Research Article
Research Article
Two complete mitochondrial genomes of the subfamily Chloroperlinae (Plecoptera: Chloroperlidae) and their phylogenetic implications
expand article infoRao-Rao Mo, Ying Wang§, Jin-Jun Cao§, Guo-Quan Wang, Wei-Hai Li§, Dávid Murányi|
‡ Guangxi University, Nanning, China
§ Henan Institute of Science and Technology, Xinxiang, China
| Eszterházy Károly Catholic University, Eger, Hungary
Open Access


Two new complete mitochondrial genomes (mitogenomes) of the subfamily Chloroperlinae, Haploperla japonica Kohno, 1946 and Sweltsa sp., were sequenced. The two species showed similar gene order, nucleotide composition, and codon usage. The Sweltsa sp. and H. japonica mitogenomes were typical circular DNA molecules, with lengths of 15,893 bp and 16,012 bp, respectively. Standard ATN start and TAN stop codons were present in most PCGs. All tRNA genes exhibited the cloverleaf secondary structure typical for metazoans except the tRNASer(AGN), which lacked the dihydrouridine arm. In both species, the secondary structure of lrRNA contained five structural domains, while the srRNA included three domains. The A+T-rich regions contained different repeat regions in each species. Phylogenetic analyses using Bayesian inference (BI) and maximum likelihood methods (ML) showed identical results. The family Chloroperlidae was sister to the Perlodidae. Our analyses inferred relationships between six of the seven Systellognatha families: (((Chloroperlidae + Perlodidae) + Perlidae) + (Styloperlidae + Pteronarcyidae)) + Peltoperlidae.


Chloroperlinae, Haploperla, mitochondrial genomes, phylogenetics, Plecoptera, Sweltsa

1. Introduction

The mitochondrial genome (mitogenome) of metazoans is 14 to 20 kb in size, and usually a circular, double-stranded molecule. It contains 13 protein coding (PCGs), 2 ribosomal RNA (rRNA) and 22 transfer RNA (tRNA) (altogether 37) genes, and an A+T-rich region which is known as the control (CR) or non-coding region (Wolstenholme 1992; Boore 1999; Gissi et al. 2008). The Plecoptera (stoneflies) constitutes an ancient order of hemimetabolous winged insects. Stoneflies include 308 existing genera in 17 families, including approximately 4,000 valid species worldwide (DeWalt et al. 2021). Chloroperlidae is one of the smaller families, with two subfamilies: Chloroperlinae Okamoto, 1912 and Paraperlinae Ricker, 1943. In the Chloroperlinae, there are three tribes and 206 described species, while in the Paraperlinae, only two genera and six species are recorded (Alexander and Stewart 1999; Judson and Nelson 2012; Chen and Du 2015a; Li et al. 2015a, 2015b). In the present study, Haploperla japonica and Sweltsa sp. were analysed; both belong to Chloroperlinae. Zwick (2000) proposed a higher classification for the world fauna of Plecoptera, which is the current generally accepted system. However, the positions of four families (Chloroperlidae, Perlidae, Perlodidae, Notonemouridae) were still not clear (Zwick 2000). So far, the phylogenetic position of Plecoptera within the Neoptera, and phylogenetic relationships among some Plecoptera families have remained controversial, and more molecular data are needed to resolve them (Stewart and Stark 1988; Uchida and Isobe 1989; Terry and Whiting 2003; Chen et al. 2016, 2018; Wang et al. 2016, 2017a, 2019; Chen and Du 2017a, 2017b; Ding et al. 2019; Wipfler et al. 2019; South et al. 2020, 2021; Cao et al. 2021). In the superfamily Perloidea, the affinities of three families: Chloroperlidae, Perlidae and Perlodidae, have been proposed as a trichotomy (Nelson 1984; Zwick 2000), while Illies (1965) proposed Perlidae as sister to Chloroperlidae + Perlodidae. Most molecular data strongly supported the latter classification (Chen et al. 2016, 2018; Wang et al. 2016, 2017b, 2019; Chen and Du 2017b, 2017c; Ding et al. 2019; South et al. 2020, 2021; Cao et al. 2021).

