A New Species of Eucnemidae (Coleoptera: Elateroidea) with Its Complete Mitogenome and Mitogenome-Based Phylogeny of Elateroidea

1. Introduction

The false click beetles (Eucnemidae) constitute a medium-sized family that has received scant research attention in China and is often misidentified in the field as true click beetles (Elateridae). The family is comparatively species-rich, with approximately 185 genera and 1,700 species [1], and its members possess a distinctive labral–clypeal complex that serves as a reliable diagnostic character. A handful of studies have shown that eucnemids represent a significant component of tropical-forest beetle diversity [2] and can function as effective indicators of forest structural heterogeneity; population declines have been linked to reduced volumes of dead wood and coarse woody debris resulting from modern forestry practices [3]. To date, only 27 species in three subfamilies have been recorded from China, and a large proportion of the fauna remains undescribed.

Research on this family has so far focused almost exclusively on morphological descriptions and taxonomic characters; molecular systematics has been almost entirely neglected. Brüstle & Muona examined Hylochares cruentatus from Finland and Russia using COI, 12S rDNA, 18S rDNA and 28S rDNA and found no genetic differentiation corresponding to morphological variation [4]. Subsequently, the phylogeny of the genus Arrhipis Bonvouloir was clarified using five molecular markers in combination with morphological data [5].

The insect mitogenome is the most thoroughly studied invertebrate genome to date. It is structurally conserved and compact, typically a 15–18 kb circular molecule that encodes 37 genes—13 protein-coding genes, 2 rRNAs, and 22 tRNAs—plus a control region (A+T-rich region). Among these, the cytochrome c oxidase subunit I (COI) gene has become the nearly exclusive data source for DNA barcoding, making mt sequences by far the most abundant insect entries in GenBank [6]. Prior to this study, however, only three complete or partial mt genomes from the family Eucnemidae were available in GenBank: Melasis buprestoides (KX087315) and two Eucnemidae sp. (MH923241 and JX412858).

Here we describe a new species, Microrhagus ziwulingensis Muona & Meng, sp. nov., Microrhagus Dejean, 1833 is widely distributed and the most prominent genus among the 12 genera in the tribe Dirhagini Reitter, 1911, which is currently represented in the Palaearctic Region by 17 described species [7,8,9]. Two Microrhagus species were previously known from China.

We present its complete, annotated mitogenome (GenBank accession OK143440). Using the 13 PCGs, we reconstructed phylogenetic relationships within Eucnemidae with the two previously published eucnemid sequences and additional representatives from the superfamily Elateroidea, thereby providing new insights into the higher-level relationships of this poorly investigated lineage and Elateroidea.

2. Materials and Methods

2.1. Sample Collection

The specimens of M. ziwulingensis sp. nov. were caught by the flight interception trap in Mt. Ziwuling (子午岭), Shanxi Province, China. They are deposited in SZPU (Shenzhen Polytechnic University, Shenzhen, Guangdong, China) and GZNU (Guizhou Normal University, Guiyang, Guizhou, China) under the collection number of SZEU-0101.

For the present study, all specimens were collected by the flight interception traps (FIT) and Malaise traps (MT) in July to August 2019. They were preserved in absolute ethanol for subsequent molecular experiments. The holotype and paratypes were prepared as dry specimens. Observations of the habitus and diagnostic characters were made using Zeiss Discovery V20 and Nikon SMZ645 microscope. Digital images were taken with Canon 800D and Helicon focus 7.6.1 system. Before dissection, the specimens were submerged in proteinase K solution (20mg/ml) in the water bath at 55 ℃ for approximately 4 hours. Genitalia was dissected, and other tissues surrounding the genitalia were removed after steeping them in proteinase K solution (20mg/ml) for 1 hour in the water bath at 55 ℃. Dissected genitalia was mounted on slides for photography. The morphological terminology follows Muona [7,10].

2.2. Sequencing, Assembly and Annotation

Total genomic DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method and applied to 500-bp paired-end library construction using the NEBNext Ultra DNA Library Prep Kit for Illumina sequencing. Sequencing was carried out on the Illumina NovaSeq 6000 platform (BIOZERON Co., Ltd., Shanghai, China). M. ziwulingensis sp. nov. genome assembly and annotation were conducted by SPAdes v3.14.1 [11] and MITOS2 WebServer (https://usegalaxy.org/) [12], respectively.

