Research Article
Research Article
Paraphyly of the subgenus Micronecta (Micronecta) Kirkaldy, 1897 (Hemiptera: Heteroptera: Micronectidae) based on mitochondrial genomes and nuclear rDNAs
expand article infoBao-Jun Xie, Ping-Ping Chen§, Jakob Damgaard|, Jie-Yi Xie, Qiang Xie, Yan-Hui Wang
‡ Sun Yat-sen University, Guangzhou, China
§ Netherlands Centre of Biodiversity Naturalis, Leiden, Netherlands
| Natural History Museum of Denmark, Zoological Museum, Copenhagen, Denmark
Open Access


The genus Micronecta Kirkaldy, 1897 is the most species-rich genus in the family Micronectidae, containing more than 160 species. Micronecta is currently divided into 11 subgenera, five of which are monotypic. Moreover, the subgenus Micronecta is an empirical mixture group. The definitions of some subgenera were based on only a few aberrant morphological features, which are specializations with few phylogenetic significances. The relationship between these subgenera remains unclear. In this study, we newly sequenced mitochondrial genomes (mitogenomes) and nuclear rDNAs (nrDNAs) for 13 Micronecta species, representing seven subgenera, and those for ten other water bugs. Our phylogenetic analyses showed that the subgenus Lundbladella represents the sister group to all other studied subgenera of Micronecta. The subgenus Unguinecta was the sister group to the clade that contains Dichaetonecta and Sigmonecta. More importantly, the subgenus Micronecta represents a paraphyletic group, which further forms a monophyletic group together with the subgenera Basileonecta and Ctenonecta. This is for the first time that the phylogeny of the genus Micronecta was investigated based on molecular data and the paraphyly of the subgenus Micronecta was revealed. Such evidence suggested the necessity of the revision of the taxonomic system of the genus in the future, and may also serve as a reference for the delimitation of subgeneric characters.

Key words

Aquatic insects, Corixoidea, Nepomorpha, water boatmen, phylogeny, subgenus

1. Introduction

Micronectidae, commonly known as pygmy water boatmen due to their minute size (0.8−5 mm), is a family of aquatic bugs (Nepomorpha) and with representatives in all temperate, subtropical and tropical biogeographical regions (Chen et al. 2005). Micronectids undergo five nymphal instars as do the majority of aquatic and semiaquatic true bugs. Most species inhabit nearly stagnant or shallow stagnant water, preferring an open sandy or clayey bottom with little or no plant debris (Chen et al. 2015). Usually, we can find a large quantity of individuals in paddy fields. While the diet and feeding habits of micronectid species are unclear, and they probably feed on fish eggs, algae, detritus, or mosquito larvae (Hädicke et al. 2017). Their complex feeding habits are likely to correlate with the modified spoon- or scoop-like “pala”. Males of micronectid species always have stridulatory structures on the right paramere and can produce sound, which are likely to play important role in mating (King 1999) and is a character distinguishing from other corixoids (Jansson 1989; Nieser 2002). In addition, Micronecta is the only nepomorphan genus besides Aphelocheirus (Aphelocheiridae) known to produce spermatophores (Andersen and Weir 2004).

