Infraorder Brachyura Research Articles

Phylogenomic analysis of brachyuran crabs using transcriptome data reveals possible sources of conflicting phylogenetic relationships within the group

Despite extensive morphological and molecular studies, the phylogenetic interrelationships within the infraorder Brachyura and the phylogenetic positions of many taxa remain uncertain. Studies that used a limited number of molecular markers have often failed to provide sufficient resolution, and may be susceptible to stochastic errors and incomplete lineage sorting (ILS). Here we reconstructed the phylogenetic relationships within the Brachyura using transcriptome data of 56 brachyuran species, including 14 newly sequenced taxa. Five supermatrices were constructed in order to exclude different sources of systematic error. The results of the phylogenetic analyses indicate that Heterotremata is non-monophyletic, and that the two Old World primary freshwater crabs (Potamidae and Gecarcinucidae) and the Hymenosomatoidea form a clade that is sister to the Thoracotremata, and outside the Heterotremata. We also found that ILS is the main cause of the gene-tree discordance of these freshwater crabs. Divergence time estimations indicate that the Brachyura has an ancient origin, probably either in the Triassic or Jurassic, and that the majority of extant families and superfamilies first appeared during the Cretaceous, with a constant increase of diversity in Post-Cretaceous-Palaeogene times. The results support the hypothesis that the two Old World freshwater crab families included in this study (Potamidae and Gecarcinucidae) diverged from their marine ancestors around 120 Ma, in the Cretaceous. In addition, this work provides new insights that may aid in the reclassification of some of the more problematic brachyuran groups.

Early life stages of common brachyuran species found in shallow waters of Tenerife Island

Brachyurans plays an important economic and ecological role worldwide, due to their relevance in marine ecosystems, acting as preys, predators, and scavengers. In the Canary Islands, several species are also considered important marine fishing resources. Despite this, little is known about its taxonomical characteristics and their early life ecology. This study utilized artificial larval collectors in the Canary Islands’ shallow rocky areas to observe juvenile settlements of main brachyuran species. In total, juvenile stages of six common species of the archipelago, belonging to five different families of the infraorder Brachyura, were collected and described. In addition, representative measures as well as high detailed photography’s of these crustaceans were taken to provide useful information for the identification at its early life stages. Furthermore, we provide the first evidence of the reproduction success of the invasive species Cronius ruber throughout direct observation of juveniles of this species in the archipelago.

Diversity of brachyuran crab (Crustacea: Decapoda) in West Coast of North Sumatra, Indonesia

The west coast of Sumatra is a water area with a high potential for fishery products. One of the areas, (i.e., Sibolga City) is a national fishery center in Indonesia. The high productivity of these waters is supported by the presence of keystone species in nature, one of which is the crab (infraorder Brachyura). This study aims to record brachyuran species on the west coast of Sumatra that are still unknown. Sampling was conducted in Central Tapanuli Regency and Sibolga City using convenience sampling. The samples obtained were 50 individuals. Based on the identification, the sample consisted of 22 species belong to 6 families. Fourteen species is first recorded in this region, and five species are new records in Indonesia.

Convergent Adaptation of True Crabs (Decapoda: Brachyura) to a Gradient of Terrestrial Environments.

For much of terrestrial biodiversity, the evolutionary pathways of adaptation from marine ancestors are poorly understood and have usually been viewed as a binary trait. True crabs, the decapod crustacean infraorder Brachyura, comprise over 7600 species representing a striking diversity of morphology and ecology, including repeated adaptation to non-marine habitats. Here, we reconstruct the evolutionary history of Brachyura using new and published sequences of 10 genes for 344 tips spanning 88 of 109 brachyuran families. Using 36 newly vetted fossil calibrations, we infer that brachyurans most likely diverged in the Triassic, with family-level splits in the late Cretaceous and early Paleogene. By contrast, the root age is underestimated with automated sampling of 328 fossil occurrences explicitly incorporated into the tree prior, suggesting such models are a poor fit under heterogeneous fossil preservation. We apply recently defined trait-by-environment associations to classify a gradient of transitions from marine to terrestrial lifestyles. We estimate that crabs left the marine environment at least 7 and up to 17 times convergently, and returned to the sea from non-marine environments at least twice. Although the most highly terrestrial- and many freshwater-adapted crabs are concentrated in Thoracotremata, Bayesian threshold models of ancestral state reconstruction fail to identify shifts to higher terrestrial grades due to the degree of underlying change required. Lineages throughout our tree inhabit intertidal and marginal marine environments, corroborating the inference that the early stages of terrestrial adaptation have a lower threshold to evolve. Our framework and extensive new fossil and natural history datasets will enable future comparisons of non-marine adaptation at the morphological and molecular level. Crabs provide an important window into the early processes of adaptation to novel environments, and different degrees of evolutionary constraint that might help predict these pathways. [Brachyura; convergent evolution; crustaceans; divergence times; fossil calibration; molecular phylogeny; terrestrialization; threshold model.].

