Variations and Evolution of Polyubiquitin Genes from Ciliates

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European Journal of Protistology 49 (2013) 40–49

Variations and evolution of polyubiquitin genes from ciliates Xihan Liua,b, Fei Shia, Jun Gonga,c,∗ aYantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China bLaboratory of Protozoology, Ocean University of China, Qingdao 266003, China cCollege of Life Sciences, South China Normal University, Guangzhou 510631, China

Received 2 January 2012; received in revised form 2 May 2012; accepted 5 May 2012 Available online 9 June 2012

Abstract

Polyubiquitin genes from seven ciliate species were amplified, cloned and sequenced. It is estimated that Strombidium sulcatum, Euplotes vannus, E. rariseta and Anteholosticha manca have a polyubiquitin gene of 3 repeats, and A. parawarreni, Paramecium caudatum and Pseudokeronopsis flava 4 repeats. The newly obtained ubiquitins mostly differ from that of humans by 1–5 residues in amino acid sequences. A neighbor-joining tree constructed based on monomeric ubiquitin genes supports the monophyly of an assemblage comprising the litostomateans and some oligohymenophoreans, but not the class Spirotrichea. The monomers from the same species are generally placed together and highly supported for the class Litostomatea, the genera Paramecium and Ichthyophthirius, but not for other species. The non-synonymous/synonymous rate ratio (dN/dS) at the protein level are less than 1, and the synonymous nucleotide differences per synonymous site (pS) from intraspecific comparisons are fairly high (0.02–0.72). These results indicate that ciliates have not only the conserved, but also some quite divergent, polyubiquitin genes and confirm that the polyubiquitin genes in ciliates evolve according to the birth-and-death mode of evolution under strong purifying selection. © 2012 Elsevier GmbH. All rights reserved.

Keywords: Ciliates; Ubiquitin; Polyubiquitin gene; Molecular evolution; Phylogenetics

Introduction The evolution of ubiquitin genes is of great interest due to their important functions in protein degradation and in the Ubiquitin is a highly conserved 76 amino-acid (aa) protein control of numerous processes including cell-cycle progres- found in all eukaryotes (Durner and Boger 1995; Nercessian sion, signal transduction, transcriptional regulation, receptor et al. 2009; Wolf et al. 1993). The ubiquitin protein sequences down-regulation and endocytosis (Finley and Chau 1991; are almost identical among animals, plants, and fungi, with Hershko and Ciechanover 1998; Seufert and Jentsch 1992). only 3 or 4 amino acid differences (identity about 94.7% or The ubiquitin genes are fusion genes of a multigene family 95%) (Hauser et al. 1991). A similar level of identity was that falls into two types: the tandem repeats of the ubiquitin also found for protozoa (Jentsch et al. 1991). Ubiquitin rivals monomer (polyubiquitin), and a single monomer followed histone H4 as the most highly conserved protein (Baker and at the 3 -terminus by an open reading frame that encodes a Board 1991; Callis et al. 1995; Sharp and Li 1987). ribosomal protein. In both cases genes are simultaneously co-transcribed and co-translated, and monomeric ubiquitin units are produced by post-translational proteolysis (Callis et al. 1995; Hershko and Ciechanover 1998; Tachikui et al. ∗Corresponding author at: Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China. 2003). The numbers of ubiquitin monomers in polyubiqui- E-mail addresses: [email protected], [email protected] (J. Gong). tin genes vary among loci and species (Krebber et al. 1994;