Using Map to Reference function in Geneious Prime 2025.1.2 (https://www.geneious.com) and quality-trimmed selecting the mitogenome of Stenothemus fukienensis Wittmer, 1974 using reference sequences in Geneious Prime 2025.1.2 with default parameters. Then, the contig was assembled and annotated into the complete circular mitogenome in a similar way also using the Geneious Prime 2025.1.2 and S. fukienensis as a reference. The annotated mitogenome was deposited in GenBank.

2.3. Sequence Analyses

Complete mitogenome sequences of M. ziwulingensis sp. nov. was deposited in GenBank and given the accession numbers OK143440. The nucleotide composition and comparison with the other two eucnemid mitogenomes (Melasis buprestoides, accession number KX087315 and Eucnemidae sp., accession number MH923241. The other retrievable Eucnemidae sp. represents only a partial mitogenome, with several coding regions missing, and was therefore excluded), as well as the calculation of relative synonymous codon usage (RSCU) values, were performed using PhyloSuite v1.2.3 [13]. Genetic distances among the mitogenomes of the three species were calculated using MEGA X [14]. Nucleotide diversity (Pi value) of 13 PCGs among three eucnemid mitogenomes was estimated by DnaSP v6.12.03 [15] using a sliding-window analysis, window size 200bp and the step size to 20bp.

2.4. Phylogenetic Analyses

To reveal the phylogenetic position of the newly described M. ziwulingensis sp. nov., we assembled 41 complete mitogenomes representing 35 elateroid species, supplemented by eight buprestoid taxa as outgroups. Within the Elateroidea dataset, duplicate mitogenomes were available for Ludioschema sulcicolle, Melanotus cribricollis, Rhagophthalmus lufengensis, Luciola kagiana, Cephalomalthinus imparicornis, and Prothemus sanguinosus (Table 1). The phylogenetic analysis was based on PhyloSuite v1.2.3. The maximum likelihood (ML) bootstrap analysis with 5000 replicates under the GTR+F+R9 model was performed using IQtree v1.6.8 [16].

3. Results

3.1. Taxonomy

Microrhagus ziwulingensis Muona & Meng sp. nov. (Figure 1, Figure 2 and Figure 3)

Zoobank registration link: urn:lsid:zoobank.org:pub:236D6F35-503F-4A39-9714-C7716E2716D3.

Type materials. Holotype, male, China, Shaanxi, Shihuigou Valley, Ziwuling national nature reserve, Yan’an City, 1256m, 27–30.VII.2019, Jian Shen, Changping Ding, and Rui Dang leg. (SZPU). Paratypes: 1 females, China, Shaanxi, Shihuigou Valley, Ziwuling national nature reserve, Yan’an City, 1256m, 6.VII.2019–1.VIII.2019, Jian Shen, Changping Ding, and Rui Dang leg. (SZPU); 1 males and 1 females, Huashugou Valley, Ziwuling national nature reserve, Yan’an City, 1256m, 6.VII.2019-13.VII.2019, Jian Shen, Changping Ding, and Rui Dang leg. (GZNU); 1 males, Chenjiahe River, Ziwuling national nature reserve, Yan’an City, 1275m, 3.VII.2019–13.VII.2019, Jian Shen, Changping Ding, and Rui Dang leg. (GZNU). Notes: 27 specimens were used for mitochondrial genome sequencing.

Distribution. China, Yan’an City.

Etymology. The species name refers to the type locality - Ziwuling.

Diagnosis. The diagnostic feature distinguishing M. ziwulingensis Muona & Meng sp. nov. from two other Chinese species M. klapperichi and M. savioi are as follows.

M. savioi (4.25 mm) is longer than M. ziwulingensis sp. nov. (3.8 mm) in body length; in M. savioi, the antennal pectinations are longer and undulate, whereas in M. ziwulingensis sp. nov. they are straight; M. savioi has a median carina on the pronotum, however, in M. ziwulingensis sp. nov. it is absent; the lateral margins of the pronotum in M. ziwulingensis are straight, with the disc lacking a pair of shallow impressions at median portion, such impressions in M. savioi caused the plate to buckle and extend, resulting in medially arched outward lateral margins.

