PATTERNS OF GENOME SIZE EVOLUTION IN TETRAODONTIFORM FISHES

Abstract We used flow cytometry to measure genome size in 15 species from seven families and subfamilies of tetraodontiform fishes. Previous studies have found that smooth pufferfishes (Tetraodontidae) have the smallest genome of any vertebrate measured to date (0.7–1.0 picograms diploid). We found that spiny pufferfishes (Diodontidae, sister group to the smooth puffers) possess a genome that is about two times larger (1.6–1.8 pg). Mola mola, a member of the sister group to Diodontidae and Tetraodontidae, also has a relatively large genome (1.7 pg). Parsimony analysis of this pattern indicates that the plesiomorphic condition for Molidae (Diodontidae, Tetraodontidae) is a genome size of 1.6–1.8 pg, and that tiny genome size is a derived character unique to smooth puffers. However, an alternative explanation is that the ancestor of Tetraodontidae acquired a heritable tendency toward decreasing genome size, such as a new or modified deletion mechanism, and genome size in all of the tetraodontid lineages has been decreasing in parallel since the split from Diodontidae. Small genome size (1.1–1.3 pg) also appears to have evolved independently in some members of Balistoidea (triggerfishes and filefishes) within Tetraodontiformes.     

SSU rRNA GENE PHYLOGENY OF MORPHOSPECIES AFFILIATED TO THE BIOASSAY ALGA “SELENASTRUM CAPRICORNUTUM” RECOVERED THE POLYPHYLETIC ORIGIN OF CRESCENT‐SHAPED CHLOROPHYTA1

The generic concept of coccoid green algae exhibiting a crescent‐shaped morphotype is evaluated using SSU rRNA gene sequence analyses and light and electron microscopical observations. These common chlorophytes evolved polyphyletically in 10 different clades of the Chlorophyceae and three clades of the Trebouxiophyceae. Six clades are assigned to known genera of Selenastraceae: Kirchneriella, Nephrochlamys, Raphidocelis, Rhombocystis, Selenastrum, and Tetranephris. Four other clades, named following their present genus designation as Ankistrodesmus‐like I and II and Monoraphidium‐like I and II, require further investigation. One crescent‐shaped morphotype, which evolved within the Trebouxiophyceae, is designated as Neocystis mucosa sp. nov. The other two lineages containing trebouxiophycean algae with this morphotype are the Elliptochloris and the Watanabea clades. The taxonomic placement of the widely used bioassay strain “Selenastrum capricornutum” NIVA‐CHL 1 in the genus Raphidocelis (species name Raphidocelis subcapitata) is indicated by molecular data.     

Morphology of the gas bladder in bumblebee catfishes (Siluriformes,Pseudopimelodidae)

The gross morphology of the gas bladder is described and compared for representatives of all valid genera of Pseudopimelodidae (Siluriformes). Cephalosilurus albomarginatus and species of Batrochoglanis, and Microglanis have the most basic form: a large, cordiform gas bladder with a simple internal T‐shaped septum. Cephalosilurus apurensis, C. fowleri, and C. nigricauda also have a large, cordiform gas bladder, but they have well‐developed trabeculae associated with the internal T‐shaped septum, and a pair of well‐developed constrictor muscles inserted on the external wall; the latter feature is present in most species of Pimelodidae, but absent in all other catfishes. The monotypic Lophiosilurus alexandri also has well‐developed constrictor muscles, and its gas bladder is moderately sized. The species of Pseudopimelodus and Cruciglanis have a diminutive gas bladder partially divided into two lateral sacs without internal communication, and lack constrictor muscles. The parapophysis of the fourth vertebra is a wide and long shelf connected to the dorsal surface of the gas bladder in most pseudopimelodid genera. However, in the species of Pseudopimelodus and Cruciglanis the parapophysis of the fourth vertebra is shorter and has its anterior ramus folded back, partially covering the gas bladder anteroventrally; and the tympanic opening is smaller than in species of the other genera. Five phylogenetic characters are proposed based on the morphology of the gas bladder and associated structures in species of Pseudopimelodidae, and the evolution of those characters in the family is discussed. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Genome Size Evaluation in Tetraodontiform Fishes from the Neotropical Region

