Tribe Acalyptaini (Hemiptera: Tingidae: Tinginae) Revisited: Can Apomorphies in Secondary and Tertiary Structures of 18S rRNA Length-Variable Regions (LVRs) Support Tribe Validity?

Simple Summary The small subunit (SSU) of the nuclear ribosomal DNA that codes 18S rRNA is one of the most frequently sequenced genes in phylogenetic analyses of true bugs (Hemiptera: Heteroptera). However, no studies have been identified that use this method with lace bugs (Tingidae). Furthermore, the secondary and tertiary structures of the 18S rRNA have not been described nor used to interpret relationships among lace bug taxa. The number of nucleotides and shapes of the 18S rRNA length-variable regions (LVRs) have been confirmed to be phylogenetically informative; therefore, we verified their usefulness in resolving the validity issues within the Acalyptaini tribe. Abstract The lace bug tribe Acalyptaini (Tingidae: Tinginae) includes five genera, Acalypta, Derephysia, Dictyonota, Kalama, and Recaredus, and it was recently resurrected based on morphological and karyological characters. We aimed to validate the distinctiveness of this tribe using 18S rDNA sequences, which have not been used in previous Tingidae phylogenomic studies. Our results confirmed the monophyly of the tribe. Moreover, the monophyly of the subfamily Cantacaderinae and its basal position within the family Tingidae were indicated, as well as the position of the tribe Litadeini as sister to all other Tinginae. In addition, we attempted to determine the apomorphic morpho-molecular characters in the secondary and tertiary structures of length-variable regions of the 18S rRNA sequences of the analysed species. The results showed that two LVRs (LVR X and LVR L) of the hypervariable region V4 exhibited significant variability in the number of nucleotides and could be considered for apomorphic recognition.

In this study, 22 terminal taxa were included in the analysis, with 19 forming the ingroup and three as the outgroup (Table S1). The ingroup contained species of the family Tingidae, including three with 18S rDNA complete sequences that were obtained from GenBank (Table S1), and 16 which were newly sequenced in this study (Table S2). Three species of other cimicomorphan families were selected as the outgroup (Table S1). Two of these represented the family Miridae, the sister group of Tingidae within the superfamily Miroidea [1,4,16], and one belonged to the superfamily Naboidea, which is usually considered a sister to Miroidea [4,16].
Taxa names, geographic origins, collector names, University of Opole (Poland) sample numbers (if applicable), and accession numbers for sequences we deposited into GenBank, and those obtained directly from GenBank are provided in Tables S1 and S2.

DNA Extraction
Ethanol-preserved specimens were used for genomic DNA extraction, except for those of Recaredus rex Distant, 1909, which were obtained from dry museum specimens. The total genomic DNA was extracted from the thorax muscle tissues of each species using the DNeasy Tissue Kit (QIAGEN Inc., Santa Clara, CA, USA), following the manufacturer's protocol. The remains of the specimens were then inserted into tubes with 96% ethanol and placed in a deep freezer at the Institute of Biology, University of Opole (for the University of Opole sample numbers, see Table S2).
The PCR reactions were conducted using an Eppendorf Master Thermocycler, following the procedure described by Lis et al. [17], with 36 cycles of denaturation at 93 • C for 1 min, annealing at 59 • C for 1 min and extension at 72 • C for 40 s, with an initial denaturation step of 93 • C for 2 min and a final extension step of 72 • C for 5 min. The quality of the final PCR products was evaluated by 1% agarose gel electrophoresis. The successful samples were purified using the Qiaquick PCR Purification Kit (QIAGEN Inc.) and eluted in 30 µL of elution buffer.
All experimental PCR runs were completed concurrent with those of the negative controls (without templating DNA). Purified amplicons were sequenced in the Health Care Center GENOMED (Warsaw, Poland) with appropriate sequencing primers. The obtained sequences were verified using BLAST searches to certify that the results were not those of contaminants. All newly obtained DNA sequences were deposited in GenBank (OR022068-OR022083), and their accession numbers are provided in Tables S1 and S2.

Phylogenetic Analysis
Sequences were aligned using ClustalW (with default parameters) in the MEGAX software [24] and then truncated at both ends to avoid the influence of missing data from incomplete sequences.
A Maximum Likelihood tree was generated using IQ-TREE [25] on the web server [26] with 10,000 replications of the Ultrafast Bootstrap method [27]. The obtained tree was visualised and edited using the online tool iTOL v5 [28] and prepared for publication with CorelDRAW 21.