In females, the lateral carina of the posterior angle extend to anterior margin in M. klapperichi by, whereas in M. ziwulingensis sp. nov., this carina does not extend to anterior margin. Additionally, the body of M. klapperichi is dark brown to blackish, appearing darker than M. ziwulingensis sp. nov. overall.

Description (holotype, male). Body fusiform, convex, dark and shiny, punctate, covered with yellowish pubescence, mandibles, antennae and legs reddish brown, tarsi yellowishbrown. body length 3.8 mm, width 1.2 mm, elytral length 2.7 mm, pronotal length 1.1 mm, antennal length 2.9 mm (Figure 1A-C). Punctation circular and dense, fused at clypeal region. (Figure 1D).

Head wider than long, inserted into prothorax, densely punctate, a longitudinal region non-punctate in front. Pubescence denser and longer towards the clypeal edge. Clypeus widest at lateral apices, about 9 times as wide as at base between antennal sockets, medially arcuate, with long setae, punctures increasingly rough and dense towards edge, eventually fused causing the surface wrinkled. Eyes medium-sized, round (Figure 1D). Mandibles stout, bidentate, densely irregular punctate, with long pubescence (Figure 1E).

Antennae long, beyond two-thirds of body; scape cylindrical, robust, about 5 times as long as antennomere 2; antennomere 2 shortest; antennomere 3 about 1.5 times as long as antennomere 4. Antennomere 4 forming subtriangular lobe at middle; Antennomeres 5 to 10 with rather long process near apex, rarely becoming longer to antennomere 8 and then becoming shorter to 10; Antennomeres 11 obviously elongate, curved, and about 2.8 times longer than antennomere 10 (Figure 1G).

Pronotum about 1.3 times wider than long (midline), widest at one forth near apices of posterior angles, lateral margins gradually narrow in the anterior third, and are parallel-sided until base of posterior angles; disc convex, anteriorly margin with finely ridge, exceeding about one-third of pronotum length on sides; absent on base of pronotum in front of scutellum, with a short, impunctate median region; disc with dense punctures separated about 0.5–1.0 times of their diameter; long pubescence protrude from the punctures and combed backwards. Posterior angles strong, subtriangularly, acute, apices toward to hind, and with finely carina extending anteriorly to about four-fifths along the lateral margin (Figure 1F). Punctates on hypomere densely, irregularly, covered with hairs pointing posterolateral; antennal grooves well-developed and defined, smooth, anterior opened. Prosternum punctures relatively sparser than disc, anterior edge of prosternum arched ridge (Figure 1E). Prosternal process convex at base, evenly depression behind procoxae, deeply insert mesosternal cavity, gradually narrowed, acutely at apex. Two pits nearby prosternal fossae more deeply and evident in males than females (Figure 1E and Figure 3B).

Scutellum slightly longer than broad, triangular, disc flat, punctates sparsely at apex than base. Base margin straightly, apex blunt (Figure 1F).

Elytra about 4.2 times as long as wide and about 2.5 times as long as pronotum, widest at humeri, sides subparallel and gradually tapering to end, covering the apex of abdomen. Elytral striae deeply impressed at base, sharp with convex interstices, sutural stria continues through, striae 2–4 vanish halfway through, and others are basal only. Integument with fine and dense punctures, interspaces between punctures on disc about 1.0–3.0 times of a puncture diameter; punctures becoming coarser and confluent on humeri and apices (Figure 2A).

Metanepisterna is about 4.0 times as long as wide, with sides subparallel. Metaventrite and metanepisterna finely and moderately densely punctate, metaventrite with punctures larger and closer to each other on laterad, evanescent on narrow region medially and apically, the distance between punctures mostly 0.5–2.0 times their diameter. Metacoxal plates abruptly expanded in medial sixth and evenly narrowed laterad, integument with fine and moderately dense punctation, the distance between punctures about 0.5–2.0 times their diameter (Figure 2B).