Smooth pufferfish of the family Tetraodontidae had become pure genomic models because of the remarkable compaction of their genome. This trait seems to be the result of DNA loss following its divergence from the sister family Diodontidae, which possess larger genomes. In this study, flow cytometry was used for estimate the genome size of four pufferfish species from the Neotropical region. Cytogenetic data and confocal microscopy were also used attempting to confirm relationships between DNA content and cytological parameters. The haploid genome size was 0.71?±?0.03 pg for Sphoeroides greeleyi, 0.34?±?0.01 pg for Sphoeroides spengleri, 0.82?±?0.03 pg for Sphoeroides testudineus (all Tetraodontidae), and 1.00?±?0.03 pg for Chilomycterus spinosus (Diodontidae). These differences are not related with ploidy level, because 46 chromosomes are considered basal for both families. The value for S. spengleri represents the smallest vertebrate genome reported to date. Since erythrocyte cell and nuclear sizes are strongly correlated with genome size, the variation in this last is considered under both adaptive and evolutionary perspectives.     

A new diminutive genus and species of catfish from Lake Tanganyika (Siluriformes: Clariidae)

The examination of material representing one of Lake Tanganyika's six previously recognized endemic catfish lineages, has revealed the presence of an additional genus of clariid, described here as Pseudotanganikallabes new genus. This genus is represented by a single species, Pseudotanganikallabes prognatha sp. nov., which is distinguished from all other clariids by its lack of an infraorbital series, the presence of multiple osseous connections between the swim bladder capsules and elements of the neurocranium, the absence of an ethmoid notch, the presence of a very large, egg‐shaped occipital fontanelle and the extension of the lower lip beyond the margin of the upper jaw. A combination of additional external and molecular characters serves to further distinguish this taxon from all currently recognized clariid species. Phylogenetic analysis of mitochondrial (cytb) and nuclear (18S‐ITS1‐5.8S‐ITS2‐28S) sequence data supports the creation of a new genus for this species, as it appears to represent an independent, monophyletic lineage within the family Clariidae.     

Comparative morphology of the gas bladder in driftwood catfishes (Siluriformes: Auchenipteridae)

The gross morphology of the gas bladder is described and illustrated for representatives of most species and all valid genera of the Auchenipteridae (Siluriformes). Although, a simple cordiform gas bladder is present in some species of the family, others are characterized by their distinctive gas‐bladder shape and diverticula disposition. An acute posterior end of the gas bladder characterizes Centromochlus heckelii and C. macracanthus, and is variably present in specimens of Auchenipterus. Tocantinsia piresi and Asterophysus batrachus have distinctive gas bladders differing in number of diverticula (two or many). The two species of Trachycorystes are diagnosed based on their gas bladder morphology: T. menezesi has a simple cordiform bladder, whereas T. trachycorystes has a pair of lateral diverticulum and, usually, a well‐developed terminal diverticulum. Species of Auchenipterichthys are characterized by having a secondary bladder with simple chamber. Short or elongate and divergent terminal diverticula are exclusive to most cis‐andine species of Trachelyopterus. Tetranematichthys and trans‐andine species of Trachelyopterus share a well‐developed secondary chamber or terminal diverticula ventrally or dorsally connected to the posterior chambers. The small‐sized species of Ageneiosus have well‐developed gas bladders with a pair of posterior diverticula, whereas large‐sized species have a reduced gas bladder with tunica externa varying from non‐, partially, or completely ossified. Eight phylogenetic characters are proposed based on the morphology of the gas bladder and associated structures in species of Auchenipteridae, and the evolution of those characters in the family discussed. J. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

Physical mapping of 5S and 45S rDNA loci in pufferfishes (Tetraodontiformes)

Chromosomal features, location and variation of the major and minor rDNA genes cluster were studied in three pufferfish species: Sphoeroides greeleyi and Sphoeroides testudineus (Tetraodontidae) and Cyclichthys spinosus (Diodontidae). The location of the major rDNA was revealed with an 18S probe in two loci for all species. The minor rDNA loci (5S rDNA) was found in one chromosome pair in tetraodontid fishes and four sites located on two distinct chromosomal pairs in C. spinosus. A syntenical organization was not observed among the ribosomal genes. Signal homogeneity for GC/AT-DNA specific fluorochromes was observed in diodontid fish except in the NORs regions, which were CMA₃-positive. Giemsa karyotypes of tetraodontid species presents 2n = 46, having the same diploid value of other Sphoeroides species that have been investigated. On the other hand, the karyotype of C. spinosus, described for the first time, shows 2n = 50 chromosomes (4m + 18sm + 12st + 16a). The foreknowledge of the karyotypic structure of this group and also the physical mapping of certain genes could be very helpful for further DNA sequence analysis.     

