68 (2018) 353–365 brill.com/ab

Marked intra-genomic variation and in the ITS1-5.8S-ITS2 rDNA of Symphurus plagiusa (Pleuronectiformes: Cynoglossidae)

Li Gong1,2, Wei Shi3,4, Min Yang3,4 and Xiaoyu Kong3,4,∗ 1 National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, Zhejiang University, 316022, Zhoushan, China 2 National Engineering Research Center for Facilitated Marine Aquaculture, Marine Science and Technology College, Zhejiang Ocean University, 316022, Zhoushan, China 3 Key Laboratory of Tropical Marine Bio-resources and , Guangdong Provincial Key Laboratory of Applied , South China Sea Institute of Oceanology, Chinese Academy of Sciences, 510000, Guangzhou, China 4 South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, 510000, Guangzhou, China Submitted: October 22, 2017. Final revision received: March 16, 2018. Accepted: April 29, 2018

Abstract The eukaryotic ribosomal DNA (rDNA) cluster consists of multiple copies of three genes (18S, 5.8S, and 28S rDNA) and two internal transcribed spacers (ITS1 and ITS2). In recent years, an increasing number of rDNA sequence polymorphisms have been identified in numerous species. In the present study, we provide 33 complete ITS (ITS1-5.8S-ITS2) sequences from two Symphurus plagiusa in- dividuals. To the best of our knowledge, these sequences are the first detailed information on ITS sequences in Pleuronectiformes. Here, two divergent types (Type A and B) of the ITS1-5.8S-ITS2 rDNA sequence were found, which mainly differ in sequence length, GC content, diversity (π), secondary structure and minimum free . The ITS1-5.8S-ITS2 rDNA sequence of Type B was speculated to be a putative according to pseudogene identification criteria. Cluster analysis showed that sequences from the same type clustered into one group and two major groups were formed. The high degree of ITS1-5.8S-ITS2 sequence polymorphism at the intra-specific level indicated that the S. plagiusa has evolved in a non-concerted evolutionary manner. These re- sults not only provide useful data for ribosomal pseudogene identification, but also further contribute to the study of rDNA in teleostean .

Keywords Blackcheek tonguefish; multigene family; non-; pseudogene; ribosomal RNA gene

∗ ) Corresponding author; e-mail: [email protected]

© Koninklijke Brill NV, Leiden, 2018 DOI 10.1163/15707563-17000134

Downloaded from Brill.com09/26/2021 07:03:23AM via free access 354 L. Gong et al. / Animal Biology 68 (2018) 353–365

Introduction

Ribosomal RNA gene (rDNA) plays a pivotal role in protein synthesis in eukary- otes and changes to these genes can profoundly affect overall function and growth of the (Weider et al., 2005). rDNA is composed of several to thousands of copies of tandemly repeated units within the genome, with each unit consisting of three rRNA genes (18S, 5.8S and 28S) and two internal transcribed spacers (ITS1 and ITS2) (Long and Dawid, 1980; Reed & Phillips, 2000). The of rDNA differs fundamentally from that of single-copy genes. Individual rDNA copies do not evolve independently but instead appear to evolve in a concerted fashion, with the result that copies are similar within a species and different between species. Although the exact mechanisms determining concerted evolution remain unclear, two fundamental mechanisms – unequal crossing over and – are thought to drive the homogenization process (Szostak & Wu, 1980; Mullins & Fultz, 1991). Nevertheless, it is important to point out that although gene homogenization appears to be common, intra-genomic variation has also been observed. Since the first cases were reported in the amphibian Xenopus laevis (Wegnez et al., 1972; Peterson et al., 1980) and in the teleost fish Misgur- nus fossilis (Mashkova et al., 1981), where two types of 5S rRNA transcripts were identified, an increasing number of studies have reported a high degree of intra- individual rDNA sequence polymorphism in many taxa (Zhuo et al., 1995; Hugall et al., 1999; Harpke & Peterson, 2006; Freire et al., 2010; Hoy & Rodriguez, 2013; Li et al., 2013). More recently, several different types of 18S rDNA and ITS1 se- quences have been found in fish species (Sajdak & Phillips, 1997; Krieger et al., 2006; Xu et al., 2009). These strikingly multiple variants suggest that the rate of ho- mogenization may be too slow to prevent significant levels of intra-genomic rDNA polymorphism. Therefore, it is considerably necessary to investigate the degree of this rDNA variation to accurately reflect polymorphism, especially since it cannot be assumed that only one type sequence exists in any given species. Among the rDNA polymorphism, pseudogenes are one of the predominant con- tributors and an increasing number of ribosomal pseudogenes have been described in various species (Márquez et al., 2003; Harpke and Peterson, 2008a; Zuriaga et al., 2015; Gong et al., 2016a). Identifying ribosomal pseudogenes is difficult due to the continuous changes between functional copies and pseudogenes; nevertheless, pseudogenes still possess characteristic features. The primary criteria for ribosomal pseudogene identification are truncated sequences combined with more indels (in- sertions and deletions), higher nucleotide diversity (π), lower GC content, a less stable secondary structure, and a lower minimum free energy (Bailey et al., 2003; Xu et al., 2009; Li et al., 2013). In the 18S rDNA of the stone flounder Kareius bi- coloratus, a pseudogene had a 15-22 bp compared to the functional gene, and the nucleotide diversity (π) was also higher (0.0052 versus 0.0012) (Xu et al., 2009). In the 5.8S rDNA of the medicinal Ophiocordyceps sinensis,the pseudogene was found to have a lower GC content (36.13% to 38.71%) than the