At present, only three chloroperlid species, Sweltsa longistyla (Wu, 1938), Suwallia bimaculata (Okamoto, 1912) and Suwallia errata Li and Li, 2021, have been completely sequenced for mitogenomes (Chen and Du 2015b; Wang et al. 2018a; Cao et al. 2019a; Li et al. 2021). This limited mitogenomic data hinders the reconstruction of accurate phylogeny (Wu et al. 2014). In the present study, two complete mitogenomes from two genera, Haploperla and Sweltsa, were sequenced. Nucleotide composition, codon usage, RNA secondary structure, the evolutionary pattern among the PCGs, and structural elements within the control regions were analysed. Furthermore, a phylogenetic tree of the infraorder Systellognatha, excluding the family Kathroperlidae was also reconstructed based on the aligned PCGs using Bayesian inference (BI) and Maximum likelihood (ML) analyses.

2. Material and Methods

2.1. Specimen sample and DNA extraction

Adult specimens of Haploperla japonica were collected from Yoshino River, Minocho, Miyoshi City, Tokushima Prefecture, Japan, and adult samples of Sweltsa sp. were collected from a forested stream along the road No.328, Kasatori Mountain, Kumakogen, Ehime Prefecture, Japan. Species-level identification of Sweltsa species is unsure, due to taxonomic problems in Japanese representatives of the genus (Shimizu et al. 2005). All specimens were fixed in 100% ethanol and stored at –20°C. The examined specimens of H. japonica (No. VHem-0114) and Sweltsa sp. (No. VHem-0116) are deposited in the Entomological Museum of Henan Institute of Science and Technology (HIST), Henan Province, China. The full genomic DNA was isolated from thoracic muscle using the QIAamp DNA Blood Mini Kit (Qiagen, Germany) and maintained at –20°C prior to PCR analysis.

2.2. Genome sequencing, assembly and annotation

The full mitogenomes of both Chloroperlidae species were amplified and sequenced as described in previous studies (Li et al. 2017; Wang et al. 2017a, 2017b; Liu et al. 2018) and uploaded in GenBank with the accession numbers: OL351265 (Haploperla japonica) and OL351266 (Sweltsa sp.) (Table 1). The program BioEdit version was used to assemble raw sequences into contigs (Hall 1999). tRNA genes were identified with the software ARWEN using default setting (Laslett and Canbäck 2008). PCG and two rRNA genes were identified using BLAST searches in NCBI (, and confirmed by alignments of homologous genes from previously published stonefly mitogenomes. MEGA 5.0 was used to calculate nucleotide composition and codon usage for PCGs (Tamura et al. 2011). Strand asymmetry was measured by the following formulas: AT skew= [A−T]/ [A+T] and GC skew= [G−C]/ [G+C] (Perna and Kocher 1995). Tandem repeats in the A+T-rich region were identified using Tandem Repeats Finder (

Table 1.

Classification of all Plecoptera species with sequenced mitogenomes, their genome lengths and accession numbers at Genbank.