Offspring sex ratio in sheep,cattle, goats and pigs: influence of season and lunar phase at conception

This study assessed the relationship between season and lunar phase at conception on offspring sex ratio of four livestock species (sheep, cattle, goats and pigs). The sex of 66,830 lambs (1995–2015); 25,546 calves (2011–2015); 5671 kids (2002–2007) and 1916 piglets was recorded. Moon phases were categorized as either new moon, crescent moon, full moon or decrescent moon. Sex ratio, expressed as proportion of males (males/males + females), was tested against the expected value of 1:1. In sheep, offspring sex ratio and lunar phase were not correlated; season had a significant (p = 0.002) effect on offspring sex ratio. The proportion of males born of spring and winter matings was significantly higher than it was among offspring born of summer (p < 0.05) or autumn (p < 0.01) conceptions. Offspring sex ratios in spring (p < 0.05), autumn (p < 0.01) and winter (p < 0.05) differed significantly from the expected. In cattle, moon phase and season did not affect the offspring sex ratio; however, the interaction effect was highly significant (p = 0.001). The overall piglet sex ratio (0.522), and the sex ratios among piglets conceived during a crescent moon and those conceived in summer differed significantly (p < 0.05) from 1:1. Research including additional factors such as hormonal treatments prior to insemination, food availability, weather and maternal and paternal factors might provide the underlying reasons for the effects of season and moon phase on offspring sex ratio in some livestock species.     

Pyramidodinium spinulosum sp. nov. (Dinophyceae), a sand‐dwelling non‐motile dinoflagellate from the seafloor (36 m deep) off Mageshima Island,Kagoshima, Japan

A new species of benthic marine dinoflagellate, Pyramidodinium spinulosum Horiguchi, Moriya, Pinto & Terada is described from the deep (36 m) seafloor off Mageshima Island, Kagoshima Prefecture, Japan in the subtropical region of the northwest Pacific. The life cycle of the dinoflagellate consists of a dominant, attached, dome‐shaped, vegetative form and short‐lasting, motile cell. Asexual reproduction takes place by the formation of two motile cells within each non‐motile cell. The released motile cells swim only for a short period and transform directly into the dome‐shaped vegetative form. The duration of the cell cycle varies and can be extremely long, ranging 5–38 days under culture conditions. The non‐motile cell is enclosed by a cell wall and its surface is covered with many (80 – 130) spines of various length. The dinoflagellate is photosynthetic and contains many (more than 50) discoidal chloroplasts. Phylogenetic analysis reveals that the dinoflagellate is closely related to the type species of the genus Pyramidodinium, P. atrofuscum which also possesses a dominant, attached, non‐motile form. However, P. spinulosum can be clearly distinguished from P. atrofuscum by the cell shape (dome‐shaped vs. pyramid‐shaped) and surface ornamentation (spines vs. wart‐like processes) of the non‐motile form. Based on these morphological differences together with molecular evidence, it was concluded that this organism from a deep water sand sample should be described as a second species of the genus Pyramidodinium, P. spinulosum.     

Three New Freshwater Cochliopodium Species (Himatismenida,Amoebozoa) from the Southeastern United States

Cochliopodium is a lens‐shaped genus of Amoebozoa characterized by a flexible layer of microscopic dorsal scales. Recent taxonomic and molecular studies reported cryptic diversity in this group and suggested that the often‐used scale morphology is not a reliable character for species delineation in the genus. Here, we described three freshwater Cochliopodium spp. from the southeastern United States based on morphological, immunocytochemistry (ICC), and molecular data. A maximum‐likelihood phylogenetic analysis and pairwise comparison of COI sequences of Cochliopodium species showed that each of these monoclonal cultures were genetically distinct from each other and any described species with molecular data. Two of the new isolates, “crystal UK‐YT2” (Cochliopodium crystalli n. sp.) and “crystal‐like UK‐YT3” (C. jaguari n. sp.), formed a clade with C. larifeili, which all share a prominent microtubule organizing center (MTOC) and have cubical‐shaped crystals. The “Marrs Spring UK‐YT4” isolate, C. marrii n. sp., was 100% identical to “Cochliopodium sp. SG‐2014 KJ569724 .” These sequences formed a clade with C. actinophorum and C. arabianum. While the new isolates can be separated morphologically, most of the taxonomic features used in the group show plasticity; therefore, Cochliopodium species can only be reliably identified with the help of molecular data.     

