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.

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

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.

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.

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.

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.

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.