Sharp and Li 1987; Swindle et al. 1988; Tachikui et al. 2003). Laboratory of Protozoology, Ocean University of China. A It has been proposed that the polyubiquitin genes undergo clone of each species was maintained in the laboratory at either concerted evolution (Nenoi et al. 1998; Sharp and Li room temperature, with rice grains added to the seawater 1987), i.e. genes from different genomic loci in one species medium as a food source to enrich bacteria. Species identifi- are more related to each other than with the homologous cation follows published references (Chen et al. 2011; Shao genes from another species, or birth-and-death evolution, a et al. 2011; Song et al. 2009). form of independent progress by means of gene duplication with some repeat genes staying in the genome for a long time DNA extraction, amplification and sequencing or becoming nonfunctional (Nei et al. 2000; Nei and Rooney 2005). Polyubiquitin insertion, with one or two amino acids One or more cells of each species clone were isolated using inserted between each monomer, was used successfully to a micropipette under a dissecting microscope. After washing clarify the relationship of Cercozoa and Foraminifera to other three times with 0.22-␮m-filtered seawater cells were trans- eukaryotic lineages (Archibald et al. 2003; Bass et al. 2005). ferred to a PCR microfuge tube with a minimum volume Ciliophora is perhaps the most morphologically differen- of water. Genomic DNA was extracted using REDExtract- tiated taxon among protists (Fleury et al. 1992; Larson et al. N-Amp Tissue PCR Kit (Sigma, St. Louis, MO) according 1991; Lynn 2008), and the genomic organization of ciliates to the manufacturer’s protocol, modified such that only 1/10 is unique among eukaryotes (Prescott 1994). Zufall et al. of the suggested volume for each solution was used (Gong (2006) compared the evolutionary rates of 6 genes (Actin, ␣-, et al. 2007). Polyubiquitin genes were amplified with primers ␤-Tubulin, EF1␣, Histone H4, and HSP90) of ciliates with (UBIQ1: 5-G GCC ATG CAR ATH TTY GTN AAR AC- animals and fungi, and proposed that genome architecture, 3, target motif MQIFVK; IUB2: 5-G ATG CCY TCY TTR including nuclear dimorphism and chromosome fragmenta- TCY TGD ATY TT-3, target motif KIQDKEGI; Archibald tion, leads to elevated rates of protein evolution in ciliates. et al. 2003). This primer set generates a ladder of ubiqui- This lead us to speculate how the most conserved protein and tin gene products ranging from a half-monomer fragment its genes vary in ciliates. to increasing numbers of tandem repeats of the polyubiqui- Currently only a few polyubiquitin gene sequences of cil- tin tract. PCR conditions were: 5 min initial denaturation at iates, mainly for model species, have been published. The 94 ◦C, followed by 35 cycles of 45 s at 92 ◦C, 1 min at 50 ◦C, presence of ubiquitin genes in ciliates was first demonstrated and 1.5 min at 72 ◦C, with a final extension of 5 min at 72 ◦C in Tetrahymena pyriformis (Neves et al. 1988). A polyubiq- (Archibald et al. 2003). To minimize sequence errors, the Ex uitin identified in T. thermophila is almost identical to that Taq (TaKaRa, Japan) which shows 4-fold higher fidelity com- found in T. pyriformis, both of which are composed of 5 iden- pared with the conventional Taq, with an error rate of about tical protein monomers and differ from human ubiquitin in 2.2 × 10−6, was used for PCR amplification. The purification 4 amino acids (positions 16, 19, 24 and 28) (Guerreiro and of PCR products, cloning and sequencing were performed Rodrigues-Pousada 1996; Neves et al. 1991). A polyubiquitin according to previous reports (Huang et al. 2010; Zhang et al. gene in the macronucleus of Euplotes eurystomus comprises 2010). Multiple positive bacterial clones were sequenced for 3 identical monomers that have different amino acids in posi- each species. tions 19, 24 and 28 (Hauser et al. 1991). Nevertheless, it was also noted that another polyubiquitin gene in T. pyriformis contains repeats which differ strikingly from that in humans Phylogenetic and sequence analyses (identity 85.5 and 89.5%) (Neves et al. 1990). In this study, we cloned and sequenced the polyubiquitin Nucleotide sequences were translated to amino acid genes from seven ciliate species. Our aims were to analyze sequences using GeneDoc 2.6.002 (Nicholas et al. 1997) the organization and variability of the genes and the proteins with translation tables of ciliate nuclear and euplotid nuclear among ciliate species and lineages, to explore the possibility genetic codes. Polyubiquitin genes of ciliates available of using ubiquitin for higher classification of ciliates, and from GenBank (Table 1) were downloaded and aligned to examine the evolutionary mode of polyubiquitin genes in with newly sequenced genes using the ClustalW pro- ciliates. gram (Larkin et al. 2007). A neighbor-joining (NJ) tree of nucleotide sequences was constructed with MEGA 4.0 (Tamura et al. 2007) based on p-distance. Sequences from Material and Methods amitochondrial protist Trichomonas vagilis were selected as outgroups. Sample source Selective pressure was measured by the non- synonymous/synonymous rate ratio (dN/dS) at the protein Seven marine ciliates, Anteholosticha manca, A. parawar- level. The values of ω = 1, <1, and >1 indicate neutral evolu- reni, Euplotes vannus, E. rariseta, Paramecium caudatum, tion, purifying selection, and positive selection, respectively Pseudokeronopsis flava, and Strombidium sulcatum, were (Yang 1997, 2007). The protein sequences were aligned obtained from the Ciliate Species Collection at the using ClustalW program (Larkin et al. 2007), and then 42 X. Liu et al. / European Journal of Protistology 49 (2013) 40–49