Molecular and Morphometric Characteristics of Ceratomyxa hamour n. sp. (Myxosporea: Bivalvulida) Infecting the Gallbladder of the Orange‐spotted Grouper Epinephelus coioides from the Arabian Gulf,Saudi Arabia

Ceratomyxa hamour n. sp. was found to infect the gallbladder of the orange‐spotted grouper, Epinephelus coioides located off the Saudi Arabian coast of the Arabian Gulf. The infection was reported as a free‐floating spore in the bile, and pseudoplasmodia were not observed. Mature spores were crescent‐shaped and measured on average 7 μm in length and 16 μm in thickness. The polar capsule, meanwhile, had length to width measurements of 4 μm and 3 μm on average. A periodical survey was conducted throughout a sampling period between December 2012 and December 2013, with the results showing that the parasite was present throughout the year with a mean prevalence of 32.6%. The objective of this study was to characterize this new species based on its morphological and molecular differences from previously described species. Molecular analysis based on the partial sequence of the SSU rDNA gene, showed the highest similarity (97.8%) to Ceratomyxa buri, reported in the cultured yellow tail Seriola quinqueradiata in Japan. Indeed, C. buri and the new species described here formed an individual cluster with a high degree of bootstrap support. This is the first reported species of genus Ceratomyxa from the Arabian Gulf fishes off Saudi Arabia.     

With eyes wide open: a revision of species within and closely related to the Pocillopora damicornis species complex (Scleractinia; Pocilloporidae) using morphology and genetics

Molecular studies have been instrumental for refining species boundaries in the coral genus Pocillopora and revealing hidden species diversity within the extensively studied global species Pocillopora damicornis. Here we formally revise the taxonomic status of species closely related to and within the P. damicornis species complex, taking into account both genetic evidence and new data on morphometrics, including fine‐scale corallite and coenosteum structure. We found that mitochondrial molecular phylogenies are congruent with groups based on gross‐morphology, therefore reflecting species‐level differentiation. However, high levels of gross morphological plasticity and shared morphological characteristics mask clear separation for some groups. Fine‐scale morphological variation, particularly the shape and type of columella, was useful for differentiating between clades and provides an excellent signature of the evolutionary relationships among genetic lineages. As introgressive hybridization and incomplete lineage sorting complicate the delineation of species within the genus on the basis of a single species concept, the Unified Species Concept may represent a suitable approach in revising Pocillopora taxonomy. Eight species are herein described (P. damicornis, P. acuta, P. aliciae, P. verrucosa, P. meandrina, P. eydouxi, P. cf. brevicornis), including a novel taxon – P ocillopora bairdi sp. nov. (Schmidt‐Roach, this study). Citation synonyms and type materials are presented. © 2014 The Linnean Society of London     

DESCRIPTION OF A NEW GENUS OF PFIESTERIA‐LIKE DINOFLAGELLATE,LUCIELLA GEN. NOV. (DINOPHYCEAE), INCLUDING TWO NEW SPECIES: LUCIELLA MASANENSIS SP. NOV. AND LUCIELLA ATLANTIS SP. NOV.1