Two subfamilies are currently recognized: Synaptogobiinae with two species of Synaptogobia Nieser and Chen 2006 from the Neotropical Region and Micronectinae with six genera and approximately 210 species predominantly from the Old World (Wróblewski 1972; Nieser and Chen 2006; Tinerella 2008, 2013). Synaptonecta Lundblad, 1933 is represented by three species in the Oriental Region, Papuanecta Tinerella, 2008 is represented by four species from New Guinea, and Austronecta Tinerella, 2013 is represented by four species in Australia. The largest and most widespread genus, Micronecta Kirkaldy, 1897 comprises 11 subgenera and more than 160 species is occurring throughout the temperate, subtropical and tropical parts of the Old World, but the fauna of Africa is poorly known and the number of species is doubtful (Nieser and Chen 2006; Ha and Tran 2021). Many species of Micronecta have excellent dispersal abilities, and some have enormous distribution ranges, fx. M. (Micronecta) ludibunda Breddin 1905, which is recorded from India, throughout South-East Asia, and eastwards to the Solomon Islands, while others seem to have a much more limited distribution, fx. M. (Micronecta) jennferae Tinerella 2008, which is recorded only from Fiji. No member of Micronectidae have been reported from further East in Oceania, and no records exist from either New Zealand or New Caledonia, both of which are inhabited by numerous species water boatmen (Larivière and Larochelle 2004; Damgaard and Zettel 2014). In the New World, Micronectinae is represented by Monogobia Nieser and Chen, 2006, including a single species from Brazil, and Tenagobia Bergroth 1899 with seven subgenera and almost 30 species distributed in South- and Central America and with a single species reaching northern Mexico (Nieser 1977; Nieser and Chen 2008). Interestingly, Micronectidae is absent from the Nearctic Region, except for two introduced Old World species from Florida (Polhemus and Rutter 1997; Polhemus and Golia 2006; Epler and Denson 2017).

Currently, the genus Micronecta is divided into 11 subgenera: Basileonecta Hutchinson, 1940, Ctenonecta Wróblewski, 1962, Dichaetonecta Hutchinson, 1940, Indonectella Hutchinson, 1940, Lundbladella Wróblewski, 1967, Mesonecta Poisson, 1938, Micronecta Kirkaldy, 1897, Micronectella Lundblad, 1933, Pardanecta Horváth, 1904, Sigmonecta Wróblewski, 1962, and Unguinecta Nieser, Chen et Yang, 2005 (Hutchinson 1940; Wróblewski 1962, 1967; Nieser et al. 2005; Ha and Tran 2021). It is worth noting that nearly half of all described species had not been formally assigned to any subgenera. Moreover, there are species that were placed tentatively into the subgenus Micronecta (Jansson 1995) or left as members of informal species group (Tinerella 2008, 2013). Eight out of the 11 subgenera have been recorded in China, except Mesonecta, Micronectella, and Pardanecta. Up to date, no study has investigated the phylogeny of the genus Micronecta and the relationships among those subgenera based on molecular data. While a robust phylogeny is vital to support both the taxonomy and biogeography.

Mitochondrial genomes (mitogenomes) have been widely used in molecular systematics and molecular evolutionary studies (Cameron 2014). The typical insect mitogenome is a circular double-strand molecule about 14-20 kb and encodes 37 genes, including 13 protein coding genes (PCGs), 22 transfer RNAs (tRNAs), and 2 ribosomal RNA genes (rRNAs) (Wolstenholme 1992; Cameron 2014). Comparing with nuclear genomes, the features of mitogenomes, e.g. fast evolutionary rates, small size, low recombination rates and conserved gene arrangements (Curole and Kocher 1999), make it frequently-used in phylogenetic studies in insects (Li et al. 2014; Wang et al. 2016; Li et al. 2017; Chang et al. 2020; Dong et al. 2022; Ye et al. 2022). Besides, nuclear ribosomal DNA (nrDNA) also plays an important role in phylogenetic studies in insects (Kjer 2004; Wang et al. 2016; Ye et al. 2022). As both mitogenomes and nrDNAs have defects in phylogenetic studies, i.e., the former is sensitive to taxon sampling while the latter is too conservative in family and lower levels, we combined the two data types to overcome these disadvantages in this study. Up to now, only one mitogenome of the species Micronecta (Dichaetonecta) sahlbergii (Zhang et al. 2018) and a few nrDNA sequences of various species of the Micronectidae have been released in public databases as of November 30, 2022.

In this study, we sequenced the mitogenomes of 13 Micronecta species covering all 37 genes, and comprehensively analyzed the characteristic of these mitogenomes. Meanwhile, ten complete mitogenomes were also sequenced for other water boatmen representing Corixidae (Corixoidea) and Diaprepocoridae (Corixoidea). We also newly provided the corresponding nrDNAs of those 23 species. The nrDNAs for Lethocerus sp. (Belostomatidae), Laccotrephes sp. (Nepidae), Enithares sp. and Notonecta sp. (both Notonectidae) were provided for the first time as well. The phylogeny of the genus Micronecta was reconstructed based on the whole mitogenomes and nrDNAs.