Table 1. Polyubiquitin gene/protein sequences from GenBank used in this study.

Group Species GenBank Accession Number Reference DNA Protein

Ciliophora Tetrahymena thermophila U46561 AAC47430 Guerreiro and Rodrigues-Pousada (1996) XM 001018500 XP 001018500* Eisen et al. (2006) Tetrahymena vorax AF003089 AAB61405 Green (unpublished) Tetrahymena pyriformis X61053 CAA43387 Neves et al. (1991) CAA84814* Neves et al. (1990) Ichthyophthirius multifiliis GL983930 EGR30931 Coyne et al. (2011) Paramecium tetraurelia XM 001451092 XP 001451129 Aury et al. (2006) Polyplastron multivesiculatum AJ965270 CAI83754 McEwan et al. (unpublished) Epidinium ecaudatum AJ965276 CAI83760 McEwan et al. (unpublished) Entodinium caudatum AJ965267 CAI83751 McEwan et al. (unpublished) Eudiplodinium maggii AJ965268 CAI83752 McEwan et al. (unpublished) Metadinium medium AJ965269 CAI83753 McEwan et al. (unpublished) Isotricha prostoma AJ965263 CAI83747 McEwan et al. (unpublished) Isotricha intestinalis AJ965261 CAI83745 McEwan et al. (unpublished) Dasytricha ruminantium AJ965257 CAI83741 McEwan et al. (unpublished) Nyctotherus ovalis AJ871355 CAI59819 Boxma et al. (2005) Sterkiella histriomuscorum AF188158 AAF00920 Quirk and Prescott (unpublished) Euplotes eurystomus M57231 AAA62225 Hauser et al. (1991) Paramecium caudatum JQ289924 This work Anteholosticha manca JQ289921 This work Anteholosticha parawarreni JQ289923 This work Pseudokeronopsis flava JQ289922 This work Strombidium sulcatum JQ289899–07 This work Euplotes rariseta JQ289920 This work Euplotes vannus JQ289908–19 This work Mammalia Homo sapiens BAA23632 Kim et al. (1998) Amphibia Xenopus laevis AAA49978 Dworkin-Rastl et al. (1984) Insecta Drosophila melanogaster P0CG69 Adams et al. (2000) Nematoda Caenorhabditis elegans AAA28154 Graham et al. (1989) Fungi Saccharomyces cerevisiae P0CG63 Ozkaynak et al. (1987) Viridiplantae Arabidopsis thaliana AEE82505 Mayer et al. (1999) Rhodophyta Griffithsia japonica AAP80690 Liu et al. (unpublished) Diplomonadida Giardia lamblia EDO79044 Morrison et al. (2007) Schizopyrinida Naegleria gruberi EFC47569 Fritz-Laylin et al. (2010) XP 002681258* Fritz-Laylin et al. (2010) Trichomonas Trichomonas vaginalis XM 001328013 XP 001328048 Carlton et al. (2007) Cercozoa Cercomonas edax AAM51212 Archibald et al. (2003) Aurigamonas solis ABB13626 Vickerman et al. (2005) Apicomplexa Plasmodium falciparum CAB59728 Horrocks and Newbold (2000) Babesia bovis XP 001610925 Brayton et al. (2007) Dinoflagellata Symbiodinium sp. ABA28993 Leggat et al. (2007) Karlodinium micrum ABV22260 Zhang et al. (2007)

