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Zagami, Giacomo, Stanković, David, Bonanzinga, Vincenzo, Costanzo, Giuseppe (2021): Paramisophria walteri a new Arietellidae (Copepoda, Calanoida) from Midway Island. Journal of Natural History 55 (25-26): 1619-1637, DOI: 10.1080/00222933.2021.1948127, URL: http://dx.doi.org/10.1080/00222933.2021.1948127

The present study is aimed at implementing the amplicon sequence variant (ASV) concept to describe meiofaunal species composition independent of morphological identification, while strongly indicating reasonable compatibility with the underlying species. A primer pair was constructed and demonstrated to PCR amplify an 18S barcode from a variety of meiofaunal taxa, compatible with the high throughput sequencing (HTS) Illumina 300 X 2 bps platform. Sixteen 18S multi-species HTS assemblies were created from meiofaunal samples and merged to one assembly of ~2,150,000 contigs. Five quality scores (q=35, 30, 25, 20, 15) were implemented to filter five 18S barcode assemblies, which served as inputs to the DADA2 software, ending with 5 reference ASV libraries. Each of them was clustered, applying 3% dissimilarity threshold and revealed an average number of 1.38±0.078 ASVs/cluster which demonstrate high level of ASV uniqueness. The libraries which were based on q ≤ 25 reached a near-asymptote number of ASVs which together with the low average number of ASVs/3% cluster, strongly indicate compatibility with the actual number of underlying species. Hence, the q=25 library was selected to be used as metabarcoding reference library. It contained 461 ASVs and 342 - 3% clusters with average number of 1.34±1.036 ASV/cluster and their BLASTN annotation elucidated the expected meiofaunal taxa. The 16 assemblies of sample-specific paired contigs were mapped to this reference library and sample ASV profiles, namely the list of ASVs and their proportional copy numbers were created and also normalized to the actual number of individuals in each sample.

Pseudo-nitzschia H. Peragallo (1900) is a globally distributed genus of pennate diatoms that are important components of phytoplankton communities worldwide. Some members of the genus produce the neurotoxin domoic acid, so regular monitoring is in place. However, the identification of toxic members in routine samplings remains problematic. In this study, the diversity and seasonal occurrence of Pseudo-nitzschia species were investigated in the Gulf of Trieste, a shallow gulf in the northern Adriatic Sea. We used time series data from 2005 to 2018 to describe the seasonal and inter-annual occurrence of the genus in the area and its contribution to the phytoplankton community. On average, the genus accounted for about 15 % of total diatom abundance and peaked in spring and autumn, with occasional outbreaks during summer and large inter-annual fluctuations. Increased water temperature and decreased salinity positively affected the presence of some members of the genus, while strong effects could be masked by an unsuitable definition of the species complexes used for monitoring purposes. Therefore, combining morphological (TEM) and molecular analyses by sequencing the ITS, 28S and rbcL markers, eight species were identified from 83 isolated monoclonal strains: P. calliantha, P. fraudulenta, P. delicatissima, P. galaxiae, P. mannii, P. multistriata, P. pungens and P. subfraudulenta. A genetic comparison between the isolated strains and other strains in the Mediterranean was carried out and rbcL was inspected as a potential barcode marker in respect to our results. This is the first study in the Gulf of Trieste on Pseudo-nitzschia time series from a long-term ecological research (LTER) site coupled with molecular data. We show that meaningful ecological conclusions can be drawn by applying integrative methodology, as opposed to the approach that only considers species complexes. The results of this work will provide guidance for further monitoring efforts as well as research activities, including population genetics and genomics, associated with seasonal distribution and toxicity profiles.

A comparison of the genetic structure of Chimaera monstrosa populations from the Atlantic Ocean and the Mediterranean Sea was carried out using mitochondrial DNA analysis. Results indicate high and significant pairwise ΦST values with no shared haplotypes between the two areas. Furthermore, migration rate estimates suggested absence of gene flow between the two basins. These findings, coupled with the species vertical distribution, suggest that the shallow depth of the Strait of Gibraltar may act as a barrier limiting the dispersal capabilities of these populations, which likely became separated at the end of the middle Pleistocene.