Downloaded from Brill.com09/26/2021 07:03:23AM via free access L. Gong et al. / Animal Biology 68 (2018) 353–365 355 functional gene (49.68% to 50.32%), as well as lower secondary structure stability (−34.82 to −25.91 kcal mol−1 versus −41.50 to −34.48 kcal mol−1) (Li et al., 2013). In addition, variations in the conserved rRNA genes – especially indels – also are useful and swift indicators of the functionality of the connective ITS re- gion. One well-known case is applying the conserved 5.8S motif and its secondary structure for the pseudogenic ITS1 and ITS2 identification in three angiosperm taxa (Araceae, Quercus and Mammillaria) (Harpke & Peterson, 2008b). To date, rDNA sequence polymorphisms have been documented in three flat- fishes, including identification of 18S-ITS1 rDNA sequence polymorphisms in the stone flounder Kareius bicoloratus (Xu et al., 2009), 18S rDNA sequence poly- morphisms in Cynoglossus lineolatus (Gong et al., 2016a) and ITS2 sequence polymorphisms in the Zanzibar tonguesole Cynoglossus zanzibarensis (Gong et al., 2016b). However, to our knowledge, there have been no studies of the ITS region (ITS1-5.8S-ITS2) in teleosts. In the present study, we investigated polymorphic rDNA sequences in the blackcheek tonguefish, Symphurus plagiusa (Pleuronecti- formes: Cynoglossidae), a small demersal flatfish widely distributed in the Western Atlantic. The results revealed remarkable intra-genomic variation in the ITS re- gion, even including the very highly conserved 5.8S rDNA. Sequence alignments identified two types of ITS1, 5.8S and ITS2 – types A and B – coexisting in the S. plagiusa genome. Further comparisons of the sequence length, indel events, GC content, secondary structure, and minimum free energy between these two sequence types suggested that the Type A genes were functional and the Type B genes were putative pseudogenes. The results presented here may provide useful data for the study of the ITS region in flatfishes and may further contribute to our understand- ing of the evolutionary mode of rDNA sequences in teleostean fishes.

Materials and methods Sampling, DNA extraction, PCR amplification, cloning and sequencing Two S. plagiusa individuals were collected from New York and Campoton, respec- tively, and were immediately stored in 95% alcohol. Total genomic DNA was ex- tracted from muscle tissue as per the instructions of a marine animal genomic DNA extraction kit (Tiangen Biotech Co., China). Based on a previous study (Kumar et al., 2013), a forward primer Z-18S-1720 (5-TCGCTACTACCGATTGGATGG TTTA-3) and a reverse primer F-28S-100 (5-GCTCTTCCCTCTTCACTCG-3) were designed for the amplification of 18S (partial)-ITS1-5.8S-ITS2-28S (partial) rDNA. In previous studies, the use of dimethyl sulfoxide (DMSO) was found to en- hance amplification diversity during PCR (Buckler et al., 1997; Gong et al., 2016b). Accordingly, 8% (generally no more than 10%) DMSO was added to the reaction system for the highest amplification efficiency. PCR was carried out in a 25 μl reaction volume containing 2.0 mM MgCl2, 0.4 mM of each dNTP, 0.5 μMof each primer, 1.0 U of Taq polymerase (TaKaRa, Beijing, China), 2.5 μlof10×