Legs moderately slender, uniformly hairy. Protibiae shorter than femora, length of meso- and metatibiae subequal to femora. Protibiae with one apical spur, with minute spine-coms on lateral side, protarsomere I with apical sex-comb (Figure 1H); meso- and metatibiae with two well-developed apical spurs, with orderly spaced spine-combs on lateral sides. Protarsus about 3/5 as long as protibia; meso- and metatarsi slightly shorter to corresponding tibiae. Claws simple (Figure 2B).

Abdomen long oval, with simple, dense punctation, distances between punctures 3.0–5.0 times puncture diameter; punctation becomes denser toward apex and base, punctures coarser and even partially confluent at apex; densely covered with long setae, directed backward; ventrite V gently rounded at apex. Tergite IX semi-oval, margin with long setae, apex with incision; tergite X small, triangular. Sternite IX semi-oval, setae slightly denser at distal portion, basal struts united with a transverse bridge (Figure 2B).

Aedeagus elongated, almost 3.6 times longer than wide, median lobe fused with lateral lobes, gently curved ventrally; lateral lobes enlarged near apex, densely clothed with elongate, fine setae; phallobase strongly constricted, 2.95 times longer than wide, approximately one-third the length of aedeagus (Figure 2C - F).

Male paratypes. Body length 3.7–3.9 mm, body width 1.1–1.3 mm, elytral length 2.6–2.7 mm, pronotal length 1.0–1.2 mm, antennal length 2.1–2.2 mm.

Female paratypes. Similar to males but shinier, pubescence comparatively sparse. The posterior median region of the pronotum is more convex, more rounded at anterior margin. Antennae and legs darker in color than in males. Body length 3.1–5.0 mm, body width 0.8–1.7 mm, elytral length 2.1–3.5 mm, pronotal length 0.8–1.3 mm, antennal length 1.8–2.8 mm (Figure 3A-E). Ovipositor extremely slender, apically cleft medially, with long setae at both sides (Figure 3F).

Natural history. The species occurred at elevations of about 1200m in the Ziwuling national nature reserve (Figure S1). At the site where the samples were collected (flight interception traps, malaise traps), the main trees were oak, white birch, aspen, and Chinese pine. The vegetation pattern belongs to the temperate deciduous broad-leaved forest, a natural secondary forest.

3.2. Nucleotide Composition

The full circular mitogenome of M. ziwulingensis sp. nov. was 15,843 bp in length. All of the typical 37 mitochondrial genes were found in the genomes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two rRNA genes (rrnS and rrnL) and one non-coding control region (D-loop). The GC content of the mt genome is 26.6%, the overall base composition was A (40.5%) > T (32.9%) > C (16.2%) > G (10.4%), and a strong A-T bias (73.4%) was detected.

The 13 concatenated protein-coding genes (PCGs) of M. ziwulingensis sp. nov. span 10 995 bp, representing 69.4% of the mitogenome. Overall, the PCG set exhibits a negative AT-skew (−0.131) and GC-skew (-0.032) (Table S2). ATP8 is the smallest gene (153 bp), whereas NAD5 is the largest (1 693 bp) (Table S1). At the third codon position, the CG content drops to 22.5%, significantly below the values observed at the first (32.4%) and second (32.4%) positions. Among the 13 PCGs, four (NAD1, NAD4, NAD4L and NAD5) are encoded on the minority (N) strand, while the other nine are situated on the majority (J) strand (Figure 4, Table S1), additionally, all PCGs initiate with the ATN start codon (ATA, or ATG), end with the putative terminal codons TAA or TAG (Table S1).

The relative synonymous codon usage (RSCU) profiling of the three eucnemids mitogenomes (Figure 5) revealed the most frequently codon preference for Leu (UUA), Ile (AUU), followed by Phe (UUU), and Met (AUA). Furthermore, all codons with bars ≥ 1.5 exclusively end in A or U (UUU, AUU, UAU, AAU, AUA, UUA, etc.), revealing a pronounced A/U bias that aligns with the ~75% genome-wide AT content.

