Evolutionary patterns of the SSU rRNA (V4 region) secondary structure in genus Euplotes (Ciliophora, Spirotrichea): insights into cryptic species and primitive traits

Sample collection and morphological study

Twelve species of Euplotes were collected from various locations in South Korea (Table 1). These specimens were cultured in Petri dishes containing water from their respective habitats. The cells were initially observed in their live state using a stereomicroscope (Nikon SMZ800, Nishioi, Shinagawa-ku, Tokyo) to assess their typical shape, movement, and behavior. For more detailed analysis, a differential interference contrast (DIC) microscope (Axio Imager A.1, Carl Zeiss, Oberkochen, Germany) was used, allowing for magnification between 100× and 1,000× for both live and stained samples.

DNA extraction, amplification, and sequencing

Genomic DNA extraction was performed using the RED Extract-N-Amp Tissue PCR Kit from Sigma (St. Louis, MO, USA), according to the manufacturer’s instructions. For polymerase chain reaction (PCR), the forward primer EUK A (5′-GAC CGT CCT AGT TGG TC-3′) and the reverse primer EUK B (5′-CTT GGA CGY CTT CCT AGT-3′) were used, as described by Medlin et al. (1988). PCR amplification was performed using the TaKaRa ExTaq DNA polymerase kit from TaKaRa Bio-medicals (Otsu, Japan) according to this specific protocol: an initial denaturation at 94 °C for 2 min, followed by 37 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 40 s, and extension at 72 °C for 4 min. This was followed by a final extension at 72 °C for 10 min (Kim et al., 2011). Sequencing was performed with bidirectional sequencing using the primers used in the PCR reaction (EUK A and EUK B).

Predicting the secondary structure of the V4 region of SSU-rRNA and CBC (compensatory base change) concept analysis

To predict the secondary structure of the SSU-rRNA, we performed an alignment of 69 SSU rRNA gene sequences from the species of genus Euplotes retrieved from GenBank (NCBI) and together with newly sequenced species from Korea (Table 1), related genera members (Certesia quadrinucleata, Aspidisca fusca, Euplotidium Rosati, Diophrys scutum, Uronychia xinjiangensis, and Discocephalus pararotatorius) selected as an outgroup. The alignment was performed using SSU-ALIGN and based on the alignment results from SSU-ALIGN, it was determined that the V4 region is a longer variable region (163–261 bp) (Table S1) and has more mutations compared to other variable regions (Fig. S1). From the SSU-ALIGN results, we isolated the V4 region of the SSU rRNA gene (Nawrocki, 2009). To generate consensus secondary structure of the V4 region of genus Euplotes member, we used RNAalifold software (Hofacker, 2008), the consensus secondary structure of V4 region used as a reference for predicting the secondary structure of the V4 region for each species. The prediction of the secondary structure for each species was achieved by using MFOLD, which calculates the minimum energy (Zuker, 2003). To guide the construction of the secondary structure using MFOLD, we used the consensus secondary structure and several criteria. First, we closed bilateral bulges (internal loops) present in published models if they could consistently form G:C pairs with non-canonical pairing bases in the stem. Second, we did not retain paired structures if multiple non-canonical base pairings occurred, instead of canonical (G:C or A:U) or wobble (G:U) base pairs. Final, in cases where multiple structures were predicted, we selected the structure with either the minimum free energy or the best compatibility with similar sequences (Řeháková et al., 2014; Voigt, Erpenbeck & Wörheide, 2008). The final secondary structure of the hypervariable region of SSU-rRNA was visualized using RNAviz software (De Rijk, Wuyts & De Wachter, 2003).

For the compensatory base change (CBC) analysis, we used 4SALE (Seibel et al., 2006) to detect CBCs between sequence-structure pairs within the alignment. The CBC analysis was applied to members of clade VI within the genus Euplotes (E. minuta, E. cf. mutabilis, E. crenosus, E. japonicum, E. cristatus, E. crassus, and E. vannus) (Fig. S2).

