Historical Zoogeography of the Eusuchian Crocodilians: A Physiological Perspective

The historical zoogeography of eusuchian crocodilians has rarelybeen reviewed in any detail and yet is of increasing interestto students of crocodilian biology as large amounts of comparativeinformation on a wide range of species come to hand. Previousinterpretations of crocodilian zoogeography have been basedon one or another of two assumptions–that the major continentalland masses have remained more or less fixed in position, andthat the eusuchians have had only very limited powers of dispersalacross marine barriers. Both of these assumptions are inappropriatein light of our present knowledge of continental drift and crocodilianphysiology. In this paper we attempt a reinterpretation of eusuchian zoogeographybased on new information on their systematic relationships,physiological capacity for marine dispersal, and fossil history.We postulate that anatomical and physiological adaptations toa marine existence have played an important role in eusuchianhistory. We propose that Gavialis and Tomistoma, now restrictedto fresh waters, may have been derived secondarily from ancestorsadapted to salt water. In the case of Tomistoma, similaritiesin lingual gland and buccal cavity anatomy to the true crocodiles(Crocodylus and Osteolaemus) suggest that marine adaptationspredated the divergence of tomistomine and crocodyline stocks.The buccal morphology of Gavialis suggests it also has a marineancestry. Its systematic affinities are uncertain, lying perhapswith Tomistoma or, on other interpretations, with the Mesosuchia.In both cases, the fossil record is not inconsistent with thispossibility. Palaeontological information now available is inadequate toreconstruct the evolutionary history of the Eusuchia in detail.However, saltwater adapted eusuchians are more common in thefossil record than is widely recognized and the likelihood ofdispersal across marine "barriers" by non-alligatorid crocodilianscannot be ignored.     

The Axial Tail Musculature of Recent Crocodiles and Its Phyletic Implications

The phylogenetic position of the Ganges Gharial (Gavalis gangeticus)among the living Crocodilia is reconsidered in the light ofnew biochemical and morphological data. The anatomical datasuggest a sister group relationship between Gavialis and allother living crocodilians. In contrast the biochemical evidenceindicates a sister group relationship between Gavialis and thefalse gavial, Tomistoma schlegelii, and the rest of the livingCrocodilia. The new morphological data from the dissectionsof the caudal musculature clearly favor the phylogenetic relationshipfor Gavialis as proposed by the comparative anatomists. In thisregard the findings of this study are concordant with thoseconcerning the skull construction of the living crocodilians.     

An Expanded Combined Evidence Approach to the Gavialis Problem Using Geometric Morphometric Data from Crocodylian Braincases and Eustachian Systems

The phylogenetic position of the Indian gharial (Gavialis gangeticus) is disputed - morphological characters place Gavialis as the sister to all other extant crocodylians, whereas molecular and combined analyses find Gavialis and the false gharial (Tomistoma schlegelii) to be sister taxa. Geometric morphometric techniques have only begun to be applied to this issue, but most of these studies have focused on the exterior of the skull. The braincase has provided useful phylogenetic information for basal crurotarsans, but has not been explored for the crown group. The Eustachian system is thought to vary phylogenetically in Crocodylia, but has not been analytically tested. To determine if gross morphology of the crocodylian braincase proves informative to the relationships of Gavialis and Tomistoma, we used two- and three-dimensional geometric morphometric approaches. Internal braincase images were obtained using high-resolution computerized tomography scans. A principal components analysis identified that the first component axis was primarily associated with size and did not show groupings that divide the specimens by phylogenetic affinity. Sliding semi-landmarks and a relative warp analysis indicate that a unique Eustachian morphology separates Gavialis from other extant members of Crocodylia. Ontogenetic expansion of the braincase results in a more dorsoventrally elongate median Eustachian canal. Changes in the shape of the Eustachian system do provide phylogenetic distinctions between major crocodylian clades. Each morphometric dataset, consisting of continuous morphological characters, was added independently to a combined cladistic analysis of discrete morphological and molecular characters. The braincase data alone produced a clade that included crocodylids and Gavialis, whereas the Eustachian data resulted in Gavialis being considered a basally divergent lineage. When each morphometric dataset was used in a combined analysis with discrete morphological and molecular characters, it generated a tree that matched the topology of the molecular phylogeny of Crocodylia.     