Downloaded from Brill.com09/26/2021 07:03:23AM via free access 356 L. Gong et al. / Animal Biology 68 (2018) 353–365

Taq buffer, and approximately 50 ng of DNA template. PCR cycling conditions in- cluded an initial denaturation at 94°C for 2 min, followed by 35 cycles at 94°C for 1 min, an annealing at 50°C for 1.5 min, and elongation at 72°C for 1 min. The PCR reaction was completed by a final extension at 72°C for 10 min. PCR products were purified using a TaKaRa Agarose Gel DNA Purification Kit (TaKaRa) and were then inserted into the pMD19-T vector (TaKaRa). Sequencing was performed on an ABI genetic analyser (Applied Biosystems, Beijing, China).

Data analyses Sequenced fragments were assembled using CodonCode Aligner (vers. 3, Codon- Code Corporation, Dedham, MA, USA). The boundaries of the coding genes and ITS region were determined using NCBI-BLAST (http://blast.ncbi.nlm.nih.gov). Sequence alignments were performed using ClustalX 2.0 (Larkin et al., 2007) and were checked manually using BioEdit v7 (Hall, 1999). GC content and poly- morphic sites were calculated using MEGA 5 (Tamura et al., 2011). Haplotype diversity (Hd) and nucleotide diversity (π) were determined using DnaSP software (Librado & Rozas, 2009). Secondary structure was predicted using the RNAfold web server (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). The minimum free en- ergy (G at 37°C) was estimated for stability comparisons. For cluster analyses of the ITS1-5.8S-ITS2 sequences, two unrooted trees were constructed in PhyML for maximum likelihood (ML) analyses (Guindon & Gascuel, 2003), and in Mr- Bayes v2.0 for Bayesian inference (BI) analyses (Huelsenbeck & Ronquist, 2001). The GTR + I + G model was adopted as the most appropriate model based on the Akaike information criterion (AIC). A bootstrap was performed (1000 replicates) to evaluate relative levels of support for various nodes in both tree topologies includ- ing bootstrap values for the ML tree and posterior probabilities for the Bayesian analysis.

Results Analysis of PCR amplification products A band at approximately 1200 bp was obtained from PCR amplification of DNA from the two S. plagiusa individuals and this band included two flanking partial 18S and 28S sequences, and the complete ITS1-5.8S-ITS2 sequence. A total of 33 isolate clones were randomly selected and determined after molecular cloning (GenBank accession numbers MG026839-MG026871) and these results showed no inter-individual variation. Excluding the two flanking fragments, the complete ITS1-5.8S-ITS2 sequence had a wide variation in length, and ranged in size from 962 bp to 1102 bp. Sequence alignment showed that two distinguishable types of ITS1-5.8S-ITS2 (Type A and B) coexisted, and each fragment (ITS1, 5.8S, and ITS2) had two types, which indicated non-concerted evolution of ITS1-5.8S-ITS2 rDNA in this species (fig. 1). A majority of the sequences (29 clones) belonged to

Downloaded from Brill.com09/26/2021 07:03:23AM via free access L. Gong et al. / Animal Biology 68 (2018) 353–365 357

Figure 1. Alignment of two types of ITS1-5.8S-ITS2 rDNA sequences in S. plagiusa. The boundaries are marked by arrows. The dots indicate nucleotide identity to the top sequence and dashes indicate alignment gaps.

Type A, while only four sequences belonged to Type B. Type A sequences pos- sessed more variations, with 28 haplotypes and 56 variable sites. However, Type B sequences were highly conserved, with only one haplotype and no variable sites. Simultaneously, the GC contents of these two types were divergent. The GC con- tent of Type A (71.6%) was much higher than that of Type B (54.2%). In addition, the minimum free energy of Type A (−565.9 kcal mol−1) was also much higher in absolute value than that of Type B (−347.1 kcal mol−1)(table1).