The symbol “*” denotes that the sequence has highly divergent monomers. these alignments were used to guide the alignment of the of nucleotide differences per site between intraspeciﬁc coding regions by tranAlign (http://mobyle.pasteur.fr/cgi- monomers. bin/portal.py?form=tranalign). From the aligned nucleotide Concerted evolution and birth-and-death evolution can sequences, we calculated the dN/dS ratios using the yn00 be easily distinguished by analysis of genes from many program in PAML 4.2 to check the natural selection (Yang pairs of closely related species (Nei et al. 2000). The high 2007). Nucleotide diversity π (Nei and Li 1979) was calcu- level of the proportion of synonymous nucleotide differ- lated based on p-distance with the software DnaSP (Librado ences per synonymous site (pS) relative to the proportion and Rozas 2009), in order to show the average number of non-synonymous differences per non-synonymous site X. Liu et al. / European Journal of Protistology 49 (2013) 40–49 43

sequences are all of 76-aa. In order to examine aa variations among ciliates and other lineages, we aligned and compared the monomers of polyubiquitins from 110 ciliates, both newly obtained from this study and from Genbank, with other rep- resentatives (e.g. human, fungi, plants and protists) (Fig. 2). Of the 110 ciliate monomeric ubiquitins, 92 sequences are not much divergent from those of animals, with only 1–5 aa differences (Fig. 2). For example, compared with the human ubiquitin, the spirotrichean taxa Sterkiella histriomuscorum, Anteholosticha manca and Pseudokeronopsis flava have the least aa substitutions (1), Euplotes spp. the largest (4–5), and other species 2–3 aa substitutions. Notably, of the 18 remain- ing monomers, those from Euplotes vannus, Anteholosticha parawarreni, Tetrahymena pyriformis and T. thermophila, are among the most divergent with 5–13 aa differences (similar- ities 82.9–93.4%) (Fig. 2). Apparently, there is no single aa residue that is specific for all ciliate ubiquitins, though D16 can be observed in 102 out of 110 monomers (Fig. 2). Residues N53 and A57 could be Fig. 1. Electrophoresis image of PCR amplified polyubiquitin very characteristic for litostomatean ubiquitins, whereas no gene fragments from seven ciliates Strombidium sulcatum (lane 1), characteristic sites can be identified for either the Spirotrichea Euplotes vannus (lane 2), E. rariseta (lane 3), Pseudokeronopsis or the Oligohymenophorea. flava (lane 4), Anteholosticha manca (lane 5), Paramecium cauda- The monomers from the same species generally encode tum (lane 6), and A. parawarreni (lane 7). Lane M indicates the identical aa sequences (Fig. 2). Nevertheless, one inter- DNA ladder. monomer substitution (D51, V44, I22) is detected in the polyubiquitins from Euplotes rariseta, E. vannus and (pN) between the individual members is diagnostic for birth- Paramecium caudatum, respectively. Similar cases also occur and-death evolution (Kubicek et al. 2008; Nei et al. 2000; in previously reported ubiquitins of P.tetraurelia and Tetrahy- Piontkivska et al. 2002). We then calculated the mean pS mena vorax (Aury et al. 2006, Green unpublished). Strikingly, and pN values from intraspecific monomers using the DnaSP the two monomers 568C6m1 and 568C6m2 (these abbrevi- (Librado and Rozas 2009). ations refer to the first and second monomers respectively obtained by sequencing bacterial clone 6 of the 568bp-long polyubiquitin fragments), which are from the same polyubiq- Results uitin locus of E. vannus and located head to tail, differ from each other by 9 aa substitutions, resulting in a protein identity Repeat number of polyubiquitin of only 88.16%.