Infraorder Superfamily Family Subfamily Species Number (bp) Accession Number
Systellognatha Perloidea Chloroperlidae Chloroperlinae Haploperla japonica 16,012 OL351265
Sweltsa sp. 15,893 OL351266
Sweltsa longistyla 16,151 KM216826
Suwallia errata 16,146 MF198253
Suwallia bimaculata 16,125 MN121757
Perlidae Acroneuriinae Acroneuria hainana 15,804 NC_026104
Acroneuria carolinensis 15,718 MN969989
Caroperla siveci 15,353 MG677942
Calineuria stigmatica 15,070 MG677941*
Flavoperla hatakeyamae 15,730 MN821010
Flavoperla sp. YW-2019 15,796 MN419916
Flavoperla sp. YZD-2020 15,805 MK905206*
Niponiella limbatella 15,924 MK686067
Perlesta teaysia 16,023 MN627432
Sinacroneuria dabieshana 15,752 MK492253
Perlinae Claassenia sp. 15,774 MN419914
Dinocras cephalotes 15,666 NC_022843
Etrocorema hochii 15,854 MK905888
Kamimuria chungnanshana 15,943 NC_028076
Kamimuria klapaleki 16,077 MN400755
Kamimuria wangi 16,179 NC_024033
Neoperlops gressitti 15,699 MN400756
Paragnetina indentata 15,885 MN627431
Neoperla sp. 15,667 KX091859*
Neoperla ignacsiveci 15,777 KX091858
Togoperla limbata 15,915 MN969990
Togoperla sp. 15,723 KM409708
Perlodidae Isoperlinae Isoperla bilineata 15,048 MF716959
Isoperla eximia 16,034 MG910457
Perlodinae Perlodes sp. 16,039 MF197377
Pseudomegarcys japonica 16,067 MG910458
Pteronarcyoidea Peltoperlidae Peltoperlinae Cryptoperla stilifera 15,633 KC952026*
Peltoperlopsis cebuano 15,790 MK387068
Soliperla sp. 15,877 MF716958
Microperlinae Microperla geei 15,216 MN096323
Pteronarcyidae Pteronarcys princeps 16,004 NC_006133
Pteronarcella badia 15,585 NC_029248
Styloperlidae Styloperla sp. 15,416 KR088971*
Styloperla spinicercia 16,129 KX845569
Cerconychia flectospina 15,188 MF100783*
Euholognatha Nemouroidea (Outgroup) Nemouridae Amphinemurinae Amphinemura yao 15,876 MH085447
Amphinemura longispina 15,709 MH085446
*Incomplete genome sequence

2.3. Phylogenetic analysis

We used forty previously published and the two newly sequenced mitogenomes for phylogenetic analyses (Table 1). Two species from the family Nemouridae, Amphinemura yao Mo, Yang, Wang and Li, 2017 and A. longispina (Okamoto, 1922), were used as outgroup taxa (Table 1). Each PCG was aligned separately using the MAFFT algorithm implemented the TranslatorX online platform (Abascal et al. 2010). Poorly aligned regions were masked from the protein alignment before back-translation to nucleotides using GBlocks (implemented in the TranslatorX). Each rRNA gene was aligned separately by MAFFT v7.0 using G-INS-I strategy (Katoh and Standley 2013), and ambiguously aligned positions masked by GBlocks v0.91b (Castresana 2000).

We assembled the “PCG13 matrix” (11,049 bp in total) for the phylogenetic analyses, including 13 PCGs. GTR+I+G was the best-fit model for the nucleotide sequence alignments (jModelTest 0.1.1, Posada 2008). Bayesian analyses were conducted on the PCG13 dataset partitioned by gene was conducted Bayesian inference (BI), using MrBayes 3.2.6 and maximum likelihood (ML) using RAxML-HPC2 8.1.11 (Stamatakis 2006; Ronquist et al. 2012). For the BI analyses, two simultaneous runs of 10 million generations were conducted, sampling every 1,000 generations and a burn-in rate of 25%. Stationarity was assessed using Tracer v.1.5 (Estimated sample size > 200). Bootstrap ML analyses of 1,000 replicates was performed with the fast ML method conducted in RAxML using the GTRGAMMA model for nucleotide sequences.

3. Results and Discussion

3.1. General characters of mitogenomes and the base composition

In this study, the full mitogenomes of two stonefly species in the subfamily Chloroperlinae: Haploperla japonica and Sweltsa sp., were studied for the first time (Table 2, Fig. 1). The new complete mitogenomes were 15,893 (Sweltsa sp.) and 16,012 bp (H. japonica) in length (Fig. 1, Table 2). Genome sizes of these mitogenomes were in the middle of the range compared to mitogenomes of other stoneflies, that range from 15,048 bp (Isoperla bilineata) (Chen et al. 2018) to 16,602 bp (Nemoura nankinensis) (Chen and Du 2017a). Control region variation is the primary factor influencing size variation in Plecoptera mitogenomes (Table 2). The typical gene content found in most insects were found in these species: 37 mitochondrial genes (13PCGs, 22tRNAs and 2 rRNAs) and a large control region (Fig. 1). Twenty-three genes (9 PCGs and 14 tRNAs) were transcribed on the majority strand (J-strand), and the remaining fourteen genes (4 PCGs, 8 tRNAs, and 2 rRNAs) were encoded on the minority strand (N-strand) (Fig. 1).

