Mitochondrial Genomes of the Genus Claassenia (Plecoptera: Perlidae) and Phylogenetic Assignment to Subfamily Perlinae

Mitochondrial genomes of three stoneflies, e.g., Claassenia magna Wu, 1948, Claassenia sp. 2 and Claassenia xucheni Chen, 2019 were sequenced in this study with 15,774, 15,777 and 15,746 bp in length, respectively. Each mitogenome contained 37 genes including 22 tRNAs, two ribosomal RNAs, 13 protein-coding genes (PCGs), and a noncoding control region (CR). In general, standard ATN start and TAN termination codons were evident in the PCGs. Although the dihydrouridine arm was absent in trnSer, the remaining 21 tRNAs displayed the characteristic cloverleaf secondary structure. Stem-loop structures were identified in the CRs of all three mitogenomes, but tandem repeats were only apparent in Claassenia xucheni. The mitogenomes of three Claassenia species were analyzed and compared with mitogenomes in 21 other stoneflies from the Perlidae and three Euholognatha species (Rhopalopsole bulbifera, Capnia zijinshana and Amphinemura longispina) as outgroups. Phylogenetic analyses using maximum likelihood and Bayesian inference. Phylogenetic analysis supported that Claassenia was recovered as the sister group of other Perlinae and Claassenia+Perlinae emerged from the paraphyletic Acroneuriinae. The final results supported that Claassenia was classified into subfamily Perlinae and proposed Claassenia represent a transitional group of the subfamilies Acroneuriinae and Perlinae. This study provided new molecular evidence for exploring the debatable taxonomic position of the genus Claassenia in Perlidae.

Plecoptera (order: Stoneflies) species are hemimetabolous insects, a small order of insects. The Claasseniini is a tribe in Perlidae that includes only the genus Claassenia containing 13 species worldwide [17][18][19]. The genus Claassenia Wu, 1934, was proposed as a replacement for Adelungia Klapálek, 1914 [20,21]. Claassenia was placed in the subfamily Acroneuriinae because of a circular hammer presented at the median near the hind margin of sternum 9, which is an important morphological characteristic of the subfamily Acroneuriinae [20]. Stark and Gaufin moved it to the subfamily Perlinae [22], because of the cleft in tergum 10 that is a synapomorphic characteristic of the subfamily Perlinae. So it is uncertain and debatable to determine the taxonomic status of Claassenia by the morphological characteristics.
Therefore, it is necessary to use mitogenomic evidence to explore the phylogenetic relationship of Claassenia in Perlidae. Up to now, although 78 species of Plecoptera have been sequenced and listed in the NCBI GeneBank, there was only one mitogenome in Claassenia 2 of 12 has been previously reported [23,24]. Chen et al. (2019) proposed that Acroneuriinae + Perlinae was a sister clade to Claassenia, based on 10 Perlidae and two Capniidae species as outgroups [23]. Based on 16 Perlidae species and two Capniidae species as outgroups, Wang et al. (2020) supported that Claassenia was a sister taxon to Acroneuriinae + Perlinae [25]. However, Wang et al. (2020) reported another phylogenetic analysis with 16 species in Perlidae and two species in Taeniopterygidae as outgroups subsequently, this analysis showed that Perlinae and Claassenia were clustered in a clade and emerged from paraphyletic Acroneuriinae [26]. The relationship of Claassenia with Acroneuriinae and Perlinae is still unstable because of the limited mitogenomes. Here, we sequenced three mitogenomes of Claassenia, downloaded almost all the mitogenomes of Perlidae species (including one Claassenia species, nine Acroneuriinae species, 11 Perlinae species) and three Euholognatha species (as outgroups) from GeneBank, these data were used to construct phylogenetic trees based on 13 PCGs to deduce the phylogenetic relationship of the genus Claassenia in Perlidae.

Sample Preparation and DNA Extraction
This study was conducted without harming protected or endangered species and all research activities were authorized. C. magna was collected from Fujian Province, China in May 2021, Claassenia sp. 2 was collected from Tibet, China in July 2020, and C. xucheni was collected from Shaanxi Province, China in May 2021; all specimens were preserved in 100% ethanol and stored at −20 • C. Genomic DNA was extracted from the legs of specimens with the Column mtDNAout Kit (Axygen Biotechnology, Hangzhou, China) as recommended by the manufacturer and stored at −20 • C until used for PCR.