DNA metabarcoding combines DNA barcoding with high-throughput sequencing to identify different taxa within environmental communities. The ITS has already been proposed and widely used as universal barcode marker for plants, but a comprehensive, updated and accurate reference dataset of plant ITS sequences has not been available so far. Here, we constructed reference datasets of Viridiplantae ITS1, ITS2 and entire ITS sequences including both Chlorophyta and Streptophyta. The sequences were retrieved from NCBI, and the ITS region was extracted. The sequences underwent identity check to remove misidentified records and were clustered at 99% identity to reduce redundancy and computational effort. For this step, we developed a script called ‘better clustering for QIIME’ (bc4q) to ensure that the representative sequences are chosen according to the composition of the cluster at a different taxonomic level. The three datasets obtained with the bc4q script are PLANiTS1 (100 224 sequences), PLANiTS2 (96 771 sequences) and PLANiTS (97 550 sequences), and all are pre-formatted for QIIME, being this the most used bioinformatic pipeline for metabarcoding analysis. Being curated and updated reference databases, PLANiTS1, PLANiTS2 and PLANiTS are proposed as a reliable, pivotal first step for a general standardization of plant DNA metabarcoding studies. The bc4q script is presented as a new tool useful in each research dealing with sequences clustering. Database URL: https://github.com/apallavicini/bc4q; https://github.com/apallavicini/PLANiTS.

Environmental DNA (eDNA) metabarcoding and metagenomics analyses can improve taxonomic resolution in biodiversity studies. Only recently, these techniques have been applied in aerobiology, to target bacteria, fungi and plants in airborne samples. Here, we present a nine-month aerobiological study applying eDNA metabarcoding in which we analyzed simultaneously airborne diversity and variation of fungi and plants across five locations in North and Central Italy. We correlated species composition with the ecological characteristics of the sites and the seasons. The most abundant taxa among all sites and seasons were the fungal genera Cladosporium, Alternaria, and Epicoccum and the plant genera Brassica, Corylus, Cupressus and Linum, the latter being much more variable among sites. PERMANOVA and indicator species analyses showed that the plant diversity from air samples is significantly correlated with seasons, while that of fungi varied according to the interaction between seasons and sites. The results consolidate the performance of a new eDNA metabarcoding pipeline for the simultaneous amplification and analysis of airborne plant and fungal particles. They also highlight the promising complementarity of this approach with more traditional biomonitoring frameworks and routine reports of air quality provided by environmental agencies.

Zooplankton biodiversity assessment is a crucial element in monitoring marine ecosystem processes and community responses to environmental alterations. In order to evaluate the suitability of metabarcoding for zooplankton biodiversity assessment and biomonitoring as a fast and more cost-effective method, seasonal zooplankton sampling was carried out in the Venice Lagoon and the nearby coastal area (Northern Adriatic Sea). The molecular analysis showed higher taxa richness compared to the classical morphological method (224 vs. 88 taxa), discriminating better the meroplanktonic component, morphologically identified only up to order level. Both methods revealed a similar spatio-temporal distribution pattern and the sequence abundances and individual counts were significantly correlated for various taxonomic groups. These results indicate that DNA metabarcoding is an efficient tool for biodiversity assessments in ecosystems with high spatial and temporal variability, where high sampling effort is required as well as fast alert systems for non-native species (NIS).

The genetic structure of European mudminnow populations throughout the species range was examined using mitochondrial DNA and seven microsatellite loci. Ten mitochondrial haplotypes were detected, suggesting three phylogeographic lineages, which likely diverged during the Early and Middle Pleistocene. These three lineages geographically correspond to three regions: the Danube drainage including the Drava system and Dniester Delta, the Sava system and the Tisza system. High genetic diversity observed using mtDNA was confirmed with microsatellite data, suggesting the existence of 14 populations in the studied area. The isolation-with-migration model showed that migration rates between populations were generally low and were highest between the Drava and its tributary Mura. According to the inferred relative population splitting times, Umbra krameri likely spread from the eastern part of the species range to the west, which also showed the highest genetic diversity and largest population size. As reported by the time-calibrated phylogeny, separation of the European and American Umbra occurred roughly at the end of Late Cretaceous and in the first half of the Paleogene (60.57 Ma with 95% highest probability density of 39.57–81.75). Taking these results into account, appropriate guidelines are proposed to conserve European mudminnow populations.