Evolutionary relationships and systematics of Atoposauridae (Crocodylomorpha: Neosuchia): implications for the rise of Eusuchia

Atoposaurids are a group of small‐bodied, extinct crocodyliforms, regarded as an important component of Jurassic and Cretaceous Laurasian semi‐aquatic ecosystems. Despite the group being known for over 150 years, the taxonomic composition of Atoposauridae and its position within Crocodyliformes are unresolved. Uncertainty revolves around their placement within Neosuchia, in which they have been found to occupy a range of positions from the most basal neosuchian clade to more crownward eusuchians. This problem stems from a lack of adequate taxonomic treatment of specimens assigned to Atoposauridae, and key taxa such as Theriosuchus have become taxonomic ‘waste baskets’. Here, we incorporate all putative atoposaurid species into a new phylogenetic data matrix comprising 24 taxa scored for 329 characters. Many of our characters are heavily revised or novel to this study, and several ingroup taxa have never previously been included in a phylogenetic analysis. Parsimony and Bayesian approaches both recover Atoposauridae as a basal clade within Neosuchia, more stemward than coelognathosuchians, bernissartiids, and paralligatorids. Atoposauridae is a much more exclusive clade than previously recognized, comprising just three genera (Alligatorellus, Alligatorium, and Atoposaurus) that were restricted to the Late Jurassic of western Europe, and went extinct at the Jurassic/Cretaceous boundary. A putative Gondwanan atoposaurid (Brillanceausuchus) is recovered as a paralligatorid. Our results exclude both Montsecosuchus and Theriosuchus from Atoposauridae. Theriosuchus is polyphyletic, forming two groupings of advanced neosuchians. Theriosuchus (restricted to Theriosuchus pusillus, Theriosuchus guimarotae, and Theriosuchus grandinaris) spanned the Middle Jurassic to early Late Cretaceous, and is known from Eurasia and North Africa. Two Cretaceous species previously assigned to Theriosuchus (‘Theriosuchus’ ibericus and ‘Theriosuchus’ sympiestodon) are shown to be nested within Paralligatoridae, and we assign them to the new genus Sabresuchus. The revised phylogenetic placement of Theriosuchus has several implications for our understanding of eusuchian evolution. Firstly, the presence of fully pterygoidean choanae, previously regarded as a defining characteristic of Eusuchia, is not found in some basal members of Eusuchia. However, eusuchians can be distinguished from Theriosuchus and other basal neosuchians in that their choanae are posteriorly positioned, with an anterior margin medial to the posterior edge of the suborbital fenestra. This feature distinguishes eusuchians from Theriosuchus and more basal neosuchians. Secondly, our refined understanding of Theriosuchus implies that this taxon possessed only amphicoelous presacral vertebrae, and therefore fully developed vertebral procoely is likely to have evolved only once in Crocodylomorpha, on the lineage leading to Eusuchia. These and other findings presented herein will provide an important framework for understanding the neosuchian–eusuchian transition.     

Gavialis from the Pleistocene of Thailand and Its Relevance for Drainage Connections from India to Java

Background

The genus Gavialis comprises a single living but endangered species, G. gangeticus, as well as fossil species recorded in the Miocene to Pleistocene deposits of the Indian subcontinent. The genus is also represented in the Pleistocene deposits of Java by the species G. bengawanicus, which was recently recognized to be valid. Surprisingly, no detailed report of the genus exists between these two provinces and the recent evolutionary history of Gavialis is not understood.

Methodology/Principal Findings

We report new material consisting of skull and mandibular remains of Gavialis from the Early Pleistocene of Khok Sung, Nakhon Ratchasima Province, northeastern Thailand. The Gavialis material described herein is attributed to Gavialis cf. bengawanicus and sheds new light on the occurrence of the genus in mainland SE Asia.

Conclusions/Significance

Comparison of this new material with other species referred to the genus Gavialis led us to preliminary restrict the content of the genus to three species, namely G. gangeticus Gmelin, G. bengawanicus Dubois and G. lewisi Lull. The occurrence of G. cf. bengawanicus in Thailand allows us to propose a scenario for the dispersal of Gavialis from Indo-Pakistan to Indonesia, thus bridging a geographical gap between these two provinces. Dispersal by sea appears a less likely possibility than dispersal through fluvial drainages.     