Figure 1. 

Map of the mitogenomes of Haploperla japonica and Sweltsa sp. Direction of gene transcription is indicated by the arrows. PCGs are shown as blue arrows, rRNA genes as purple arrows, tRNA genes as red arrows and CR as gray arrows. tRNA genes are labeled according to single-letter IUPAC-IUB abbreviations (L1: UUR, L2: CUN, S1: AGN, S2: UCN). The GC content is plotted using a black sliding window, as the deviation from the average GC content of the entire sequence. GC skew is plotted as the deviation from the average GC skew of the entire sequence.

Table 2.

The size of nucleotide composition of the Haploperla japonica and Sweltsa sp. mitogenomes.

Family Subfamily Species Genome PCGs tRNAs lrRNA srRNA Control region
Chloroperlidae Chloroperlinae Haploperla japonica 16012 11247 1483 1324 776 1118
Sweltsa sp. 15893 11244 1488 1325 799 1011

Overlap between gene regions ranged from one to eight bp in length. Both species had seven bp overlaps in (ATP6–ATP8) and (ND4ND4L), “ATGATAA” and “TTAACAT”, respectively. This phenomenon was also found in the mitogenome of many insects (Yang et al. 2017; Cao et al. 2019a). An intergenic spacer sequence was found to be located between tRNALeu(UUR) and COII in the two species, ranging from 12 bp in Sweltsa sp. to 31 bp in Haploperla japonica (Table 3).

Table 3.

Mitogenome structures of Haploperla japonica and Sweltsa sp.

Gene Haploperla japonica Sweltsa sp.
Direction Location Size IGN Location Size IGN
tRNAIle F 1–67 67 1–67 67
tRNAGln R 65–133 69 –3 65–133 69 –3
tRNAMet F 139–207 69 5 136–204 69 2
ND2 F 208–1242 1035 0 205–1239 1035 0
tRNATrp F 1241–1308 68 –2 1238–1305 68 –2
tRNACys R 1301–1367 67 –8 1298–1365 68 –8
tRNATyr R 1368–1433 66 0 1366–1433 68 0
COI F 1426–2982 1557 –8 1426–2982 1557 –8
tRNALeu(UUR) F 3003–3068 66 20 2995–3060 66 12
COII F 3100–3783 688 31 3073–3760 688 12
tRNALys F 3788–3858 71 0 3761–3831 71 0
tRNAAsp F 3858–3926 69 –1 3831–3899 69 –1
ATP8 F 3927–4088 162 0 3900–4058 159 0
ATP6 F 4082–4759 678 –7 4052–4729 678 –7
COIII F 4759–5547 789 –1 4729–5517 789 –1
tRNAGly F 5550–5615 66 2 5517–5582 66 2
ND3 F 5616–5969 354 0 5583–5936 354 0
tRNAAla F 5968–6033 66 –2 5935–6000 66 –2
tRNAArg F 6034–6099 66 0 6001–6066 66 0
tRNAAsn F 6103–6168 66 3 6071–6136 66 4
tRNASer(AGN) F 6169–6235 67 0 6137–6203 67 0
tRNAGlu F 6236–6301 66 0 6204–6269 66 0
tRNAPhe R 6316–6380 65 14 6275–6339 65 5
ND5 R 6381–8115 1735 0 6340–8074 1735 0
tRNAHis R 8116–8182 67 0 8075–8142 68 0
ND4 R 8187–9527 1341 4 8148–9488 1341 5
ND4L R 9521–9817 297 –7 9482–9778 297 –7
tRNAThr F 9820–9886 67 2 9781–9848 68 2
tRNAPro R 9888–9955 68 1 9850–9917 68 1
ND6 F 9957–10481 525 1 9919–10443 525 1
CytB F 10481–11617 1137 –1 10443–11579 1137 –1
tRNASer(UCN) F 11616–11685 70 –2 11579–11648 70 –1
ND1 R 11706–12656 951 20 11670–12620 951 21
tRNALeu(CUN) R 12658–12723 66 1 12622–12687 66 1
lrRNA R 12724–14047 1324 0 12688–14012 1325 0
tRNAVal R 14048–14118 71 0 14013–14083 71 0
srRNA R 14119–14894 776 0 14084–14882 799 0
A+T-rich region 14895–16012 1118 0 14883–15893 1011 0