Phylomitogenomics elucidates the evolution of symbiosis in Thoracotremata (Decapoda: Cryptochiridae, Pinnotheridae, Varunidae).

Thoracotremata belong to the large group of "true" crabs (infraorder Brachyura), and they exhibit a wide range of physiological and morphological adaptations to living in terrestrial, freshwater and marine habitats. Moreover, the clade comprises various symbiotic taxa (Aphanodactylidae, Cryptochiridae, Pinnotheridae, some Varunidae) that are specialised in living with invertebrate hosts, but the evolutionary history of these symbiotic crabs is still partially unresolved. Here we assembled and characterised the complete mitochondrial genomes (hereafter mitogenomes) of three gall crab species (Cryptochiridae): Kroppcarcinus siderastreicola, Opecarcinus hypostegus and Troglocarcinus corallicola. A phylogenetic tree of the Thoracotremata was reconstructed using 13 protein-coding genes and two ribosomal RNA genes retrieved from three new gall crab mitogenomes and a further 72 available thoracotreme mitogenomes. Furthermore, we applied a comparative analysis to characterise mitochondrial gene order arrangement, and performed a selection analysis to test for selective pressure of the protein-coding genes in symbiotic Cryptochiridae, Pinnotheridae, and Varunidae (Asthenognathus inaequipes and Tritodynamia horvathi). The results of the phylogenetic reconstruction confirm the monophyly of Cryptochiridae, which clustered separately from the Pinnotheridae. The latter clustered at the base of the tree with robust branch values. The symbiotic varunids A. inaequipes and T. horvathi clustered together in a clade with free-living Varunidae species, highlighting that symbiosis in the Thoracotremata evolved independently on multiple occasions. Different gene orders were detected in symbionts and free-living species when compared with the ancestral brachyuran gene order. Lastly, the selective pressure analysis detected two positively selected sites in the nad6 gene of Cryptochiridae, but the evidence for positive selection in Pinnotheridae and A. inaequipes and T. horvathi was weak. Adaptive evolution of mitochondrial protein-coding genes is perhaps related to the presumably higher energetic demands of a symbiotic lifestyle.

What Lies Inside a Dead Coral: Diversity of Reef-Associated Cryptic Crustacean (Crabs: Anomura and Brachyura) from Different Habitat Characteristics in Tidung Islands, Indonesia

Highlight Research The very first attempt to investigated and revealing biodiversity of crustacean crab in coral reef ecosystem at Tidung Island, Indonesia Conservation area (Tidung Kecil) has more abundant crab species than tourism area (Tidung Besar). A microhabitat that suffers anthropogenic pressure may alter the composition of crustacean biodiversity, especially in crab group. Different characteristics and complexity of coral habitat affect the dynamics of cryptic crustacean communities Abstract Recent studies have been widely carried out on the important role and ecological function of coral reefs. However, little attention has been given to small and cryptic crustaceans organisms living within different microhabitat in dead corals. This study aimed to reveal the diversity of crustaceans that inhabit dead corals in Tidung Islands, Indonesia. A total of 134 individuals from 41 species in 13 families were recorded within eight dead corals taken from two different sites. Furthermore, the species identified from Tidung Kecil were significantly different and more diverse than Tidung Besar. It was suggested that differences in anthropogenic pressure between Tidung Besar and Kecil might have an important role in the spread of crab crustaceans’ organisms. Xanthidae from infraorder Brachyura were observed to be the most dominant family, while Galatheidae and Porcellanidae from infraorder Anomura spread almost equally in both sites. Additionally, Psaumis cavipes and Chlorodiella nigra were species with the largest contribution of crab crustaceans’ communities in the death of coral’s ecosystem in the Tidung Islands. This result indicated that dead corals might play a key role in supporting and providing a habitat for crustacean cryptic.