The polyubiquitin gene fragments from the seven ciliate species were successfully amplified (Fig. 1). As expected, Evolutionary analyses based on nucleotide the lengths of these gene fragments are 340, 568 and 796 bp, sequences which correspond to about 1.5, 2.5 and 3.5 repeat units of a monomeric ubiquitin, respectively. No fragments compris- Although the ubiquitin protein sequences are highly con- ing 4.5 or more repeat units were amplified, indicating that served, their nucleotide sequences are relatively divergent, the repeat numbers of ubiquitin in any polyubiquitin loci as the nucleotide diversity for species ranges from 0.702% are no more than 4 for the ciliates we studied. There are to 18.519% (Table 2). The nucleotide diversity of ubiquitins no introns within these fragments. Based on the sizes of is much higher in species of the class Spirotrichea (mean the longest fragments, it can be deduced that Strombidium 14.444%, n = 7) than those in Oligohymenophorea (mean sulcatum, Euplotes vannus, E. rariseta and Anteholosticha 4.272%, n = 6) and Litostomatea (mean 3.039%, n = 7). manca each has a polyubiquitin gene containing 3 repeats, A. A neighbor-joining tree was constructed to infer the parawarreni, Paramecium caudatum and Pseudokeronopsis phylogenetic relationships among ciliate ubiquitin genes flava each has a polyubiquitin of 4 repeats. (Fig. 3). At the class level, only the litostomatean sequences are strongly supported as a monophyletic Variations of ubiquitin at amino acid level group (bootstrap value 98%). The relationship between the Ichthophthirius–Paramecium and Tetrahymena clades For the full-length ubiquitin monomers that we have of ubiquitin sequences (Fig. 3) does not agree with obtained from the seven ciliate species, their protein the currently accepted phylogeny and classification of 44 X. Liu et al. / European Journal of Protistology 49 (2013) 40–49

Fig. 2. Comparison of ubiquitin monomers from different species. The newly obtained sequences are shown in bold. Dots indicate residues with identity to the first ubiquitin. “Abnormal” ubiquitin monomers of Euplotes vannus, Anteholosticha parawarreni, Tetrahymena thermophila and Naegleria gruberi are also presented here to show their high divergence. oligohymenophorean ciliates based on morphology and SSU values (0.06–0.19). Paramecium tetraurelia and Ichthyoph- rRNA gene sequences (Lynn 2008). Phylogenetic relation- thirius multifiliis seem to have exceptionally low pS values, ships of spirotrichean ubiquitin sequences are completely i.e. 0.02 and 0.08 respectively, for oligohymenophoreans. unresolved in the tree. The monomers of the same species generally cluster as monophyletic groups with moderate to high bootstrap Discussion support (61–100%) in the classes Litostomatea and Oligo- hymenophorea (when Tetrahymena spp. are excluded). In this study, we employed a PCR strategy in order to obtain However, no monophyletic assemblages can be recognized polyubiquitin genes from ciliate genomes. Beside the poly- for any spirotrichean species (Fig. 3). For instance, there meric ubiquitin genes, the primer pairs possibly also amplify are three separate clades for Euplotes vannus, and two for a fragment (ca. 112 bp) of the monomeric ubiquitin that fuses Strombidium sulcatum (Fig. 3). with ribosomal proteins. We only sequenced the polyubiqui- The calculation of dN and dS shows that dN/dS 1, indi- tin gene fragments, and did not sequence the short fragments cating that the polyubiquitin gene in ciliates is under strong due to their limited information. Furthermore, the fusion purifying selection (Fig. 4). The estimations of pN are effec- ubiquitin genes lack conservation in several phyla (Catic and tively zero owing to few amino acid differences detected Ploegh 2005). It thus can be concluded that the sequence vari- within species (Fig. 2). The comparison of mean pS val- ations we observed are indeed for the polyubiquitin genes. ues from intraspecific monomers showed similar divergence It is estimated that only 0.03 nucleotide errors occurred in with our phylogenetic tree. Spirotrichea has relatively high the 12.944 kb DNA sequences we obtained, which indicates pS values (0.22–0.72), Oligohymenophorea intermediate pS that the variations we observed are basically due to gene values (0.02–0.30) and Litostomatea has most of the low pS variations. X. Liu et al. / European Journal of Protistology 49 (2013) 40–49 45

Table 2. The mean values of synonymous sequences differences per site (pS × 100), and nucleotide diversity ␲ (%) for ciliate species.

Class Species pS × 100 ␲ (%) Spirotrichea Anteholosticha manca 72 18.421 Euplotes rariseta 49 13.158 Pseudokeronopsis ﬂava 63 16.520 Sterkiella histriomuscorum 72 18.519 Euplotes eurystomus 54 14.181 Euplotes vannus 56 9.855 Strombidium sulcatum 22 10.683 Oligohymenophorea Tetrahymena thermophila 24 6.053 Tetrahymena pyriformis 25 6.228 Tetrahymena vorax 30 7.895 Ichthyophthirius multiﬁliis 8 2.120 Paramecium caudatum 10 2.632 Paramecium tetraurelia 2 0.702 Litostomatea Polyplastron multivesiculatum 9 2.193 Epidinium ecaudatum 19 4.532 Isotricha prostoma 16 4.167 Dasytricha ruminantium 9 1.901 Entodinium caudatum 18 4.386 Eudiplodinium maggii 13 3.216 Isotricha intestinalis 6 0.877