Crocodilians and Their Helminth Parasites: Macroevolutionary Considerations

Crocodilian relationships supported by the phylogenetic relationshipsof digenean and nematode parasites are compared with currentestimates of crocodilian phylogeny. The parasite data support(1) the placement of Gavialis as the sister-group of the alligatoridsand the crocodylids, (2) the monophyly of alligators and caimans,(3) the placement of Caiman (as a monophyletic group) as thesister-group of Melanosuchus plus Paleosuchus, and (4) ancientorigins of Crocodylus consistent with patterns of continentaldrift. The parasite data do not support the monophyly of Crocodylus,but the "misplaced" species (C. palustris and Osteolaemus) havehad few parasites reported from them. There is evidence of widespreadhost-switching, but most of the ambiguity appears to resultfrom uneven representation of parasite groups in host species.This is probably due both to uneven sampling by parasitologistsand to parasite extinctions associated with crocodilian extinctions.     

Complete Nucleotide Sequence of the Chloroplast Genome from the Tasmanian Blue Gum, Eucalyptus globulus (Myrtaceae)

The complete nucleotide sequence of the chloroplast genome ofthe hardwood species Eucalyptus globulus is presented and comparedwith chloroplast genomes of tree and non-tree angiosperms andtwo softwood tree species. The 160 286 bp genome is similarin gene order to that of Nicotiana, with an inverted repeat(IR) (26 393 bp) separated by a large single copy (LSC) regionof 89 012 bp and a small single copy region of 18 488 bp. Thereare 128 genes (112 individual gene species and 16 genes duplicatedin the inverted repeat) coding for 30 transfer RNAs, 4 ribosomalRNAs and 78 proteins. One pseudogene (-infA) and one pseudo-ycf(-ycf15) were identified. The chloroplast genome of E. globulusis essentially co-linear with that of another hardwood treespecies, Populus trichocarpa, except that the latter lacks rps16and rpl32, and the IR has expanded in Populus to include rps19(part of the LSC in E. globulus). Since the chloroplast genomeof E. globulus is not significantly different from other treeand non-tree angiosperm taxa, a comparison of hardwood and softwoodchloroplasts becomes, in essence, a comparison of angiospermand gymnosperm chloroplasts. When compared with E. globulus,Pinus chloroplasts have a very small IR, two extra tRNAs andfour additional photosynthetic genes, lack any functional ndhgenes and have a significantly different genome arrangement.There does not appear to be any correlation between plant habitand chloroplast genome composition and arrangement.     

Convergence and functional evolution of longirostry in crocodylomorphs

During the Mesozoic, Crocodylomorpha had a much higher taxonomic and morphological diversity than today. Members of one particularly successful clade, Thalattosuchia, are well‐known for being longirostrine: having long, slender snouts. It has generally been assumed that Thalattosuchia owed their success in part to the evolution of longirostry, leading to a feeding ecology similar to that of the living Indian gharial, Gavialis. Here, we compare form and function of the skulls of the thalattosuchian Pelagosaurus and Gavialis using digital reconstructions of the skull musculoskeletal anatomy and finite element models to show that they had different jaw muscle arrangements and biomechanical behaviour. Additionally, the relevance of feeding‐related mandibular traits linked to longirostry in the radiation of crocodylomorph clades was investigated by conducting an evolutionary rates analysis under the variable rates model. We find that, even though Pelagosaurus and Gavialis share similar patterns of stress distribution in their skulls, the former had lower mechanical resistance. This suggests that compared to Gavialis, Pelagosaurus was unable to process large, mechanically less tractable prey, instead operating as a specialized piscivore that fed on softer and smaller prey. Secondly, innovation of feeding strategies was achieved by rate acceleration of functional characters of the mandible, a key mechanism for the diversification of certain clades like thalattosuchians and eusuchians. Different rates of functional evolution suggest divergent diversification dynamics between teleosaurids and metriorhynchids in the Jurassic.     

