Introduction

While assessing the polyphyletic subfamily Tenebrioninae Latreille, 1802, Kamiński et al. (2021) suggested the Central-Asian tribe Dissonomini Medvedev, 1968 (2 genera, 26 species, Iwan et al. 2020, Makhan 2018) as a potential member of the resurrected subfamily Blaptinae Leach, 1815. The inclusion of Dissonomini was suggested strictly based on morphological traits due to a lack of molecular data on the tribe at the time. Adult representatives in the tribe possess a reduced scutellar shield, aedeagal tegmen without ancorae (see Lumen & Kamiński 2023a), and ‘blaptoid’ habitus, all of which indicate a close morphological affiliation with the tribes Blaptini Leach, 1815 and Platyscelidini Lacordaire, 1859 (Medvedev 1968). Between these two tribes, Dissonomini appeared to share more adult morphological traits with Platyscelidini, such as widened pro- and mesotarsi (Medvedev 1968). However, when considering larval morphological characteristics, Dissonomini seemed to share more features in common with other Blaptinae, specifically Dendarini Mulsant & Rey, 1854, Pedinini Eschscholtz, 1829, and Platynotini Mulsant & Rey, 1853 (Medvedev 1968). Most larvae of those tribes are equipped with four enlarged apical spines on the terminal segment of the abdomen (Kamiński et al. 2019). As a result of conflicting morphology and the lack of molecular data, Kamiński et al. (2021) decided not to place Dissonomini within Blaptinae formally, pending additional data.

To investigate the phylogenetic position of Dissonomini, a historical museum specimen of Dissonomus tibialis Reitter, 1904 was sequenced using targeted enrichment with a hybridization capture probe set (after Swichtenberg et. al. 2023). Phylogenetic analyses were then conducted using the recovered genetic loci for this species and representatives from the current Blaptinae tribes, as well as taxa representing four additional tenebrionid subfamilies.

Material and methods

Sequence assembly and analysis

Read quality was assessed with FastQC v.0.11.9 (Andrews 2010). Reads with an average sequence quality across any 4 bases below 20 were removed with Trimmomatic (Bolger et al. 2014). HybPiper v. 2.0 pipeline (Johnson et al. 2016), operating in DIAMOND mode (Buchfink et al. 2015), was used to assemble reads using the set of orthologs designed by Kanda (2017). The implemented files contained sequence information for 618 protein-coding genetic loci acquired from transcriptomes of the following species: Clamoris americana (Horn, 1874) (voucher code: KKRNA00020) representing Phrenapatinae, Eleodes delicata Blaisdell, 1929 (KKRNA00086), and Eulabis bicarinata Eschscholtz, 1829 (KKRNA00037) both representing Tenebrioninae. This dataset is hereafter referred to as the Tenebrionidae bait probe markers (Tps). To ensure that mapped sequences of the museum sample were not biased by the reference (Smith et al. 2021), data acquired for Dissonomus tibialis were also mapped to a more phylogenetically distant probeset designed for the subfamily Pimeliinae (for details see Kanda 2017) – referred hereafter as Pps. All data used in the mapping process are available as supplementary material (see Kamiński et al. 2024). The resulting amino acid sequences were then aligned using MAFFT with the L-INS-I algorithm (Katoh et al. 2005). Low-quality amino acid sequence sites were masked, and sequences with over 50% gaps per locus were trimmed using trimAl (Capella-Gutiérrez et al. 2009). SendSketch script, as implemented in BBMap/ 36.92, was used to characterize the metagenomic profile of assembled contigs. In the case of Dissonomus tibialis, data concerning two loci (OrthoMCL8004, OrthoMCL7212) were excluded from further analysis due to contamination detection. Surviving sequences were concatenated with FASConCAT (Kück and Meusemann 2010) into a single partitioned dataset including a total of 548 loci spanning 178,309 amino acids. All sequence assembly analyses were performed on Purdue University's Bell community cluster, within Rosen Center for Advanced Computing (McCartney et al. 2014).

Table 1.

Characteristics of samples analyzed in the present study.

Results

Bioinformatic analysis did not reveal any obvious taxonomic sensitivity for the used probeset (Table 1). In particular, the genotyping success rates were relatively consistent between treated subfamilies, and seemed to be dependent on sample quality. This also included the outgroup (Cis sp.) for which more than 90% of targeted loci were at least partly (>50%) recovered (Fig. S1 in Kamiński et al. 2024). Furthermore, this study revealed a great degree of convergence between the molecular methodologies of Ragionieri et al. (2023) and Swichtenberg et al. (2023).

After sequence trimming and quality assessment, the final matrix contained a total of 548 loci (17,309 amino acids). Only a small portion of the total recovered loci for the matrix was recovered for Dissonomus tibialis (15 loci, 2047 amino acids) (Table S1 in Kamiński et al. 2024). The number of mapped reads for Dissonomus tibialis was similar regardless of the used bait probe markers in the bioinformatic analysis (Tps/Pps). In the case of the Tenebrionidae probes (Tps), 103,826 reads (12.0% of total recovered reads) were mapped, while 102,082 reads (11.8%) were mapped using Pimeliinae probes (Pps). Overlapping sequences obtained in both mapping approaches were identical, indicating that they were not subject to the mapping bias (Fig. 1).

Regardless of the used bait probe markers and phylogenetic inference method (ML/BI), Dissonomus tibialis was recovered sister to both analyzed Blaps species, and clustered with Eleodes nigrina (Fig. 1). The Dissonomus+Blaps+Eleodes clade (i.e. ‘blaptinoid clade’) constitutes a sister grouping to the opatrinoid clade composed of members of Dendarini (Neoisocerus ferrugineus), Opatrini (Opatrum sabulosum, Stenolamus reichenspergeri), Pedinini (Pedinus sp.), and Platynotini (Asiopus aciculatus, Eurynotus capensis). All taxa above represent the subfamily Blaptinae, and statistical support for the majority of recovered clades was absolute (Fig. 1).