In M. ziwulingensis sp. nov. mitogenome, 14 gene overlaps ranging from 1 to 7 bp collectively span 32 bp, with the longest interval (7 bp) located at the nad4-nad4L junction; seven intergenic spacers of 1–73 bp add up to 107 bp, with the largest (73 bp) lying between control-region and trnI, and the second-largest (17 bp) located between nad1 and trnaL1. Consistent patterns are observed in two known mitogenomes: Eucnemidae sp. exhibits 14 overlaps (1–8 bp; 43 bp total) and 10 spacers (2–568 bp; 737 bp total), the longest spacer (568 bp) also being between control-region and trnI, and the second-largest (45 bp) between NAD5 and trnaH, whereas M. buprestoides harbors 19 overlaps (1–7 bp; 39 bp total) and 11 spacers (1–35 bp; 97 bp total), with the greatest intergenic distance (35 bp) situated between trnY and COX1 (Table S1).

3.3. Transfer and Ribosomal RNA Genes

The positions of all 22 typical transfer RNA genes (tRNAs) were located in M. ziwulingensis sp. nov. (Table S1). In which, 14 tRNAs are encoded on the majority (J) strand and the remaining eight on the minority (N) strand. The total length of the 22 tRNAs was 1425 bp in M. ziwulingensis sp. nov., accounting for 8.99% of their whole genomes, respectively. The sizes of the 22 tRNAs range from 61 (trnR, trnY) to 71 bp (trnK). All 22 tRNAs indicated a negative AT-skew (-0.028) and a positive GC-skew (0.344) (Table S2).

The 22 tRNA genes all folded into the canonical clover-leaf secondary structure comprising four length-conserved arms: the aminoacyl (acceptor) stem, the dihydrouridine (DHU) stem, the anticodon stem that determines tRNA isotype, and the TΨC stem. The only deviation was observed in trnS1, which lacks the DHU arm—a condition that is ubiquitous across metazoans [6]. The anticodon loops of all tRNAs were highly conserved, each containing 7 bp, whereas the DHU and TΨC loops exhibited length heterogeneity (Figure 6). In addition to canonical AU and GC pairs, 21 GU wobble pairs were identified. A single unpaired nucleotide was also present within the aminoacyl stem.

Both rRNA genes (rrnL and rrnS) are encoded on the minority (N) strand, with a combined length of 2,078 bp. The large-subunit rRNA (rrnL) is interposed between trnL1 and trnV and spans 1,272 bp, whereas the small-subunit rRNA (rrnS) lies between trnV and the control region and comprises 806 bp (Table S1). Both genes exhibit a pronounced AT bias, reaching 81.5% (Table S2). Additionally, the rRNA region displays a negative AT-skew (−0.028) and a positive GC-skew (0.344) (Table S2).

3.4. Nucleotide Diversity Analysis

The sliding window analysis concerning the nucleotide diversity (Pi values) of the 13 aligned PCGs among the three Eucnemidae mitogenomes: Eucnemidae sp., M. buprestoides and M. ziwulingensis sp. nov. are shown in Figure 7. This exhibits the high degree of nucleotide variation within different genes. Nucleotide diversity values range from 0.176 (COX1) to 0.348 (ATP8) in these three species. In all PCGs, the ATP8 (Pi = 0.348) presents the highest variability next to NAD6 (Pi = 0.308), NAD2 (Pi = 0.271) and NAD3 (Pi = 0.253) showing the comparatively high nucleotide diversity values. The NAD1 (Pi = 0.200), NAD4L (Pi = 0.200), сOX3 (Pi = 0.192) and COX1 (Pi = 0.176) with relatively low nucleotide diversity values indicate that they are relatively conserved genes in 13 PCGs (Figure 7).

3.5. Phylogenetic Relationships

Maximum-likelihood (ML) phylogeny recovered the designated ingroup as monophyletic: all Elateroidea specimens formed a single, strongly supported clade. Within this superfamily, the traditionally recognized families Eucnemidae, Elateridae, Lampyridae, Lycidae and Cantharidae were clearly resolved, congruent with previous molecular hypotheses [34,35]. Meanwhile, Rhagophthalmidae and Lampyridae emerged as sister taxa, as did the clade Phengodidae + Lycidae with Cantharidae. All internal nodes of Elateroidea received moderate to high bootstrap support. Duplicate mitogenomes for several species (e.g., Ludioschema sulcicolle, Melanotus cribricollis, Rhagophthalmus lufengensis, Luciola kagiana, Cephalomalthinus imparicornis and Prothemus sanguinosus) formed terminal sister pairs, confirming intraspecific concordance and data reliability (Figure 8).