2. Methods

2.1. Sampling and DNA extraction

Our taxon sampling included 31 species, of which 13 species of Micronecta were in-groups and 10 species of other water boatmen and 8 species of the remaining true water bugs were out-groups (Table 1). The 13 species covered seven out of the 8 subgenera distributed in China. They were all preserved in 100% ethanol under –20°C until used for DNA extraction. These species of Micronecta were identified using morphological characteristic provided by Nieser et al. (2005) and Ha and Tran (2021). Whole genomic DNA was extracted from the heads and thoraces using the CTAB method (Reineke et al. 1998).

Table 1.

Taxon sampling used in this study.

Family Genus Subgenus Species GenBank Accession
Mitogenome 18S nrDNA 28S nrDNA
Micronectidae Micronecta Basileonecta Micronecta orientalis * OQ606211 OQ598531 OQ598681
Ctenonecta Micronecta jaczewskii * OQ606210 OQ598532 OQ598676
Dichaetonecta Micronecta sahlbergii * OQ606212 OQ598530 OQ598687
Lundbladella Micronecta guttatostriata * OQ606215 OQ598533 OQ598675
Micronecta Micronecta wui wui * OQ581713 OQ598526 OQ598678
Micronecta Micronecta anatolica * OQ606213 OQ598529 OQ598679
Micronecta Micronecta vietnamica* OQ606216 OQ598534 OQ598684
Micronecta Micronecta drepani * OQ606217 OQ598535 OQ598685
Micronecta Micronecta erythra * OQ606218 OQ598536 OQ598683
Micronecta Micronecta tuberculata * OQ606219 OQ598537 OQ598682
Micronecta Micronecta ornitheia * OQ606220 OQ598538 OQ598680
Micronecta Micronecta griseola OP850016(COI) OP850221(16S) / OP849810
Micronecta Micronecta minutissima OP849995(COI) OP850197(16S) / OP849786
Micronecta Micronecta poweri OP849996(COI) OP850198(16S) / OP849787
Sigmonecta Micronecta quadristrigata * OQ587936 OQ598527 OQ598686
Unguinecta Micronecta melanochroa * OQ606214 OQ598528 OQ598677
Unguinecta Micronecta khasiensis OP849907(COI) OP850107(16S) / OP849696
Tenagobia Incertagobia Tenagobia incerta * OR545228 OR544013 OR552402
Corixidae Sigara Sigara striata * OQ606224 OQ598548 OQ598671
Paracorixa Paracorixa concinna * OQ606223 OQ598547 OQ598672
Cymatia Cymatia coleopterata * OQ606225 OQ598542 OQ598668
Callicorixa Callicorixa praeusta * OQ606221 OQ598543 OQ598673
Corixa Corixa punctata * OQ606226 OQ598544 OQ598670
Glaenocorisa Glaenocorisa propinqua * OQ606222 OQ598545 OQ598674
Hesperocorixa Hesperocorixa linnaei * OQ606227 OQ598546 OQ598669
Diaprepocoridae Diaprepocoris Diaprepocoris barycephalus * OQ612738 OQ598549 OQ598666
Diaprepocoris zealandiae * OQ612739 OQ598550 OQ598667
Belostomatidae Diplonychus Diplonychus rusticus FJ456940 KJ461265 KJ461227
Lethocerus Lethocerus indicus KM588201 OQ598541* OQ598663*
Nepidae Laccotrephes Laccotrephes sp. FJ456948 OQ598540* OQ598662*
Gelastocoridae Nerthra Nerthra indica NC012838 KJ461313 KJ461276
Ochteridae Ochterus Ochterus marginatus FJ456950 KJ461251 KJ461315
Notonectidae Enithares Enithares sp. FJ456949 OQ598539* OQ598664*
Notonecta Notonecta sp. KX034086 FJ372662 OQ598665*
Aphelocheiridae Aphelocheirus Aphelocheirus ellipsoideus FJ456939 KJ461184 KJ461297
* Species with newly sequenced mitogenomes and nrDNAs, or newly sequenced nrDNAs in the present study.