PCR Amplification and Sequencing
Mitochondrial genome was amplified using LA-PCR and continuous specific PCR amplification as the following conditions: perform initial denaturation at 93 • C for 2 min, and then perform 40 cycles at 92 • C for 10 s; annealing at 54 • C for 30 s; and stretching at 68 • C (20 cycles) for 8 min Elongation rate, which increases by 20 s/cycle in the last 20 cycles; the final extension is 10 min at 68 • C. PCR products were purified with Axygen DNA Gel Extraction Kit (Axygen Biotechnology, Hangzhou, China) [16], and quality control was subsequently carried out on the purified DNA samples. The quality of DNA was assessed using qubit3.0 and 1% agarose gel electrophoresis.
High qualified DNA samples were applied to 500-bp paired-end library construction using the NEBNext Ultra DNA Library Prep Kit for Illumina sequencing. Sequencing was carried out on the Illumina NovaSeq 6000 platform (BIOZERON Co., Ltd., Shanghai, China). De novo assembly with GetOrganelle v1.6.4 referencing mitogenome of closely related species produced contigs of mitogenome. A number of potential mitochondrion reads were extracted from the pool of Illumina reads using BLAST searches against mitogenomes of related species and the GetOrganelle results. The mitochondrion Illumina reads were obtained to perform complete mitogenome de novo assembly using the SPAdes-3.13.1 package. The GetOrganelle assembly contig was optimized by the scaffolds from SPAdes-3.13.0 result. Finally, the assembled sequences were reordered and oriented according to the reference mitogenome, thus generating the final assembled mitochondrion genomic sequence (BIOZERON Co., Ltd., Shanghai, China).

Mitogenome Annotation and Base Composition
Mitogenomes of C. magna and Claassenia sp. 2 and C. xucheni were circular DNA molecules consisting of 15,774, 15,777 and 15,746 bp, respectively, which are ranges consistent with mitogenomes in other stoneflies [36]. The three mitogenomes encoded a large noncoding control region and two rRNAs, 22 tRNAs and 13 PCGs. Twenty-three genes (14 tRNAs and nine PCGs) were located on the majority J-strand and 14 genes (two rRNAs, eight tRNAs, and four PCGs) were on the minority N-strand ( Figure 1, Tables S1-S3). The order of genes in the three Claassenia mitogenomes was conserved with other stoneflies and identical to the ancestral mitogenome of Drosophila yakuba [37]. In C. magna, 70 overlapping nucleotides were located in 11 pairs of neighboring genes; whereas 89 overlapping nucleotides were found in 14 pairs of neighboring genes in Claassenia sp.2 and C. xucheni contained 69 nucleotide overlaps with 13 gene pairs.   Table 2). In C. magna, the highest and lowest A+T content was 76.12% In C. magna, Claassenia sp. 2 and C. xucheni, the A+T content was as follows: 61.46%, 65.81% and 62.89% (whole mitogenomes); 59.40%, 64.12% and 60.62% (PCGs); 66.98%, 68.70% and 67.40% (tRNAs); 64.15%, 69.04% and 67.06% (rRNAs); and 71.51%, 74.45% and 73.95% (CRs), respectively (Table 2). In C. magna, the highest and lowest A+T content was 76.12% for trnAsp (D) and trnthr (T) and 53.72% for cox1. In Claassenia sp. 2, the highest and lowest A+T content was 78.26% for trnAsp (D) and 55.55% for trnVal (V); whereas trnthr (T) was 78.46% and cox3 was 55.09% in C. xucheni, respectively (Tables S1-S3). The A+T contents of whole mitogenomes, PCGs, tRNAs, rRNAs and CRs genes in Claassenia sp. 2 were all the highest. This phenomenon may be related to its distribution in the Qinghai Tibet Plateau where the environment was harsh. In view of this phenomenon, it is necessary to collect more specimens from different environments and extract more molecular data for more accurate exploration.

Protein-Coding Genes
The 13 PCGs of the three Claassenia mitogenomes were similar in size and A + T content ( Table 2). The majority of the PCGs in all three mitogenomes initiated with the standard start codon ATN (ATT, ATC, ATA and ATG); however, cox1 in C. magna started with CAA and nad1, nad4 and nad5 used GTG as a start codon. In Claassenia sp. 2, nad1 and nad5 initiated with TTG and GTG, respectively; whereas nad1 started with GTG in C. xucheni (Tables S1-S3). Most PCGs had standard stop codons (TAA or TAG); however, cox2 and nad5 in both C. magna and Claassenia sp. 2 and cox2, nad4, nad5 in C. xucheni contained a truncated termination codon ('T'), which is likely completed by post-transcriptional polyadenylation [38]. Some PCG genes used nonstandard start codons or stop codons, these phenomena are common in Plecoptera [6][7][8].