3.2. Nucleotide composition

The A+T content in the Haploperla japonica and the Sweltsa sp. mitogenome is detailed in Table 4. The A+T content of Sweltsa sp. is similar to the base compositional biases reported for other chloroperlid species, that range from 66.7% (Suwallia talalajensis Zhiltzova, 1976) to 69.6% (Sweltsa longistyla (Wu, 1938)) (Chen and Du 2015b; Wang et al. 2018a), whereas A+T content in H. japonica is larger than other chloroperlids. The four major genome partitions (PCGs, tRNAs, rRNAs, and CR) and the whole mitogenome of both Chloroperlinae species were distinctly biased towards A and T. Among the four major partitions across Chloroperlinae species, PCGs consistently had the lowest A+T content, while the control region had the highest. A+T content of PCGs encoded on the minority strand (PCGs–N) was also higher than that of PCGs encoded on the majority strand (PCGs–J) (Table 4).

Table 4.

The nucleotide composition of the Haploperla japonica and Sweltsa sp. mitogenomes.

Region H. japonica Sweltsa sp.
A+T% 69.7 68.2
AT-skew 0.065 0.087
GC-skew –0.223 –0.254
PCGs A+T% 68.2 66.5
AT-skew –0.173 –0.164
GC-skew –0.009 –0.047
PCGs–J A+T% 66.4 64.5
AT-skew –0.096 –0.063
GC-skew –0.179 –0.235
PCGs–N A+T% 71.0 69.6
AT-skew –0.289 –0.312
GC-skew 0.307 0.305
tRNAs A+T% 69.3 68.0
AT-skew –0.007 –0.022
GC-skew 0.083 0.109
rRNAs A+T% 72.4 72.1
AT-skew –0.100 –0.119
GC-skew 0.283 0.318
CR A+T% 80.6 80.0
AT-skew 0.083 0.068
GC-skew –0.235 –0.257

Both Chloroperlinae species showed positive AT-skew and negative GC-skew for the whole mitogenome. Total mitogenome of Haploperla japonica, the AT-skew and GC-skew were 0.065 and –0.223, respectively, whereas in Sweltsa sp., the AT-skew and GC-skew were 0.087 and –0.254, respectively. Slight T-skews and moderate C-skews were found in the majority strand PCGs while the reverse phenomenon was found for minority strand PCGs: both T-skews and G-skews were marked. For tRNAs and rRNAs, G-skews varied from slight to marked, while T-skews were slight to moderate. Modest A-skew and marked C-skew were found in the CR (Table 4).

For the majority strand PCGs, most known metazoan mitogenomes have positive AT-skew and negative GC-skew (Wei et al. 2010a). However, both Chloroperlinae species had negative AT-skews and negative GC-skews for the majority strand PCGs (Table 4). This difference has been noted for other published stonefly mitogenomes (Wu et al. 2014; Chen and Du 2017a, 2017c; Wang et al. 2017a, 2017b), as well as in other insects such as Philopteridae (bird lice), Aleyrodidae (whiteflies) and Braconidae (wasps); this phenomenon may be a result from replication direction charges (Wei et al. 2010a). For both Chloroperlinae species, the four major partitions (PCGs, tRNAs, rRNAs and CR) all had the same trends in the A+T content, AT and GC-skews which displayed similar patterns and were consistent with the common base composition biases of insect mitogenomes.

