Adaptive sequence evolution in a color gene involved in the formation of the characteristic egg-dummies of male haplochromine cichlid fishes
© Salzburger et al; licensee BioMed Central Ltd. 2007
Received: 20 September 2007
Accepted: 15 November 2007
Published: 15 November 2007
The exceptionally diverse species flocks of cichlid fishes in East Africa are prime examples of parallel adaptive radiations. About 80% of East Africa's more than 1 800 endemic cichlid species, and all species of the flocks of Lakes Victoria and Malawi, belong to a particularly rapidly evolving lineage, the haplochromines. One characteristic feature of the haplochromines is their possession of egg-dummies on the males' anal fins. These egg-spots mimic real eggs and play an important role in the mating system of these maternal mouthbrooding fish.
Here, we show that the egg-spots of haplochromines are made up of yellow pigment cells, xanthophores, and that a gene coding for a type III receptor tyrosine kinase, colony-stimulating factor 1 receptor a (csf1ra), is expressed in egg-spot tissue. Molecular evolutionary analyses reveal that the extracellular ligand-binding and receptor-interacting domain of csf1ra underwent adaptive sequence evolution in the ancestral lineage of the haplochromines, coinciding with the emergence of egg-dummies. We also find that csf1ra is expressed in the egg-dummies of a distantly related cichlid species, the ectodine cichlid Ophthalmotilapia ventralis, in which markings with similar functions evolved on the pelvic fin in convergence to those of the haplochromines.
We conclude that modifications of existing signal transduction mechanisms might have evolved in the haplochromine lineage in association with the origination of anal fin egg-dummies. That positive selection has acted during the evolution of a color gene that seems to be involved in the morphogenesis of a sexually selected trait, the egg-dummies, highlights the importance of further investigations of the comparative genomic basis of the phenotypic diversification of cichlid fishes.
Haplochromine egg-dummies begin to form in the juvenile stage of males and start out from the edge of the anal fin , though they only begin to brighten when the young males reach sexual maturity. Haplochromine species differ greatly in egg-spot number, arrangement and morphology. Anal fin egg-dummies are, however, not an exclusive male characteristic. In some species, females also show ovoid markings on their anal fins, but these are typically much less conspicuous than the egg-spots of males. A typical haplochromine egg-dummy is characterized by a conspicuous yellow to reddish central area and a more or less transparent outer ring [11, 12, 14]. This type of egg-dummy is found in most riverine and rock-dwelling haplochromines, while other species, such as more ancestral riverine taxa or pelagic and sand-dwelling species in Lake Malawi, sometimes show a more amorphic blotch pattern. Additionally, a small number of extant haplochromines lack egg-spots entirely, and it has been suggested that they have lost their dummies secondarily .
Yellow blotches that also act as egg-dummies are also known from a few species of the Lake Tanganyika tribe Ectodini, which are only distantly related to the haplochromines (see e.g., ). However, the ectodines' egg-dummies are morphologically less complex than those of the haplochromines and they are found on the paired pelvic fins instead of the unpaired anal fins as in haplochromines. Finally, some mouthbrooding Tilapia species have evolved filamentous arborescent appendages at their genital papillae, so-called genital tassels, which act as egg-dummies [11, 14].
Here, we report on the identification of a gene that is likely to play a role in the development of the yellow xanthophores in the haplochromines' egg-spots. We hypothesized that a previously isolated xanthophore-related color gene might be involved in the formation of xanthophores in the egg-spots: The colony-stimulating factor 1 receptor a (csf1ra) gene, coding for a type III receptor tyrosine kinase [23, 24], is known to be expressed by cells of the xanthophore lineage in zebrafish; it is essential for recruiting xanthophores from their precursors, and it is indirectly involved in the organization of the dark melanophores [25, 26]. Zebrafish mutants for csf1ra (panther) exhibit disrupted stripe patterns and lack xanthophores , and it has been shown that some species in the genus Danio vary in their csf1ra pathway during pigment pattern formation . csf1ra is, thus, not only an important marker for the xanthophore lineage in zebrafish, but is also involved in xanthophore development and, possibly, color patterning. In addition to its role in body coloration, csf1ra is expressed in the macrophage and osteoclast cell lineage in zebrafish . We performed RT-PCR and in situ hybridization experiments in several haplochromine cichlid species in order to confirm the expression of csf1ra in the males' egg-spots. For detailed evolutionary analyses, we determined the DNA sequence of this gene locus in 19 East African cichlid species and tested for the signature of adaptive evolution in the haplochromine lineage. Finally, we also tested for csf1ra expression in the ectodine species Ophthalmotilapia ventralis, which has egg-dummies at the end of the paired pelvic fins.
