Genome composition and GC content influence loci distribution in reduced representation genomic studies.

BACKGROUND: Genomic architecture is a key evolutionary trait for living organisms. Due to multiple complex adaptive and neutral forces which impose evolutionary pressures on genomes, there is a huge variability of genomic features. However, their variability and the extent to which genomic content determines the distribution of recovered loci in reduced representation sequencing studies is largely unexplored. RESULTS: Here, by using 80 genome assemblies, we observed that whereas plants primarily increase their genome size by expanding their intergenic regions, animals expand both intergenic and intronic regions, although the expansion patterns differ between deuterostomes and protostomes. Loci mapping in introns, exons, and intergenic categories obtained by in silico digestion using 2b-enzymes are positively correlated with the percentage of these regions in the corresponding genomes, suggesting that loci distribution mostly mirrors genomic architecture of the selected taxon. However, exonic regions showed a significant enrichment of loci in all groups regardless of the used enzyme. Moreover, when using selective adaptors to obtain a secondarily reduced loci dataset, the percentage and distribution of retained loci also varied. Adaptors with G/C terminals recovered a lower percentage of selected loci, with a further enrichment of exonic regions, while adaptors with A/T terminals retained a higher percentage of loci and slightly selected more intronic regions than expected. CONCLUSIONS: Our results highlight how genome composition, genome GC content, RAD enzyme choice and use of base-selective adaptors influence reduced genome representation techniques. This is important to acknowledge in population and conservation genomic studies, as it determines the abundance and distribution of loci.

Identification of potential invasive alien species in Spain through horizon scanning.

Invasive alien species have widespread impacts on native biodiversity and ecosystem services. Since the number of introductions worldwide is continuously rising, it is essential to prevent the entry, establishment and spread of new alien species through a systematic examination of future potential threats. Applying a three-step horizon scanning consensus method, we evaluated non-established alien species that could potentially arrive, establish and cause major ecological impact in Spain within the next 10 years. Overall, we identified 47 species with a very high risk (e.g. Oreochromis niloticus, Popillia japonica, Hemidactylus frenatus, Crassula helmsii or Halophila stipulacea), 61 with high risk, 93 with moderate risk, and 732 species with low risk. Many of the species categorized as very high or high risk to Spanish biodiversity are either already present in Europe and neighbouring countries or have a long invasive history elsewhere. This study provides an updated list of potential invasive alien species useful for prioritizing efforts and resources against their introduction. Compared to previous horizon scanning exercises in Spain, the current study screens potential invaders from a wider range of terrestrial, freshwater, and marine organisms, and can serve as a basis for more comprehensive risk analyses to improve management and increase the efficiency of the early warning and rapid response framework for invasive alien species. We also stress the usefulness of measuring agreement and consistency as two different properties of the reliability of expert scores, in order to more easily elaborate consensus ranked lists of potential invasive alien species.

To denoise or to cluster, that is not the question: optimizing pipelines for COI metabarcoding and metaphylogeography.

BACKGROUND: The recent blooming of metabarcoding applications to biodiversity studies comes with some relevant methodological debates. One such issue concerns the treatment of reads by denoising or by clustering methods, which have been wrongly presented as alternatives. It has also been suggested that denoised sequence variants should replace clusters as the basic unit of metabarcoding analyses, missing the fact that sequence clusters are a proxy for species-level entities, the basic unit in biodiversity studies. We argue here that methods developed and tested for ribosomal markers have been uncritically applied to highly variable markers such as cytochrome oxidase I (COI) without conceptual or operational (e.g., parameter setting) adjustment. COI has a naturally high intraspecies variability that should be assessed and reported, as it is a source of highly valuable information. We contend that denoising and clustering are not alternatives. Rather, they are complementary and both should be used together in COI metabarcoding pipelines. RESULTS: Using a COI dataset from benthic marine communities, we compared two denoising procedures (based on the UNOISE3 and the DADA2 algorithms), set suitable parameters for denoising and clustering, and applied these steps in different orders. Our results indicated that the UNOISE3 algorithm preserved a higher intra-cluster variability. We introduce the program DnoisE to implement the UNOISE3 algorithm taking into account the natural variability (measured as entropy) of each codon position in protein-coding genes.  This correction increased the number of sequences retained by 88%. The order of the steps (denoising and clustering) had little influence on the final outcome. CONCLUSIONS: We highlight the need for combining denoising and clustering, with adequate choice of stringency parameters, in COI metabarcoding. We present a program that uses the coding properties of this marker to improve the denoising step. We recommend researchers to report their results in terms of both denoised sequences (a proxy for haplotypes) and clusters formed (a proxy for species), and to avoid collapsing the sequences of the latter into a single representative. This will allow studies at the cluster (ideally equating species-level diversity) and at the intra-cluster level, and will ease additivity and comparability between studies.