The origin of modern crocodyliforms: new evidence from the Cretaceous of Australia

While the crocodyliform lineage extends back over 200 million years (Myr) to the Late Triassic, modern forms-members of Eusuchia-do not appear until the Cretaceous. Eusuchia includes the crown group Crocodylia, which comprises Crocodyloidea, Alligatoroidea and Gavialoidea. Fossils of non-crocodylian eusuchians are currently rare and, in most instances, fragmentary. Consequently, the transition from Neosuchia to Crocodylia has been one of the most poorly understood areas of crocodyliform evolution. Here we describe a new crocodyliform from the mid-Cretaceous (98-95 Myr ago; Albian-Cenomanian) Winton Formation of Queensland, Australia, as the most primitive member of Eusuchia. The anatomical changes associated with the emergence of this taxon indicate a pivotal shift in the feeding and locomotor behaviour of crocodyliforms-a shift that may be linked to the subsequent rapid diversification of Eusuchia 20 Myr later during the Late Cretaceous and Early Tertiary. While Laurasia (in particular North America) is the most likely ancestral area for Crocodylia, the biogeographic events associated with the origin of Eusuchia are more complex. Although the fossil evidence is limited, it now seems likely that at least part of the early history of Eusuchia transpired in Gondwana.     

Morphology, fossils, divergence timing, and the phylogenetic relationships of Gavialis

Although morphological data have historically favored a basal position for the Indian gharial (Gavialis gangeticus) within Crocodylia and a Mesozoic divergence between Gavialis and all other crocodylians, several recent molecular data sets have argued for a sister-group relationship between Gavialis and the Indonesian false gharial (Tomistoma schlegelii) and a divergence between them no earlier than the Late Tertiary. Fossils were added to a matrix of 164 discrete morphological characters and subjected to parsimony analysis. When morphology was analyzed alone, Gavialis was the sister taxon of all other extant crocodylians whether or not fossil ingroup taxa were included, and a sister-group relationship between Gavialis and Tomistoma was significantly less parsimonious. In combination with published sequence and restriction site fragment data, Gavialis was the sister taxon of all other living crocodylians, but the position of Tomistoma depended on the inclusion of fossil ingroup taxa; with or without fossils, preferred morphological and molecular topologies were not significantly different. Fossils closer to Gavialis than to Tomistoma can be recognized in the Late Cretaceous, and fossil relatives of Tomistoma are known from the basal Eocene, strongly indicating a divergence long before the Late Tertiary. Comparison of minimum divergence time from the fossil record with different measures of molecular distance indicates evolutionary rate heterogeneity within Crocodylia. Fossils strongly contradict a post-Oligocene divergence between Gavialis and any other living crocodylian, but the phylogenetic placement of Gavialis is best viewed as unresolved.     

Life history evolution and genome size in subtribe Oncidiinae (Orchidaceae)

• Background and Aims Within Oncidiinae, there are severalgroups of species that are effectively annuals, and we wishedto see if these species had smaller genome sizes than averagefor the subtribe. • Methods Fifty-four genome size estimates (50 of whichare new) for species in subtribe Oncidiinae (Orchidaceae) wereexamined for the first time in a phylogenetic context to evaluatehypotheses concerning genome sizes and life history traits. • Results and Conclusions Within the limits of still relativelysparse sampling, the species that are effectively annuals doappear to have smaller genome sizes than average. However, thegenome sizes of their immediate sister group are also small,indicating that changes in genome size preceded the change inlife history traits. Genome sizes and chromosome numbers alsodo not correlate; some slowly growing species have lower chromosomenumbers but large genomes and vice versa. Based on a surveyof the literature on orchids, it is also clear that epiphyticspecies have smaller genome sizes than do terrestrial species,which could be an effect of different water relations or thefact that most terrestrial orchids are geophytic or have distinctgrowth and dormancy phases.     

Mosaic nature in the skeleton of East Asian crocodylians fills the morphological gap between “Tomistominae” and Gavialinae

Crocodylian systematics has long been confounded by conflicting hypotheses of higher level relationships—although molecular data sets strongly supported the sister-taxon relationship of Tomistoma and Gavialis, morphological data sets placed Gavialis as sister to all other living taxa. One of the perceived difficulties in interpreting morphological character evolution on the molecular tree is the extensive character reversal occurring in Gavialinae, the mechanism of which has yet to be explained. Here, we provide evidence of gavialine-specific atavistic characters from East Asian “tomistomines” Penghusuchus pani and Toyotamaphimeia machikanensis. These taxa exhibit a mosaic assembly of “tomistomine” and gavialine features, which fill the gap between the two longirostrine groups. Although the parsimony analysis of morphological data (69 taxa, 254 characters) still supports the previous morphological hypothesis, the alternative tree that was forced to fit the molecular hypothesis was insignificantly (5/954 steps; 0.52%) longer than the unconstrained tree, suggesting that morphological evolution can also be interpreted on the molecular tree. Although the problem of stratigraphic gaps remains, future studies may be directed to resolving the interrelationships within Gavialoidea, a large longirostrine group of crocodylians, in the molecular tree context.     