Gene expression assays
Phylogenetic and molecular evolutionary analyses of the csf1ralocus
The ancestral state reconstructions using a consensus tree on the basis of this newly-generated phylogenetic hypothesis (Figure 4d) and a previously published mitochondrial phylogeny  revealed that both the particular mating system and the egg-dummies on male anal fins are likely to have evolved only once in the ancestral lineage of the haplochromines (see Figure 1a). Thus, the lack of egg-spots in a few haplochromine species is most likely due to secondary loss.
We then tested for the existence of a signal of adaptive sequence evolution in the coding region of csf1ra. This gene consists of 21 exons with a combined length of 2 928 bp, and is made up of a cysteine-rich extracellular ligand-binding domain composed of five immunoglobulin-like domains, a transmembrane domain, and an intracellular domain with two separate tyrosine kinase domains [23, 29] (Figure 4a, c). A sliding window analysis with DNASP  uncovered several sections in csf1ra of haplochromines with a dN/dS ratio greater than one (Figure 4b), which would indicate that positive selection has acted to shape the protein. These regions were primarily located in the first 1 551 bp of the gene (amino acid positions 1 to 517) corresponding to the extracellular ligand-binding domain of Csf1ra. In this domain, there are in total more non-synonymous substitutions than synonymous ones in the haplochromines (see amino acid alignment in Additional file 2), but not in the more basal cichlid lineages that are without anal fin egg-dummies. The maximum likelihood reconstructions of dN/dS ratios in this domain of csf1ra revealed that the only internal branch with a dN/dS >> 1 is the one representing the common ancestor of the haplochromines (dN/dS > 5; Figure 4e). Such a signal of adaptive sequence evolution specific to the haplochromine lineage could not be detected in a segment of the extracellular domain of kita, another type III receptor tyrosine kinase with a known function in pigment patterning , which we have sequenced in the 19 cichlid species as a control. Also, in kita there was no indication that the segment of the extracellular domain would have evolved under a different selection regime as compared to a segment in the intracellular domain (Additional file 3).
We also analyzed a genomic region of approximately 1 000 bp upstream of csf1ra in the 19 cichlid species, to investigate sequence differences in putative gene regulatory regions. The only mutation common to all haplochromines, distinguishing them from the more ancestral and less species-rich cichlid lineages, involves a mutation in a putative transcription factor-binding site, a TATA box, about 130 bp upstream of csf1ra.
csf1rais expressed in egg-dummies of haplochromine cichlids
Egg-dummies on the male anal fins play an important role during the breeding behavior of the female mouthbrooding haplochromines (see e.g., [7, 13, 14]). Phylogenetic and character state reconstructions corroborate that these egg-dummies evolved only once in the ancestral lineage of haplochromines  (Figure 1a), and that the absence of ovoid markings on the anal fins of some haplochromine species is due to secondary loss, e.g. because of reasons of camouflage or as an adaptation to the deep-water habitat and visual environment where these markings would not be easily visible.
Haplochromine egg-spots vary in size, shape, number and arrangement in different species. Figure 3 provides some examples of the diversity of egg-spots found in haplochromines. A typical egg-dummy consists of a conspicuous yellow to reddish central area and a more or less transparent outer ring [11, 12, 14] (see Figure 3b, f), although a number of species only show amorphic blotches (see e.g., Figure 3j). The brightly colored inner circle is, as we have shown (Figure 2), made up of xanthophores. Yellowish, orange, or reddish xanthophores also occur in cichlid fins other than the anal fin (see e.g., Figure 3q–u) and in skin tissue, where substantial differences in densities – depending on coloration patterns – can be found (Clabaut, Salzburger and Meyer, unpublished results). However, nowhere (not even in yellow colored fish) could we identify a higher density of xanthophores than in the egg-dummies of haplochromine males.