Two new populations of the European mudminnow (Umbra krameriWalbaum, 1792) were discovered in the Sava River system, one in its middle part (Bosnia and Herzegovina) and the other in a tributary to the Kupa River (Croatia). The Croatian population is the most upstream mudminnow discovery in the Sava River system known to date. The genetic structure of the newly recorded mudminnow populations was examined using mitochondrial DNA and microsatellite loci. By adding these new populations to the analysis of the population genetic structure of mudminnow from the Sava River system, previously unresolved relationships between the populations from the upper and the lower Sava were clarified: the middle Sava populations were shown to be well outside the hybridisation zone with the Danubian clade, meaning the upstream boundary of this zone is confined to the lower Sava. The results also suggest that mudminnow populations in the Sava River system are less isolated than previously believed. Namely, intermediate gene-flow was detected when comparing the uppermost Sava population with the lower Sava populations. Taking these results into account, appropriate guidelines are proposed to preserve mudminnow populations from the Sava River system.

A previous study detected mixing of two deeply split mtDNA clades (Clade I and Clade II) for Atlantic and Mediterranean populations of the medusozoanPelagia noctiluca. The north hemisphere glaciations and the Messinian salinity crisis have been proposed as the two main biogeographic events related to the isolation between the Atlantic Ocean and the Mediterranean Sea. We tested if the splitting time between Clade I and Clade II ofP. noctilucawas associated with one of these geological events. Our study was based on DNA sequence data of mitochondrial (COI and 16S ribosomal RNA) and nuclear (18S ribosomal RNA, internal transcribed spacer 1 and 5.8S ribosomal RNA) genes from populations of the Atlantic and Pacific Ocean and the Mediterranean Sea. The rise of the Isthmus of Panama was used to calibrate substitution rates for COI. This calibration was based on the detection of a shallow but significant genetic structure betweenP. noctilucapopulations from the Pacific and the Atlantic Oceans. Considering our calibration for COI, we refute a possible origin of Clades I and II during the Messinian salinity crisis. Our estimates suggest the origin for a putative common ancestor of Clades I and II around 2.57 Ma (with 95% 2.91–2.22 HPD), roughly corresponding to the Gelasian stage of the early Pleistocene. These alterations include changes in the sea level and oceanic currents at the Strait of Gibraltar and other regions of the Mediterranean basin, and could explain the origin of the twoP. noctilucaclades.

Fungal spores and mycelium fragments are particles which become and remain airborne and have been subjects of aerobiological studies. The presence and the abundance of taxa in aerobiological samples can be very variable and impaired by changeable climatic conditions. Because many fungi produce mycotoxins and both their mycelium fragments and spores are potential allergens, monitoring the presence of these taxa is of key importance. So far data on exposure and sensitization to fungal allergens are mainly based on the assessment of few, easily identifiable taxa and focused only on certain environments. The microscopic method used to analyze aerobiological samples and the inconspicuous fungal characters do not allow a in depth taxonomical identification. Here, we present a first assessment of fungal diversity from airborne samples using a DNA metabarcoding analysis. The nuclear ITS2 region was selected as barcode to catch fungal diversity in mixed airborne samples gathered during two weeks in four sites of North-Eastern and Central Italy. We assessed the taxonomic composition and diversity within and among the sampled sites and compared the molecular data with those obtained by traditional microscopy. The molecular analyses provide a tenfold more comprehensive determination of the taxa than the traditional morphological inspections. Our results prove that the metabarcoding analysis is a promising approach to increases quality and sensitivity of the aerobiological monitoring. The laboratory and bioinformatic workflow implemented here is now suitable for routine, high-throughput, regional analyses of airborne fungi.