Seed Protein Electrophoresis in Agropyron junceum (L.) P.B. Complex

Agropyron striatulum (Elymus striatulus Run.) 2n = 14, A. rechingeriRun. 2n = 28, A. junceum (L.) P.B. subsp. boreoatlanticum simonetet Guinochet (A. junceiforme Löve and Löve) 2n = 28,A. junceum (L.) P.B. subsp. mediterraneum Simonet (A. junceum(L.) P.B.) 2n = 42 and A. diae (Elymus diae Run.) 2n = 56 werestudied by isoelectric focusing of seed soluble proteins. The electrophoretic phenotypes obtained from the five materialsshowed a striking degree of similarity. The typical proteinprofile was recognized to consist of 40 bands. No qualitativeprotein phenotypic differences were found and all observed variationconcerned the intensities of some particular bands. The data of the present study combined with cytological informationprovided by other workers indicate that in the composition ofthe polyploid taxa another genome besides that of the diploidA. striatulum is not likely to participate. Consequently thepolyploid taxa of A. junceum (L.) P.B. complex can be characterizedas segmental allopolyploids with the same basic genome moreor less modified at different ploidy levels. Agropyron junceum (L.) P.B. complex, seed storage proteins, protein profile, isoelectric focusing, genomic constitution, segmental allopolyploids     

Structure and Expression of the Tobacco Nuclear Gene Encoding RNA-binding Protein RZ-1: The Existence of an Intron in the 3'-Untranslated Region

We have previously characterized a tobacco cDNA encoding a noveltype RNA-binding protein (RZ-1), which contains a zinc fingermotif in addition to a consensus sequence-type RNA-binding domainand is localized in the nucleus. Here we isolated its genomicclone from a Nicotiana sylvestris genomic library. Southernblot analysis suggested that RZ-1 is coded for by a single locusper haploid genome. Comparison of the cDNA and genomic sequencesindicated that the RZ-1 gene contains two introns, one in thecoding region and another in the 3'-untranslated region. RT-PCRand ribonuclease protection analyses showed that splicing ofRZ-1 pre-mRNA occurs efficiently. The RZ-1 protein is activelysynthesized in rapidly dividing tobacco cells, as demonstratedby immunoblot analysis.     

Nuclear DNA Amounts in Angiosperms

Bennett and Smith (Philosophical Transactions of the Royal Societyof London B274:227-274; B334: 309-345) and Bennett, Smith andHeslop-Harrison (Proceedings of the Royal Society of London,B216: 179-199) published lists of nuclear DNA amounts estimatedfor 1612 angiosperm species collected from 163 sources datedbetween 1951 and 1986. Subsequently, interest in genome sizein angiosperms and its significance has continued, and manynew DNA estimates were published during 1986-1994. Their inaccessibility,and the flow of enquiries for such information, shows that afurther compilation is needed. This paper presents a supplementarylist of nuclear DNA C-values for 105 sources for 899 angiospermspecies not listed in the above-mentioned compilations, plus284 additional estimates for 208 species already listed by them.The data are assembled primarily for reference purposes, withspecies listed in alphabetical order, rather than by any taxonomicscheme. Some advantages and limitations of flow cytometry, nowincreasingly used to quantify DNA C-values in plants, are reviewed.Recent reports regarding the occurrence and extent of intraspecificvariation in genome size are also discussed. While some examplesare real, others reflect technical shortcomings. Work has begunto combine the genome size data compiled in this and the above-mentionedpapers into a unified data base, and to present the informationin separate lists, with species in alphabetical and systematicorders, respectively. DNA C-values are now known for 1% of theworld's angiosperm flora, but improved representation of taxonomicgroups, geographical regions and plant life forms is urgentlyneeded.Copyright 1995, 1999 Academic Press Angiosperm DNA amounts, DNA C-values, nuclear genome sizes, intraspecific variation, flow cytometry, reference lists, genome size database     