Marine biomonitoring with eDNA: Can metabarcoding of water samples cut it as a tool for surveying benthic communities?

In the marine realm, biomonitoring using environmental DNA (eDNA) of benthic communities requires destructive direct sampling or the setting-up of settlement structures. Comparatively much less effort is required to sample the water column, which can be accessed remotely. In this study we assess the feasibility of obtaining information from the eukaryotic benthic communities by sampling the adjacent water layer. We studied two different rocky-substrate benthic communities with a technique based on quadrat sampling. We also took replicate water samples at four distances (0, 0.5, 1.5, and 20 m) from the benthic habitat. Using broad range primers to amplify a ca. 313 bp fragment of the cytochrome oxidase subunit I gene, we obtained a total of 3,543 molecular operational taxonomic units (MOTUs). The structure obtained in the two environments was markedly different, with Metazoa, Archaeplastida and Stramenopiles being the most diverse groups in benthic samples, and Hacrobia, Metazoa and Alveolata in the water. Only 265 MOTUs (7.5%) were shared between benthos and water samples and, of these, 180 (5.1%) were identified as benthic taxa that left their DNA in the water. Most of them were found immediately adjacent to the benthos, and their number decreased as we moved apart from the benthic habitat. It was concluded that water eDNA, even in the close vicinity of the benthos, was a poor proxy for the analysis of benthic structure, and that direct sampling methods are required for monitoring these complex communities via metabarcoding.

Enjoying the warming Mediterranean: Transcriptomic responses to temperature changes of a thermophilous keystone species in benthic communities.

Information about the genomic processes underlying responses to temperature changes is still limited in non-model marine invertebrates. In this sense, transcriptomic analyses can help to identify genes potentially related to thermal responses. We here investigated, via RNA-seq, whole-transcriptomic responses to increased and decreased temperatures in a thermophilous keystone sea urchin, Arbacia lixula, whose populations are increasing in the Mediterranean. This species is a key driver of benthic communities' structure due to its grazing activity. We found a strong response to experimentally induced cold temperature (7°C), with 1,181 differentially expressed transcripts relative to the control condition (13°C), compared to only 179 in the warm (22°C) treatment. A total of 84 (cold treatment) and three (warm treatment) gene ontology terms were linked to the differentially expressed transcripts. At 7°C the expression of genes encoding different heat shock proteins (HSPs) was upregulated, together with apoptotic suppressor genes (e.g., Bcl2), genes involved in the infection response and/or pathogen-recognition (e.g., echinoidin) and ATP-associated genes, while protein biosynthesis and DNA replication pathways were downregulated. At 22°C neither HSPs induction nor activation of the previously mentioned pathways were detected, with the exception of some apoptotic-related activities that were upregulated. Our results suggest a strong transcriptional response associated with low temperatures, and support the idea of low water temperature being a major limitation for A. lixula expansion across deep Mediterranean and northern Atlantic waters.

From metabarcoding to metaphylogeography: separating the wheat from the chaff.