Reconstruction of Secondary Structures
The secondary structure of the 18S rRNA for each species was constructed according to the models provided for Heteroptera [15,22,29]. The three hypervariable regions (V2, V4, V7), which are considered crucial for recovering the phylogenetic relationships among higher-level Heteroptera taxa [15,22,29], were analysed.
Thirteen length-variable regions (LVRs) were identified in the heteropteran 18S rRNA secondary structure models [15,22,29], and those which could potentially serve as morphomolecular apomorphies (synapomorphies or autapomorphies) were considered for further analysis: three LVRs (E, F, G) in the V2 region, one (L) in the V4 region, and two (S, T) in the V7 region [15,22,29] (Figure 1).   LVR L was the longest and most variable region, and it presented the most appropriate length-variable region for phylogenetic relationship analyses [15,22,29]. Therefore, it was thoroughly examined, and its secondary structures were predicted using the computer program RNAstructure ver. 6.3 [30]. The three-step procedure described by Lis [15] was applied to the comparative sequence analysis. RNAstructure ver. 6.3 suggested a species that exhibited a secondary structure common to two or more sequences, and this was considered the "consensus species" for these sequences [15].
The hypervariable region numbering, the numbering system for the length-variable regions (LVRs), and the nucleotide numbering of the entire gene sequences followed that of Yu et al. [23], Wu et al. [29], and Lis [15]. Subdividing of the secondary structures of LVR L into subregions was performed according to Lis [15].
LVR L tertiary structures were predicted using RNAComposer (http://rnacomposer. ibch.poznan.pl, accessed on 10 May 2023), which is a fully automated RNA structure modelling server [32,33]. Twenty 3D RNA models were generated for each LVR sequence, and the best model with the lowest free energy was selected for analysis. The tertiary structural images were visualised using PyMol software ver. 2.4.0 [34].
To recognise the apomorphies in the predicted LVR tertiary structures of the analysed 18S rRNA sequences, a two-step procedure recently proposed by Lis [15] was applied. Only tertiary structures which had their distinctness confirmed at the level of secondary structures were considered apomorphic [15].

Sequence Analysis and Tree Topology
The 18S rDNA genes of 16 of the 17 species analysed were successfully amplified and sequenced; only the extraction and amplification of the dry museum specimen of Recaredus rex Distant, 1909 failed (Table S2).
The final 18S rDNA alignment contained 1931 sites. The number of conserved and variable sites were 1536 and 383, respectively, while 211 sites were parsimony-informative, and 172 were singletons. The alignment file used for the phylogenetic analysis is provided in the Supplementary Materials (File S1).
ModelFinder in the IQ-TREE [24] has tested 88 DNA models for this set of sequences, and the TNe + I + G4 substitution model was chosen as the best fit according to the Bayesian Information Criterion. The IQ-TREE generated 98 initial parsimony trees; the ML consensus tree is shown in Figure 2.
Our analyses showed that the tribe Acalyptaini conceived by Golub et al. [9] was a monophyletic group with a node support of 68% ML bootstrap value (MBLv) ( Figure 2). However, the species representing the tribe Tingini formed two independent lineages on the tree, the first including species of the Tingis, Oncochila, and Physatocheila genera, and the second consisted of Stephanitis, Metasalis, Pseudacysta, Corythucha, Copium, Dictyla, and Lasiacantha species.
Nobarnus signatus (Litadeini) was indicated as belonging to the sister group to Acalyptaini + Tingini, and two species of Cantacaderinae were recovered as a strongly supported monophyletic clade with a node support of 99% MBLv. What is essential is that the clade was retrieved as a sister to all species of the subfamily Tinginae. The latter clade (including Acalyptaini, Tingini, and Litadeini) had a ML bootstrap value of 100% ( Figure  2).