A new genus of Pfiesteria‐like heterotrophic dinoflagellate, Luciella gen. nov., and two new species, Luciella masanensis sp. nov. and Luciella atlantis sp. nov., are described. These species commonly occur with other small (<20 μm) heterotrophic and mixotrophic dinoflagellates in estuaries from Florida to Maryland and the southern coast of Korea, suggesting a possible global distribution. An SEM analysis indicates that members of the genus Luciella have the enhanced Kofoidian plate formula of Po, cp, X, 4′, 2a, 6″, 6c, PC, 5+s, 5?, 0p, and 2″″. The two four‐sided anterior intercalary plates are diamond shaped. The genus Luciella differs from the other genera in the Pfiesteriaceae by a least one plate in the plate tabulation and in the configuration of the two anterior intercalary plates. An SSU rDNA phylogenetic analysis confirmed the genus as monophyletic and distinct from the other genera in the Pfiesteriaceae. The morphology of Luciella masanensis closely resembles Pfiesteria piscicida Steid. et J. M. Burkh. and other Pfiesteria‐like dinoflagellates in size and shape, making it easily misidentified using LM. Luciella atlantis, in contrast, has a more distinctive morphology. It can be distinguished from L. masanensis and other Pfiesteria‐like organisms by a larger cell size, a more conical‐shaped epitheca and hypotheca, larger rhombic‐shaped intercalary plates, and an asymmetrical hypotheca. The genus Luciella is assigned to the order Peridiniales and the family Pfiesteriaceae based on plate tabulation, plate pattern, general morphology, and phylogenetic analysis.     

The significance of wing pattern diversity in the Lycaenidae: mate discrimination by two recently diverged species

Closely related species of lycaenid butterflies are determinable, in part, by subtle differences in wing pattern. We found that female wing patterns can act as an effective mate‐recognition signal in some populations of two recently diverged species. In field experiments, we observed that males from a Lycaeides idas population and an alpine population of L. melissa preferentially initiate courtship with conspecific females. A morphometric study indicated that at least two wing pattern elements were important for distinguishing the two species: hindwing spots and orange crescent‐shaped pattern elements called aurorae. We deceived male L. idas into initiating courtship with computer generated paper models of heterospecific females when these pattern elements were manipulated, indicating that the wing pattern elements that define the diversity of this group can be effective mate recognition signals.     

A comparative study of the adrenergic innervation of the teleost heart

Summary An adrenergic cardiac innervation has been found in the following teleost species:Platycephalus bassensis (Platycephalidae);Atopomycterus nicthemerus (Diodontidae);Aracana ornata (Ostraciontidae);Torquiginer glaber (Tetraodontidae);Aldrichetta forsteri (Mugilidae);Anguilla australis occidentalis (Anguillidae). In contrast, no evidence for an adrenergic cardiac innervation was found in the pleuronectid,Rhombosolea tapirina.Fluorescence histochemical studies indicated that adrenergic nerves in the sinus venosus and atrium entered via the vagus, whereas those to the ventricle passed mainly along the coronary vasculature. No fluorescent nerves were observed in the heart ofRhombosolea.Transmural stimulation of these adrenergic nerves increased the force of beat in the atrium and ventricle ofPlatycephalus, Atopomycterus andAracana, and in the atrium only ofTorquiginer, Aldrichetta andAnguilla. In addition stimulation of the abdominal vagus nerve in the presence of hyoscine increased the force of beat and heart rate in spontaneously beating sinus-atrium preparations of all species exceptRhombosolea. Applied catecholamines increased the force of beat and heart rate in the spontaneously beating heart of all species includingRhombosolea.     

Systematics and cladistics of a new Naupactini genus (Coleoptera: Curculionidae: Entiminae) from the Andes of Colombia and Ecuador

A new genus of broad‐nosed weevils belonging to the tribe Naupactini (Coleoptera: Curculionidae) is described, based on six species from the Páramo–Puna subregion (Andean region) of South America. Three species are new and three have been transferred from the genus Asymmathetes (Asymmathetes nigrans, Asymmathetes rugicollis, and Asymmathetes vulcanorum). The new genus is recognized by the black, denuded, and shiny tegument, the well‐developed pre‐epistome, the elytral base curved backwards on middle, the reduction of the hindwings, and the widely separated procoxae, closer to the anterior than to the posterior margin of the prosternum. A cladistic analysis of the six species of the new genus plus five out‐groups (Amitrus, Amphideritus, Asymmathetes, Melanocyphus, and Trichocyphus), using 49 morphological characters, resulted in a single cladogram. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166 , 54–71.     