Sentience in decapod crustaceans: A general framework and review of the evidence

We outline a framework for evaluating scientific evidence of sentience, focusing on pain experience. It includes eight neural and cognitive-behavioural criteria, with confidence levels for each criterion reflecting the reliability and quality of the evidence. We outline the rationale for each criterion and apply our framework to a controversial sentience candidate: decapod crustaceans. We have either high or very high confidence that true crabs (infraorder Brachyura) satisfy five criteria, amounting to strong evidence of sentience. Moreover, we have high confidence that both anomuran crabs (infraorder Anomura) and astacid lobsters/crayfish (infraorder Astacidea) meet three criteria—substantial evidence of sentience. The case is, as yet, weaker for other infraorders, such as penaeid shrimps, highlighting important research gaps. Having demonstrated our framework’s application to decapod crustaceans, we hope that future research will apply it to other taxa.

Systematic assessment of the Panopeidae and broader Eubrachyura (Decapoda: Brachyura) using mitochondrial genomics

Abstract This study provides a broad phylogenetic analysis for the Eubrachyura, with the inclusion of three new Panopeidae mitochondrial genomes: Eurypanopeus depressus (flatback mud crab) (15,854bp), Panopeus herbstii (Atlantic mud crab) (15,812bp) and Rhithropanopeus harrisii (Harris, or ‘white-fingered’ mud crab) (15,892bp). These new mitogenomes were analyzed alongside all available brachyuran mitochondrial genomes (n = 113), comprising 80 genera from 29 families, to provide an updated phylogenetic analysis of the infra-order Brachyura (“true crabs”). Our analyses support the subsection Potamoida within the Eubrachyura as the sister group to Thoracotremata. The family Panopeidae aligns with the family Xanthidae to form the Xanthoidea branch, which is supported by current morphological and genetic taxonomy. A unique gene arrangement termed ‘XanGO’ was identified for the panopeids and varies relative to other members of the subsection Heterotremata (within the Eubrachyura) via a transposition of the trnV gene. This gene arrangement is novel and is shared between several Xanthoidea species, including Etisus anaglyptus (hairy spooner crab), Atergatis floridus (brown egg crab), and Atergatis integerrimus (red egg crab), suggesting that it is a conserved gene arrangement within the Xanthoidea superfamily. Our study further reveals a need for taxonomic revision of some brachyuran groups, particularly the Sesarmidae. The inclusion of panopeid mitogenomes into the greater brachyuran phylogeny increases our understanding of crab evolution and higher level Eubrachyuran systematics.

A network analysis of crab metamorphosis and the hypothesis of development as a process of unfolding of an intensive complexity

Development has intrigued humanity since ancient times. Today, the main paradigm in developmental biology and evolutionary developmental biology (evo-devo) is the genetic program, in which development is explained by the interplay and interaction of genes, that is, by the action of gene regulatory networks (GRNs). However, it is not even clear that a GRN, no matter how complex, can be translated into a form. Therefore, the fundamental enigma of development still remains: how is a complex organism formed from a single cell? This question unfolded the historical drama and the dialectical tension between preformation and epigenesis. In order to shed light on these issues, I studied the development of crabs (infraorder Brachyura), as representative of the subphylum Crustacea, using network theory. The external morphology of the different phases of brachyuran metamorphosis were modeled as networks and their main characteristics analyzed. As one could expect, the parameters usually regarded as indicative of network complexity, such as modularity and hierarchy, increased during development. However, when more sophisticated complexity measures were tested, it was evidenced that whereas a group of complexity measures increased during development, another group decreased. This led to consider that two kinds of complexities were being measured. I called them intensive and extensive complexity. In view of these results, I propose that crab development involves a passage from an intensive to an extensive complexity. In other words, crab development can be interpreted as a process of unfolding of an intensive, preexistent complexity.