2.2. Low-coverage genomic sequencing and assembly, annotation, and analysis

An independent DNA library was constructed for each species with an insert size of 250 base pairs (bp), and then sequenced with a 150 bp paired end (PE) using the Illumina HiSeq 4000 Platform at Biomarker Technologies (Qingdao, China). The purified reads were filtered from raw data by removing adaptor contamination and low-quality sequences. To better distinguish the repeat fragment brought by the assemble process, two approaches were employed to assemble the complete mitogenome for each species. For the first method, SOAPDENOVO2 (Luo et al. 2012) were applied to conduct de novo assembly under different settings respectively (-k = 61&71). Then the mitogenome and nrDNA assemblies were identified using the program BLAST+ (Camacho et al. 2009) against local databases. All the reference sequences of mitogenome and nrDNA used for constructing local databases were downloaded from the GenBank database. For the second method, MITOBIM (Hahn et al. 2013) was employed to bait and assemble mitogenomes directly referring to the mitogenomes of closely related species. As for the nrDNAs, only the first method under different k values was employed.

The online webserver of MITOS (Bernt et al. 2013) was used to annotate each mitogenome, as well as predict and determine tRNA structures with invertebrate mitochondrial genetic codes. The boundary of protein-coding genes (PCGs) were re-confirmed through Open Reading Frame Finder (ORF Finder) ( and verified manually by an alignment with homologous genes from published heteropteran mitogenomes. The boundaries of 12S and 16S rRNAs were delimitated by the boundaries of tRNA-Leu (L1) and tRNA-Val (V) and compared with homologous regions of known nepomorphan mitogenomes. Boundary definitions of 18S and 28S nrDNAs were also realized by alignment with homologous genes.

Base composition and relative synonymous codon usage (RSCU) were calculated using MEGA 11 (Tamura et al. 2021). Base compositional skews were measured using the formulae AT-skew = (A−T)/(A+T) and GC-skew = (G−C)/(G+C) (Perna and Kocher 1995). DNASP v5 (Librado and Rozas 2009) was used to calculate the rate of non-synonymous substitutions (Ka) and synonymous substitutions (Ks), and the ratio of Ka/Ks for each PCG, in order to evaluate the evolutionary rate of micronectid mitochondrial PCGs. ALIGROOVE (Kück et al. 2014) was used to analyze the compositional heterogeneity across sequences.

2.3. Phylogenetic analyses

Phylogenetic relationships of Micronecta were reconstructed based on 37 genes from mitochondrion and 18S and 28S nrDNAs. Individual genes were aligned using MUSCLE integrated in MEGA. The ambiguously aligned sites from both protein and nucleotide alignments were removed using GBlocks (Talavera and Castresana 2007). Then all individual matrixes were concatenated into three datasets for phylogenetic analyses: (1) the PCGNTRNA matrix, including nucleotide sequences of 13PCGs, 22 tRNAs, and two nrDNAs (File S1: PCGNTRNA); (2) the PCGNT12RNA matrix, including the first two codons of nucleotide sequences of 13PCGs, 22 tRNAs, and two nrDNAs (File S2: PCGNT12RNA); (3) the PCGAARNA matrix, comprising amino-acid sequences of 13PCGs and nucleotide sequences of 22 tRNAs and two nrDNAs (File S3: PCGAARNA).

