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Article

Phylogenetic and Comparative Analysis of Cryptochironomus, Demicryptochironomus and Harnischia Inferred from Mitogenomes (Diptera: Chironomidae)

by
Wenbin Liu
,
Chengyan Wang
,
Jingyuan Wang
,
Yaning Tang
,
Wenxuan Pei
,
Xinyu Ge
and
Chuncai Yan
*
Tianjin Key Laboratory of Conservation and Utilization of Animal Diversity, Tianjin Normal University, Tianjin 300387, China
*
Author to whom correspondence should be addressed.
Insects 2024, 15(9), 642; https://doi.org/10.3390/insects15090642
Submission received: 17 July 2024 / Revised: 23 August 2024 / Accepted: 23 August 2024 / Published: 26 August 2024
(This article belongs to the Section Insect Systematics, Phylogeny and Evolution)
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Abstract

:

Simple Summary

Chironomidae is ecologically an important family within the order Diptera. With the rapid advances in next-generation sequencing technologies, an increasing amount of mitochondrial genome data for chironomids has been published. The phylogenetic relationships between the three relatively large and morphologically similar genera within the Harnischia generic complex, namely Cryptochironomus, Demicryptochironomus, and Harnischia, have yet to be thoroughly investigated. The comprehensive mitotic genomes of 11 species within this generic complex were sequenced. Our findings indicate that the monophyly of the genus Harnischia is strongly supported across all topologies, revealing that Cryptochironomus is a sister taxon to Demicryptochironomus. This represents a new insight into the systematic study of the Harnischia generic complex.

Abstract

(1) Background: Mitochondrial genomes have been extensively employed as a crucial marker in numerous dipteran families for understanding phylogenetics and systematics relations, thereby playing a pivotal role in molecular biology studies. The phylogenetic relationship of the Harnischia generic complex remains contentious due to the paucity of taxonomic and molecular data. Specifically, the evolutionary relationships among Cryptochironomus, Demicryptochironomus, and Harnischia are still unclear. (2) Methods: In this study, Polypedilum and Endochironomus were used as outgroups to analyze phylogenetic relationships among Cryptochironomus, Demicryptochironomus, and Harnischia, mitogenomes of four Cryptochironomus, two Demicryptochironomus, two Harnischia, and two Cladopelma were newly sequenced. Subsequently, we conducted a thorough analysis of the nucleotide composition, sequence length, and evolutionary rate. (3) Results: All mitogenomes exhibited structural conservation, with all genes consistently arranged in the identical order as that of the ancestral mitogenome. Nucleotide composition varied significantly among different genes, and the control region displayed the highest A + T content. All protein-coding genes undergo rigorous purification selection, with the ATP8 gene exhibiting the most rapid evolutionary rate among them. Utilizing Bayesian Inference (BI) and Maximum Likelihood (ML) methods across various databases, we reconstructed the phylogenetic relationships among the genera within the Harnischia generic complex, drawing insights from an analysis of 14 mitochondrial genomes. (4) Conclusions: Our results showed that the monophyly of the genera Harnischia was well supported in all topologies; Cryptochironomus is sister to Demicryptochironomus.