The Chinese mitten crab genome provides insights into adaptive plasticity and developmental regulation

The infraorder Brachyura (true or short-tailed crabs) represents a successful group of marine invertebrates yet with limited genomic resources. Here we report a chromosome-anchored reference genome and transcriptomes of the Chinese mitten crab Eriocheir sinensis, a catadromous crab and invasive species with wide environmental tolerance, strong osmoregulatory capacity and high fertility. We show the expansion of specific gene families in the crab, including F-ATPase, which enhances our knowledge on the adaptive plasticity of this successful invasive species. Our analysis of spatio-temporal transcriptomes and the genome of E. sinensis and other decapods shows that brachyurization development is associated with down-regulation of Hox genes at the megalopa stage when tail shortening occurs. A better understanding of the molecular mechanism regulating sexual development is achieved by integrated analysis of multiple omics. These genomic resources significantly expand the gene repertoire of Brachyura, and provide insights into the biology of this group, and Crustacea in general.

Cell proliferation in the central nervous system of an adult semiterrestrial crab.

Neurogenesis occurs in adults of most organisms, both vertebrates and invertebrates. In semiterrestrial crabs of the infraorder Brachyura, the deutocerebrum, where neurogenesis occurs, processes the olfactory sensory information from the antennae. The deutocerebrum is composed of a pair of olfactory lobes associated with cell clusters 9 and 10 (Cl 9 and Cl 10), containing proliferating cells. Because the location of the neurogenic niche in brachyuran semiterrestrial crabs has not been defined, here we describe a neurogenic niche in the central olfactory system of the crab Ucides cordatus and report two types of glial cells in the deutocerebrum, based on different markers. Serotonin (5-hydroxytryptamine) labeling was used to reveal neuroanatomical aspects of the central olfactory system and the neurogenic niche. The results showed a zone of proliferating neural cells within Cl 10, which also contains III beta-tubulin (Tuj1)+ immature neurons, associated with a structure that has characteristics of the neurogenic niche. For the first time, using two glial markers, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), we identified two types of astrocyte-like cells in different regions of the deutocerebrum. This study adds to the understanding of neurogenesis in a brachyuran semiterrestrial crustacean and encourages comparative studies between crustaceans and vertebrates, including mammals, based on shared aspects of both mechanisms of neurogenesis and regenerative potentials.

Molecular Cloning, Structure and Phylogenetic Analysis of a Hemocyanin Subunit from the Black Sea Crustacean Eriphia verrucosa (Crustacea, Malacostraca)

Hemocyanins are copper-binding proteins that play a crucial role in the physiological processes in crustaceans. In this study, the cDNA encoding hemocyanin subunit 5 from the Black sea crab Eriphia verrucosa (EvHc5) was cloned using EST analysis, RT-PCR and rapid amplification of the cDNA ends (RACE) approach. The full-length cDNA of EvHc5 was 2254 bp, consisting of a 5′ and 3′ untranslated regions and an open reading frame of 2022 bp, encoding a protein consisting of 674 amino acid residues. The protein has an N-terminal signal peptide of 14 amino acids as is expected for proteins synthesized in hepatopancreas tubule cells and secreted into the hemolymph. The 3D model showed the presence of three functional domains and six conserved histidine residues that participate in the formation of the copper active site in Domain 2. The EvHc5 is O-glycosylated and the glycan is exposed on the surface of the subunit similar to Panulirus interruptus. The phylogenetic analysis has shown its close grouping with γ-type of hemocyanins of other crustacean species belonging to order Decapoda, infraorder Brachyura.

Checklist of decapod crustaceans from the coast of the São Paulo state (Brazil) supported by integrative molecular and morphological data: III. Infraorder Brachyura Latreille, 1802.

This checklist is the third part of a series derived from a long-term multidisciplinary project on the biodiversity of decapod crustaceans from marine and coastal environments of São Paulo state (Brazil). We integrated molecular techniques (DNA markers) and morphological analyses of adult specimens for accurate identifications. We compilated 185 species from the literature, but we confirmed the presence of 168 species: 130 of which we sampled, analyzed and obtained sequences (COI and/or 16S totalizing 113 sequences) and 38 that were not directly collected but were confirmed by analyses. In addition, 27 had their distribution removed from São Paulo due to uncertainties, and absence of material as voucher. Five species were reported for the first time on the coast of São Paulo (Calappa ocellata, Neohelice granulata, Teleophrys pococki, Teramnonotus monodi, Tetraxanthus rathbunae) and one on the Brazilian coast (Pseudomedaeus agassizi). Most of the non-sampled species previously reported on the coast of São Paulo might be considered doubtful records stablished in the past by inaccurate analyses, which assumed extended distribution to the area and/or misidentifications. At this time and based on our refined compilation, we can estimate the brachyuran diversity on the coast of São Paulo in 168 species. This detailed inventory contributes to the knowledge on the local decapod fauna by checking available dataset, adding new species records in São Paulo and new sequences to GenBank database. These data may serve as baseline for future identifications and studies on conservation, population genetics, biogeography and phylogenetics, which might flag species that deserve further investigations and concerns.