Metabarcoding is by now a well-established method for biodiversity assessment in terrestrial, freshwater, and marine environments. Metabarcoding data sets are usually used for α- and ß-diversity estimates, that is, interspecies (or inter-MOTU [molecular operational taxonomic unit]) patterns. However, the use of hypervariable metabarcoding markers may provide an enormous amount of intraspecies (intra-MOTU) information-mostly untapped so far. The use of cytochrome oxidase (COI) amplicons is gaining momentum in metabarcoding studies targeting eukaryote richness. COI has been for a long time the marker of choice in population genetics and phylogeographic studies. Therefore, COI metabarcoding data sets may be used to study intraspecies patterns and phylogeographic features for hundreds of species simultaneously, opening a new field that we suggest to name metaphylogeography. The main challenge for the implementation of this approach is the separation of erroneous sequences from true intra-MOTU variation. Here, we develop a cleaning protocol based on changes in entropy of the different codon positions of the COI sequence, together with co-occurrence patterns of sequences. Using a data set of community DNA from several benthic littoral communities in the Mediterranean and Atlantic seas, we first tested by simulation on a subset of sequences a two-step cleaning approach consisting of a denoising step followed by a minimal abundance filtering. The procedure was then applied to the whole data set. We obtained a total of 563 MOTUs that were usable for phylogeographic inference. We used semiquantitative rank data instead of read abundances to perform AMOVAs and haplotype networks. Genetic variability was mainly concentrated within samples, but with an important between seas component as well. There were intergroup differences in the amount of variability between and within communities in each sea. For two species, the results could be compared with traditional Sanger sequence data available for the same zones, giving similar patterns. Our study shows that metabarcoding data can be used to infer intra- and interpopulation genetic variability of many species at a time, providing a new method with great potential for basic biogeography, connectivity and dispersal studies, and for the more applied fields of conservation genetics, invasion genetics, and design of protected areas.

Spatio-temporal patterns of genetic variation in Arbacia lixula, a thermophilous sea urchin in expansion in the Mediterranean.

The genetic structure of 13 populations of the amphiatlantic sea urchin Arbacia lixula, as well as temporal genetic changes in three of these localities, were assessed using ten hypervariable microsatellite loci. This thermophilous sea urchin is an important engineer species triggering the formation of barren grounds through its grazing activity. Its abundance seems to be increasing in most parts of the Mediterranean, probably favoured by warming conditions. Significant genetic differentiation was found both spatially and temporally. The main break corresponded to the separation of western Atlantic populations from those in eastern Atlantic and the Mediterranean Sea. A less marked, but significant differentiation was also found between Macaronesia (eastern Atlantic) and the Mediterranean. In the latter area, a signal of differentiation between the transitional area (Alboran Sea) and the rest of the Mediterranean was detected. However, no genetic structure is found within the Mediterranean (excluding Alboran) across the Siculo-Tunisian Strait, resulting from either enough gene flow to homogenize distance areas or/and a recent evolutionary history marked by demographic expansion in this basin. Genetic temporal variation at the Alboran Sea is as important as spatial variation, suggesting that temporal changes in hydrological features can affect the genetic composition of the populations. A picture of genetic homogeneity in the Mediterranean emerges, implying that the potential expansion of this keystone species will not be limited by intraspecific genetic features and/or potential impact of postulated barriers to gene flow in the region.

A phylogenomic framework and timescale for comparative studies of tunicates.

BACKGROUND: Tunicates are the closest relatives of vertebrates and are widely used as models to study the evolutionary developmental biology of chordates. Their phylogeny, however, remains poorly understood, and to date, only the 18S rRNA nuclear gene and mitogenomes have been used to delineate the major groups of tunicates. To resolve their evolutionary relationships and provide a first estimate of their divergence times, we used a transcriptomic approach to build a phylogenomic dataset including all major tunicate lineages, consisting of 258 evolutionarily conserved orthologous genes from representative species. RESULTS: Phylogenetic analyses using site-heterogeneous CAT mixture models of amino acid sequence evolution resulted in a strongly supported tree topology resolving the relationships among four major tunicate clades: (1) Appendicularia, (2) Thaliacea + Phlebobranchia + Aplousobranchia, (3) Molgulidae, and (4) Styelidae + Pyuridae. Notably, the morphologically derived Thaliacea are confirmed as the sister group of the clade uniting Phlebobranchia + Aplousobranchia within which the precise position of the model ascidian genus Ciona remains uncertain. Relaxed molecular clock analyses accommodating the accelerated evolutionary rate of tunicates reveal ancient diversification (~ 450-350 million years ago) among the major groups and allow one to compare their evolutionary age with respect to the major vertebrate model lineages. CONCLUSIONS: Our study represents the most comprehensive phylogenomic dataset for the main tunicate lineages. It offers a reference phylogenetic framework and first tentative timescale for tunicates, allowing a direct comparison with vertebrate model species in comparative genomics and evolutionary developmental biology studies.

Feeding cessation alters host morphology and bacterial communities in the ascidian Pseudodistoma crucigaster.