1. Introduction

Recently, the insect mitochondrial genomes have garnered increased attention in research, exhibiting a remarkable degree of conservation in their mitogenome structure [1,2,3,4,5]. This conservation is evident in the similar gene arrangement they maintain, which is comparable to that of their ancestral insect counterparts [6,7]. The insect’s typical mitochondrial genome, known as the mitogenome, is a double-stranded circular molecule that ranges in size from 14 to 20 kilobases (kb) [8]. This genome encodes a comprehensive set of genetic components, including 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a control region (CR) [1]. Furthermore, due to its small genome size, maternal inheritance, low sequence recombination, and rapid evolutionary rate, the insect mitogenome is deemed a highly effective marker for molecular identification and phylogenetic analysis across various taxonomic levels within Diptera [9,10,11,12,13].
Chironomidae is an ecologically important family within the order Diptera, also known as one of the most abundant and diverse groups in freshwater ecosystems and an important indicator for environmental monitoring [14,15,16,17]. At present, the accurate identification of Chironomidae species and the phylogenetic relationships among some genera are still challenging [18]. With the rapid advances in next-generation sequencing technologies, an increasing amount of mitochondrial genome data for Chironomids has been published [19,20,21,22,23,24]. The structural characteristics of the mitochondrial genome offer valuable insights and evidence for morphological classification, significantly contributing to the study of the phylogeny of Chironomidae [25].
The Harnischia generic complex, belonging to the subfamily Chironominae, encompasses 20 genera and over 300 species worldwide [26,27,28]. Initially, the earliest species of the Harnischia generic complex were categorized into four distinct species groups under the genus Tendipes, T. (C.) Paracladopelma species group, T. (C.) Harnischia species group, T. (C.) Cryptochironomus species group and T. (C.) Parachironomus species group [29]. Subsequently, systematic research on Chironomidae in North America led to the promotion of these four groups to two separate genera [30]. Beck and Beck established the Harnischia generic complex in 1969, and soon afterward, numerous scholars conducted systematic studies on this complex or its internal genera, leading to the establishment of several genus-level taxonomic units [31,32,33,34,35,36].
Drawing upon morphological characteristics of female adults, the subfamily Chironominae is classified into three tribes, with robust support for the monophyly of the Harnischia generic complex, which forms a sister-group relationship with the Xenochironomus [36]. Sasa (1989), focusing on specimens from East Asia, further divided the tribe Chironomini into three generic complexes: Chironomus generic complex, Harnischia generic complex, and Polypedilum generic complex [37]. A comprehensive morphological study of Chironominae’s phylogeny, utilizing 119 morphological traits from 62 genera, reaffirmed the monophyly of the Harnischia generic complex [38]. Subsequently, Andersen et al. (2017) encoded 119 morphological traits from 70 genera and constructed a phylogenetic tree for Chironominae, further validating the monophyly of the Harnischia generic complex [39,40].
Some species within Harnischia generic complex are tolerant to organic pollution, serving as indicator species of eutrophication [41]. A few species are pests to rice crops, damage water supply, or can cause mass outbreaks to emerge that impact human life [42]. Within the Harnischia generic complex, although a significant portion of species can be precisely categorized, there remains a level of ambiguity regarding the taxonomic positioning and phylogenetic relationships of certain genera, necessitating further validation and verification [36,43,44]. For the immature stages of these three genera (Cryptochironomus, Demicryptochironomus, and Harnischia), their habitats exhibit considerable similarity, predominantly occupying the bottoms of lakes and large rivers, while a minority can be found inhabiting small streams [45]. In terms of larval structure, the number of antennal flagellomeres, the shape of lateral teeth, and the form of the mentum can readily distinguish these three genera [42]. The pupal characteristics of Cryptochironomus and Harnischia are relatively similar, and both possess the same pattern of armature on the tergites, whereas Demicryptochironomus can be easily distinguished from these two genera by its unique features such as the thoracic horns and the pedes spurii B [46]. The adult insects of these three genera are relatively more difficult to distinguish from each other. From the perspective of the genitalia characteristics of male adults, Cryptochironomus and Harnischia are more similar, whereas in terms of female characteristics, Cryptochironomus and Demicryptochironomus are more alike [36,47].
The phylogenetic relationships between the three relatively large and morphologically similar genera within the Harnischia generic complex, namely Cryptochironomus, Demicryptochironomus, and Harnischia, have yet to be thoroughly investigated. In this study, we have sequenced, assembled, and annotated the mitogenomes of four Cryptochironomus species, two Demicryptochironomus species, two Harnischia species, and two Cladopelma species. Additionally, we incorporated four previously published mitogenomes into our analysis to delve deeper into the characteristics of these mitogenomes. Utilizing Bayesian Inference (BI) and Maximum Likelihood (ML) methods across various databases, we reconstructed the phylogenetic relationships among the genera within the Harnischia generic complex, drawing insights from an analysis of 14 mitochondrial genomes. Our findings indicate that the monophyly of the genus Harnischia is robustly supported in all topological configurations, further clarifying that Cryptochironomus is a sister group to Demicryptochironomus.

2. Materials and Methods

2.1. Taxon Sampling and Sequencing

Our analysis incorporated four Cryptochironomus species, two Demicryptochironomus species, two Harnischia species, and two Cladopelma species originating from China (Table 1). Furthermore, for comparative mitogenomic analysis and phylogeny reconstruction, we retrieved the mitogenomes of Microchironomus tabarui (Sasa, 1987), Microchironomus tener (Kieffer, 1918), Polypedilum yongsanensis (Ree and Kim, 1981), Endochironomus albipennis Meigen, 1830 in our analysis from GenBank [19,20,21]. Drawing upon extensive previous phylogenetic research on Chironomidae, Polypedilum yongsanensis (Ree and Kim, 1981), and Endochironomus albipennis (Meigen, 1830) were selected to function as outgroups in our analysis. Prior to DNA extraction and morphological examination, all samples were immersed in a solution of 85% to 95% ethanol at a temperature of −20 °C.
For the extraction of total genomic DNA, the Qiagen DNA Blood and Tissue Kit was utilized. The voucher specimens have been deposited at the College of Life Sciences, Tianjin Normal University, located in Tianjin, China (TJNU), for future reference and analysis. All whole genomes were submitted to Berry Genomics, located in Beijing, China, for sequencing. The Truseq Nano DNA HT Sample Preparation Kit from Illumina (San Diego, CA, USA) was utilized to prepare the sequencing libraries. DNA fragments with an insert size of 350 bp were sequenced using the Illumina Nova 6000 platform (PE150, Illumina), employing a paired-end strategy. Following the trimming of raw reads using Trimmomatic, the resulting clean reads were retained for subsequent downstream analysis [48].