Comparative analysis of the two types of ITS1, 5.8S, and ITS2 rDNA In this study, two distinguishable types of ITS1, 5.8S and ITS2 sequences were ob- tained. The ITS1 region, the longest sequence among these three fragments, had a relatively wide variation in length, ranging from 515 bp to 589 bp in length, which was mainly due to multiple deletions in the Type B sequence. Sequence alignment

Table 1. Nucleotide diversity of different types of ITS1-5.8S-ITS2 sequences in S. plagiusa.

ITS1 5.8S ITS2 ITS1-5.8S-ITS2 Type A Type B Type A Type B Type A Type B Type A Type B

No. of sequences 29 4 29 4 29 4 29 4 Length (bp) 584-589 515 160 159 353 288 1097-1102 962 No. of haplotypes 19 1 6 1 9 1 28 1 Haplotype diversity (Hd) 0.956 0 0.320 0 0.820 0 0.998 0 Variable sites 32 0 5 0 10 0 56 0 Parsimony-informative sites 19 0 0 0 4 0 23 0 Nucleotide diversity (π) 0.01208 0 0.00216 0 0.00452 0 0.00877 0 GC content (%) 73.5 54.4 57.6 56.6 74.9 52.4 71.6 54.2 − Minimum free energy (kcal mol 1) −310.8 −131.8 −65.5 −53.6 −179.3 −99.2 −565.9 −347.1

Downloaded from Brill.com09/26/2021 07:03:23AM via free access 358 L. Gong et al. / Animal Biology 68 (2018) 353–365 revealed that a total of 277 nucleotide substitutions were found between Type A and B sequences and the sequence divergence was 47.4% (277/585). Further anal- yses showed that the majority of the substitutions were CG to TA in the Type B sequences; we also found 61 C → T transitions, 31 C → A transversions, 31 G → A transitions and 22 G → T transversions. Consequently, although only about a 70-bp deletion, the GC content of the Type B sequence (54.4%) was signif- icantly lower than that of Type A (73.5%). Similarly, the secondary structures were also divergent (fig. 2a, b) and the minimum free energy of Type B (−131.8 kcal mol−1) was much lower than that of Type A (−310.8 kcal mol−1). In addition, the haplotype diversity (Hd), number of variable sites and nucleotide diversity (π)– representing the sequence variations – were also calculated. The values of these measures for Type A were 0.956, 32 and 0.01208, respectively, while those for Type B sequences were zero, indicating total conservation. In most cases, 5.8S rDNA is very highly conserved. However, in this study, two 5.8S rDNA sequence types were found. Although the sequence length was rela- tively conserved (159-160 bp), the two sequences showed obvious mutations and deletions (fig. 1). The sequence alignment indicated that a total of 27 nucleotide substitutions were found between Type A and B sequences and the sequence di- vergence was 16.9% (27/160) and included 15 C → T transitions, three A → G transitions, four A → T transversions, three G → T transversions and two A → C transversions. Slight differences were observed in GC content and minimum free energy between the two types, with Type A having 57.6% GC content and −65.5 kcal mol−1 minimum free energy and Type B having 56.6% GC content and −53.6 kcal mol−1 minimum free energy. In addition to these differences, the secondary structures were also divergent. The 5.8S of Type A was folded into a con- served structure as in other (Hribova et al., 2011; Bargues et al., 2014: Rampersad, 2014), while that of Type B showed a relatively large degree of varia- tion (fig. 2c, d). Likewise, ITS2 sequences showed two distinct types that not only differed in length polymorphism but also in nucleotide variation. The Type B sequence (288 bp) was 65 bp shorter than the Type A sequence (353 bp) due to multiple dele- tions. According to the sequence alignment, a total of 163 nucleotide substitutions were found between the Type A and B sequences and the sequence divergence was up to 45.9% (163/355). As in the ITS1 region, most of the substitutions were also in CG to TA mutations in the Type B sequences, including 41 C → T transitions, 11 C → A transversions, 22 G → A transitions and 13 G → T transversions. As a result, although differing by only a short deletion, the GC content of the Type B (52.4%) sequence was significantly lower than that of the Type A sequence (74.9%). Interestingly, these two type sequences had similar secondary structures (fig. 2e, f). However, the minimum free energy of the Type B sequence (−99.2 kcal mol−1) was much lower than that of Type A (−179.3 kcal mol−1). As in the ITS1 sequence, the haplotype diversity (Hd), number of variable sites and nucleotide diversity (π)

Downloaded from Brill.com09/26/2021 07:03:23AM via free access L. Gong et al. / Animal Biology 68 (2018) 353–365 359

Figure 2. Inferred secondary structures of two types of rDNA sequences in S. plagiusa.(A,B)ITS1 of Type A and Type B; (C, D) 5.8S of Type A and Type B; (E, F) ITS2 of Type A and Type B.