BACKGROUND: Ascidians can associate with abundant and diverse consortia of microbial symbionts, yet these communities remain unexamined for the majority of host ascidians and little is known about host-symbiont interactions. METHODS: We coupled electron microscopy and 16S rRNA gene tag pyrosequencing to investigate the bacterial communities associated with the colonial ascidian Pseudodistoma crucigaster, a species endemic to the Mediterranean Sea that has a life cycle with two phases: actively-filtering (active) and non-filtering (resting) forms. RESULTS: Resting colonies exhibited a reduced branchial sac (feeding apparatus) and a thickened cuticle. Electron microscope images also suggested higher abundance of colonizing microorganisms on surfaces of resting colonies. Accordingly, bacterial sequences associated with environmental sources (sediment and biofilms, >99 % similarity) were detected exclusively in resting colonies. Bacterial communities of P. crucigaster colonies (active and resting) were dominated by 3 core taxa affiliated (>94 % similarity) with previously described symbiotic Alphaproteobacteria in marine invertebrates. Shifts in rare bacteria were detected when ascidians entered the resting phase, including the appearance of strictly anaerobic lineages and nitrifying bacterial guilds. CONCLUSIONS: These findings suggest that physical (thickened cuticle) and metabolic (feeding cessation) changes in host ascidians have cascading effects on associated bacteria, where modified oxygen concentrations and chemical substrates for microbial metabolism may create anaerobic microhabitats and promote colonization by environmental microorganisms.

Biotechnological Production of Docosahexaenoic Acid Using Aurantiochytrium limacinum: Carbon Sources Comparison And Growth Characterization.

Aurantiochytrium limacinum, a marine heterotrophic protist/microalga has shown interesting yields of docosahexaenoic acid (DHA) when cultured with different carbon sources: glucose, pure and crude glycerol. A complete study in a lab-scale fermenter allowed for the characterization and comparison of the growth kinetic parameters corresponding to each carbon source. Artificial Marine Medium (AMM) with glucose, pure and crude glycerol offered similar biomass yields. The net growth rates (0.10-0.12 h(-1)), biomass (0.7-0.8 g cells/g Substrate) and product (0.14-0.15 g DHA/g cells) yields, as well as DHA productivity were similar using the three carbon sources. Viable potential applications to valorize crude glycerol are envisioned to avoid an environmental problem due to the excess of byproduct.

Molecular and morphological discrimination between an invasive ascidian, Ascidiella aspersa, and its congener A. scabra (Urochordata: Ascidiacea).

The solitary ascidian Ascidiella aspersa (Müller, 1776) has sometimes been regarded as conspecific with A. scabra (Müller, 1776), although previous detailed morphological comparisons have indicated that the two are distinguishable by internal structures. Resolution of this taxonomic issue is important because A. aspersa has been known as a notoriously invasive ascidian, doing much damage to aquaculture e.g. in Hokkaido, Japan. We collected many specimens from European waters (including the Swedish coast, near the type localities of these two species) and Hokkaido, Japan (as an alien population) and made molecular phylogenetic analyses using the mitochondrial cytochrome c oxidase subunit I (COI) gene, and found that in terms of COI sequences all the analyzed specimens were clustered into two distinct groups, one of which is morphologically referable to A. aspersa and the other to A. scabra. Thus, these two species should be regarded as distinct from each other.

A new sampling device for metabarcoding surveillance of port communities and detection of non-indigenous species.

Metabarcoding techniques are revolutionizing studies of marine biodiversity. They can be used for monitoring non-indigenous species (NIS) in ports and harbors. However, they are often biased by inconsistent sampling methods and incomplete reference databases. Logistic constraints in ports prompt the development of simple, easy-to-deploy samplers. We tested a new device called polyamide mesh for ports organismal monitoring (POMPOM) with a high surface-to-volume ratio. POMPOMS were deployed inside a fishing and recreational port in the Mediterranean alongside conventional settlement plates. We also compiled a curated database with cytochrome oxidase (COI) sequences of Mediterranean NIS. COI metabarcoding of the communities settled in the POMPOMs captured a similar biodiversity than settlement plates, with shared molecular operational units (MOTUs) representing ca. 99% of reads. 38 NIS were detected in the port accounting for ca. 26% of reads. POMPOMs were easy to deploy and handle and provide an efficient method for NIS surveillance.

