Introduction

Although the importance of larval morphology for understanding the systematics of different groups of Coleoptera has been postulated by many researchers (e.g., Beutel et al. 1999; Grebennikov and Scholtz 2004; Lawrence et al. 2011), for the vast majority of currently known species, there are no available larval descriptions. This also holds true for darkling beetles (Tenebrionidae). The most comprehensive overview of larval morphology within the family was done by Watt (1974), who partly based the modern classification of this group on immature stages. Other important contributions were published by Lawrence (1991) and Matthews et al. (2010). Despite this, comparative analyses of larval features at the lower taxonomic levels (subfamily, tribe, and subtribe) are relatively scarce (however, see Schulze 1963, 1968; Smith et al. 2014). In the majority of cases, such analyses are inhibited by the lack of available larval descriptions or well-preserved material.

Currently, the larval morphology of about 6% of described species representing the ‘Opatrinoid’ clade is known (139 out of ~ 2325 spp.; present estimation). The available data do, however, appear to cover most of the currently designated subtribes (Table 1). The majority of available descriptions are of the subtribe Opatrina Brullé, 1832 (54 species). Platynotina Mulsant and Rey, 1853 ranks second (36), and is followed by Helopinina Lacordaire, 1859 (12), Pedinina Eschscholtz, 1829 (9) Stizopina Lacordaire, 1859 (8), Dendarina Mulsant and Rey, 1854 (5), Blapstinina Mulsant and Rey, 1853 (1, plus 3 described below), Ammobiina Desbrochers des Loges, 1902 (3), Leichenina Mulsant, 1854 (2), Eurynotina Mulsant and Rey, 1854 and Melambiina Mulsant and Rey, 1854 (both 1 and 1 described below), and Heterotarsina Blanchard, 1845 (1). No larval descriptions are available for Neopachypterina Bouchard et al. 2007 and Sclerina Lacordaire, 1859 (Table 1). It should be noted that, in the case of Platynotini (i.e., Eurynotina and Platynotina), some of the contributions concern early instar larvae extracted from the bursa copulatrix (Tschinkel 1978; Iwan 2005).

Table 1 Checklist of described larvae of the species representing the ‘Opatrinoid’ clade
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The most comprehensive discussion of opatrinoid larval characters in a wider phylogenetic context was presented by Iwan and Bečvář (2000), who largely focused on the evaluation of different classification concepts developed for tenebrionidae larvae (Skopin 1962, 1964; Watt 1974). Valuable contributions were also published by Medvedev (1968) and Schulze (1963, 1969). However, due to a lack of phylogenetic studies concerning opatrinoid beetles, all of the above-mentioned papers have dealt with slightly different, paraphyletic entities [Opatrinae sensu Koch (1956) or Medvedev (1968)]. A phylogeny-based classification of the ‘Opatrinoid’ clade was introduced recently (Iwan and Kamiński 2016; Kamiński et al. 2018a, 2019). This reclassification provided an opportunity to revise the available knowledge on larval morphology in a new context. Moreover, recent rearing efforts provided larvae of taxa representing subtribes for which few, or no immature stages were previously described.

The main aim of this paper is to summarize current morphological knowledge on the larval stages representing the ‘Opatrinoid’ clade, and to discuss its relevance for understanding the phylogeny of the group. Moreover, based on some newly accessed material, larvae of the following five species are described: Zadenos mulsanti Koch, 1956 (Dendarini: Melambiina; South Africa), Blapstinus histricus Casey, 1890 (Opatrini: Blapstinina; North America), Blapstinus longulus LeConte, 1851 (Opatrini: Blapstinina; North America), Trichoton sordidum (LeConte, 1851) (Opatrini: Blapstinina; North America), and Eurynotus rudebecki Koch, 1955 (Platynotini: Eurynotina; South Africa).

Materials and methods

This is the second and final paper of a series focused on immature stages of beetles representing the ‘Opatrinoid’ clade (Coleoptera: Tenebrionidae). For part one, concerning the pupal stages, and the phylogenetic background see Kamiński et al. (2018b).

Material

The larval specimens of the two analysed Afrotropical species (Eurynotus rudebecki Koch, 1955 and Zadenos mulsanti Koch, 1956) were bred from imagines collected in the surroundings of Mossel Bay (South Africa) (see Fig. 1 in Kamiński et al. 2018b). Adults of both species were placed in terraria filled to a depth of ~ 10 cm with sand. Leaves of red clover were provided as a food resource. Terraria were watered every 2 weeks. Larvae were collected after 2 months from the beginning of the experiment. Obtained specimens were stored in 98% ethanol in − 20 °C. In the case of Z. mulsanti, a single specimen pupated (Kamiński et al. 2018b). The above-mentioned breeding efforts were conducted by MJK.

Fig. 1
figure 1

Morphology of the selected Trichoton sensu novum species: a, bT. sordidum and cT. villosum. Image b illustrates a newly proposed diagnostic character (glabrous and depressed basal angles of fifth abdominal ventrite)

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Trichoton sordidum larvae were bred in two attempts. For the first, approximately 100 individuals (not sorted by sex) were introduced into containers (22 cm × 40 cm × 30 cm) filled to a depth of ~ 5 cm with a 50/50 mix of Imagitarium Calcium Sand (from PetCo) and fine-grain, baked Mojave Desert Sand. Two medium sized, flat rocks were provided as sites for beetles to hide. The colony was watered 2–3 times per week. Zucchini was provided as a food resource (replaced when consumed or dried up). Ambient room temperature was kept in the range of 21.6–23.8 °C. Substrate was sifted once per month. Although many mating specimens were immediately observed, no larvae were observed for 2 months. The first larvae were detected after approximately 3–4 months. Specimens were taken for preservation, as survivorship to pupation/adulthood was uncertain. No larvae survived or pupated.