Downloaded from Brill.com09/26/2021 07:03:23AM via free access 360 L. Gong et al. / Animal Biology 68 (2018) 353–365 were zero in the Type B sequence, while for the Type A sequence, these values were 0.820, 10 and 0.00452, respectively.

Cluster analysis of ITS1-5.8S-ITS2 rDNA

To explore the relationship between the different sequence types, based on the 33 ITS1-5.8S-ITS2 rDNA sequences two unrooted trees were constructed using ML and BI methods. Both trees were largely congruent with each other; consequently, only one topology (ML) with both support values is displayed, including both the bootstrap values for the ML tree and the posterior probability for the Bayesian analysis (fig. 3). According to the tree topology, two major groups were formed and sequences from the same type clustered into one group, which reflected the non- concerted mode of evolution of ITS1-5.8S-ITS2 rDNA in the S. plagiusa genome from another aspect.

Figure 3. Maximum likelihood (ML) tree based on the ITS1-5.8S-ITS2 rDNA dataset (1000 bootstrap replicates). Bootstrap support values for maximum likelihood above the branch and Bayesian posterior probabilities below the branch.

Downloaded from Brill.com09/26/2021 07:03:23AM via free access L. Gong et al. / Animal Biology 68 (2018) 353–365 361

Discussion Based on the potential features of pseudogenes mentioned above, in the present study ITS1-5.8S-ITS2 rDNA sequences of Type B were identified as putative pseu- dogenes. Our reasoning was as follows: firstly, due to more than a hundred dele- tions, the length of the Type B sequence was about 87% of the length of the Type A. These deletions included 69-74-bp deletions in ITS1, a 1-bp deletion in 5.8S, and 65-bp deletions in ITS2. Secondly, the ITS1 and ITS2 regions usually show high GC contents in vertebrates (Coleman & Vacquier, 2002; Grimm & Denk, 2008; Chow et al., 2009). Here, the Type A sequence did in fact show a high GC content – i.e., 73.5% GC content in ITS1 and 74.9% in ITS2. However, the GC content of the Type B sequence was much lower – i.e., 54.4% GC content in ITS1 and 52.4% in ITS2. Thirdly, the minimum free energy of the Type B sequence was much lower than those of Type A. For example, minimum free energy of the ITS1 and ITS2 sequences of the Type B sequence was only 42.4% (131.8/310.8) and 55.3% (99.2/179.3) of that of Type A, respectively. Fourthly, 5.8S rDNA is a highly conserved region that does not allow large sequence variation (especially deletions) or incorrect folding structure; as a result, variations in sequence and secondary structure of the conserved 5.8S also can act as a swift indicator for the entire ITS pseudogene identification. Here, the 5.8S of Type B had a 1-bp deletion and relative large variations compared to Type A. Moreover, the secondary structure of the Type B sequence significantly differed from those typical of other organisms (Hribova et al., 2011; Bargues et al., 2014; Rampersad, 2014). Taken together, these results pro- vided convincing evidence that the ITS1-5.8S-ITS2 rDNA of Type B in S. plagiusa genome may be a pseudogene. According to previous studies, the proportion of pseudogenes present in a species widely fluctuates among different species: 26.3% in fungus, 33% in dinoflag- ellate, 58.8% in stone flounder (Xu et al., 2009), 82.3% in Cynoglossus lineolatus (Gong et al., 2016a), and as high as 97% in cactus species (Harpke & Peterson, 2007). Unfortunately, few reports have identified potential factors causing the pro- portion of pseudogenes present in a given system. For example, in one study of species in the cactus genus Mammillaria (Harpke & Peterson, 2007), the author attributed the fact that they contain >97% pseudogenic ITS to the fact that they also have an overabundance of pseudogenic copies in their genome in general. In a different system, Li et al. (2013) speculated that a 26.3% pseudogenic ITS1 was possibly due to pseudogenic copies being far less abundant than in and . In the present study, the abundance of pseudogenes identified using ran- dom sequencing was much lower (12.1%) than the abundance of functional genes (87.9%). Here, two underlying causes are discussed. The first is that, as mentioned above, the genome is less rich in pseudogenes than in functional genes. The second reason may be related to the experimental method. Theoretically, pseudogenes are more likely to be amplified by ordinary PCR methods due to the fact that pseudo- genes have less stable secondary structures. However, in specific PCR, the addition of DMSO is able to reduce or destroy the secondary structures of functional genes