3.3. Protein-coding genes and codon usage

The total length of PCGs in the studied mitogenomes was 11,247 bp (Haploperla japonica) and 11,244 bp (Sweltsa sp.) (Table 2). In both mitogenomes from Chloroperlinae, typical start codons ATN (Met/Ile) were the most commonly used start codons. Additionally, TTG was inferred as a start codon for ND1, and GTG as the start codon for ND5 in both species. This phenomenon has been found in other stoneflies, and TTG is used in other insect species (Bae et al. 2004; James and Andrew 2006; Sheffield et al. 2008; Wei et al. 2010b; Elbrecht and Leese 2015; Elbrecht et al. 2015; Huang et al. 2015; Sproul et al. 2015; Chen et al. 2016; Chen and Du 2017c; Wang et al. 2017a, 2017b; Cao et al. 2019b). In both species, ATP6, ATP8, COII, COIII, CYTB, ND2, ND4 and ND4L started with ATG (Met), while COI, ND3 and ND6 started with ATT (Ile) (Table 5).

The most commonly used stop codon in both Chloroperlinae species was TAA, found in ATP6, ATP8, COI, COIII, ND2, ND4, ND4L and ND6. In both species, the stop codon TAG was used by CYTB, ND1 and ND3 (except CYTB of Sweltsa sp., which used TAA). COII and ND5 used a partial stop codon T in both species (Table 5). This phenomenon has also been recorded from other published stoneflies (James and Andrew 2006; Qian et al. 2014; Elbrecht and Leese 2015; Elbrecht et al. 2015; Huang et al. 2015; Wu et al. 2014; Chen et al. 2016; Chen and Du 2017c; Wang et al. 2017a, 2017b; Cao et al. 2019b). Truncated stop codons occur in many arthropod mitogenomes, and it is corrected by post-transcriptional polyadenylation (Ojala et al. 1981; Wolstenholme 1992). The two species had the same start and stop codons in ten genes (COI, COII, COIII, ATP8, ATP6, ND1, ND2, ND4, ND4L and ND5), while the remaining three genes (ND3, ND6 and CYTB) only shared either the same start or stop codon (Table 5).

Table 5.

The start and stop codons of the Haploperla japonica and Sweltsa sp. mitogenomes.

Start Stop Start Stop Start Stop Start Stop Start Stop Start Stop Start Stop
Species ND5 ND4 ND4L ND6 CYTB ND1
Start Stop Start Stop Start Stop Start Start Start Stop Start Stop

The genome-wide AT bias is reflected in codon usage. RSCU (relative synonymous codon usage) was calculated to identify the predominant synonymous codon (Grantham et al. 1980; Meganathan et al. 2012) (Fig. 2). The high AT base compositional biases was shown in the RSCU statistics for both Chloroperlinae mitogenomes. Codons ending with an A or U were favoured both four-fold and two-fold degenerate codons. RSCU demonstrates that codon usage patterns of these two stonefly species was consistent with other published Plecoptera species. All most commonly used codons: AAU (Asn), AUU (Ile), UUA (Leu1), AAA (Lys), AUA (Met), UUU (Phe) and UAU (Tyr), were always biased towards A and T at third codon positions (Fig. 2, Table 5).

Figure 2. 

Relative synonymous codon usage (RSCU) in Haploperla japonica and Sweltsa sp. mitogenomes. Codon families are provided on the X-axis.

3.4. Transfer and ribosomal RNAs

In the present study, the tRNA genes in both species ranged from 65 to 71 bp, and comprised 9.26% (1,483 bp in H. japonica) and 9.36% (1,488 bp in Sweltsa sp.) of the complete mitogenomes (Tables 2 and 3). tRNASer(AGN), which could not be detected by ARWEN software, was identified by alignment with homologous genes from other published stoneflies (James and Andrew 2006; Chen et al. 2016; Wang et al. 2017a, 2018a, 2018b; Cao et al. 2019b; Guo et al. 2021). Most tRNAs had the typical cloverleaf structure, except tRNASer(AGN) that lacks the dihydrouridine (DHU) arm that formed a simple loop. In most insect mitogenomes, it is common for tRNASer(AGN) to lack the DHU stem (Cameron and Whiting 2007; Wan et al. 2013). The length of the different nucleotides of tRNAs between H. japonica and Sweltsa sp. ranged from 1 bp (tRNASer(AGN), tRNASer(UCN), tRNAVal) to 13 bp (tRNAHis). By comparison, the tRNACys and tRNAHis had the most variation with more than ten indels or substitutions; the nucleotide insertion-deletion appears in tRNACys, tRNAHis, tRNAPhe, tRNAThr, tRNATyr (Fig. 3).