Navigating spatio-temporal microbiome dynamics: Environmental factors and trace elements shape the symbiont community of an invasive marine species.

The proliferation of marine invasive species is a mounting concern. While the role of microbial communities in invasive ascidian species is recognized, the role of seasonal shifts in microbiome composition remains largely unexplored. We sampled five individuals of the invasive ascidian Styela plicata quarterly from January 2020 to October 2021 in two harbours, examining gills, tunics, and surrounding water. By analysing Amplicon Sequence Variants (ASVs) and seawater trace elements, we found that compartment (seawater, tunic, or gills) was the primary differentiating factor, followed by harbour. Clear seasonal patterns were evident in seawater bacteria, less so in gills, and absent in tunics. We identified compartment-specific bacteria, as well as seasonal indicator ASVs and ASVs correlated with trace element concentrations. Among these bacteria, we found that Endozoicomonas, Hepatoplasma and Rhodobacteraceae species had reported functions which might be necessary for overcoming seasonality and trace element shifts. This study contributes to understanding microbiome dynamics in invasive holobiont systems, and the patterns found indicate a potential role in adaptation and invasiveness.

Chromosome-level genome assembly and annotation of the black sea urchin Arbacia lixula (Linnaeus, 1758).

The black sea urchin (Arbacia lixula) is a keystone species inhabiting the coastal shallow waters of the Mediterranean Sea, which is a key driver of littoral communities' structure. Here, we present the first genome assembly and annotation of this species, standing as the first Arbacioida genome, including both nuclear and mitochondrial genomes. To obtain a chromosome-level assembly, we used a combination of PacBio high fidelity (HiFi) reads and chromatin capture reads (Omni-C). In addition, we generated a high-quality nuclear annotation of both coding and non-coding genes, by using published RNA-Seq data from several individuals of A. lixula and gene models from closely related species. The nuclear genome assembly has a total span of 607.91 Mb, being consistent with its experimentally estimated genome size. The assembly contains 22 chromosome-scale scaffolds (96.52% of the total length), which coincides with its known karyotype. A total of 72,767 transcripts were predicted from the nuclear genome, 24,171 coding, and 48,596 non-coding that included lncRNA, snoRNA, and tRNAs. The circularized mitochondrial genome had 15740 bp comprising 13 protein-coding genes, 2 rRNA, and 22 tRNA. This reference genome will enhance ongoing A. lixula studies and benefit the wider sea urchin scientific community.

Sources of secondary metabolite variation in Dysidea avara (Porifera: Demospongiae): the importance of having good neighbors.

Several studies report temporal, geographical, and intra-individual variation in sponge metabolite yields. However, the internal and/or external factors that regulate the metabolite production remain poorly understood. Dysidea avara is a demosponge that produces sesquiterpenoids (avarol and derivatives) with interesting medical properties, which has prompted addressed studies to obtain enough amounts of these metabolites for research on drug discovery. Within this framework, specimens of Dysidea avara from a population of the Northwest Mediterranean were sampled and their secondary metabolites quantified to assess their variability and the possible relationship with external (seasonality, interactions with neighbors) and internal (reproductive stages) factors. The results show a variation of the amount of both avarol and its monoacetate derivative with time, with no clear relationship with seawater temperature. A trade-off with sponge reproduction was not found either. However, our results showed for the first time that sponges are able to increase production or accumulation of secondary metabolites in their peripheral zone depending on the nature of their neighbors. This finding could explain part of the high variability in the amount of secondary metabolites usually found in chemical ecology studies on sponges and opens new biotechnological approaches to enhance the metabolite yield in sponge cultures.

Multidimensional variability of the microbiome of an invasive ascidian species.

Animals, including invasive species, are complex entities consisting of a host and its associated symbionts (holobiont). The interaction between the holobiont components is crucial for the host's survival. However, our understanding of how microbiomes of invasive species change across different tissues, localities, and ontogenetic stages, is limited. In the introduced ascidian Styela plicata, we found that its microbiome is highly distinct and specialized among compartments (tunic, gill, and gut). Smaller but significant differences were also found across harbors, suggesting local adaptation, and between juveniles and adults. Furthermore, we found a correlation between the microbiome and environmental trace element concentrations, especially in adults. Functional analyses showed that adult microbiomes possess specific metabolic pathways that may enhance fitness during the introduction process. These findings highlight the importance of integrated approaches in studying the interplay between animals and microbiomes, as a first step toward understanding how it can affect the species' invasive success.