2.2. Assembly, Annotation and Composition Analyses

The mitogenome sequences were assembled de novo using NOVOPlasty v3.8.3 (Brussels, Belgium) [49], employing the COI gene as the seed sequence and a range of k-mer sizes from 23 to 39 bp to facilitate the mitogenome assembly process. The annotation of the mitogenome was performed following the methodology described previously by Zheng et al. (2020) [50]. The secondary structure of tRNAs was determined using the MITOS 2 WebServer. The annotation of rRNAs and PCGs was manually carried out in Geneious, utilizing the Clustal Omega algorithm [51]. The analysis of nucleotide composition bias and the nucleotide composition of each gene was conducted using SeqKit v0.16.0, developed in Chongqing, China [52]. The mitogenome map was created utilizing the CGView server, accessible at https://cgview.ca/ (accessed on 25 March 2024). The nucleotide composition, codon usage, and relative synonymous codon usage of the mitogenome were determined using MEGA 11 (Temple University, Philadelphia, PA, USA) [53]. The bias in nucleotide composition was quantified using the AT-skew, calculated as (A − T)/(A + T), and the GC-skew, determined as (G − C)/(G + C). Additionally, the synonymous (Ks) and non-synonymous substitution rates (Ka) of 13 PCGs were computed using DnaSP 6.0 [54].

2.3. Phylogenetic Analyses

For phylogenetic analysis, 2 rRNAs and 13 PCGs genes were selectively extracted from 14 mitochondrial genomes. To achieve this, MAFFT (Osaka, Japan) was employed to carry out batch alignment of both nucleotide and protein sequences, utilizing the L-INS-I method to eliminate regions of ambiguous alignment. Trimming was conducted utilizing Trimal v1.4.1 (Barcelona, Spain), followed by the execution of phylogenetic analysis. This analysis was based on five distinct data matrices generated by FASconCAT-G v1.04 (Santa Cruz, CA, USA), specifically: (1) PCG: Including all three codon positions of the 13 protein-coding genes (PCGs); (2) PCG_RNA: Encompassing all three codon positions of the 13 PCGs as well as the two ribosomal RNA (rRNAs); (3) PCG12_RNA: Incorporating the first and second codon positions of the 13 PCGs along with the 2 rRNAs; (4) PCG12: Solely focusing on the first and second codon positions of the 13 PCGs; (5) PCG_AA: Utilizing the amino acid sequences derived from the 13 PCGs. To assess the heterogeneity among the various matrices, AliGROOVE v1.06 (Bonn, Germany) was employed, referencing prior works by Katoh et al. (2013), Capella-Gutiérrez et al. (2009), and Kück et al. (2014) [55,56,57]. Subsequently, the maximum likelihood (ML) and Bayesian inference (BI) trees were constructed using IQ-tree v2.0.7 and Phylobayes-MPI v1.8, respectively.

3. Results and Discussion

3.1. Mitogenomic Organization

The newly obtained sequences exhibited a length range spanning from 15,662 bp in Cladopelma edwardsi to 17,642 bp in Cryptochironomus rostratus, with the primary source of this variation being the fluctuating size of the control region (CR; range of sizes from 110 bp in Cryptochironomus rostratus to 2450 bp in Endochironomus albipennis) (Table 2). All newly assembled mitogenomes encompassed a standardized set of genetic components comprising one control region (CR) and 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs, Figures S1–S10), and 2 ribosomal RNAs (rRNAs; Figure 1). Notably, the lengths of most of these newly assembled mitogenomes were comparable to those of previously published Chironomidae mitogenomes. The sequence characteristics of the represented species are illustrated in Figure 1.