Downloaded from Brill.com09/26/2021 07:03:23AM via free access 362 L. Gong et al. / Animal Biology 68 (2018) 353–365 and hence enhance the amplification of functional rDNA copies (Buckler et al., 1997; Álvarez & Wendel, 2003). This may be a reason why a low proportion of pseudogenes was detected. Currently, both concerted and non-concerted evolution of rDNA have been docu- mented in organism genomes (Johansen et al., 2006; Eickbush and Eickbush, 2007; Xu et al., 2009; Li et al., 2013; Naidoo et al., 2013). rDNA was originally viewed as undergoing strict concerted evolution. However, an increasing number of excep- tions have been found, suggesting that rDNA polymorphisms are common in the genomes of many organisms. Which mode of evolution occurs in which organisms largely depends on the evolutionary dynamics between rates and homog- enization processes. In concerted evolution, for instance, multiple units of rDNA evolve at a uniform rate in a coordinated fashion, resulting in sequence consistency in all units. On the contrary, in non-concerted evolution, some units are not sub- jected to homogenizing processes to the same degree and have different mutation rates. This results in the production of multiple sequence variations in the genome. As in the S. plagiusa genome, a high degree of intra-genomic polymorphism of the ITS1, ITS2 and even the extremely conserved 5.8S sequence was found. These se- quences showed remarkable differences in sequence length, GC content, secondary structure, and minimum free energy, which suggests that the mutation rate of these units far exceeds the homogenization processes, resulting in non-concerted evolu- tion. Recently, with the rapid development of sequencing technology and the great reduction of cost, scanning the copies of tandemly repeated rDNA units using next-generation sequencing (NGS) is no longer a difficult approach. For example, Symonová et al. (2017) witnessed an extreme amplification of 5S rDNA reaching up to tens of thousands of copies in two European Esox species using whole genomic sequencing technology combined with cytogenetic and molecular approaches. In this case, the copies of tandemly repeated rDNA units can be estimated, and intra- genomic homogeneity or heterogeneity of the organism can be revealed, which will provide a powerful tool for rDNA evolution in future studies.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (31272273 and 41706176) and the Scientific Research Foundation for the Introduc- tion of Talent of Zhejiang Ocean University.

Downloaded from Brill.com09/26/2021 07:03:23AM via free access L. Gong et al. / Animal Biology 68 (2018) 353–365 363