Characterizing coral reef biodiversity: genetic species delimitation in brachyuran crabs of Palmyra Atoll, Central Pacific

Coral reefs are highly threatened ecosystems, yet there are numerous challenges in conducting inventories of their vanishing biodiversity, partly because many taxa remain difficult to detect and describe. Genetic species delimitation methods provide a standardized means for taxonomic classification including of cryptic, rare, or elusive groups, but results can vary by analytical method and genetic marker. In this study, a combination of morphological and genetic identification methods was used to estimate species richness and identify taxonomic units in true crabs (Infraorder Brachyura; n = 200) from coral reefs of Palmyra Atoll, Central Pacific. Genetic identification was based on matches between mitochondrial 16S ribosomal RNA (16S rRNA) and/or cytochrome c oxidase subunit I (COI) sequences to GenBank data, while morphological work relied on the taxonomic literature. Broad agreement in the number of candidate species delimited by genetic distance thresholds and tree-based approaches was found, although the multi-rate Poisson tree process (mPTP) was less appropriate for this dataset. The COI sequence data identified 30–32 provisional species and the 16S data revealed 34–35. The occurrence of 10 families, 20 genera, and 19 species of brachyurans at Palmyra was corroborated by at least two methods. Diversity levels within Chlorodiella laevissima indicated possible undescribed or cryptic species in currently lumped taxa. These results illustrate the efficacy of DNA sequences in identifying organisms and detecting cryptic variation, and underscore the importance of using appropriate genetic markers and multiple species delimitation analyses, with applications for future species descriptions.

A New Record of Symbiotic Crab (Harrovia elegans de Man, 1887) from the Gulf of Mannar, India

The present study deals with the first record of symbiotic crab Harrovia elegans de Man 1887 (Crustacea: Decapoda: Pilumnidae) collected from Tuticorin and Mandapam regions of Gulf of Mannar during February 2019. This crab species affiliates to family Pilumnidae, under the Decapoda infra-order Brachyura and is for the first time reported from the Indian mainland. The present specimens (One adult male, one adult female and a sub-adult) were collected from a depth of 5 m and showing symbiotic association with the crinoids, Comanthus wahlbergii. Although widely distributed in the Indo-Pacific region, this is the first report of this species from the Indian mainland.

The Unfolding of Complexity: Network Hierarchy, Modularity and Topology in Crab Evolutionary Development

Development has intrigued humanity since ancient times. Today, evolutionary developmental biology (Evo-Devo) is primarily a branch of molecular biology in which development is explained by the interplay and interaction of gene regulatory networks (GRNs). However, it is not even clear that a GRN, no matter how complex, can be translated into a form. Therefore, the fundamental enigma of evolutionary development still remains: how a complex organism is formed from a single cell? This question unfolded the historical drama and the dialectical tension between preformation and epigenesis. In order to shed light on these issues, I studied the evolutionary development of crabs (infraorder Brachyura), as representative of the subphylum Crustacea, using network theory. The morphology of the different phases of brachyuran development were modeled as networks and their main characteristics analyzed. As one could expect, the parameters usually regarded as indicative of network complexity, such as modularity and hierarchy, increased during development. However, when more sophisticated complexity measures were tested, it was evidenced that whereas a group of complexity measures increased during development, another group decreased. This led to consider that two kinds of complexities were being measured. I called them intensive and extensive complexity. In view of these results, I propose that evolutionary development involves a passage from an intensive to an extensive complexity. In other words, evolutionary development can be interpreted as an unfolding of an intensive, preexistent complexity.