3.2. Protein-Coding Genes, Codon Usage, and Evolutionary Rates

Across the various species, there was no significant variation in the size of the tRNA, PCGs, and rRNAs. Specifically, the total length of the 13 PCGs in the acquired mitogenomes ranged narrowly from 11,220 to 11,232 base pairs. When we combined and contrasted our findings with published Chironomidae data, a noteworthy trend emerged: the AT content at the third codon positions of the protein-coding genes (PCGs) was significantly elevated compared to the first and second positions (Figure 2). Strikingly, the majority of the 14 mitogenomes exhibited a negative GC-skew in their PCGs, while each of them displayed a negative AT-skew in the same genes, ranging from −0.200 in P. yongsanensis to −0.168 in D. spatulatus. The AT content, expressed as a percentage, spanned from 73.86 in D. spatulatus to 77.57 in D. minus, while the GC content ranged from 22.43 in D. minus to 26.14 in D. spatulatus (detailed information is presented in Table 2).
All 13 protein-coding genes (PCGs) in the acquired mitogenomes possessed the standard start codon ATN, which closely aligns with the typical insect mitochondrial start codon [18]. However, variations were observed in other genes. Specifically, the COI gene in 10 species utilized TTG as its start codon, while one species employed ATG. The ATP8 gene started with ATT in five species and ATC in five species. The ND1 gene consistently utilized TTG as its start codon in all species. Additionally, the ND2, ND3, and ND6 genes consistently started with ATT, while the ND5 gene uniquely started with GTG in all species (Figure 3) and provides information on these start codons. Regarding stop codons, 13 PCGs primarily used TAA, with exceptions being COX2 and ND3, which had one TAG.
The Ka/Ks value is a commonly employed metric to quantify the rate of sequence evolution under natural selection [20]. Our findings align closely with those reported in other insect species, revealing that the Ka/Ks ratio for all 13 protein-coding genes (PCGs) was consistently below one, spanning a range from 0.025 (COX1) to 0.287 (ATP8) (Figure 4). The evolutionary rates of these PCGs can be ranked as follows: ATP8 > ND6 > ND5 > ND4 > ND2 > ND1 > ND3 > ND4L > CYTB > APT6 > COX3 > COX2 > COX1. Notably, our results suggest that in many cases, genes under purifying selection evolved to eliminate deleterious mutations, operating under varying selection pressures. Specifically, the low ω values observed for COX2 (0.039) and COX1 (0.025) suggest a stringent selection environment, while the high ω values for ATP8 (0.287), ND6 (0.183), and ND5 (0.110) indicate a relatively relaxed purifying selection pressure (Figure 4).
The lengths of the 14 mitochondrial tRNAs varied from 1483 to 1510 base pairs (bp), with AT content ranging from 78.81% (in M. tabarui) to 81.56% (in E. albipennis). All tRNAs exhibited a positive AT-skew, spanning values from 0.008 to 0.049. In contrast, the GC content ranged from 18.91% (D. spatulatus) to 21.19% (M. tabarui), and the GC-skew varied significantly, from 9.51 (C. sp.) to 17.33 (M. tener).
Regarding the rRNA sequences, their lengths ranged from 2171 bp in C. edwardsi to 2288 bp in P. yongsanensis. The AT content remained consistently high, varying from 83.81% to 84.89%. The GC content, on the other hand, ranged from 15.11% to 16.20%. Notably, the GC-skew of all mitogenomes was significantly positive, ranging from 0.325 to 0.412. While most mitogenomes exhibited a negative AT-skew (−0.028 to −0.001), three species, H. angularis, C. rostratus, and C. maculus, displayed positive values, with AT-skews of 0.005, 0.005, and 0.013, respectively. For a more comprehensive overview of these findings, please refer to Table 2.