Secondary Structure Models
The secondary structure models of the entire 18S rRNA gene were predicted for 15 species, including all consensus species (File S2). The prediction included four species of the tribe Acalyptaini, seven of Tingini, one of Litadeini, one of the subfamily Cantacaderinae, and three species of the outgroup. Although the secondary structure models were similar in their general outlines, local differences were found within certain hypervariable regions (V) and length-variable regions (LVRs) (File S2). The secondary structure model of 18S rRNA of Acalypta sauteri Drake, 1942, showing the positions of these regions, is presented in Figure 1.
The nucleotides in the A. sauteri sequence formed 584 pairs (61.2% of all nucleotides in the secondary structure model), with the standard canonical pairs (G-C and A-U) as the most common (450 pairs, 77.1%). The wobble G:U pairs were approximately six times less commonly formed than the standards between the paired nucleotides (83 pairs, 14.2%). The A:G, A:C and other non-canonical pairs were observed rarely (51 pairs, 8.7%). The number of nucleotides that formed particular pair types varied insignificantly among the studied sequences (i.e., ±1.0-1.5% in the standard canonical, ±1.5-3.0% in the wobble Nobarnus signatus (Litadeini) was indicated as belonging to the sister group to Acalyptaini + Tingini, and two species of Cantacaderinae were recovered as a strongly supported monophyletic clade with a node support of 99% MBLv. What is essential is that the clade was retrieved as a sister to all species of the subfamily Tinginae. The latter clade (including Acalyptaini, Tingini, and Litadeini) had a ML bootstrap value of 100% ( Figure 2).

Secondary Structure Models
The secondary structure models of the entire 18S rRNA gene were predicted for 15 species, including all consensus species (File S2). The prediction included four species of the tribe Acalyptaini, seven of Tingini, one of Litadeini, one of the subfamily Cantacaderinae, and three species of the outgroup. Although the secondary structure models were similar in their general outlines, local differences were found within certain hypervariable regions (V) and length-variable regions (LVRs) (File S2). The secondary structure model of 18S rRNA of Acalypta sauteri Drake, 1942, showing the positions of these regions, is presented in Figure 1.
The nucleotides in the A. sauteri sequence formed 584 pairs (61.2% of all nucleotides in the secondary structure model), with the standard canonical pairs (G-C and A-U) as the most common (450 pairs, 77.1%). The wobble G:U pairs were approximately six times less commonly formed than the standards between the paired nucleotides (83 pairs, 14.2%). The A:G, A:C and other non-canonical pairs were observed rarely (51 pairs, 8.7%). The number of nucleotides that formed particular pair types varied insignificantly among the studied sequences (i.e., ±1.0-1.5% in the standard canonical, ±1.5-3.0% in the wobble G:U, and ±4.5-7.5% in all other non-canonical pairs). This range of variability was consistent with those of previous data for other Heteroptera species [23,29,37]. The alignment results of the entire 18S rRNA sequences analysed (File S1) confirmed the existence of the three hypervariable regions (V2, V4, and V7), which have been described in Heteroptera [15,23,29,37]. The sequence length of the V4 region was highly diverse (299-323 nucleotides), whereas those of the V2 and V7 regions were less variable (198-202 and 90-91 nucleotides, respectively) (File S1, Table 1). However, when considering all analysed species (not only the consensus species), the V4 hypervariable region sequence length (Files S1, Table 1) was stable within the Acalyptaini (at 321 nucleotides) and the Cantacaderinae (at 299 nucleotides). In contrast, this region was highly variable within the Tingini (305-321 nucleotides). The positions of the LVRs within the gene sequences are shown for Acalypta sauteri ( Figure 1) and all analysed consensus species (in File S2). The LVR G, which has been detected in some heteropterans [15,29], was absent in all analysed species. The five LVRs (M, T, U, R, and W) displayed the same number of nucleotides for each region, while six others (B, D, E, F, S, and X) exhibited only insignificant variations in length (one to three nucleotide differences) ( Table 2). All 11 of these LVRs were short (three to thirteen nucleotides). In contrast, LVR L was relatively long (from 57 to 81 nucleotides) and showed distinct variations in the sequence lengths (Table 2). Therefore, as suggested in the recent analyses of the 18S rRNA secondary structures in the heteropteran superfamily Pentatomoidea [15], this region was subdivided into subregions to compare the homologous fragments in analysed sequences (Figures 3 and S1). The number of nucleotides for each subregion resulting from this comparative analysis is provided in Table 3. Two subregions, L2 and LA (LA1 + LA2), showed little variability (a single nucleotide). All remaining subregions were variable, with nucleotide numbers ranging from 19-24 in LB, 7-12 in LC, 10-14 in LD, and 9-16 in LE (Table 3).