Reconstruction of ancestral state

For the ancestral state analysis of the V4 (SSU-rRNA) secondary structure, we used representative species from the genus Euplotes and related genera (Certesia quadrinucleata, Aspidisca fusca, Euplotidium rosati, Diophrys scutum, Uronychia xinjiangensis, and Discocephalus pararotatorius) (Table S1). The presence or absence of two additional helices on the extension helix E23_8 (Type I vs. Type II) (Fig. 1) was mapped onto the best-likelihood tree generated by RAxML analysis on the CIPRES platform (Miller, Pfeiffer & Schwartz, 2011). The character matrix and subsequent ancestral state reconstruction were performed using the parsimony model in Mesquite software (version 3.70) (Maddison & Maddison, 2021) (Table S1).

Significant characters of SSU rRNA secondary structure

In this study, we focused on the secondary structures of the V4 region. This choice was prompted by the observation of a significant number of mutations in this region, as indicated by the results of SSU-Align and primary sequence alignment. The consensus secondary structure predicted for the V4 region by the RNAalifold software identified four major helices (Fig. 1). In general, the secondary structure of each member of the genus Euplotes consists of four helices, with variations observed specifically in helix E23_8. These variations include two additional helices or hairpin loops at the end of the helix, highlighting the structural diversity within the genus Euplotes in the V4 region (Fig. 1).

Each clade depicted in the phylogenetic tree (Fig. S2) has a distinct secondary structure pattern. Members of clade I display a single large hairpin loop composed of 21 nucleotides in helix E23_8 (Fig. 2). This structural feature is also shared by the members of clade II, but smaller in size, consisting of 13 nucleotides (Fig. 2). Clade III is divided into two groups based on the characteristics of the V4 secondary structure. The first group include species with a hairpin loop consisting of five nucleotides (Fig. 2). Members of this group include E. curdsi, E. dominicanus, E. estuarianus, E. nobili, E. raikovi, and E. shii (Fig. 3). The second group is characterized by a long helix in E23_8 and the presence of two additional helices (E23_11 & E23_12) (Fig. 2). This group includes E. bergeri, E. elegans, E. qatarensis, and E. wuhanensis (Fig. 3). All members of clade IV share a V4 secondary structure characterized by the presence of two additional helices (E23_11 & E23_12), with the helix E23_8 shorter compared to the second group of clade III (Fig. 2). All members of clade V have two additional helices, except for three species that have a single hairpin loop at the end of helix E23_8 (Fig. 3). These three species are E. trisulcatus (hairpin loop consisting of 32 nucleotides), E. trisulcatus (hairpin consisting of 17 nucleotides) and E. shini (hairpin loop consisting of 29 nucleotides) (Fig. 2). In clade VI, members share a common pattern of a single large hairpin loop composed of 25–29 nucleotides. Interestingly, E. minuta, a member of clade VI, deviates from this pattern and show two additional helices (E23_11 & E23_12) (Fig. 3 and Fig. S2).

In addition to secondary structure, we applied compensatory base change (CBC)analysis to the members of clade VI, focusing specifically on the genus Euplotes vannus-minuta-crassus complex. The CBC shows that E. minuta has one CBC compared to other members of clade VI (Fig. 4).

Ancestral state of V4 secondary structure within genus Euplotes

The ancestral state was analyzed using the V4 secondary structure within the genus Euplotes to discern the evolutionary pattern of molecular characters, with particular focus on helix E23_8 (hairpin loop vs. two additional helices) (Fig. 1). The V4 secondary structure type I represents a molecular feature inherited from the ancestor of the genus Euplotes, and it is retained in members of clade I and II (Fig. 3). The ancestral state analysis shows that Type II of the V4 secondary structure, indicating the addition of two helices, is evolve feature compared to Type I of the V4 secondary structure (Fig. 3). Type II V4 secondary structure is commonly observed in almost all members of clade III to V with the exceptions of a few species that have Type I of the V4 secondary structure as their molecular character. These species include E. dominicanus, E. estuarinus, E. curdsi, E. nobilii, and E. shii in clade III, and E. trisulcatus, E. bisulcatus and E. shini in clade V (Fig. 3).

An interesting observation arises in the members of clade VI, where all members except E. minuta have Type I of the V4 secondary structure as their molecular character. Clade VI appears to represent a relatively recent clade in the phylogenetic tree of the genus, and these results suggest a remark reappearance of primitive characters reappearing in the most advanced species within this clade (Fig. 3).