3.3. Phylogenetic Relationships

The analysis of heterogeneity divergence differences provides a window into the similarities existing in mitochondrial gene sequences across distinct species [58]. Notably, owing to the degeneracy of codons, the dataset AA exhibited the least heterogeneity, whereas the PCG12_rRNA dataset displayed a relatively higher degree of heterogeneity (Figure 5). This observation suggests that the mutation rate of the third codon in protein-coding genes (PCG) surpassed that of the first and second codons. Consequently, the positions of the third codons were deemed unsuitable for reconstructing the phylogenetic relationship among the three genera. Furthermore, it is evident that the heterogeneity observed in the outgroup species of Polypedilum and Endochironomus is notably lower compared to that found in the ingroup.
Both BI and ML analyses of these five datasets concurred in revealing a consistent topological pattern among the phylogenetic trees despite variations in branch lengths and statistical support (Figures S11–S19). Notably, the monophyly of the genus Harnischia was robustly supported in all topologies. Furthermore, our data revealed that within this group, Cryptochironomus emerged as a sister taxon to Demicryptochironomus, providing valuable insights into their evolutionary history (Figure 6).
There is relatively limited and contradictory research on the systematic studies of these three genera [58]. Previously, a TNT (Tree analysis using New Technology) generated tree based on 74 female characteristics supported Cryptochironomus and Demicryptochironomus as sister groups, with the rebuilt Harnischia sister to (Cryptochironomus + Demicryptochironomus) [58]. Subsequently, a TNT tree was constructed based on the comprehensive morphological characteristics, encompassed features from the larval, pupal, and adult stages, of 119 species belonging to 70 genera within the Chironominae subfamily, revealing that Harnischia and Cryptochironomus form sister groups, while Demicryptochironomus occupies a basal position within the Harnischia generic complex [41]. In the phylogenetic analysis of Chironomidae, using molecular data from fragments of 18SrRNA, 28SrRNA, CAD1, CAD4, and mtCOI analyzed by mixed-model Bayesian and maximum likelihood inference methods, Harnischia and (Cryptochironomus + (Cryptotendipes + Parachironomus)) were found to be sister groups [40].
However, due to the lack of molecular information for Harnischia and the inclusion of only one species per genus, the evolutionary relationships among these three genera based on molecular sequences remain unknown at present, not resolving the traditional morphological analysis as whether Cryptochironomus and Harnischia are sister groups, or Harnischia and Demicryptochironomus are sister groups. According to traditional morphological analysis, Cryptochironomus and Harnischia are sister groups, or Harnischia and Demicryptochironomus are sister groups. Our findings reveal that Cryptochironomus is a sister taxon to Demicryptochironomus and indicate the monophyly of the genus Harnischia. This study holds significant value as it offers the inaugural molecular data, specifically the mitogenome, for a species belonging to the Harnischia generic complex. To further refine and accurately assess the phylogenetic relationships within the Harnischia generic complex, mitogenomes from additional species spanning a broader range of genera are imperative.
The nucleotide composition of the newly reported mitogenomes exhibited similarity across the samples (Table 2), reflecting the characteristic AT-biased composition that is typical of Chironomidae and other insect lineages. The AT content of the mitochondrial genomes varied significantly, ranging from 76.31% in M. tabarui to 80.34% in C. rostratus (Figure 2; Table 2). Notably, the CR exhibited the highest AT content, spanning from 93.25% in D. spatulatus to 98.18% in E. albipennis. In contrast, the AT content in tRNAs and PCGs was relatively lower than that in rRNAs (Table 2). All newly assembled mitogenomes exhibited a negative GC-skew, indicating a bias toward cytosine, while most displayed a positive AT-skew, reflecting an abundance of adenine and thymine. The GC-skew ranged from −0.240 in P. yongsanensis to −0.169 in D. minus, while the AT-skew spanned from 0.013 in D. minus to 0.050 in P. yongsanensis. The GC content itself varied from 19.66% in C. rostratus to 23.69% in M. tabarui, providing further insights into the nucleotide composition of these mitogenomes (Table 2).