Towards resolving the evolutionary history of Caucasian pears (Pyrus,Rosaceae)—Phylogenetic relationships,divergence times and leaf trait evolution

With approximately 25 endemic species, the genus Pyrus (pears) is highly diverse in the Caucasus ecoregion. The majority of Caucasian pears inhabit xerophytic open woodlands or similar habitats, to which they display morphological adaptations, such as narrow leaves. The other species, both Caucasian and non‐Caucasian taxa, mainly inhabit mesophytic forests and display broad leaves. Using a representative taxon sampling of Pyrus from the Caucasus, Europe and Asia, we reconstruct phylogenetic relationships in the genus based on multiple plastid regions. We also estimate the divergence times of major clades in Pyrus, reconstruct the evolution of leaf shapes, and discuss the emergence of xeromorphic leaf traits. Our results confirm the monophyly of Pyrus and the existence of two major clades: (a) an E Asian clade with a crown group age of 15.7 (24.02–8.37 95% HPD) My, and (b) a W Eurasian clade that comprises species from Europe, SW Asia and the Caucasus and that displays a slightly younger crown group of 12.38 (19.02–6.41 95% HPD) My. The existing infrageneric classification of Pyrus was found partially incongruent with the inferred phylogenetic trees. Several currently accepted species were not recovered as monophyletic, indicating that current species limits require re‐evaluation. Ancestral character state reconstructions revealed several independent transitions from broad‐ to narrow‐shaped leaves in Pyrus, probably via intermediate‐shaped leaves.     

Evolutionary and biogeographic history of the subfamily Neoplecostominae (Siluriformes: Loricariidae)

Freshwater fish evolution has been shaped by changes in the earth's surface involving changes in the courses of rivers and fluctuations in sea level. The main objective of this study is to improve our knowledge of the evolution of loricariids, a numerous and adaptive group of freshwater catfish species, and the role of geological changes in their evolution. We use a number of different phylogenetic methods to test the relationships among 52 representative taxa within the Neoplecostominae using 4676 bps of mitochondrial and nuclear DNA. Our analysis revealed that the subfamily Neoplecostominae is monophyletic, including Pseudotocinclus, with three lineages recognized. The first lineage is composed of part of Pareiorhina rudolphi, P. cf. rudolphi, and Pseudotocinclus; the second is composed of Isbrueckerichthys, Pareiorhaphis, Kronichthys, and the species Neoplecostomus ribeirensis; and the third is composed of Pareiorhina carrancas, P. cf. carrancas, Pareiorhina sp. 1, a new genus, and all the species of the genus Neoplecostomus, except N. ribeirensis. The relaxed molecular clock calibration provides a temporal framework for the evolution of the group, which we use for a likelihood‐based historical biogeographic analysis to test relevant hypotheses on the formation of southeast Brazil. We hypothesize that headwater capture events and marine regressions have shaped the patterns of distribution within the subfamily Neoplecostominae throughout the distinct basins of southeast Brazil.     

MOLECULAR PHYLOGENY OF THE GREEN ALGAL ORDER PRASIOLALES (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

The systematics of the Prasiolales was investigated by phylogenetic inference based on analyses of the rbcL and 18S rRNA genes for representatives of all four genera currently attributed to this order (Prasiococcus, Prasiola, Prasiolopsis, Rosenvingiella), including all type species. The rbcL gene had higher sequence divergence than the 18S rRNA gene and was more useful for phylogenetic inference at the ranks of genus and species. In the rbcL gene phylogeny, three main clades were observed, corresponding to Prasiola, Prasiolopsis, and Rosenvingiella. Prasiococcus was nested among species of Prasiola occurring in subaerial and supralittoral habitats. Trichophilus welckeri Weber Bosse, a subaerial alga occurring in the fur of sloths in Amazonia, was closely related to Prasiolopsis ramosa Vischer. The species of Prasiola were grouped into three well‐supported clades comprising (i) marine species, (ii) freshwater and terrestrial species with linear blades, and (iii) terrestrial species with rounded or fan‐shaped blades. Sequence divergence was unexpectedly low in the marine group, which included species with different morphologies. For the 18S rRNA gene, the phylogenetic analyses produced several clades observed for the rbcL gene sequence analysis, but, due to very little sequence variation, it showed considerably lower resolution for inference at the species and genus levels. Due to the low support of some internal branches, the results of the analyses did not allow an unambiguous clarification of the origin and the early evolution of the Prasiolales.