Reverse evolutionary pattern of the V4 secondary structure within the genus Euplotes

The Type I of V4 secondary structure is regarded as simpler compared to Type II, as it shares structural similarities to the V4 secondary structure found in prokaryotes. In prokaryotes, the E23 (V4) region typically lacks an additional helix, indicating a primitive feature compared to eukaryotes (Lee & Gutell, 2012). This primitive feature is observed in members of the genus Euplotes and outgroup genera used as the earliest divergence group at the base of the phylogenetic tree, as supported by molecular clock analysis (Fig. S3). The primitive character of the V4 secondary structure was also observed in some later diverging species, particularly in Group VI (Fig. 3 & Fig. S1).

The secondary structure of V4 in the common ancestor of the genus Euplotes is characterized by Type I, which later evolved into the more complex Type II structure (Fig. 3). Interestingly, in certain evolved species there was a reduction in the E23_11 and E23_12 helices, resulting in a reversion to the Type I V4 secondary structure (Fig. 3). This pattern is suggestive of reverse evolution, where a character state changes to resemble the ancestral state, involving reversals and regressions that reflect evolutionary patterns of reversion to earlier or simplified forms after initially becoming more complex (Porter & Crandall, 2003; Teotónio & Rose, 2001).

The structural variations observed in the V4 secondary structure result from deletions or insertions in the V4 SSU rRNA region (Fig. S4). This pattern suggests that the occurrence of insertions or deletions in this region implies its lack of conservation and limited relevance to ribosome function, and thus the high degree of evolutionary change in this region is unlikely to have a significant impact on ribosomal function (Wuyts, Van de Peer & De Wachter, 2001). Although this region may not directly affect ribosome function, it is important because of the exceptionally high mutation rate within the SSU rRNA gene sequence. This region is likely to play a critical role in maintaining the free energy level to support the conservation of the SSU rRNA secondary structure. This study shows that the Type II of the V4 secondary structure has a more favorable free energy profile when compared to Type I (Table S1). The reverse evolution of the V4 secondary structure is not driven by the energy favorability, but is likely due to deletions that occur within this specific region.

The evolutionary pattern of V4 secondary structure supports the primitive nature of the basal clade in the genus Euplotes

The genus Euplotes shows distinct groupings, with a basal group (clade I) containing species such as E. huizhouensis, E. petzi, and E. sinicus, and subsequent divergent groups (clades II to VI) (Fig. 3). The evolutionary pattern of the V4 secondary structure shows that clade I, as a basal group, has primitive or ancestral characteristics compared to the later evolved groups. This pattern extends beyond molecular characters, morphological characters also follow a similar trend. Specifically, the basal group (clade I) displays a distinctive double-pattern argyrome character, representing the ancestral state of the genus Euplotes. In addition, these species display 10 fronto-ventral cirri (FVC), another character considered primitive or plesiomorphic in the genus Euplotes. In summary, molecular evidence from the V4 secondary structure supports the idea that species in the basal group (clade I) of the genus Euplotes are characterized by primitive traits compared to species in later diverging groups (clades II to VI), as reflected in their morphological characters (Syberg-Olsen et al., 2016; Zhao et al., 2018).

Cryptic species within the genus Euplotes in clade VI are revealed by the CBC concept in the V4 secondary structure

In this study, the CBC concept is applied to the members within clade VI, that potentially contain cryptic species. The presence of cryptic species makes it difficult to distinguish the species by morphological characters. By examining the secondary structure of V4, it is clear that E. minuta has a different structure compared to other members of clade VI. The CBC shows changes in nucleotide bonds in E. minuta compared to E. crassus, E. vannus, E. cf. mutabilis, and E. japonicum. To date, cryptic species have been identified among E. minuta, E. crassus, and E. vannus, which can be distinguished mainly by cell size alone (Dini, 1984; Valbonesi, Ortenzi & Luporini, 1988). However, the presence of CBC in E. minuta allows us to reasonably conclude that E. minuta represents a separate species from E. crassus and E. vannus. Furthermore, the CBC concept in E. crassus and E. vannus is consistent with evidence that both species are capable of mating under laboratory conditions (Valbonesi, Ortenzi & Luporini, 1988). This mating compatibility results from their morphological and chemical compatibility, in particular their pheromones. Considering both mating compatibility and the CBC concept, it is plausible that these two species are more appropriately classified as a single species.