Figure 1.

Phylogenetic placement of Dissonomini within the subfamily Blaptinae. Strict consensus tree derived from the maximum likelihood analysis of the concatenated matrix. Gray taxon names represent subfamilies outside Blaptinae, and Dissonomini representatives are blue. Branch support is displayed as an ultrafast bootstrap (above). Missing values indicate full branch support. All displayed nodes were fully supported in the Bayesian analysis (Posterior probability = 1.0). Dissonomus tibialis samples: Tps – sample mapped to Tenebrionidae probeset; Pps – sample mapped to Pimeliinae probeset. Abbreviations: BC – ‘blaptinoid clade’, OC – ‘opatrinoid clade’.

Figure 2.

Morphology of species representing Dissonomini. (A–H) Bradyus pygmaeus, (I–K) Dissonomus tibialis. (A) dorsum of head, (B) antenna, (C) prosternal process in lateral view, (D) scutellum, (E) epipleuron, (F) male protarsus, (G) female protarsus, (H–J) ovipositor, (K) genital tubes. Abbreviations: bc – bursa copulatrix, c1–4 – coxites, p – paraproct, sp – spermatheca, ag – accessory gland.

Figure 3.

Male terminalia of (A) Dissonomus tibialis and (B, C, D) Bradyus pygmaeus. (B) Spiculum gastrale, (C) ventral and (D) dorsal views of tegmen.

Discussion

The analyses here provide novel data necessary to address the neglected phylogenetic position of Dissonomini, which were recovered deeply embedded within Blaptinae (Fig. 1). Their position close to Blaptini within the ‘blaptinoid clade’ appears to be morphologically justified, as representatives of Dissonomini lack ancorae (Lumen & Kamiński 2023a) and opatrinoid trochanters (Iwan & Kamiński 2016) – precluding a closer relationship with Dendarini, Pedinini, Platynotini, and Opatrini (Iwan & Kamiński 2016, Kamiński & Iwan 2017, Kamiński et al. 2021, Lumen & Kamiński 2023b). The lack of ancorae also distinguishes Dissonomini from the Nearctic tribe Amphidorini (Johnston et al. 2022), which also clustered within the ‘blaptinoid clade’ in these (Fig. 1), and previous analyses (Kamiński et al. 2021). Both tribes can be further separated by the ovipositor structure, which in Amphidorini is composed of fused apical coxites (4-lobed in Dissonomini, Fig. 2H–J).

Figure 4.

Morphology of selected representatives of Blaptini and Platyscelidini. (C, F) Blaps putrida, (A, B, D, I, J) Oodoscelis tibialis, and (E, G, H, K) Platyscelis striata. (A) Head in dorsal view, (B, C) mentum, (D) scutellum, (E, F) epipleura, (G, I) spiculum gastrale, (H) aedeagal tegmen, (J, K) ovipositor. Abbreviations: c1–4 – coxites, g – gonostylus, p – paraproct, sc – scutellum.

Dissonomini is easily distinguished from Blaptini by the widened protarsi in males (Medvedev 1968, Chigray et al. 2020) (Fig. 2F). While this study lacked sequences of Platyscelidini, Dissonomini is morphologically separable inter alia by the notched epistoma (Fig. 2A). Dissonomini is also distinct from both Blaptini and Platyscelidini by their sharply keeled menta (flat in Blaptini or broadly keeled at most in Platyscelidini, Fig. 4B and C) and epipleura terminating at abdominal ventrite V (epipleura ending later in Platyscelidini and continuing to apex in Blaptini) (Figs 2E and 4E and F) (see also Egorov 2004).

Although the monophyly of Dissonomini was not tested here by molecular data, several unique morphological features strongly suggest that Bradyus and Dissonomus are linked (Figs 2, 3, Medvedev 1968): concealed scutellum (Figs. 1 and 2D), antennal shape (short, robust, with apical antennomeres forming a loose club (Fig. 2B); longer in Blaptini and Platyscelidini where the third antennomere is often also elongated,) termination of the epipleura (reaching the level of the base of fifth ventrite) (Fig. 2E), notched epistoma (Fig. 2A), configuration of the aedeagi (ventrally without clear opening for the penis) (Fig. 3C), formation of the spiculum gastrale (weakly sclerotized, U-shaped with reduced ‘stem’ (Fig. 3A and B); Platyscelidini and Blaptini both have strongly sclerotized spiculae with or without a long stem and large, paddle-shaped terminations of the ‘arms’) (Fig. 4G and I) (see also Chigray et al. 2020), and the structure of the ovipositor (4-lobed coxities, lack of gonostyli, and overlapping coxites 3 and 4) (Fig. 2I and J).

In conclusion, the molecular data recovered here are consistent with morphological data revealed for adult forms (Figs 1–4). The taxonomic distinctiveness of Dissonomini appears well-grounded, and its phylogenetic position regarding Platyscelidini should be investigated further (for recent treatments within Platyscelidini see Bai et al. 2019a, b, Bai & Ren 2019). Based on the results presented here, Dissonomini is incorporated within the borders of Blaptinae where it becomes the eighth tribe of the subfamily. The definition of Blaptinae remains unmodified (Kamiński et al. 2021): Adults: antennae lacking compound/stellate sensoria (except Stenolamus Gebien, 1920 – Kamiński et al. 2023); procoxal cavities externally and internally closed, intersternal membrane of abdominal ventrites 3–5 visible; paired abdominal defensive glands present, elongate, not annulated. Larvae: prolegs enlarged (adapted for digging); ninth tergite lacking urogomphi.