Ciliates have two distinct genomes within every cell, one in for example ingested prey or symbiotic eukaryotes of the cil- the “somatic” macronucleus and the other in the “germline” iate hosts. However, in E. vannus, the divergent monomer is micronucleus, which is unique among eukaryotes. One may directly linked with the “normal” monomer in tandem, which question whether the gene fragments of polyubiquitins we indicates that the highly divergent polyubiquitin genes exist obtained are from the micronuclei or from the macronu- in the genome of E. vannus. These “abnormal” polyubiquitin clei? It is well known that the micronuclei are diploid, have genes, which have an identity of more than 80% to normal many internally excised sequences (IESs) that interrupt cod- ubiquitin, could be ubiquitin-like genes, or pseudogenes, as ing regions, while the developed macronuclei are polyploid, some high divergent polyubiquitin pseudogenes have been with 950–15,000 times amplified chromosomes, and have detected in humans (Baker and Board 1987) and in the plant no IESs (Klobutcher and Herrick 1997; Prescott 1994). It is Arabidopsis thaliana (Callis et al. 1995). therefore very likely that the polyubiquitin gene fragments we Polyubiquitin insertion has been used successfully to clar- have obtained by PCR were from the macronuclear genomes. ify the relationships of higher taxa such as Cercozoa and Foraminiferan (Archibald et al. 2003; Bass et al. 2005), which led us to ask whether there are residues specific Conservation and variation of ubiquitin for the phylum Ciliophora, and the superphylum Alveolata. Our analyses show that there are no such aa residues for We compared all the full-length ciliate ubiquitin monomers either Ciliophora or the Alveolata, indicating that polyu- currently available and found that most (92, about 83.6%) biquitins are too conserved among eukaryotes to be a good monomers of ciliate polyubiquitins have 1–5 aa differences marker for higher classifications. Futhermore, the phylogeny relative to the human ubiquitin (Fig. 2), which is comparable of ubiquitin genes is inconsistent with phylogenies based on to the level of ubiquitin conservation (no more than 4 substi- character-rich genes (e.g. SSUrRNA), which results from the tutions) reported for animals, plants, and fungi (Hauser et al. small amount of variation in the short ubiquitin sequences. 1991). Nevertheless, we also demonstrated that there are a Thus, the phylogeny of ubiquitin cannot be used to infer the number of “abnormal” ciliate polyubiquitin genes that hypo- phylogeny of taxa. thetically encode some extremely divergent ubiquitins (5–13 differences relative to human ubiquitin). It should be noted that these “abnormal” genes were found Evolution of polyubiquitin genes not only from the two species we examined using the PCR and cloning approach, but also from existing genomic data The phylogenetic tree inferred from nucleotide sequences for Tetrahymena pyriformis (Neves et al. 1990) and T. ther- shows some signs for both concerted evolution and birth- mophila (Eisen et al. 2006). There is a possibility that these and-death evolution. Among litostomatean sequences the divergent polyubiquitins could be derived from contaminants, intraspecific monomers are more closely related than 46 X. Liu et al. / European Journal of Protistology 49 (2013) 40–49

Fig. 3. Neighbor-Joining tree from monomeric genes of polyubiquitin in ciliates based on p-distances. The bootstrap values of 50 percent or more are shown at the nodes. Two monomers of Trichomonas vaginalis were selected as the outgroup. Significantly supported clades of ubiquitins from Euplotes vannus and Strombidium sulcatum are blocked in grey. interspecific ones, indicating concerted evolution. However, polyubiquitin genes, we calculated intraspecific pS values spirotrichean sequences are evolving independently with (Table 2). The mean pS values among ciliate ubiquitin relatively little gene conversion, supporting the birth-and- genes are rather high (0.02–0.72), as previously shown for death evolution. In order to clarify the evolution of ciliate fungi, plants, and animals (0–0.738) (Nei et al. 2000). As X. Liu et al. / European Journal of Protistology 49 (2013) 40–49 47

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