Phylogenetic analyses were conducted using MRBAYES 3.2.6 (Ronquist et al. 2012) for Bayesian inference (BI) and RAXML 8.2.12 in PThreads version (Stamatakis 2014) for Maximum likelihood (ML). We used IQ-TREE (Nguyen et al. 2015) to obtain the best matched substitution model and partitioning schemes. For the BI inference with PCGAARNA matrix, a “mixed” substitution model for amino-acids and a GTR model for nucleotides were employed with a discrete gamma model (G) allowing for a proportion of invariable sites (I). While for the ML analysis with PCGAARNA matrix, the substitution model GTR+G+I for nrDNAs, rRNAs, and tRNAs; amino acid substitution models mtArt+G+I for COI and mtZOA+G+I for the remaining PCGs turned out to be the most appropriate ones. For phylogenetic analyses with matrixes PCGNTRNA and PCGNT12RNA, the substitution model GTR+G+I was employed. In BI analyses, we conducted 2,000,000 generations with sampling every 100 generations. The generations with values of the standard deviation greater than 0.01 were discarded. The numbers of burned generations were also checked with the help of Tracer (available at In ML analyses, the best ML tree and bootstrap trees were assessed by 1,000 rapid bootstrap replicates (-f a option).

3. Results

3.1. Genome organization and nucleotide composition

In this study, lengths of the 13 newly obtained mitogenomes of Micronecta species range from 14,825 bp to 15,405 bp (Table 2). The mitogenomes of M. (Micronecta) wui wui, M. (Unguinecta) melanochroa, M. (Micronecta) anatolica, M. (Micronecta) vietnamica, and M. (Micronecta) ornitheia were complete, and the rest mitogenomes were nearly complete with a partial control region (CR). All mitogenomes included 37 genes (13 PCGs, 22 tRNAs, and 2 rRNAs) and a control region, sharing the same strand distribution pattern of coding genes: 23 genes located on the majority strand; the remaining 14 genes located on the minority strand (Fig. 1, Fig. S1). Comparison of the mitogenomes of Micronecta species indicated that the PCGs, tRNAs, and rRNAs are relatively conservative in length (14,367–14,482 bp). Detailed statistics for the mitogenomes of the remaining water boatmen were showed in the supplementary Table S1.

Table 2.

Length of Micronecta mitochondrial genomes, AT-skew and GC-skew were measured for the 37 genes except the control regions.

Species PCGs (bp) tRNAs (bp) 12S rRNA (bp) 16S rRNA (bp) CR (bp) Total (bp) AT-skew GC-skew
Micronecta (Basileonecta) orientalis 11029 1425 730 1127 681 15163 0.16 –0.14
Micronecta (Ctenonecta) jaczewskii 11044 1425 761 1226 590 14998 0.16 –0.13
Micronecta (Dichaetonecta) sahlbergii 11017 1425 759 1241 664 15074 0.22 –0.22
Micronecta (Lundbladella) guttatostriata 10996 1430 768 1242 944 15311 0.22 –0.27
Micronecta (Micronecta) wui wui 11033 1425 761 1224 589 14992 0.15 –0.12
Micronecta (Micronecta) anatolica 10981 1423 761 1226 589 14990 0.15 –0.11
Micronecta (Micronecta) vietnamica 11024 1425 762 1223 666 15072 0.16 –0.14
Micronecta (Micronecta) drepani 11018 1428 759 1271 939 15405 0.21 –0.14
Micronecta (Micronecta) erythra 11023 1427 761 1224 777 15195 0.23 –0.16
Micronecta (Micronecta) tuberculata 11023 1425 761 1225 414 14825 0.14 –0.12
Micronecta (Micronecta) ornitheia 11028 1424 760 1262 592 15032 0.16 –0.14
Micronecta (Sigmonecta) quadristrigata 11014 1431 761 1248 578 15000 0.16 –0.25
Micronecta (Unguinecta) melanochroa 11187 1428 763 1244 519 14948 0.19 –0.23
Figure 1. 

Circular diagram of the Micronecta (Micronecta) ornitheia mitogenome. The transcriptional direction is denoted by arrows.

The nucleotide composition of Micronecta mitogenomes biased toward A/T, with A+T contents ranging from 69.65% to 74.0% (Fig. S2). The mitogenomes of M. (Dichaetonecta) sahlbergii and M. vietnamica exhibit the lowest and highest A+T contents, respectively. The AT skew and GC skew present similar patterns in all Micronecta mitogenomes, with positive AT skews (from 0.14 to 0.23) and negative GC skews (from –0.27 to –0.11) (Table 2).