We applied a candidate gene approach in order to test whether the previously isolated xanthophore-related color gene csf1ra [25, 26] is expressed in haplochromine egg-spots. Our in situ hybridization experiments indeed corroborate csf1ra expression in the egg-dummies of all tested haplochromine species (Figure 3). We detected csf1ra expression in the younger and still growing egg-spots of A. burtoni (Figure 3a–d), in the single egg-dummy of P. sp. 'bicolor' (Figure 3e–h), and in the relatively unstructured male anal fin blotches of T. brauschi (Figure 3i–k), which is a member of an ancestral riverine haplochromine clade. The simple orange blotches of T. brauschi (Figure 3j) again illustrate (see above) that not all types of egg-dummies show a clear-cut separation into a brightly colored inner circle and a more or less transparent outer ring. In this specific case, it could, however, be argued that because of the phylogenetic position of this species, the undifferentiated spots of T. brauschi represent an intermediate character state in the evolution of egg-dummies. That csf1ra is expressed in egg-spots of younger males and the smaller and still developing egg-spots of adult males might indicate that csf1ra is required for xanthophore recruitment from pigment cell precursors during egg-spot formation, just as has been reported for the formation of stripes in zebrafish [25, 26]. This would need to be tested in future experiments.
In view of the fact that csf1ra is expressed in both patterns, the comparative in situ hybridization experiments also seem to support the earlier suggestion that egg-spots of haplochromines are likely to be derived from the 'Perlfleckmuster' found in unpaired fins of haplochromines and many other cichlid species [11, 12]. Specifically, we show that csf1ra is expressed in pearly spots on dorsal fins of A. burtoni and P. multicolor (Figure 3q–u). As such pearly spots are also found on anal fins of some (ancestral) haplochromine lineages (see Figure 3l for csf1ra expression in the pearly spots on the posterior part of the anal fin of T. brauschi), a co-option of at least some aspects of the molecular basis of the pearly blotch pattern for the formation of egg-dummies appears likely.
Adaptive sequence evolution in the extracellular domain of csf1rain haplochromines
In order to investigate the molecular evolutionary history of csf1ra we sequenced large fractions of the locus in 19 representative cichlid species (Figure 4a). The comparison of haplochromine with non-haplochromine species revealed that several regions in csf1ra show a dN/dS ratio greater than one (Figure 4b), indicating that positive selection (adaptive sequence evolution) has acted to change the protein in haplochromines. Positive Darwinian selection often only acts on particular domains of a gene, whereas other sections remain subject to purifying selection. Also, adaptive evolution is expected to act only at particular times during the evolution of a lineage. In our case, we found that the regions showing a dN/dS > 1 are located in the part of the gene that encodes the extracellular domain. The Csf1ra protein functions as membrane spanning cell surface receptor [29, 32], and is characterized by a cysteine-rich extracellular ligand-binding domain composed of five immunoglobulin-like domains containing growth factor binding sites, a transmembrane domain, and two separate tyrosine kinase domains [23, 29] (Figure 4c). A maximum likelihood reconstruction of dN/dS ratios revealed that the extracellular domain of csf1ra underwent adaptive evolution in the common ancestor of the haplochromines (Figure 4e) – simultaneous to when the egg-dummies are likely to have evolved. The occurrence of adaptive changes in the amino acid sequence in the ligand-binding portion of csf1ra (Figure 4e, Additional file 2) seems to suggest that novel modifications of existing signal transduction mechanisms evolved in the haplochromine lineage that were associated with the evolution of egg-spots, or, possibly, other color patterns involving xanthophores. Functional assays (see e.g., ) are now required to test whether the observed differences in the coding sequence of csf1ra have any effect on ligand-receptor interactions. Similarly, a more thorough comparative analysis of the upstream region of csf1ra is necessary to test the possibility that regulatory elements also underwent evolutionary changes in the ancestor of haplochromines, as is suggested by the observed haplochromine-specific mutation in a putative transcription factor binding site and the differential expression of csf1ra between haplochromine and non-haplochromine cichlids. In addition, csf1ra expression should also be investigated in other tissues and cell lineages. Although it is not apparent how e.g., macrophages or osteoclasts (in which csf1ra is also expressed ) could contribute to the evolutionary success of haplochromines but not of other cichlids, the remote possibility remains that the signal of adaptive sequence evolution is due to functions other than coloration.