Based on the secondary structures of tRNA, eight mismatched base pairs are found in H. japonica, which are U-U (2 bp), A-A (3 bp) and A-C (3 bp) (Fig. 3). And they have 24 G-U pairs, 7 located in the AA, 7 in the DHU, 8 in the AC and 2 in the TΨC stem. In Sweltsa sp., there are nine mismatches in tRNAs: U-U (1 bp), A-A (5 bp), C-C (1 bp) and A-C (2 bp) (Fig. 3); the 29 G-U pairs are located in the AA (5 bp), the DHU (8 bp), the AC (12 bp) and the TΨC stem (4 bp).

Figure 3. 

Secondary structures of 22 tRNAs of Haploperla japonica and Sweltsa sp. All tRNAs are labeled with the abbreviations of their corresponding amino acids. Dashes (–) indicate Watson-Crick base pairing and dots (•) indicate G-U base pairing. Both species are represented in gray; blue circle only on H. japonica, yellow only on Sweltsa sp.; green means not on H. japonica, red means not on Sweltsa sp.

Similar to the mitogenomes of most other insects, the large and small rRNA subunits (lrRNA and srRNA) in both species were located between tRNALeu(CUN)tRNAVal (lrRNA) and tRNAVal – the control region (srRNA) (Fig. 1, Table 3). The lengths of lrRNA and srRNA of Haploperla japonica were 1324 bp and 776 bp, and the lengths of lrRNA and srRNA of Sweltsa sp. were 1325 bp and 799 bp (Table 2). Secondary structures of both lrRNA and srRNA were highly similar between these species (Pair-wise sequence identity was 87.82% (lrRNA) and 87.98% (srRNA)).

Haploperla japonica was used as model for comparison of rRNA secondary structures. lrRNA consists of 5 structural domains (I–II, IV–VI), while domain III was absent, as usual in arthropods (Fig. 4) (Cannone et al. 2002). Domains I, II and IV were variable between these species, while eight helices (H2064, H2347, H2395, H2455, H2507, H2520, H2547 and H2588) within domain V had high similarity (Wang et al. 2017a).

srRNA consists of three domains (Fig. 5). Domains I and II were more variable than domain III in both species, while Helix 1399 region was the most conserved (Wang et al. 2017a). Furthermore, Sweltsa sp. possessed 21 more bp nucleotides in Helix 577 than Haploperla japonica.

Figure 4. 

Predicted secondary structure of the lrRNA in Haploperla japonica and Sweltsa sp. Roman numerals represent the conserved domain structures. Dashes (–) indicate Watson-Crick base pairings and dots (•) indicate G-U base pairing. Different nucleotides of the two species are shown in red. Missing nucleotides in H. japonica are indicated by red arrows, missing nucleotides in Sweltsa sp. are shown in blue.

Figure 5. 

Predicted secondary structure of the srRNA in Haploperla japonica and Sweltsa sp. Roman numerals denote the conserved domain structure. Dashes (–) indicate Watson-Crick base pairing and dots (•) indicate G-U base pairing. Different nucleotides of the two species are shown in red. Missing nucleotides in H. japonica are indicated by red arrows, missing nucleotides in Sweltsa sp. are shown in blue.

3.5. The control region

In a previous study, the A+T-rich region (CR) was found to contain essential elements for the initiation of transcription and for replication (Zhang and Hewitt 1997). Haploperla japonica mitogenome had an A+T-rich region of 1,118 bp, with an A+T content of 80.6%; while Sweltsa sp. it was 1,011 bp long, with an A+T content of 80.0% (Tables 2 and 4). The A+T-rich region of both species is located between the srRNA and tRNAIle gene cluster (Fig. 1). The following structural elements were found in the control region of H. japonica and Sweltsa sp. (Fig. 6): (1) a leading sequence (588 bp (H. japonica), 496 bp (Sweltsa sp.)) adjacent to srRNA; (2) two large tandem repeats (TRs), while (3) the remaining control region was 321 bp (H. japonica) and 372 bp (Sweltsa sp.).