The total length of all 13 PCGs ranges from 10,981 bp in M. anatolica to 11,187 bp in M. melanochroa (Table 2). The A+T content of the 13 PCGs ranges from 68.54% (M. vietnamica) to 73.63% (M. sahlbergii). The majority of the PCGs in the thirteen Micronecta mitogenomes initiate with conventional star codons (ATN), except for ND2, ND4L, ND5, which use TTG as the star codon in several species. The most frequently used stop codon is TAA, followed by T and TAG. Meanwhile, the most prevalent codons are UUA(L), AUU(I), UUU(F), AUA(M), UAU(Y) and AAU(N), whereas AGG(S), CGC(R) and CGG(R) are rarely used (Fig. S3).

The Ka/Ks ratio is used to evaluate the evolutionary rate of 13 PCGs of the Micronecta species (Fig. S4). The results showed that the average Ka/Ks ratios are lower than 1, indicating that these PCGs evolved likely under the purifying selection (Hurst 2002; Ye et al. 2021). Among which the COI had the lowest evolutionary rate (0.020), while ATP8 had the highest evolutionary rate (0.471). The average Ka/Ks ratios of COI, COII, COIII, and CYTB (Ka/Ks<0.1) are lower than that of the remaining genes, indicating these four genes are usually under stronger selection and constraints.

There are 22 tRNA genes in the Micronecta mitogenomes, as observed in other heteropteran mitogenomes. All tRNAs display the classic clover-leaf secondary structure except tRNA-Ser (GCU), with the dihydrouridine (DHU) stem simply forms a loop (Fig. S5). The A+T content of tRNAs ranges from 72.54% (M. (Unguinecta) quadristrigata) to 76.0% (M. vietnamica).

The 12S and 16S rRNA genes in Micronecta species are encoded on the J-strand and located at conserved positions between trnL1 and trnV and between trnV and control region, respectively. The length of 12S rRNA varies from 730 bp in M. (Basileonecta) orientalis to 768 bp in M. (Lundbladella) guttatostriata, with A+T content from 71.22% in M. guttatostriata to 76.61% in M. (Micronecta) tuberculata. The length of 16S rRNA ranges from 1,127 bp in M. orientalis to 1,271 bp in M. (Micronecta) drepani, with A + T content from 73.67% in M. guttatostriata to 78.09% in M. vietnamica. Hence, there is no substantial size variation in 12S and 16S rRNA among the mitogenomes of the thirteen Micronecta species (Table 2).

Heterogeneous composition of amino-acid or nucleotide sequences may bias results of likelihood based tree reconstructions. The AliGROOVE analyses showed a low heterogeneity in both nucleotide sequences and amino-acid sequences of PCGs (Fig. 2). Thus, our phylogenetic results were hardly influenced by sequence heterogeneity.

Figure 2. 

The compositional heterogeneity of mitochondrial sequences used in phylogenetic analyses. The mean similarity score between sequences is represented by a colored square, based on the AliGROOVE scores from -1, indicating great differences in rates from the remainder of the datasets (red), to +1, indicating rates match all other comparisons (blue).

3.2. Phylogenetic analyses

Phylogenetic analyses using both BI and ML approaches based on different datasets produced a congruent and well-resolved tree (Fig. 3, Figs S6–S11). All families of Corixoidea, i.e., Micronectidae, Corixidae and Diaprepocoridae, were consistently recovered as monophyletic groups.

Figure 3. 

Phylogenomic relationships of Micronecta. The tree was constructed using the PCGNT12RNA dataset with Bayesian analysis. The bootstrap values of maximum-likelihood analyses and posterior probabilities of Bayesian analyses are summarized and labelled around each node. Higher taxa are indicated as taxon labels on the right of the tree.