To date, evidence for accelerated protein evolution in haplochromines has been found in the bone morphogenetic protein 4 (bmp4) that is hypothesized to be involved in jaw formation (; see also [5, 35]), in a color perception gene, the long wavelength-sensitive (LWS) opsin , and in a putative color gene, the F-box-WD-repeat hagoromo . The finding of signatures of adaptive sequence evolution in a jaw-related gene, as well as in color and color-perception genes, seems to corroborate the hypothesis that both the particular architecture of the cichlids' jaw apparatus and the haplochromines' mating system are important traits that have contributed to the evolutionary success of cichlid fishes in general and of haplochromines in particular [7, 13, 14, 38]. The adaptive advantage of the mating system of the haplochromines (with coloration and egg-spots as sexual advertisement) might be the facilitation of sexual selection through female choice (see e.g., [7, 16, 21]). Sexual selection has been suggested as a major cause for the explosive origin of new species of cichlids in species flocks [3, 13, 39, 40].
csf1rais also expressed in egg-dummies of ectodine cichlids
One of the most fascinating aspects of cichlid evolution is the repeated occurrence of evolutionary parallelisms [1, 41–43]. This has led to the question of whether natural selection alone is sufficient to produce parallel morphologies or whether a developmental or genetic bias has influenced the direction of diversification . Because of their independent origin in at least two lineages of mouthbrooding cichlids (not counting the genital tassels of some Tilapia species), egg-dummies on cichlid fins are likely to represent another example of evolutionary parallelism in the adaptive radiations of cichlids in East Africa – in this case involving a rather complex ethomorphological trait.
The function of egg-dummies in mimicking eggs to attract females is known from haplochromines and also from ectodines such as O. ventralis  (Figure 3v). Nevertheless, the dummies of O. ventralis (and its congeners) and those of haplochromines are of independent evolutionary origin, and they show different degrees of complexity. Most importantly, the two kinds of egg-markings are found on different anatomical structures, leading to substantial differences in the spawning behavior. In male haplochromines, the often numerous egg-spots are situated on the anal fin and, hence, are in close proximity to the genital opening to which the female's mouth is supposed to be guided (Figure 1). In O. ventralis (and its congeners) two blotches each are found on the tassels at the tips of the paired pelvic fins, which are conspicuously elongated (Figure 3v, w). Spawning in O. ventralis takes place in huge sand bowers of a diameter of up to half a meter in size, which are built by the territorial males in order to attract females. An interested female lays a few eggs in the center of the bower and picks them up into her mouth, after which the male displays its ventral fins with their yellow markings at the egg-laying spot. The female takes up the tassels into her mouth. The tips of the pelvic fins are put into close proximity to the male's genital opening, which discharges sperm .
Here, we provide evidence that the same gene is expressed in both kinds of egg-dummies. Just as in the haplochromine species examined, csf1ra is expressed in the yellow blotches on the tassels at the tips of the paired ventral fins of males of O. ventralis, whereas females do not show csf1ra expression (Figure 3y, z). This observation primarily indicates that both kinds of egg dummies are made up of xanthophores, for which csf1ra is a good marker gene. The two kinds of independently evolved egg dummies might, in the future, serve as model system to test whether the same genetic pathways are involved in the morphogenesis of a complex ethomorphological trait.
List of specimens used in this study and taxonomic information. The tribe names follow the nomenclature of [57,58]. Note that the 'modern haplochromines' are a monophyletic subgroup of the haplochromine cichlids and contain the entire species flocks of Lake Malawi and the Victoria region, the Tanganyikan Tropheini, as well as some riverine and lacustrine haplochromines in East Africa . Species that exhibit egg-dummies on male anal fins (AF) or pelvic fins (PF) are indicated.
East Africa, Lake Victoria region
Lake Tanganyika area
East Africa, rivers
East Africa, rivers and lakes
Pseudotrophus sp. 'bicolor'
Lake Kanyaboli, Lake Victoria
Fluorescence visualization of xanthophores
For fluorescence visualization of xanthophores in cichlid fins, we used a modified version of the method described in . Amputated anal, dorsal and ventral fins of haplochromine cichlids were mounted in 5% methylcellulose (500 μl) with dilute ammonia (25 μl), and β-mercaptoethanol, pH 10 (1 μl). Digital images were taken with a Zeiss AxioCam Mrc digital camera using a Zeiss Axioplan2 stereomicroscope (Zeiss, Jena, Germany).