4. Conclusions

For the first time, we present a new insight into the genomics of Harnischia complex genomics by obtaining the comprehensive mitotic genomes of eleven species spanning four genera within the Harnischia generic complex, including four species from the genus Cryptochironomus, two from Demicryptochironomus, two from Harnischia, and two from Cladopelma. Additionally, these genomes amalgamated with the previously published mitogenomes of two Microchironomus species, enabling us to conduct natural phylogenetic analyses. All newly sequenced mitogenomes displayed strikingly similar structural traits and nucleotide compositions, closely aligning with previously published Chironomidae data.
Our study first offers a mitochondrial genomic perspective on the evolutionary history of the Harnischia generic complex. Our findings indicate that the monophyly of the genus Harnischia is strongly supported across all topologies, revealing that Cryptochironomus is a sister taxon to Demicryptochironomus, which constitutes a novel insight into the systematics of the Harnischia complex.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/insects15090642/s1, Figure S1. Putative secondary structures of the 22 tRNA genes identified in the mitogenome of Cryptochironomus maculus. Figure S2. Putative secondary structures of the 22 tRNA genes identified in the mitogenome of Cryptochironomus sp. Figure S3. Putative secondary structures of the 22 tRNA genes identified in the mitogenome of Cryptochironomus defectus. Figure S4. Putative secondary structures of the 22 tRNA genes identified in the mitogenome of Cryptochironomus rostratus. Figure S5. Putative secondary structures of the 22 tRNA genes identified in the mitogenome of Demicryptochironomus spatulatus. Figure S6. Putative secondary structures of the 22 tRNA genes identified in the mitogenome of Demicryptochironomus minus. Figure S7. Putative secondary structures of the 22 tRNA genes identified in the mitogenome of Harnischia angularis. Figure S8. Putative secondary structures of the 22 tRNA genes identified in the mitogenome of Harnischia turgidula. Figure S9. Putative secondary structures of the 22 tRNA genes identified in the mitogenome of Cladopelma edwardsi. Figure S10. Putative secondary structures of the 22 tRNA genes identified in the mitogenome of Cladopelma virescens. Figure S11. ML phylogenetic tree of Cryptochironomus, Demicryptochironomus, and Harnischia based on the analysis PCG_AA with Partition model in IQTREE. Support values on nodes indicate SHaLRT/UFBoot2, respectively. Figure S12. ML phylogenetic tree of Cryptochironomus, Demicryptochironomus, and Harnischia based on the analysis PCG with Partition model in IQTREE. Support values on nodes indicate SHaLRT/UFBoot2, respectively. Figure S13. ML phylogenetic tree of Cryptochironomus, Demicryptochironomus, and Harnischia based on the analysis PCG_rRNA with Partition model in IQTREE. Support values on nodes indicate SHaLRT/UFBoot2, respectively. Figure S14. ML phylogenetic tree of Cryptochironomus, Demicryptochironomus, and Harnischia based on the analysis PCG12 with Partition model in IQTREE. Support values on nodes indicate SHaLRT/UFBoot2, respectively. Figure S15. BI phylogenomic tree of Cryptochironomus, Demicryptochironomus, and Harnischia based on the analysis PCG_AA with CAT +GTR model in phylobayes. Support values on nodes indicate Bayesian posterior probabilities. Figure S16. BI phylogenomic tree of Cryptochironomus, Demicryptochironomus, and Harnischia based on the analysis PCG with CAT +GTR model in phylobayes. Support values on nodes indicate Bayesian posterior probabilities. Figure S17. BI phylogenomic tree of Cryptochironomus, Demicryptochironomus, and Harnischia based on the analysis PCG_rRNA with CAT +GTR model in phylobayes. Support values on nodes indicate Bayesian posterior probabilities. Figure S18. BI phylogenomic tree of Cryptochironomus, Demicryptochironomus, and Harnischia based on the analysis PCG12 with CAT +GTR model in phylobayes. Support values on nodes indicate Bayesian posterior probabilities. Figure S19. BI phylogenomic tree of Cryptochironomus, Demicryptochironomus, and Harnischia based on the analysis PCG12_rRNA with CAT +GTR model in phylobayes. Support values on nodes indicate Bayesian posterior probabilities.