In the present study, the described M. ziwulingensis sp. nov. formed a strongly supported clade with the other two eucnemid species. The relationships recovered here—Rhagophthalmidae + Lampyridae and Phengodidae + Lycidae with Cantharidae differ from those reported by Bocakova et al.[34], Kundrata and Bocak [35], Bocak et al. [36], and Kusy et al. [37,38] . We attribute this discrepancy to the fact that the latter study focused on superfamily-level relationships and included a broader sampling of distantly related families, whereas our dataset is restricted primarily to within Elateroidea. Future efforts should therefore prioritize obtaining additional mitogenomes from across Eucnemidae and expanding the taxonomic breadth of the ingroup sample; this will provide a more robust phylogenetic framework within which to test and refine the topologies reported here.

4. Discussion

Genus Microrhagus Dejean, 1833 is a large group with 156 described species [39]. Kovalev considered that this genus needs a revision due to its diversity and heterogeneity. In this study, we assembled the mitochondrial genome of M. ziwulingensis sp. nov. and performed phylogenetic analyses amongst Elateroidea.

The phylogenetic tree was constructed using maximum-likelihood (ML) method based on 13 protein-coding genes (PCGs) of the mitochondrial genome. 42 Elateroidea species were selected as ingroups and three Byrrhoidea and five Buprestoidea species as outgroups. To establish a robust phylogenetic framework, this study aimed to achieve broad taxonomic coverage across most families of Elateroidea. However, complete mitochondrial genomes were available for species limited within partial families of this superfamily. Nevertheless, the sampling in this study remains insufficient, representing a major limitation of the current analysis. In present work, branching structure of the phylogenetic tree differs from that of previous studies. In the studies of Bocakova et al.[34], Kundrata & Bocak [35], and Kusy et al. [37], Phengodidae + Rhagophthalmidae consistently formed a monophyletic group. Furthermore, in Kusy et al., Lampyridae, Phengodidae, Rhagophthalmidae, and Sinopyrophoridae together constituted the lampyroid clade. In contrast, two sister-group relationships, Phengodidae + Lycidae and Rhagophthalmidae + Lampyridae are reconstructed herein. Additionally, our phylogenetic tree does not reflect the previously established conclusions that Drilidae, Omalisidae, and Plastoceridae are embedded within Elateridae due to the absence of species for these three families in our dataset. All families exhibit strong monophyly, as does the entire Elateroidea, which forms a large monophyletic group. As noted by Kusy et al. [38]. and Muona & Taräväinen [40], when considering the extreme scenario that prioritizes monophyly alone, the entire Elateroidea could be treated as a single family.

The above-mentioned discrepancy may be attributed to differences in sequence fragments and species coverage. In present study, 13 PCGs were utilized, which differs from the gene fragments used in previous studies. Given the currently limited availability of mitogenome data, the present study can only offer a preliminary phylogeny of Elateroidea; nevertheless, these limitations highlight the need for broader taxonomic sampling and additional mitogenome data for Eucnemidae and other lineages in future studies, which would contribute to more robust phylogenetic frameworks.

5. Conclusions

Herein, we describe a new species of the genus Microrhagus Dejean, 1833 namely Microrhagus ziwulingensis Muona & Meng, sp. nov., from Ziwuling National Nature Reserve in Shaanxi Province, China. This discovery significantly expands the known distribution of the genus Microrhagus in China.

We further sequenced and assembled the complete mitochondrial genome of M. ziwulingensis. The circular mitogenome is 15,843 bp in length and contains the typical set of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), and two ribosomal RNA genes (rrnL and rrnS), along with a control region (Figure 4, Table S1). The mitochondrial genome is highly conserved in base composition, genome size and gene order, codon usage, and tRNA secondary structure relative to previously published eucnemid sequences. Maximum-likelihood phylogenetic analysis based on 13 protein-coding genes yielded strong support for most branches, providing a robust dataset for future phylogenetic investigations within Eucnemidae and Elateroidea.