Tertiary Structure Models
The tertiary structure models of the entire 18S rRNA genes were predicted for all five consensus species (Figure 4).

Tertiary Structure Models
The tertiary structure models of the entire 18S rRNA genes were predicted for all five consensus species (Figure 4).   When the tertiary structures of the consensus species were aligned ( Figure 5A), the three species, representing the subfamily Tinginae (A. sauteri, N. signatus, and T. matsumurai) appeared comparable ( Figure 5B). This similarity involved the location of the hypervariable regions V2, V4, and V7. However, the general shape of the 18S rRNA tertiary structure for Cantacader lethierryi of the subfamily Cantacaderinae differed significantly, especially when the V7 hypervariable region was considered ( Figure 5C). When the tertiary structures of the consensus species were aligned ( Figure 5A), the three species, representing the subfamily Tinginae (A. sauteri, N. signatus, and T. matsumurai) appeared comparable ( Figure 5B). This similarity involved the location of the hypervariable regions V2, V4, and V7. However, the general shape of the 18S rRNA tertiary structure for Cantacader lethierryi of the subfamily Cantacaderinae differed significantly, especially when the V7 hypervariable region was considered ( Figure 5C). LVR L, which was the longest segment (57 to 81 nucleotides) of the hypervariable region V4, differed in its level of visibility. This region was indistinct and mainly hidden in the core of the entire tertiary structure in Adelphocoris lineolatus (the outgroup) and Cantacader lethierryi (the subfamily Cantacaderinae of Tingidae) ( Figure 6A-B). However, in three consensus species of the subfamily Tinginae (Acalypta sauteri, Nobarnus signatus and Tingis matsumurai), the LVR L was well recognisable (Figure 6C-E).
The predicted tertiary structures of the LVR Ls for all five consensus species are presented in Figures 7 and 8, with all subregions recovered in four of these (A. lineolatus, A. sauteri, N. signatus and T. matsumurai). For C. lethierryi, subregion LB was missing what can be considered its autapomorphy. The other fragments that could serve as morphomolecular autapomorphies are indicated by the arrows that are colour-coded to the particular LVR L subregion. LVR L, which was the longest segment (57 to 81 nucleotides) of the hypervariable region V4, differed in its level of visibility. This region was indistinct and mainly hidden in the core of the entire tertiary structure in Adelphocoris lineolatus (the outgroup) and Cantacader lethierryi (the subfamily Cantacaderinae of Tingidae) ( Figure 6A-B). However, in three consensus species of the subfamily Tinginae (Acalypta sauteri, Nobarnus signatus and Tingis matsumurai), the LVR L was well recognisable (Figure 6C-E).
The predicted tertiary structures of the LVR Ls for all five consensus species are presented in Figures 7 and 8, with all subregions recovered in four of these (A. lineolatus, A. sauteri, N. signatus and T. matsumurai). For C. lethierryi, subregion LB was missing what can be considered its autapomorphy. The other fragments that could serve as morpho-molecular autapomorphies are indicated by the arrows that are colour-coded to the particular LVR L subregion. Insects 2023, 14, x FOR PEER REVIEW 1      the particular subregion, indicate the fragments that can serve as potential morpho-molecular derived characters (autapomorphies). All sequences are aligned to the outgroup (A. lineolatus) sequence. The numbers above the arrows show the autapomorphic number of nucleotides in the subregion.

18S rRNA Secondary and Tertiary Structures of Tingidae
The 18S rRNA secondary structure models predicted for the four Tingidae consensus species were similar (File S2). Differences were identified within certain hypervariable regions (V2, V4 and V7) ( Table 1), especially when modifications in the LVRs were considered (Table 2, Figure 3). Among the LVRs analysed, LVR L appeared the most variable, which corroborates the results of previous studies on 18S rRNA secondary structures in Heteroptera [15,23,29].
Despite similarities among secondary structures of the analysed species, their predicted tertiary structures differed, sometimes substantially (Figures 4 and 5). In particular, the specific tertiary structure of Cantacader lethierryi of the subfamily Cantacaderinae differed significantly from those of all other species representing the subfamily Tinginae (Figures 4-6).
Our results support the recent opinion [15] that probable in vivo tertiary configurations of the 18S rRNA are not predictable using existing software (3dRNA v2.0 Web Server). However, small nucleolar RNAs (snoRNAs) activities could have modified the ribosomal RNA tertiary structures [38][39][40][41][42]. The impact of these activities on such structures has not been identified in heteropteran studies; therefore, further research is required in this area [15].