Transfer RNA Genes
The typical set of 22 tRNA genes was predicted from the three mitogenomes. The lengths of C. magna, Claassenia sp. 2 and C. xucheni tRNA genes were 1493 bp, 1492 bp and 1491 bp, and the A+T content of tRNA genes was 66.98%, 68.70% and 67.40%, respectively ( Table 2). Most tRNAs had a typical cloverleaf secondary structure (Figure 3, Figures S3 and S4); however, in trnSer (AGN), the dihydrouridine (DHU) arm was missing in the three species, which is common in mammals and some insects [39]. The anticodons of the 22 tRNAs in the three Claassenia species were identical to other stoneflies. The tRNAs contained mismatched base pairs, and most of these were G-U pairs (Figure 3, Figures S3 and S4).

Transfer RNA Genes
The typical set of 22 tRNA genes was predicted from the three mitogenomes. The lengths of C. magna, Claassenia sp. 2 and C. xucheni tRNA genes were 1493 bp, 1492 bp and 1491 bp, and the A+T content of tRNA genes was 66.98%, 68.70% and 67.40%, respectively ( Table 2). Most tRNAs had a typical cloverleaf secondary structure (Figures 3,S3 and S4); however, in trnSer (AGN), the dihydrouridine (DHU) arm was missing in the three species, which is common in mammals and some insects [38]. The anticodons of the 22 tRNAs in the three Claassenia species were identical to other stoneflies. The tRNAs contained mismatched base pairs, and most of these were G-U pairs (Figures 3,S3 and S4).

Ribosomal RNA Genes
There were two rRNAs predicted in each mitogenome, and their total length and A+T content were basically similar ( Table 2). Like other Plecoptera species, the two rRNA genes mapped in the conserved location between trnLeu (CUN) and the control region (Figure 1, Figures S1 and S2). The rrnL gene was 1,365 bp with an A+T content of 65.27% in C. magna, 1392 bp with an A+T content of 69.68% in Claassenia sp. 2 and 1371 bp with an A+T content of 67.98% in C. xucheni. Meanwhile, the small ribosomal RNA (rrnS) gene was 836 bp with an A+T content of 62.32% in C. magna, 830 bp with an A+T content of 67.95% in Claassenia sp. 2 and 833 bp with an A+T content of 65.54 % in C. xucheni (Tables S1-S3).

The Non-Coding Control Region
The mitogenome control regions are highly variable with respect to length and nucleotide composition. The A + T content of CR in C. magna, Claassenia sp. 2 and C. xucheni was 71.51%, 74.45% and 73.95%, respectively (Table 2), the differences were not obvious and within the scope of all sequenced stoneflies [36]. The CR in C. magna, Claassenia sp. 2 and C. xucheni mapped between rrnS and trnIle, which is a relatively conserved position in stoneflies (Figure 1, Figures S1 and S2).
The  (Figure 4). The stem-loops structure was a single vertical root, there were (TA) n structure on the left and G (A) nT, GT (A)n, GCAT, CAT, or C (T) nA structures on the right. These SL structures were considered to be related to the initiation of mitogenome replication and transcription [40]. There was only one tandem repeat between 15,378-15,421 bp in C. xucheni, the absence of tandem repeats in the other two species maybe can be accounted for insertion and deletion events, and differences in variable domain length. However, their structural patterns, variations, and functions are still indistinct, although great quantity stonefly CRs data have been provided [16].  were (TA) n structure on the left and G (A) nT, GT (A)n, GCAT, CAT, or C (T) nA structures on the right. These SL structures were considered to be related to the initiation of mitogenome replication and transcription [39]. There was only one tandem repeat between 15,378-15,421 bp in C. xucheni, the absence of tandem repeats in the other two species maybe can be accounted for insertion and deletion events, and differences in variable domain length. However, their structural patterns, variations, and functions are still indistinct, although great quantity stonefly CRs data have been provided [16].