References Álvarez, I. & Wendel, J.F. (2003) Ribosomal ITS sequences and phylogenetic inference. Mol. Phylogenet. Evol., 29, 417-434. Bailey, C.D., Carr, T.G., Harris, S.A. & Hughes, C.E. (2003) Characterization of angiosperm nrDNA polymorphism, paralogy, and pseudogenes. Mol. Phylogenet. Evol., 29, 435-455. Bargues, M.D., Zuriaga, M.A. & Mas-Coma, S. (2014) Nuclear rDNA pseudogenes in Chagas dis- ease vectors: evolutionary implications of a new 5.8S+ITS-2 paralogous sequence marker in triatomines of north, central and northern South America. Infect. Genet. Evol., 21, 134-156. Buckler, E.S., Ippolito, A. & Holtsford, T.P. (1997) The evolution of ribosomal DNA: divergent par- alogues and phylogenetic implications. , 145, 821-832. Chow, S., Ueno, Y., Toyokawa, M., Oohara, I. & Takeyama, H. (2009) Preliminary analysis of length and GC content variation in the ribosomal first internal transcribed spacer (ITS1) of marine ani- mals. Mar. Biotechnol., 11, 301-306. Coleman, A.W. & Vacquier, V.D. (2002) Exploring the phylogenetic utility of ITS sequences for animals: a test case for abalone (Haliotis). J. Mol. Evol., 54, 246-257. Eickbush, T.H. & Eickbush, D.G. (2007) Finely orchestrated movements: evolution of the ribosomal RNA genes. Genetics, 175, 477-485. Freire, R., Arias, A., Méndez, J. & Insua, A. (2010) Sequence variation of the internal transcribed spacer (ITS) region of ribosomal DNA in Cerastoderma species (Bivalvia: Cardiidae). J. Mollus- can Stud., 76, 77-86. Gong, L., Shi, W., Yang, M., Si, L. & Kong, X. (2016a) Long duplication of 18S ribosomal DNA in Cynoglossus lineolatus (Pleuronectiformes: Cynoglossidae): novel molecular evidence for unequal crossing over model. Acta Oceanol. Sin., 35, 38-50. Gong, L., Shi, W., Yang, M., Si, L. & Kong, X. (2016b) Non-concerted evolution in ribosomal ITS2 sequence in Cynoglossus zanzibarensis (Pleuronectiformes: Cynoglossidae). Biochem. Syst. Ecol., 66, 181-187. Grimm, G.W. & Denk, T. (2008) ITS evolution in Platanus (Platanaceae): homoeologues, pseudo- genes and ancient hybridization. Ann. Bot., 101, 403-419. Guindon, S. & Gascuel, O. (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol., 52, 696-704. Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser., 41, 95-98. Harpke, D. & Peterson, A. (2006) Non-concerted ITS evolution in Mammillaria (Cactaceae). Mol. Phylogenet. Evol., 41, 579-593. Harpke, D. & Peterson, A. (2007) Quantitative PCR revealed a minority of ITS copies to be functional in Mammillaria (Cactaceae). Int. J. Plant Sci., 168, 1157-1160. Harpke, D. & Peterson, A. (2008a) Extensive 5.8 S nrDNA polymorphism in Mammillaria (Cactaceae) with special reference to the identification of pseudogenic internal transcribed spacer regions. J. Plant Res., 121, 261-270. Harpke, D. & Peterson, A. (2008b) 5.8S motifs for the identification of pseudogenic ITS regions. , 86, 300-305. Hoy, M.S. & Rodriguez, R.J. (2013) Intragenomic sequence variation at the ITS1-ITS2 region and at the 18S and 28S nuclear ribosomal DNA genes of the New Zealand mud snail, Potamopyrgus antipodarum (Hydrobiidae: Mollusca). J. Molluscan Stud., 79, 205-217. Hribova, E., Cizkova, J., Christelova, P., Taudien, S., de Langhe, E. & Dolezel, J. (2011) The ITS1-5.8S-ITS2 sequence region in the Musaceae: structure, diversity and use in molecular phy- logeny. PLoS One, 6, e17863.

Downloaded from Brill.com09/26/2021 07:03:23AM via free access 364 L. Gong et al. / Animal Biology 68 (2018) 353–365