18S rRNA Secondary and Tertiary Structures of Tingidae
The 18S rRNA secondary structure models predicted for the four Tingidae consensus species were similar (File S2). Differences were identified within certain hypervariable regions (V2, V4 and V7) ( Table 1), especially when modifications in the LVRs were considered (Table 2, Figure 3). Among the LVRs analysed, LVR L appeared the most variable, which corroborates the results of previous studies on 18S rRNA secondary structures in Heteroptera [15,23,29].
Despite similarities among secondary structures of the analysed species, their predicted tertiary structures differed, sometimes substantially (Figures 4 and 5). In particular, the specific tertiary structure of Cantacader lethierryi of the subfamily Cantacaderinae differed significantly from those of all other species representing the subfamily Tinginae (Figures 4-6).
Our results support the recent opinion [15] that probable in vivo tertiary configurations of the 18S rRNA are not predictable using existing software (3dRNA v2.0 Web Server). However, small nucleolar RNAs (snoRNAs) activities could have modified the ribosomal RNA tertiary structures [38][39][40][41][42]. The impact of these activities on such structures has not been identified in heteropteran studies; therefore, further research is required in this area [15].

Potential Apomorphies in Secondary and Tertiary Structures of LVRs
Our analysis demonstrated that the unique nucleotide numbers of two LVRs potentiate these regions as autapomorphies for a particular taxon. LVR X had a unique number of nucleotides (three to four), which is a potential autapomorphy for the tribe Acalyptaini (Table 2, Figure 1). All other analysed species have five to six nucleotides for this LVR (Table 2, File S2).
However, the most effective method for recovering apomorphic morpho-molecular characters was the comparative analyses of LVR L secondary and tertiary structures (Figures 3 and 6-8). These results showed the occurrence of several synapomorphies and autapomorphies related to the number of nucleotides in specific subregions ( Table 3).
The nineteen nucleotides in the LA subregion, eight in the LE subregion, and ten in the LD subregion can be considered synapomorphies for the Acalyptaini, Litadeini and Tingini of the subfamily Tinginae (Table 3). In addition, an equal number of nucleotides (16) in the LE subregion for Acalyptaini and Litadeini could indicate the synapomorphy of these tribes.
Among the analysed taxa, Cantacaderinae had the highest number of autapomorphies (four) in the specific subregions (except for L2 and LA). Tingini had two autapomorphies (in L2 and LB), while a single autapomorphy was revealed for Acalyptaini and Litadeini ( Table 3). The most noteworthy autapomorphy was the absence of the entire subregion LB in the Cantacaderinae; such an extensive deletion in the 18S rDNA sequence has not been recorded in Heteroptera. Strict morpho-molecular autapomorphies in the LVR L tertiary structures were found in four subregions, LB, LC, LD, and L2 (Figures 7 and 8). However, most morpho-molecular autapomorphies involved the LB subregion (Figures 7 and 8) and unique nucleotide numbers for certain tribes (21 for Acalyptaini, 22 for Tingini and 24 for Litadeini). A single morpho-molecular autapomorphy was recovered for the four other subregions (Figures 7 and 8): one in L2 for the Tingini, and one each in LC, LD, and E for Cantacaderinae (Figures 7 and 8). These results indicate that the subfamily Cantacaderinae exhibited the highest number of morpho-molecular autapomorphies in the secondary and tertiary structures of the 18S rRNA. In addition, these results agree with our phylogenomic analysis, which identified the Cantacaderinae as sister to all other Tingidae used by us ( Figure 2). Unfortunately, we could not study any representative of the subfamily Vianaidinae, usually considered the sister group to Tingidae (sensu stricto) (Tinginae + Cantacaderinae), based on morphological analyses [2,43,44].
Because the species of Vianaidinae were never included in any known molecular phylogenetic studies, we must bear in mind that the sister relationship between Cantacaderinae and Tinginae presented here cannot be fully supported without the addition of the Vianaidinae into the molecular phylogenetic analysis of the entire Miroidea.