Phylogenetic Analyses
The phylogenetic analyses were constructed by using concatenated sequences of 13 PCGs from 27 stoneflies. These mitogenomes included nine species from subfamily Acroneuriinae, 11 from Perlinae, one Claassenia sp. 1 (GenBank accession no. MN419914), and Claassenia magna, Claassenia sp. 2, and Claassenia xucheni which were sequenced by this study. Three Euholognatha species (Rhopalopsole bulbifera, Capnia zijinshana and Amphinemura longispina) were included as outgroups (Table 1). Tree structures were similar for dendrograms generated by Bayesian inference (BI) and maximum likelihood (ML) analyses, and species grouped with high support values ( Figure 5).
In the two analyses, Claassenia was recovered as the sister group of other Perlinae, they were grouped together and emerged from a paraphyletic Acroneuriinae. So our results supported that the genus Claassenia was classified into subfamily Perlinae at present based on molecular data, this corresponds with the current taxonomic position of Claassenia based on morphological characteristics. In addition, Claassenia was monophyletic in the phylogenetic tree, we proposed that the genus Claassenia was a transitional group of the two subfamilies Acroneuriinae and Perlinae.
However, in the phylogenetic tree, the Acroneuriini species Calineuria stigmatica grouped with the Kiotinini species Perlesta teaysia ( Figure 5). They belong to different tribes, but both of them are distributed in North America and eastern Asia [17]. Maybe it can be explained by animal geography. However, due to the limitations of mitochondrial genes, their relationship is still unclear, more gene sequencing is necessary to explore this problem. the two subfamilies Acroneuriinae and Perlinae.
However, in the phylogenetic tree, the Acroneuriini species Calineuria stigmatica grouped with the Kiotinini species Perlesta teaysia ( Figure 5). They belong to different tribes, but both of them are distributed in North America and eastern Asia [17]. Maybe it can be explained by animal geography. However, due to the limitations of mitochondrial genes, their relationship is still unclear, more gene sequencing is necessary to explore this problem.

Discussion
In this study, we sequenced three Claassenia mitogenomes and downloaded all the data of Perlidae species (except one Togoperla sp.) from GenBank to present phylogenetic analyses. The results showed that Claassenia was monophyletic and grouped with Perlinae. Acroneuriinae was paraphyletic and Perlinae + Claassenia emerged from Acroneuriinae. The final relationship was listed as follows: (Perlinae + Claassenia) + Acroneuriinae ( Figure 5). However, Chen et al (2019) resulted that Acroneuriinae + Perlinae was a sister clade to Claassenia, based on 10 Perlidae and two Capniidae species as outgroups [23]. The difference between our results may come from his use of limited mitogenomic data. Based on 16 Perlidae species and two Capniidae species as outgroups, Wang et al (2020) supported that Claassenia was a sister taxon to Acroneuriinae + Perlinae [25], which was consistent with Chen (2019). Subsequently, Wang et al (2020) reported another phylogenetic analysis with 16 species in Perlidae and two species in Taeniopterygidae as outgroups. This analysis showed that Perlinae and Claassenia were clustered in a clade and emerged from paraphyletic Acroneuriinae [26], this result was consistent with us. The two inconsistent results of Wang may be caused by the different choices of outgroups. The selection of outgroups is very crucial in phylogenetic analysis.
To sum up, the prior assignments of genus Claassenia in the Perlidae were inconsistent based on mitogenomes and uncertain based on morphology due to Claassenia having the identical derived characters of the subfamily Perlinae and the important morphological characteristic of the subfamily Acroneuriinae. Besides, Duran et al (2020) found that mitogenomes were inconsistent with genomic divergences and species-level taxonomy, and consequently, taxon identifications based on mitogenomes (e.g., DNA barcodes) may be misleading [41]. So more molecular evidence other than mitogenomes can be considered for reference and morphological characteristics of more eggs, nymphs and adults of the genus are needed to deduce its relationship with Perlinae and Acroeuriinae.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/genes12121986/s1, Table S1: Organization of the C. magna mitogenome; Table S2: Organization of the Claassenia sp. 2 mitogenome; Table S3: Organization of the C. xucheni mitogenome; Figure S1: Mitochondrial maps of Claassenia sp. 2. Genes outside the map are transcribed clockwise, whereas genes inside the map are transcribed counterclockwise. The interior circles show GC content and the GC skew, and these are plotted as the deviation from the average value of the entire sequence; Figure S2: Mitochondrial maps of C. xucheni. Genes outside the map are transcribed clockwise, whereas genes inside the map are transcribed counterclockwise. The interior circles show GC content and the GC skew, and these are plotted as the deviation from the average value of the entire sequence; Figure   Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data will be available on reasonable request.