Reverse transcriptase mediated PCR
Prior to in situ hybridization experiments, we confirmed expression of csf1ra in the haplochromine egg-spots by means of a reverse transcriptase mediated PCR. We amplified and sequenced a 540-bp fragment of csf1ra from cDNA that was transcribed from mRNA extracted from A. burtoni egg-spot tissue, using the primers F_1986 5'-GCTGCCCTACAATGAAAAGTG-3' and R_2186 5'-TTGACGATGTTCTGGTGGTGA-3'.
In situhybridization experiments
A 1 233-bp fragment of csf1ra was amplified by PCR from A. burtoni cDNA using primers F_1986 5'-GCTGCCCTACAATGAAAAGTG-3' and R_3199 5'-AYTGRTAGTTRTTGGKCTTCA-3'. The amplified fragment was cloned into the pCRII vector using the TA Cloning Dual Promoter Kit (Invitrogen, Karlsruhe, Germany). The orientation of ligated inserts with respect to Sp6 and T7 promoters was determined by direct sequencing on an ABI 3100 capillary sequencer using the BigDye terminator reaction chemistry (Applied Biosystems, Darmstadt, Germany). For in situ hybridization experiments, DIG-labeled (Roche) antisense RNA was transcribed from a linearized fragment using DIG-labeled dNTPs.
Four rounds of in situ hybridization experiments were performed. In total, five male anal fins (plus two female anal fins) were studied from P. sp. 'bicolor' and seven male anal fins (plus one female and one dorsal fin) from A. burtoni. Furthermore, we used six anal fins (plus one dorsal fin) from P. multicolor, three male anal fins from T. brauschi, and three male plus two female ventral fins from O. ventralis. Amputated fins were fixed in 4% paraformaldehyde in phosphate buffer saline (PFA/PBS) at 4°C overnight, washed twice in PBTw (0.1% Tween-20 in PBS, 0.01 DEPC), and stored in 100% methanol. Then, fins were rehydrated (PBTw washed and post fixed in 4% PFA/PBS) and treated with proteinase K (Roche) for 10 min at a final concentration of 14 μg/ml (see ). After PBTw washing, cichlid fins were prehybridized (50% formamide, 5 × SSC, 1 mg/ml tRNA, 50 μg/ml heparin, 0.1 % Tween-20, 9 mM citric acid, pH 6.0) and hybridized at 69°C overnight in hybridization buffer plus 1/10 volume of labeled probe. Fins were gradually transferred to PBTw and blocking solution (Boehringer Mannheim, Mannheim, Germany). Anti-(DIG-AP) antibody (Roche) in 0.5% blocking solution and BCIP/X-phos (Roche) were used to visualize target RNA. The tissue was finally fixed in 4% PFA/PBS and stored in 70% glycerol/PBS. As negative control, we applied labeled sense RNA to male fins of A. burtoni and P. sp. 'bicolor'. Photos of live fins and stained tissue were taken with a Zeiss AxioCam Mrc digital camera using a Zeiss Axioplan2 and Stemi SV11 APO stereomicroscopes. Photos were processed with AxioVision 3.1 (Zeiss) and Photoshop 7.0 (Adobe) software; the background of images was modified with Photoshop 7.0 (Adobe, San Jose, California, USA).
Polymerase chain reaction and DNA sequencing: the csf1ralocus
Primers used for sequencing of the csf1ra locus in 19 cichlid specimens
Primers used for cDNA amplification and sequencing
Molecular evolutionary analyses: the csf1ralocus
DNA sequences were quality trimmed with Phred  and assembled with Sequencher 3.0 . Sequences have been deposited in GenBank under the accession numbers EU042675–EU042749 (csf1ra) and EU042637–EU042674 (kita) (note that the segment spanning exons 17 to 19 in csf1ra could not be amplified for T. unimaulatum). Exon/intron boundaries were identified using a reference sequence from A. burtoni (DQ386648) and checked by homology comparison with reference sequences from pufferfish (U63926), zebrafish (AF240639) and trout (AJ417832). The protein structure of Csf1ra and the functional domains were identified from homologous sequence motifs in zebrafish (using the Protein families database Pfam ) and pufferfish . MatInspector from Genomatix  was used to identify putative promoter modules in the upstream regions of csf1ra.