Huelsenbeck, J.P. & Ronquist, F. (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioin- formatics, 17, 754-755. Hugall, A., Stanton, J. & Moritz, C. (1999) Reticulate evolution and the origins of ribosomal internal transcribed spacer diversity in apomictic Meloidogyne. Mol. Biol. Evol., 16, 157-164. Johansen, T., Repolho, T., Hellebø, A. & Raae, A.J. (2006) Strict conservation of the ITS regions of the ribosomal RNA genes in Atlantic cod (Gadus morhua L.). Dna Seq., 17, 107-114. Krieger, J., Hett, A.K., Fuerst, P.A., Birstein, V.J. & Ludwig, A. (2006) Unusual intraindividual varia- tion of the nuclear 18S rRNA gene is widespread within the Acipenseridae. J. Hered., 97, 218-225. Kumar, R., Singh, M., Kushwaha, B., Nagpure, N., Mani, I. & Lakra, W. (2013) Molecular character- ization of major and minor rDNA repeats and genetic variability assessment in different species of mahseer found in north India. Gene, 527, 248-258. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J. & Higgins, D.G. (2007) Clustal W and Clustal X version 2.0. , 23, 2947-2948. Li, Y., Jiao, L. & Yao, Y.-J. (2013) Non-concerted ITS evolution in fungi, as revealed from the impor- tant medicinal fungus Ophiocordyceps sinensis. Mol. Phylogenet. Evol., 68, 373-379. Librado, P. & Rozas, J. (2009) DnaSP v5: a software for comprehensive analysis of DNA polymor- phism data. Bioinformatics, 25, 1451-1452. Long, E.O. & Dawid, I.B. (1980) Repeated genes in . Annu. Rev. Biochem., 49, 727-764. Márquez, L.M., Miller, D.J., MacKenzie, J.B. & van Oppen, M.J. (2003) Pseudogenes contribute to the extreme diversity of nuclear ribosomal DNA in the hard coral Acropora. Mol. Biol. Evol., 20, 1077-1086. Mashkova, T., Serenkova, T., Mazo, A., Avdonina, T., Timofeyeva, M.Y. & Kisselev, L. (1981) The primary structure of oocyte and somatic 5S rRNAs from the loach Misgurnus fossilis. Nucleic Acids Res., 9, 2141-2152. Mullins, J. & Fultz, P. (1991) Evidence for biased gene conversion in concerted evolution of ribosomal DNA. Science, 251(4991), 308-310. Naidoo, K., Steenkamp, E.T., Coetzee, M.P., Wingfield, M.J. & Wingfield, B.D. (2013) Concerted evolution in the ribosomal RNA cistron. PLoS One, 8, e59355. Peterson, R.C., Doering, J.L. & Brown, D.D. (1980) Characterization of two Xenopus somatic 5S and one minor oocyte-specific 5S DNA. Cell, 20, 131-141. Rampersad, S.N. (2014) ITS1, 5.8S and ITS2 secondary structure modelling for intra-specific dif- ferentiation among species of the Colletotrichum gloeosporioides sensu lato species complex. SpringerPlus, 3, 684. Reed, K.M. & Phillips, R.B. (2000) Structure and organization of the rDNA intergenic spacer in lake trout (Salvelinus namaycush). Res.,8,5-16. Sajdak, S.L. & Phillips, R.B. (1997) Phylogenetic relationships among Coregonus species inferred from the DNA sequence of the first internal transcribed spacer (ITS1) of ribosomal DNA. Can. J. Fish. Aquat. Sci., 54, 1494-1503. Symonová, R., Ocalewicz, K., Kirtiklis, L., Delmastro, G.B., Pelikánová, Š., Garcia, S. & Kovarík,ˇ A. (2017) Higher-order organisation of extremely amplified, potentially functional and massively methylated 5S rDNA in European pikes (Esox sp.). BMC , 18, 391. Szostak, J.W. & Wu, R. (1980) Unequal crossing over in the ribosomal DNA of Saccharomyces cere- visiae. , 284(5755), 426-430. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol., 28, 2731-2739.

Downloaded from Brill.com09/26/2021 07:03:23AM via free access L. Gong et al. / Animal Biology 68 (2018) 353–365 365

Wegnez, M., Monier, R. & Denis, H. (1972) Sequence heterogeneity of 5 S RNA in Xenopus laevis. FEBS Lett., 25, 13-20. Weider, L.J., Elser, J.J., Crease, T.J., Mateos, M., Cotner, J.B. & Markow, T.A. (2005) The functional significance of ribosomal (r) DNA variation: impacts on the evolutionary ecology of organisms. Annu. Rev. Ecol. Evol. Syst., 36, 219-242. Xu, J., Zhang, Q., Xu, X., Wang, Z. & Qi, J. (2009) Intragenomic variability and pseudogenes of ribosomal DNA in stone flounder Kareius bicoloratus. Mol. Phylogenet. Evol., 52, 157-166. Zhuo, L., Reed, K.M. & Phillips, R.B. (1995) Hypervariability of ribosomal DNA at multiple chro- mosomal sites in lake trout (Salvelinus namaycush). Genome, 38, 487-496. Zuriaga, M.A., Mas-Coma, S. & Bargues, M.D. (2015) A nuclear ribosomal DNA pseudogene in triatomines opens a new research field of fundamental and applied implications in Chagas disease. Mem. Inst. Oswaldo Cruz, 110, 353-362.

Downloaded from Brill.com09/26/2021 07:03:23AM via free access