Following its introduction in the late nineteenth century, rainbow trout gradually spread to become one of the most widespread non-native fishes. Nevertheless, despite continuous introductions, they have become self-sustaining in only a limited number of European rivers, most of which are located in the Prealps. There is no consensus on the reasons for the absence of self-sustaining populations over most of Europe, though genetic differences have been suggested as a potential underlying factor. We conducted a population genetic study of the structure and origin of European populations to help resolve these questions. We examined variation in mitochondrial D-loop and the Y-linked marker of 41 self-sustaining, stocked, and hatchery populations from Europe and compared them to native and hatchery populations in North America. High levels of genetic diversity but little inter-population genetic variation suggests that European populations are derived from multiple sources, with a great majority of parental populations belonging to steelhead and coastal rainbow trout from Central and Northern California. Our study revealed no considerable genetic differences among European self-sustaining, stocked, and hatchery populations. The mitochondrial DNA data do not allow evaluation of selection and adaptive traits and thus cannot explain potential population differences arising in their naturalization success.

Zooplankton plays a pivotal role in marine ecosystems and the characterisation of its biodiversity still represents a challenge for marine ecologists. In this study, mesozooplankton composition from 46 samples collected in summer along the western Adriatic Sea, was retrieved by DNA metabarcoding analysis. For the first time, the highly variable fragments of the mtDNA COI and the V9 region of 18S rRNA genes were used in a combined matrix to compile an inventory of mesozooplankton at basin scale. The number of sequences retrieved after quality filtering were 824,148 and 223,273 for COI and 18S (V9), respectively. The taxonomical assignment against reference sequences, using 95% (for COI) and 97% (for 18S) similarity thresholds, recovered 234 taxa. NMDS plots and cluster analysis divided coastal from offshore samples and the most representative species of these clusters were distributed according to the dominant surface current pattern of the Adriatic for the summer period. For selected sampling sites, mesozooplankton species were also identified under a stereo microscope providing insights on the strength and weakness of the two approaches. In addition, DNA metabarcoding was shown to be helpful for the monitoring of non-indigenous marine metazoans and spawning areas of commercial fish species. We defined pros and cons of applying this approach at basin scale and the benefits of combining the datasets from two genetic markers.

Lichen thalli harbor complex fungal communities (mycobiomes) of species with divergent trophic and ecological strategies. The complexity and diversity of lichen mycobiomes are still largely unknown, despite surveys combining culture-based methods and high-throughput sequencing (HTS). The results of such surveys are strongly influenced by the barcode locus chosen, its sensitivity in discriminating taxa, and the depth to which public sequence repositories cover the phylogenetic spectrum of fungi. Here, we use HTS of the internal transcribed spacer 2 (ITS2) to assess the taxonomic composition and diversity of a well-characterized, alpine rock lichen community that includes thalli symptomatically infected by lichenicolous fungi as well as asymptomatic thalli. Taxa belonging to the order Chaetothyriales are the major components of the observed lichen mycobiomes. We predict sequences representative of lichenicolous fungi characterized morphologically and assess their asymptomatic presence in lichen thalli. We demonstrated the limitations of metabarcoding in fungi and show how the estimation of species diversity widely differs when ITS1 or ITS2 are used as barcode, and particularly biases the detection of Basidiomycota. The complementary analysis of both ITS1 and ITS2 loci is therefore required to reliably estimate the diversity of lichen mycobiomes. Electronic supplementary material The online version of this article (10.1007/s11557-018-1415-4) contains supplementary material, which is available to authorized users.

The rainbow trout (Oncorhynchus mykiss) is probably the most widely introduced fish species in the world. Since the first translocation outside of the range of its natural distribution, the species has been introduced into at least 99 countries and has established reproducing populations in many different parts of the world. The present review aims to synthesize the existing information on these translocations, with special emphasis on self-sustaining populations in Europe, where continuous introductions have in general not led to naturalization. Our survey produced a list of more than 130 confirmed or potential self-sustaining populations across 16 European countries. The highest abundance of such populations was observed in the Alpine foothills of central Europe where naturalization is not limited to modified waters less suitable for native salmonids but also occurs commonly in pristine and near-natural waters. There is no consensus on the reasons for the absence of self-sustaining populations of rainbo...