Author Contributions

Conceptualization, W.L. and X.G.; Software, X.G. and J.W.; Investigation, J.W. and W.P.; Data curation, C.W., W.P., and Y.T. Writing—original draft, W.L., C.W., and Y.T.; Writing—review & editing, C.Y.; Visualization, W.L.; supervision, C.Y.; Funding acquisition, C.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (32370489, 32170473, 32400357) and Natural Science Foundation of Tianjin Science and Technology Correspondent (23KPHDRC00240, 22KPXMRC00070).

Data Availability Statement

The following information was supplied regarding the availability of DNA sequences: The new mitogenomes are deposited in GenBank of NCBI and the accession numbers are in Table 2.

Acknowledgments

We thank Xiaolong Lin (Shanghai Ocean University) for providing information on some species.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The mitogenome map depicted the distinctive mitochondrial genome attributes of various representative species spanning four genera within the Harnischia generic complex. The arrow served as a guide, pointing to the orientation of gene transcription. We adhered to standardized abbreviations to denote Protein-Coding Genes (PCGs) and ribosomal RNAs (rRNAs), while single-letter abbreviations were chosen for transfer RNAs (tRNAs). The second circle highlighted the GC content of the entire mitogenome, whereas the third circle revealed the GC-skew. The innermost circle encapsulated the length of the entire mitogenome, providing a comprehensive visualization of its characteristics.
Figure 1. The mitogenome map depicted the distinctive mitochondrial genome attributes of various representative species spanning four genera within the Harnischia generic complex. The arrow served as a guide, pointing to the orientation of gene transcription. We adhered to standardized abbreviations to denote Protein-Coding Genes (PCGs) and ribosomal RNAs (rRNAs), while single-letter abbreviations were chosen for transfer RNAs (tRNAs). The second circle highlighted the GC content of the entire mitogenome, whereas the third circle revealed the GC-skew. The innermost circle encapsulated the length of the entire mitogenome, providing a comprehensive visualization of its characteristics.
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Figure 2. Difference in AT content of protein-coding genes of Harnischia generic complex mitogenomes. (A) First-codon positions; (B) second-codon positions; (C) third-codon positions; (D) first/second-codon positions.
Figure 2. Difference in AT content of protein-coding genes of Harnischia generic complex mitogenomes. (A) First-codon positions; (B) second-codon positions; (C) third-codon positions; (D) first/second-codon positions.
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Figure 3. Start codons of protein-coding genes among Harnischia generic complex mitogenomes.
Figure 3. Start codons of protein-coding genes among Harnischia generic complex mitogenomes.
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Figure 4. Evolution rate of 13 PCGs of the Harnischia generic complex mitogenomes. Ka refers to non-synonymous nucleotide substitutions, Ks refers to synonymous nucleotide substitutions, Ka/Ks refers to the selection pressure of each PCG. The abscissa represented 13 PCGs, and the ordinate represented Ka/Ks values.
Figure 4. Evolution rate of 13 PCGs of the Harnischia generic complex mitogenomes. Ka refers to non-synonymous nucleotide substitutions, Ks refers to synonymous nucleotide substitutions, Ka/Ks refers to the selection pressure of each PCG. The abscissa represented 13 PCGs, and the ordinate represented Ka/Ks values.
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Figure 5. The assessment of the heterogeneity among the mitogenomes of 13 species belonging to the Harnischia generic complex focused on their Protein-Coding Genes (PCGs), amino acid sequences, and ribosomal RNAs (rRNAs). The degree of sequence similarity was visually represented through colored blocks, utilizing AliGROOVE scores that span a spectrum from −1 (indicating substantial heterogeneity between datasets, represented by red) to +1 (signaling minimal heterogeneity between datasets, depicted in blue). The lighter hue of each dataset’s-colored block corresponds to a higher degree of heterogeneity, while the darker tone signifies reduced heterogeneity.
Figure 5. The assessment of the heterogeneity among the mitogenomes of 13 species belonging to the Harnischia generic complex focused on their Protein-Coding Genes (PCGs), amino acid sequences, and ribosomal RNAs (rRNAs). The degree of sequence similarity was visually represented through colored blocks, utilizing AliGROOVE scores that span a spectrum from −1 (indicating substantial heterogeneity between datasets, represented by red) to +1 (signaling minimal heterogeneity between datasets, depicted in blue). The lighter hue of each dataset’s-colored block corresponds to a higher degree of heterogeneity, while the darker tone signifies reduced heterogeneity.
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Figure 6. Phylogenetic tree of Harnischia generic complex, ML tree based on analysis PCG12_rRNA in partition.
Figure 6. Phylogenetic tree of Harnischia generic complex, ML tree based on analysis PCG12_rRNA in partition.
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Table 1. Collection information of newly sequenced species in this study.