C21orf5, a New Member of Dopey Family Involved in Morphogenesis, Could Participate in Neurological Alterations and Mental Retardation in Down Syndrome

Availability of the human genome sequence promises importantprogress in the understanding of human pathologies, particularlyfor multifactorial diseases. Among these, Down syndrome (DS)is the most frequent genetic cause of mental retardation. Acritical region of chromosome 21, the Down syndrome ChromosomalRegion-1 (DCR-1), is responsible for many features of the DSphenotype including mental retardation. We studied DCR-1 C21orf5as a new candidate gene for DS considering its restricted expressionin key brain regions altered in DS patients and involved inlearning and memory processes. To elucidate C21orf5 molecularfunction, we performed a comparative study of protein sequencesin several species and showed that C21orf5 represents a newmember of the Dopey leucine zipper-like family. The C21orf5C-termini contains two highly conserved leucine-like zipperdomains in invertebrate and vertebrate species. Evolution analysisindicated a common ancestral origin of these protein sequencesalso suggesting a conserved function of this gene throughoutphylogenesis. Mutations of the known C21orf5 homologous genesAspergillus nidulans DopA, Saccharomyces cerevisiae Dop1 andCaenorhabditis elegans pad1, determine morphological abnormalities.We studied transgenic mice carrying the human C21orf5 gene andwe showed that this gene is overexpressed in brain regions byin situ hybridization and by real-time RT–PCR experiments.Interestingly, we also showed that these transgenic mice havean increased density of cortical cells overexpressing C21orf5.Similarly, DS patients have an altered lamination pattern intheir cortex. Considering together our and previous findings,we suggest that the human dopey family member, C21orf5, couldplay a role in brain morphogenesis and, when overexpressed,it could participate in neurological features and mental retardationobserved in DS patients.     

黄鼠巢和宿主密度的年间动态关系

根据内蒙古自治区正镶白旗乌宁巴图苏木1981～1993年达乌尔黄鼠Citellus dauricus密度和巢蚤指数监测资料进行分析,得到如下结果。共检巢蚤10种,其中方形黄鼠蚤蒙古亚种Citellophilus tesquorum mongolicus(45.4%)为优势蚤种,光亮额蚤Frontopsylla lucu lenta(25.0%)和阿巴盖新蚤Neopsylla abagaitui(21.3%)为次优势蚤种。二齿新蚤N.Bidentatiformis和角尖眼蚤Ophthalmopsylla praefecta为常见种,余为少见种。鼠密度与巢蚤指数 的关系不显著(p>0.10)、与巢染蚤率关系显著(p<0.05),不同年份的蚤类多样性和均匀性比较稳定。巢蚤和体蚤指数的关系是显著的(PP<0.005)。     

Genome Size inAllium: In Quest of Reproducible Data

ComparingAlliumgenome size measurements of different authors,we noticed that the estimates for certain species diverge morestrongly than one would have expected in view of the methodologicaladvantages of the material. As the matter has theoretical significancefor explaining the biological role of genome size variation,we measured, by Feulgen densitometry, 28 species and altogether57 accessions or cultivars. Flow cytometric measurements supplementedthese data. The current hypothesis of a discontinuous and step-wisedistribution of DNA amounts inAlliumseems questionable, as mostof our DNA values did not appear in the corresponding DNA groupas proposed previously. On the other hand, we can confirm thatthere is a significant negative correlation between genome sizeof a species and its first month of flowering, but only in diploids,or in diploids and polyploids if only the basic genome size(2Cxlevel) is considered. We compared our results with thoseof nine other publications. Only 29 of 60 2C values publishedpreviously deviate less than 10% from our data, the others deviatemore strongly, from 0.44- to 1.44-fold. The more comprehensivedata sets of various authors were compared by correlation analysiswith our data. Positive and mostly significant correlationswere seen in all tests, but nevertheless the degree of incongruencebetween studies was unsatisfactory in view of the much betterintra-laboratory reproducibility of the present data. The presentwork highlights the need for generally agreed improvements instandardization and preparative procedures of cytophotometricgenome size determination.Copyright 1999 Annals of Botany Company Allium, genome size, Feulgen densitometry, flow cytometry, discontinuous DNA content variation, nucleotype hypothesis, flowering time, data reproducibility, correlation analysis.