Taxonomic identity of Distapliastylifera (Tunicata, Ascidiacea), a new arrival to the eastern Pacific displaying invasive behavior in the Gulf of California, Mexico.

A colonial ascidian of the genus Distaplia caused a mass mortality of the pen shell Atrinamaura (Sowerby, 1835) during June 2016 in the southwest of the Gulf of California (Mexico), with a significant socio-economic cost. Tentatively identified in previous works as Distapliacf.stylifera, a precise taxonomic determination was still lacking. In the present work, based on a detailed morphological study, it is confirmed that this aggressive species is Distapliastylifera (Kowalevsky, 1874). Originally described from the Red Sea, the species currently has a wide circumtropical distribution (with the exception of the Eastern Pacific to date) and is reported as introduced in parts of its range. The present account thus represents an important range extension of this species. However, when revising the original description and later observations, the reported variability of several characters makes it likely that the binomen is in fact a complex of species, as is common in other ascidians with wide distributions. A complete morphological and genetic study including populations from the entire range of distribution would be necessary to settle the status of D.stylifera. Taxonomic uncertainties hinder a correct interpretation of biogeographical patterns and inference on the origin of the studied population. Nevertheless, the known introduction potential of the species, coupled with an explosive growth in an anthropized environment, and the lack of any previous reports in the Eastern Pacific, strongly suggest that the investigated population represents yet another instance of ascidian introduction. From the point of view of management, its invasive behavior is cause for great concern and warrants mitigation measures.

Analysis of the P. lividus sea urchin genome highlights contrasting trends of genomic and regulatory evolution in deuterostomes.

Sea urchins are emblematic models in developmental biology and display several characteristics that set them apart from other deuterostomes. To uncover the genomic cues that may underlie these specificities, we generated a chromosome-scale genome assembly for the sea urchin Paracentrotus lividus and an extensive gene expression and epigenetic profiles of its embryonic development. We found that, unlike vertebrates, sea urchins retained ancestral chromosomal linkages but underwent very fast intrachromosomal gene order mixing. We identified a burst of gene duplication in the echinoid lineage and showed that some of these expanded genes have been recruited in novel structures (water vascular system, Aristotle's lantern, and skeletogenic micromere lineage). Finally, we identified gene-regulatory modules conserved between sea urchins and chordates. Our results suggest that gene-regulatory networks controlling development can be conserved despite extensive gene order rearrangement.

Stability of sponge-associated bacteria over large seasonal shifts in temperature and irradiance.

Complex microbiomes reside in marine sponges and consist of diverse microbial taxa, including functional guilds that may contribute to host metabolism and coastal marine nutrient cycles. Our understanding of these symbiotic systems is based primarily on static accounts of sponge microbiota, while their temporal dynamics across seasonal cycles remain largely unknown. Here, we investigated temporal variation in bacterial symbionts of three sympatric sponges (Ircinia spp.) over 1.5 years in the northwestern (NW) Mediterranean Sea, using replicated terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of bacterial 16S rRNA gene sequences. Bacterial symbionts in Ircinia spp. exhibited host species-specific structure and remarkable stability throughout the monitoring period, despite large fluctuations in temperature and irradiance. In contrast, seawater bacteria exhibited clear seasonal shifts in community structure, indicating that different ecological constraints act on free-living and on symbiotic marine bacteria. Symbiont profiles were dominated by persistent, sponge-specific bacterial taxa, notably affiliated with phylogenetic lineages capable of photosynthesis, nitrite oxidation, and sulfate reduction. Variability in the sponge microbiota was restricted to rare symbionts and occurred most prominently in warmer seasons, coincident with elevated thermal regimes. Seasonal stability of the sponge microbiota supports the hypothesis of host-specific, stable associations between bacteria and sponges. Further, the core symbiont profiles revealed in this study provide an empirical baseline for diagnosing abnormal shifts in symbiont communities. Considering that these sponges have suffered recent, episodic mass mortalities related to thermal stresses, this study contributes to the development of model sponge-microbe symbioses for assessing the link between symbiont fluctuations and host health.