Europe’s obligate cave-dwelling amphibian Proteus anguinus inhabits subterranean waters of the north-western Balkan Peninsula. Because only fragments of its habitat are accessible to humans, this endangered salamander’s exact distribution has been difficult to establish. Here we introduce a quantitative real time polymerase chain reaction-based environmental DNA (eDNA) approach to detect the presence of Proteus using water samples collected from karst springs, wells or caves. In a survey conducted along the southern limit of its known range, we established a likely presence of Proteus at seven new sites, extending its range to Montenegro. Next, using specific molecular probes to discriminate the rare black morph of Proteus from the closely related white morph, we detected its eDNA at five new sites, thus more than doubling the known number of sites. In one of these we found both black and white Proteus eDNA together. This finding suggests that the two morphs may live in contact with each other in the same body of groundwater and that they may be reproductively isolated species. Our results show that the eDNA approach is suitable and efficient in addressing questions in biogeography, evolution, taxonomy and conservation of the cryptic subterranean fauna.

Biomarkers have a wide application in research and clinic, they help to choose the correct treatment for diseases. Recent studies, addressing the vaginal microbiome using next generation sequencing (NGS), reported the involvement of bacterial species in infertility. We compared the vaginal microbiome of idiopathic infertile women with that of healthy, including bacterial vaginosis affected women and non-idiopathic infertile women, to identify bacterial species suitable as biomarkers. Information on microorganisms was obtained from the V3-16S rDNA sequencing of cervical-vaginal fluids of 96 women using the Ion Torrent platform. Data were processed with QIIME and classified against the Vaginal 16S rDNA Reference Database. The analysis revealed a significant beta-diversity variation (p

Catarino, Diana ... et. al.-- 19 pages, 5 figures, 4 tables, data accessibility http://dx.doi.org/10.5061/dryad.ss368, supporting information http://dx.doi.org/10.1111/mec.13453, Knowledge of the mechanisms limiting connectivity and gene flow in deep-sea ecosystems is scarce, especially for deep-sea sharks. The Portuguese dogfish (Centroscymnus coelolepis) is a globally distributed and near threatened deep-sea shark. C. coelolepis population structure was studied using 11 nuclear microsatellite markers and a 497-bp fragment from the mtDNA control region. High levels of genetic homogeneity across the Atlantic (Φ = -0.0091, F = 0.0024, P > 0.05) were found suggesting one large population unit at this basin. The low levels of genetic divergence between Atlantic and Australia (Φ = 0.0744, P < 0.01; F = 0.0015, P > 0.05) further suggested that this species may be able to maintain some degree of genetic connectivity even across ocean basins. In contrast, sharks from the Mediterranean Sea exhibited marked genetic differentiation from all other localities studied (Φ = 0.3808, F = 0.1149, P < 0.001). This finding suggests that the shallow depth of the Strait of Gibraltar acts as a barrier to dispersal and that isolation and genetic drift may have had an important role shaping the Mediterranean shark population over time. Analyses of life history traits allowed the direct comparison among regions providing a complete characterization of this shark's populations. Sharks from the Mediterranean had markedly smaller adult body size and size at maturity compared to Atlantic and Pacific individuals. Together, these results suggest the existence of an isolated and unique population of C. coelolepis inhabiting the Mediterranean that most likely became separated from the Atlantic in the late Pleistocene, This study was performed under the framework of ReDEco (FP6 ERA-NET, MARIN-ERA/MAR/0003/2008) and Hermione (FP7-ENV-2008-1, nr 226354) projects. Samples were collected under other scientific projects: DEECON (ESF, 06-EuroDEEP-FP-008 & SFRH-EuroDEEP/0002/2007); CONDOR (EEA Grants, PT0040/2008); SMaRT (M.2.1.2/029/2011) and DEMERSAIS (Azorean Government); OASIS (FP5, EVK3-CT-2002-00073-OASIS); PescProf I (INTERREG III B, MAC/4.2/M12); BIOFUN (ESF, CTM2007-28739-E); DOS MARES (CTM2010-21810-C03-03); ADIOS (EVK3-CT-2000-00035). DC is a PhD student funded by FCT (SFRH/BD/65730/2009); HK is cofunded by the University of Agder and the University of Oslo (CEES); AV is a postdoctoral fellow funded by FCT (POPH/FSE; SFRH/BPD/77487/2011); DS is a postdoctoral fellow funded by the DIANET programme (FP1527385002); and SS is a research fellow supported by the Marie Curie grant cofunded by the EU under the FP7-People-2012-COFUND; Cofunding of Regional, National and International Programmes, GA n.600407 and the Bandiera Project RITMARE. MARE is funded by FCT through the strategic project UID/MAR/04292/2013