3.6. Phylogenetic relationships

Phylogenetic relationships were reconstructed for the clade Systellognatha using 42 species: 36 complete (including the two species herein studied) and 6 nearly complete mitogenome sequences (Table 1). Amphinemura yao Mo, Yang, Wang and Li, 2017 and A. longispina (Okamoto, 1922) were used as outgroup taxa. Bayesian inference (BI) and maximum likelihood (ML) analyses, of the aligned 13 PCGs dataset, generated trees with identical topologies (Fig. 7).

Figure 6. 

Structure elements found in the control region of Haploperla japonica and Sweltsa sp.

Figure 7. 

Phylogenetic tree inferred from the sequences of the mitogenomes of 40 Systellognatha species and 2 outgroup species. Numbers at the nodes are Bayesian posterior probabilities (left) and ML bootstrap values (right).

Systellognatha includes seven families: superfamily Perloidea with Chloroperlidae, Kathroperlidae, Perlidae, Perlodidae; and superfamily Pteronarcyoidea with Peltoperlidae, Pteronarcyidae, Styloperlidae; all except Kathroperlidae (no mitogenomes yet sequenced) were represented in our study. We obtained all families as monophyletic (colors in Fig. 7). Our analyses strongly supported Peltoperlidae as the sister group of a clade containing the five other families. Similar relationships were recovered in previous phylogenetic analyses (Chen et al. 2018; South et al. 2020, 2021; Cao et al. 2021). A sister-group relationship between Perloidea (monophyletic herein: Fig. 7) and Pteronarcyoidea was not supported, as the latter was obtained as paraphyletic, which is inconsistent with morphological hypotheses (Zwick 2000). Phylogenetic analyses based on morphological characters proposed a clade Peltoperlidae + Styloperlidae, with Pteronarcyidae as their sister group (Zwick 2000). According to the present studies, Styloperlidae is sister to Pteronarcyidae rather than Peltoperlidae albeit with poor nodal support (as in Ding et al. 2019 Wang et al. 2019; Cao et al. 2019a; 2021). Perlidae was sister to Chloroperlidae + Perlodidae with high support (Fig. 7) (confirming Ricker 1952; Illies 1965; Chen et al. 2018; Wang et al. 2019, 2018c; South et al. 2020, 2021). Our analyses found relationships within Systellognatha to be (((Chloroperlidae + Perlodidae) + Perlidae) + (Styloperlidae + Pteronarcyidae)) + Peltoperlidae (Fig. 7). Recent phylogenetic analyses based on mitochondrial data have inferred the same topology (Chen et al. 2018; Cao et al. 2021), however, our results differ from the older morphology-based classification. The different results may be due to the limited mitogenomic data yet published about the Systellognatha, being especially scarce in Pteronarcyidae and Styloperlidae, and lacking in Kathroperlidae.

At the generic level, one pattern is the disjunct phylogenetic positions for Asian and North American members of the same genus (Fig. 7). For example, regarding Isoperla Banks, 1906, Pseudomegarcys japonica Kohno, 1946 was sister to the Asian I. eximia Zapekina-Dulkeit, 1975, rather than the North American I. bilineata (Say, 1823), the type species of genus Isoperla. Additionally, regarding Acroneuria Pictet, 1841, Sinacroneuria dabieshana Li and Murányi, 2014 was sister to the Asian species A. hainana Wu, 1938, rather than the North American A. carolinensis (Banks, 1905). Regarding their morphology, both Pseudomegarcys and Sinacroneuria are rather distinctive genera, in addition, Pseudomegarcys belongs to the subfamily Perlodinae Klapálek, 1909, while Isoperla belongs to Isoperlinae Frison, 1942. Inclusion of these genera in Isoperla and Acroneuria, respectively, is not likely, and additional molecular studies are needed to clarify their phylogenetic position.

4. Competing Interests

The authors have declared that no conflict of interest exists.

5. Acknowledgments

The research was partially supported by the National Natural Science Foundation of China (No. 31970402), the Graduate Education Innovation Training Base Project of Henan Province in 2021 (107020221005) and the Program for Science & Technology Innovation Talents in Universities of Henan Province (No. 21HASTIT042).

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