyofaunistic surveys in the Atlantic Rainforest have been published in relatively few works, in spite of the major biological importance of this once vast biome which is rapidly vanishing due to disordered human population growth and natural resources overexploitation. The present study aimed to access the fish fauna of a relatively well preserved basin between the cities of Santos and Cubatão (SP), an area highly modified by human activities where recent ichthyofaunistic surveys are still missing. Collections were made during three field trips in Rio Jurubatuba, a medium sized costal river, and Riacho Sabão, one of its main tributaries. A total of 2773 specimens were sampled, representing 25 species from 14 families. Six species were primary marine using the upper reaches of Rio Jurubatuba. Twelve of the 19 freshwater species are endemic of the Atlantic Rainforest and four are present in regional lists of endangered species. Only five species occurred in both Rio Jurubatuba and Riacho Sabão. The most diverse family was Characidae, followed by Poeciliidae, Rivulidae and Heptapteridae. Phalloceros caudimaculatus was the most abundant species, followed by Poecilia vivipara and Geophagus brasiliensis. The study area is considered well preserved and due to its critical location, urges for conservation policies to protect its fish diversity.

The ichthyofauna of upper rio Capivari: defining conservation strategies based on the composition and distribution of fish species

Although the rio Capivari basin is recognized as an area of great importance for the ichthyofauna, it lacks virtually every basic requirement for the definition of appropriate conservation strategies, since not even its species composition is known. The objective of this work is to determine the composition and distribution of fish species in the upper rio Capivari basin, relating them to the local physical features, and to evaluate the effectiveness of proposed conservations units, delimited based on areas of native vegetation remains, on the conservation of local ichthyofauna. During 2007, 50 different watercourses were sampled with gillnets, cast nets and kick nets. A total of 1308 individuals belonging to 41 species were captured. Degree of conservation, altitude and width were the parameters that best explained fish species richness. Considering the recently proposed boundaries for potential conservation units in the region only 15 or 20 out of 41 species would be found in the State Park and Environmental Protection Area respectively. In practice, the proposed conservation units would not be effective tools for fish conservation, since it would be located in mountainous areas of high altitude, of headwaters streams and where few species are found. In such context, the conservation of specific stretches of larger rivers is critical.