Maximum likelihood and maximum parsimony phylogenetic analyses with 19 cichlid taxa were performed with Paup * 4.0b10 . The appropriate model parameters for the maximum likelihood analysis were determined by means of a likelihood ratio test with Modeltest version 3.6 . For the maximum likelihood tree search based on the non-coding section of the csf1ra locus (4 171 bp), we used the general time reversible model of molecular evolution (six types of substitutions) with a proportion of invariable sites of 0.4714 and a gamma substitution correction (α = 0.8669). An unweighted heuristic maximum parsimony search was performed with the same dataset (50 replicates). Bootstrap analyses were performed with 100 replicates under the maximum likelihood criterion and with 1 000 replicates for maximum parsimony analysis. Alternative branching orders at critical branches within the haplochromines were evaluated by means of a nonparametric Shimodaira-Hasegawa test under a resampling-estimated log likelihood with 1 000 bootstrap replicates as implemented in Paup*: The optimal maximum likelihood topology with Pseudocrenilabrus multicolor as sistergroup to Thoracochromis brauschi and all remaining haplochromines was tested against trees in which T. brauschi or Astatoreochromis alluaudi were forced to occupy the most ancestral position within the haplochromines (see also ).
Sliding window analyses for calculating the nucleotide diversity (π) in the coding region of csf1ra in haplochromines versus non-haplochromines were performed with DNASP 4.0 . We used a window size of 50 bp and an overlap of 10 bp. We also used DNASP for the calculation of the dN/dS ratio in haplochromines compared to non-haplochromines using the same 50-bp windows (overlap: 10 bp). We then used HyPhy  for the reconstruction of dN/dS ratios, based on maximum likelihood, on the branches of the phylogeny obtained before (see above), analyzing the entire dataset as well as the first 1 551 bp only, which correspond to the extracellular ligand-binding domain of csf1ra. We applied a site-to-site variation model with two independent gamma distributions and the MG94 model, and tested for relevant internal branches in the tree. The reconstructed dN/dS ratios were visualized in form of a branch-scaled tree applying the Suzuki-Gojobori derived adaptive selection tool implemented in HyPhy. We also used DNASP for sliding window analyses in kita and HyPhy to plot dN/dS ratios for the two segments of kita on the maximum likelihood tree.
Character state reconstructions
The characteristic egg-dummies of the haplochromines have been identified as a potential key evolutionary innovation that might be directly related to the evolutionary success of this most species-rich group of cichlid fishes  (but see ). As some ancestral (but also some derived) species of haplochromines do not show egg-dummies on male anal fins, we intended to reconstruct the evolutionary origin of these markings based on the new phylogenetic and comparative morphological data now available. Specifically, we wanted to evaluate the hypothesis that these characteristic egg-spots evolved only once in the ancestor of the haplochromines and that the missing ovoid markings on anal fins of males of some species are due to secondary loss. We used Mesquite 1.03  for maximum likelihood and maximum parsimony ancestral state reconstructions of the evolutionary origin of egg-dummies on male anal fins, on the basis of a consensus phylogeny of East African cichlid fishes. We also mapped the evolutionary origin of the characteristic polygynous mating system with maternal mouthbrooding involving egg-spots on that phylogeny. The consensus tree was built using a previous mitochondrial phylogeny  as well as the present phylogenetic hypothesis that is based on nuclear DNA. Note that the ancestral polytomy between the Pseudocrenilabrus, the Astatoreochromis, the Congolese/South African lineage, and the modern haplochromines  remained unresolved in all available phylogenetic hypotheses.
We thank C Chang-Rudolf, Y Gibert, N Offen, N Sigel and T Wirth for technical assistance; M Barluenga, G Begemann, P Bunje, B Egger, H Hoekstra, H Hofmann, M Nachmann, D Parichy, T Wirth and two anonymous reviewers for discussion and valuable suggestions on the manuscript. WS was supported by a Marie Curie Fellowship of the EU, and grants from the Landesstiftung-Baden Württemberg gGmbH and the Center for Junior Research Fellows, University of Konstanz; AM was supported by the Deutsche Forschungsgemeinschaft (DFG) and the University of Konstanz.
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