Analysis of complete mitochondrial genome of Etisus anaglyptus (Arthropoda, Decapoda, Xanthidae) with phylogenetic consideration

The complete mitochondrial genome is sequenced and analyzed from a xanthid crab Etisus anaglyptus, which is the first complete mitochondrial genome for the genus. The mitochondrial genome length of E. anaglyptus is 16,435 bp and it is composed of 13 protein coding genes, two ribosomal RNA genes, and 22 tRNA genes. The structure and gene orientation of the mitochondrial genome is identical with the other brachyuran records. Furthermore, phylogenetic relationships of the infraorder Brachyura evaluated by mitochondrial protein coding genes. The phylogenetic study showed that E. anaglyptus is positioned in the superfamily Xanthoidea and the closest species to E. anaglyptus is Leptodius sanguineus.

Complete mitochondrial genome of xanthid crab Atergatis floridus (Linnaeus, 1767)

The complete mitochondrial genome sequenced from the floral egg crab Atergatis floridus (Linnaeus, 1767) and the determination of the position of the species in the reconstructed phylogenetic tree of the infraorder Brachyura using the protein coding mitochondrial genes are presented. Results show the mitochondrial genome length of A. floridus is 16,435 bp with nucleotide distribution as 33.4% A, 20.3% C, 10.5% G and 35.8% T. The structure of the complete mitochondrial genome of the species is the same as with the previous xanthid record. The result of the phylogenetic analysis suggests that A. floridus is the closest species to other Xanthidae species in the brachyuran records. This is the first complete mitochondrial genome record from the genus Atergatis.

The carcinofauna found in stomach contents of the flying gurnard (Dactylopterus volitans) on the continental shelf of the Campos Basin, Brazil

ABSTRACTA total of 3109 crustaceans belonging to 50 taxa distributed in 42 families were found in 117 analysed stomachs of flying gurnard (Dactylopterus volitans). Samples were obtained in April 2008 by the R/V Gyre using a bottom trawl towed in 12 stations at 14–100 m depth on the continental shelf of the Campos Basin, Brazil. The carcinofauna was analysed and the order Calanoida (Copepoda) found to be the most important item in terms of relative abundance and frequency of occurrence, followed by the order Amphipoda (Peracarida), the infraorder Brachyura (Decapoda), the order Stomatopoda and the subclass Myodocopa (Ostracoda). In the order Calanoida, the species Pontellopsis cf. villosa (Pontellidae) represented 98.04% of total crustacean abundance. The diet of Dactylopterus volitans varied according to fish size, with higher diversity of Crustacea at smaller size classes, decreasing in larger fishes. A similar pattern regarding depth was obtained, with greater diversity of taxa in gurnard stomachs caught at shallower depths. Flying gurnard is considered a generalized carnivore of invertebrates, eating mobile macrobenthic organisms, such as crustaceans, and its diet varies with its life stage, without any specific group as its main food source.

Anomuran and Brachyuran Symbiotic Crabs in Coastal Areas between the Southern Ryukyu arc and the Coral Triangle.

Parinya Limviriyakul, Li-Chun Tseng, Jiang-Shiou Hwang, and Tung-Wei Shih (2016) Symbiotic anomuran and brachyuran crabs were identified after extensive surveys of reef zones, especially from sponges, scleractinian corals, crinoids, and sea urchins, in the coastal areas of the southern East China Sea. Twenty-nine species belonging to 17 genera, 9 families, and 2 infraorders were identified (3 were identified to the generic level). More crabs belonged to the infraorder Brachyura (82.8%) than to Anomura (17.2%). Two anomuran symbionts (Allogalathea elegans and Petrolisthes virgatus) and 5 brachyuran symbionts (Tetralia glaberrima, Tetralia rubridactyla, Trapezia cymodoce, Trapezia septata, and Cymo melanodactylus) are common in this area. Two species of Anomura (Lauriea simulata, Petrolisthes virgatus) and 3 of Brachyura (Gonatonotus nasutus, Tetralia aurantistellata and Tetralia nigrolineata) were identified for the first time from waters adjacent to Taiwan. These records represent the northernmost recorded of L. simulata and T. aurantistellata. The occurrence of P. virgatus is the second in the western Pacific Ocean. This study revealed the geospatial distribution of symbiotic crabs, which connects the region from the southern Ryukyu arc to the Coral Triangle, and provides the supporting taxonomic account of symbiotic anomuran and brachyuran crab fauna inhabiting the reef zone in northern Taiwan.