Within Micronectidae, the genus Micronecta was strongly supported as a monophyletic group and split into three well-supported clades. subgenus Lundbladella was recovered as the sister group to all other Micronecta by all analyses. Subgenus Unguinecta were supported as the sister group to subgenera Dichaetonecta and Sigmonecta. Subgenus Micronecta together with subgenera Ctenonecta and Basileonecta formed a monophyletic clade. Subgenus Micronecta were recovered as paraphyly based on both BI and ML analyses. For this clade, the relationship among the three groups, i.e., (M. vietnamica + M. drepani + M. (Micronecta) erythra), (M. orientalis + M. tuberculata), and (M. ornitheia, M. wui wui, M. anatolica, M. (Ctenonecta) jaczewskii, M. poweri, M. griseola, M. minutissima) are controversial among different analyses (Fig. S6–S11). The results of all BI analyses support the sister relationship between (M. orientalis + M. tuberculata) and (M. ornitheia, M. wui wui, M. anatolica, M. jaczewskii, M. poweri, M. griseola, M. minutissima), while the ML analyses exhibit different topologies.

4. Discussion

Our study presents 13 newly sequenced mitogenomes of the genus Micronecta, 1 that of the genus Tenagobia (Micronectidae) and ten those of the remaining water boatmen (Corixidae, Diaprepocoridae). All mitogenomes exhibited the similar putative pattern as in other heteropteran insects (Cameron et al. 2014; Ye et al. 2021).

Phylogenetic trees based on mitogenomes and nrDNAs are largely congruent among different analyses, which laid a foundation for further phylogenetic analyses and taxonomic studies. Before this study, only two works involved the phylogenetic relationships between micronectid genera of continental Australia based on morphological characters (i.e., Tinerella, 2008, 2013), in which the subgenera Dichaetonecta and Sigmonecta were also recovered as sister groups. They share the same shape of the left paramere shaft, which is long, straight and narrow.

Among the 11 nominated subgenera, male individuals of three subgenera, i.e., Lundbladella, Indonectella, Micronectella, lack the strigil structure on abdominal tergite VI (Wróblewski 1967; Ha and Tran 2021). While within Micronectidae, both the genera Monogobia and Tenagobia lack this structure as well. Presence and absence of strigil is likely a secondary character, which cannot serve as the evidence for the close relationship among the three subgenera mentioned above. Both the subgenera Lundbladella and Indonectella are monotypic subgenus, while Micronectella include two species. Unfortunately, it was not possible to analyze representatives of subgenera Indonectella and Micronectella. The status of the subgenus Lundbladella as sister group to all other Micronecta in this study needs to be verified with more taxa sampling from other subgenera, especially Indonectella and Micronectella.

According to the identification key provided by Hutchinson (1940) and Ha and Tran (2021), diagnostic features of current subgenera of Micronecta were only applicable to male specimens, i.e., the shape of the palar claw, the setae of seventh abdominal sternite, the free lobe of eighth abdominal tergite and the morphology of the left paramere, some of which are potentially homoplasious characters. For example, the free lobe of subgenus Dichaetonecta and Micronecta, is nearly rectangular. The left paramere of subgenera Basileonecta and Ctenonecta is styliform. As the phylogenetic results shown, both the subgenera Basileonecta and Ctenonecta imbedded within the subgenus Micronecta and therefore their subgeneric status is questionable. It probably need more stable characteristics to identify or redefine current subgenera.

The genus Micronecta is the most diverse and speciose group of Micronectidae, which is the same condition with the subgenus Micronecta. Although 11 subgenera have been proposed to accompany the extant species of the genus Micronecta, there are still some species which do not fit any known subgenus. As a result, they were just placed tentatively into the subgenus Micronecta (see Jansson 1995). There are also some species which do not fit any known subgenus were left as incertae sedis or species groups (Ha and Tran 2021). In future, a more comprehensive taxon sampling including all subgenera even those species that were not assigned to any subgenera is still expected via a broad range of international collaborations.

As a result, the current taxonomy of Micronecta does not yet satisfactorily reflect natural relationships among subgeneric taxa. As more and more species are being describ, it is necessary to redefine the subgenus Micronecta or split it into more subgenera. The taxon sampling might not be that complete, although the paraphyly of the subgenus Micronecta can be revealed convincingly.