Table 1. Collection information of newly sequenced species in this study.
SpeciesSample IDLocationLongitude and LatitudeDataCollector
Cryptochironomus defectusKTM004Tianmu Mountain, Zhejiang, China30°31′23″ N; 119°44′59″ E29.VII.2011Shu-Li Li
Cryptochironomus sp.KKH002Kaihua, Zhejiang, China29°25′00″ N; 118°09′25″ E8.VII.2017Wei Cao
Cryptochironomus maculusWGL002Zunyi, Guizhou, China28°94′48″ N; 107°88′80″ E29.VII.2021Wei Cao
Cryptochironomus rostratus2BW005Bawangling National Forest Park, Hainan, China19°08′86″ N; 109°12′92″ E3.IV.2008Qian Wang
Demicryptochironomus minusWTS110Tongren, Guizhou, China28°31′73″ N; 109°19′95″ E29.VII.2021ChunCai Yan
Demicryptochironomus spatulatusKJL001Kowloon National Wetland Park, Zhejiang, China28°39′48″ N; 118°84′58″ E5.VIII.2011Xiao-Long Lin
Harnischia angularisDTH95Yueyang, Hunan, China28°99′21″ N; 112°89′07″ E30.XII.2021Fei-Xiang He
Harnischia turgidulaKTD484Taizhou, Zhejiang, China28°48′67″ N; 121°89′96″ E20.V.2011Xing Qi
Cladopelma edwardsiKHZ175Hangzhou, Zhejiang, China30°25′51″ N; 120°14′15″ E23.IV.2011Xiao-Long Lin
Cladopelma virescensXTC002Tengchong, Yunnan, China25°00′67″N; 98°45′82″E23.IV.2018Wei Cao
Table 2. Nucleotide composition of 14 mitogenomes.
Table 2. Nucleotide composition of 14 mitogenomes.
SpeciesWhole GenomePCGtRNA
Length (bp)AT%AT-SkewGC%GC-SkewLength (bp)AT%AT-SkewGC%GC-SkewLength (bp)AT%AT-SkewGC%GC-Skew
Cladopelma edwardsi15,66277.550.036 22.45−0.21111,22076.36 −0.186 23.64 −0.049 149379.100.016 20.9014.10
Cladopelma virescens15,99777.970.036 22.03−0.21911,22076.50 −0.184 23.50 −0.070 148379.160.014 20.8413.92
Cryptochironomus defectus15,820 78.35 0.022 21.65−0.18511,22977.15 −0.187 22.85 −0.024 149580.270.023 19.7315.25
Cryptochironomus sp.17,33179.850.025 20.1−0.199 11,22376.90 −0.175 23.10 −0.037 149179.540.037 20.469.51
Cryptochironomus maculus16,24378.770.035 21.23−0.192 11,22076.92 −0.173 23.08 −0.050 149480.190.030 19.8111.49
Cryptochironomus rostratus17,642 80.340.032 19.66−0.202 11,22977.54 −0.174 22.46 −0.058 149280.630.013 19.3714.88
Demicryptochironomus minus15,90379.050.013 20.95−0.169 11,22377.57 −0.177 22.43 −0.037 149880.770.008 19.2315.28
Demicryptochironomus spatulatus16,85878.850.016 21.14−0.235 11,22076.16 −0.168 23.84 −0.100 149181.090.049 18.9112.77
Harnischia angularis15,90178.420.021 21.57−0.182 11,22076.66 −0.183 23.33 −0.041 149480.120.014 19.8814.48
Harnischia turgidula15,94278.340.02821.66−0.195 11,22076.73 −0.183 23.27 −0.053 149380.040.011 19.9613.42
Microchironomus tabarui15,66776.310.04223.69−0.203 11,22074.72 −0.182 25.27 −0.067 149678.810.030 21.1911.67
Microchironomus tener15,79177.730.02722.28−0.206 11,22275.64 −0.182 24.36 −0.073 149479.920.020 20.0817.33
Endochironomus albipennis15,91679.800.02820.20−0.19811,22976.93−0.17023.07−0.230149781.560.03018.4414.49
Polypedilum yongsanensis16,22677.010.05022.99−0.24011,23273.86−0.20026.14−0.035151080.000.03020.0014.57
SpeciesrRNACRGenBank AccessionReference
Length (bp)AT%AT-SkewGC%GC-SkewLength (bp)AT%AT-SkewGC%GC-Skew
Cladopelma edwardsi217183.99 −0.02816.02 0.380 52194.24−0.027 5.76 −0.33 PQ014460This Study
Cladopelma virescens217183.81 −0.02716.20 0.393 79994.75−0.044 5.13 −0.22 PQ014464This Study
Cryptochironomus defectus2200 84.21 −0.00315.79 0.386 61295.59−0.046 4.41 −0.48 PQ014461This Study
Cryptochironomus sp.222484.74 −0.00115.27 0.369 203194.98−0.039 4.73 −0.50 PQ014463This Study
Cryptochironomus maculus222484.43 0.01315.57 0.386 89394.850.020 5.16 −0.43 PQ014454This Study
Cryptochironomus rostratus220884.68 0.00515.34 0.369 245094.860.034 5.10 −0.20 PQ014455This Study
Demicryptochironomus minus221084.89 −0.00915.11 0.325 65696.34−0.089 3.66 −0.08 PQ014456This Study
Demicryptochironomus spatulatus225284.09 −0.00915.92 0.412 133493.25−0.122 6.60 −0.36 PQ014457This Study
Harnischia angularis220884.57 0.00515.44 0.362 74196.09−0.048 3.91 −0.17 PQ014458This Study
Harnischia turgidula221384.21 −0.00615.79 0.368 73994.99−0.046 5.00 −0.24 PQ014459This Study
Microchironomus tabarui218284.51 −0.02015.50 0.377 54894.71−0.044 5.29 −0.38 MZ261913[21]
Microchironomus tener219384.25 −0.00215.76 0.375 67495.11−0.020 4.90 −0.45 ON975027[20]
Endochironomus albipennis225286.01−0.0114.000.34011098.18−0.1001.821.00OP950227[19]
Polypedilum yongsanensis228884.88−0.0215.120.35331295.51−0.1004.49−0.43OP950222[19]
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Liu, W.; Wang, C.; Wang, J.; Tang, Y.; Pei, W.; Ge, X.; Yan, C. Phylogenetic and Comparative Analysis of Cryptochironomus, Demicryptochironomus and Harnischia Inferred from Mitogenomes (Diptera: Chironomidae). Insects 2024, 15, 642. https://doi.org/10.3390/insects15090642

AMA Style

Liu W, Wang C, Wang J, Tang Y, Pei W, Ge X, Yan C. Phylogenetic and Comparative Analysis of Cryptochironomus, Demicryptochironomus and Harnischia Inferred from Mitogenomes (Diptera: Chironomidae). Insects. 2024; 15(9):642. https://doi.org/10.3390/insects15090642

Chicago/Turabian Style

Liu, Wenbin, Chengyan Wang, Jingyuan Wang, Yaning Tang, Wenxuan Pei, Xinyu Ge, and Chuncai Yan. 2024. "Phylogenetic and Comparative Analysis of Cryptochironomus, Demicryptochironomus and Harnischia Inferred from Mitogenomes (Diptera: Chironomidae)" Insects 15, no. 9: 642. https://doi.org/10.3390/insects15090642

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