Identity and Systematic Position of the Tribe Acaltyptaini within the Subfamily Tinginae
Although the tribe Acalyptaini has only recently been validated [9], three of its genera (Acalypta, Dictyonota, and Kalama) have already been included in the first and only phylogenomic analysis of the entire family Tingidae [8]. Five loci were used, including four from mitochondrial genes (COI, Leu-tRNA, COII, and 16S rRNA) and one from a nuclear gene (28S rRNA). That study [8] indicated, among others, that these three genera (Acalypta, Dictyonota, and Kalama) form a monophyletic clade. However, the clade was identified a part of the broadly conceived Tingini [8], which is contrary to our present findings.
Our results confirmed the monophyly of the tribe Acalyptaini, based on an analysis of 18S rDNA sequences, which has not been used in previous phylogenomic studies of Tingidae [8]. We could not include the DNA sequences of Recaredus rex, which was recently included in the tribe Acalyptaini [11], due to the lack of freshly collected specimens. Therefore, placing this genus within the tribe must be based only on morphological characters [11].
In addition, the present study indicated that the tribe Tingini was not monophyletic. The Litadeini, placed within the tribe Tingini by Guilbert et al. [8], was suggested in our study as sister to the clade, which consisted of the Acalyptaini and Tingini. In both molecular analyses [8], in the present study, the tribe Litadeini was represented only by New Caledonian endemic taxa (two genera with three species, and one genus with a single species, respectively). Moreover, the tribe range is considered pantropical [7]. Therefore, all these could be a reason for explaining the different Litadeini position on the phylogenetic tree of Tingidae obtained in both surveys [8] in the present study.

1.
The results of the present molecular analyses (phylogenetic and structural) validated the recognition of the tribe Acalyptaini within the subfamily Tinginae.

2.
The monophyly of the subfamily Cantacaderinae and its basal position within the family Tingidae were indicated, as well as the position of the tribe Litadeini as sister to all other Tinginae. 3.
The structural analysis of the predicted tertiary structures of the entire 18S rRNA confirmed the proposed hypothesis that the in vivo configuration of this gene is likely not predictable using only secondary structure models and existing software. However, this may result from small nucleolar RNAs (snoRNAs) activities that can affect changes in the tertiary structures of ribosomal genes.

4.
The results showed that two LVRs (LVR X and LVR L) of the hypervariable region V4 exhibited significant variability in the number of nucleotides and could be considered for apomorphic recognition. 5.
LVR L appeared to be the most appropriate for phylogenetic relationship analysis within the family Tingidae when considering the secondary and tertiary structure models suitable for identifying morpho-molecular apomorphies. 6.
The subfamily Cantacaderinae exhibited the highest number of morpho-molecular autapomorphies in the secondary and tertiary structures of the 18S rRNA. In particular, the absence of the entire subregion LB in this subfamily is the first example of such an extensive deletion in the 18S rDNA sequence in Heteroptera. 7.
The tertiary structure of the 18S rRNA exhibited evolutionary properties, which were not detectable in the primary or secondary structures. Therefore, the results of rRNA tertiary structure analyses for phylogenetic considerations are promising. Therefore, including the methods of rRNA tertiary structure analyses in the phylogenetic evaluations in other groups of Heteroptera is strongly suggested.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/insects14070600/s1, Table S1 [45,46]: List of specimens used in the phylogenetic analysis, their geographic origin (if provided), GenBank accession numbers, and the sources for the sequences downloaded from GenBank. Table S2: List of specimens with 18S used for extraction and amplification during the present study. Their geographic origin, GenBank accession numbers, University of Opole sample numbers, and names of the persons who provided the specimens for analyses are provided. Table S3: Primers used for PCR amplification and sequencing of the nuclear 18S rDNA gene. File S1: The alignment file of the 18S rDNA dataset used for the phylogenetic analysis. File S2: The predicted secondary structure models of the 18S rRNA gene for analysed consensus species. Figure S1: Funding: This research received no external funding.

Data Availability Statement:
The data presented in this study are available in the article and supplementary material.