5. Conclusion

In this study, we investigated the phylogenetic relationships concerning the genus Micronecta based on the species sampled in China. This is the first time that the subgeneric relationships among Micronecta were investigated based on molecular evidence. Our main findings are the paraphyly of the subgenus Micronecta, the status of the subgenus Lundbladella as sister group to all other studied Micronecta, and the sister relationship between the subgenera Dichaetonecta and Sigmonecta. This study provided a chance to redefine the subgenera level classification of the genus Micronecta and laid a foundation for further molecular studies with complete taxon sampling to fully resolve the phylogeny of Micronecta via a broad range of international collaborations.

6. Authors’ contributions

Conceptualization, Y.W., Q.X.; funding acquisition, Y.W.; formal analysis, B.X., Y.W. and J.X.; writing—original draft preparation, B.X., Y.W., Q.X.; writing—review and editing, J.D., P.C. and Q.X. All authors have read and agreed to the published version of the manuscript.

7. Acknowledgements

We are grateful to Dr. Jiu-Yang Luo (Yancheng Teachers University, China) and Dr. Yu Men (Zhaoqing University, China) for collecting specimens and providing helpful assistance during phylogenetic analyses. We appreciate Dr. Michael Raupach (Zoologische Staatssammlung München) and Dr. Anh Duc Tran (Vietnam National University) for valuable suggestions to improve the quality of our manuscript. This work was supported by the National Natural Science Foundation of China (grant number: 32370468). The authors have declared that no competing interests exist.

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Supplementary materials

Supplementary material 1 

Figures S1–S11

Xie B-J, Chen P-P, Damgaard J, Xie J-Y, Xie Q, Wang Y-H (2024)

Data type: .docx

Explanation notes: Figure S1. Circular diagram of the mitochondrial genomes of Micronecta spp. and Tenagobia incerta. — Figure S2. The A+T content of Micronecta spp. mitochondrial genomes. — Figure S3. Relative synonymous codon usage (RSCU) of mitochondrial genomes of Micronecta spp. — Figure S4. Average evolutionary rate of Micronecta mitochondrial PCGs. — Figure S5. Universal models of Micronecta mitochondrial tRNAs. — Figure S6. Phylogenetic tree inferred from PCGNTRNA matrix using ML analysis. Numbers at the nodes are bootstrap values. — Figure S7. Phylogenetic tree inferred from PCGNTRNA matrix using BI analysis. Numbers at the nodes are Bayesian posterior probabilities. — Figure S8. Phylogenetic tree inferred from PCGNT12RNA matrix using ML analysis. Numbers at the nodes are bootstrap values. — Figure S9. Phylogenetic tree inferred from PCGNT12RNA matrix using BI analysis. Numbers at the nodes are Bayesian posterior probabilities. — Figure S10. Phylogenetic tree inferred from PCGAARNA matrix using ML analysis. Numbers at the nodes are bootstrap values. — Figure S11. Phylogenetic tree inferred from PCGAARNA matrix using BI analysis. Numbers at the nodes are Bayesian posterior probabilities.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (12.91 MB)
Supplementary material 2 

Tables S1, S2

Xie B-J, Chen P-P, Damgaard J, Xie J-Y, Xie Q, Wang Y-H (2024)

Data type: .docx

Explanation notes: Table S1. Mitochondrial genome statistics for the other water boatmen. AT-skew and GC-skew were measured for the 37 genes except the control regions. Only partial mitogenome of Diaprepocoris zealandiae was available, so it was not included in this table. — Table S2. Locality data for each Micronecta species used in this study.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (18.18 kb)
Supplementary material 3 

Files S1–S3

Xie B-J, Chen P-P, Damgaard J, Xie J-Y, Xie Q, Wang Y-H (2024)

Data type: .zip

Explanation notes: File S1. PCGNTRNA matrix. — File S2. PCGNT12RNA matrix. — File S3. PCGAARNA matrix.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (246.72 kb)
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