Blapstinus longulus, B. histricus, and a second attempt of Trichoton sordidum larvae were reared following the methods of Zilkowski and Cossé (2015). Trichoton sordidum adults (ca. 70–80 specimens) from the first attempt were used for the second. Blapstinus longulus and B. histricus were collected and introduced into similarly prepared containers. Halved apples were provided for food and as a water source (wet side down) on the surface of the wood shavings, and changed out every 7–9 days to prevent molding. Containers were checked every 2 weeks for larvae. After 3 weeks, larvae were detected in both Blapstinus containers, and representatives were taken for preservation on the fifth week. Trichoton larvae were detected after a month, and specimens were taken for preservation immediately, as survivorship was low in the previous attempt. Specimens selected for the molecular analysis were stored in 98% ethanol in − 20 °C, other individuals were boiled in water briefly and were then preserved with 98% ethanol. Breeding efforts concerning the representatives of Blapstinina were done by RL.

Adult specimens used for breeding were identified using Koch (1954, 1955, 1956), Davis (1970), and Arnett and Thomas (2002).

Molecular association

Because E. rudebecki and Z. mulsanti were bred in a single terrarium, molecular techniques were required to separate the obtained larvae. As well, to confirm the identity of the Blapstinus larvae, the same approach was employed. The larvae of Trichoton sordidum were not included in molecular analyses due to scarcity of the obtained larvae (see below). However, adults of T. sordidum were processed to test the phylogenetic position of Trichoton. Finally, as there is now a more recent molecular phylogeny of the ‘Opatrinoid’ clade to compare to, the molecular data obtained here can be integrated for further study of the group (Fig. 2).

Fig. 2
figure 2

Phylogenetic relations within the ‘Opatrinoid’ clade: a variability of the selected larval features presented on the simplified phylogeny. b–d Maximum likelihood trees of concatenated data set confirming the association of the studied larvae: b Blapstinina, c Eurynotina, and d Melambiina. (1) Rounded (in dorsal view) pygidium with several short spines [state commonly observed in Opatrini and Amphidorini, Fig. 5b, f; (2) with four or six elongated spines [common in Dendarini, Pedinini. Platynotini, Figs. 4g,6i; (3) without spines, with a rim at the lateral and basal edges [state characteristic for the genus Gonopus]; (4) triangular pygidium with several short spines (common in Opatrini, Fig. 5k); (tu) protarsungulus, (tib) protibia, (fem) profemur, (tr) protrochanter; Asterik indicates maximum likelihood bootstrap percentage of 100% boot support of a particular clade (other percentage values are indicated by numbers on particular branches). Newly added OTUs were marked with black. For phylogenetic details see Kamiński et al. (2019)

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DNA was extracted from ethanol-preserved specimens using DNeasy Blood and Tissue kits (Qiagen), following the manufacturer’s protocols. Extractions were performed on disarticulated specimens. Efforts were taken to amplify and sequence DNA for six gene fragments: 12S rRNA (12S, 354 bp), 28S rRNA (28S, 1057 bp) D1–D3, cytochrome c oxidase subunit II (COII, 654 bp), wingless (wg, 435 bp), CAD/rudimentary (CAD2, 723 bp), and arginine kinase (ArgK, 669 bp). This set of markers fully corresponds to those used in the only available phylogeny of the ‘Opatrinoid’ clade (Kamiński et al. 2019). For primers’ specification and details concerning the PCR reactions, see Kamiński et al. (2019) and Kanda et al. (2015, 2016). Molecular analysis of the South African species was done at the Museum and Institute of Zoology of the Polish Academy of Sciences (MIIZ), while the representatives of Blapstinina were processed at the Northern Arizona University (NAU). Sequences were aligned using Mesquite (Maddison and Maddison 2016). All nucleotide alignments were combined into a single concatenated data set for phylogenetic analyses. Optimal data partitions and models of molecular evolution were inferred using PartitionFinder v. 2.1.1 (Lanfear et al. 2012). The phylogenetic tree was constructed under Maximum Likelihood estimation using RAxML v. 8 (Stamatakis 2014). Analysis was conducted on servers maintained by the CIPRES Scientific Gateway (Miller et al. 2010).

Larval descriptions

Nomenclature and descriptions follow that of Smith et al. (2014). However, modifications were made to reflect the specific morphology of the ‘Opatrinoid’ clade larvae (see Schulze 1968; Iwan 1995; Iwan & Bečvář 2000). Descriptions were based only on mature larvae, i.e., large specimens (one to two instars prior the pupation). Total length (TL) of analysed specimens was measured from the anterior edge of the clypeus to the apex of abdominal segment IX. Head capsule width (HW) was measured dorsally across the widest portion of the head. Prothoracic width (PW) and length (PL) were also measured dorsally across the widest and longest points on the segment, respectively. Images were taken using a Canon 1000D body with accordion bellows and a Canon Macro Lens EF 100 mm, and with a Hitachi S-3400 N SEM at MIIZ.

Results

DENDARINI: Melambiina


Genus Zadenos Laporte, 1840


Zadenos mulsanti Koch, 1956


Material examined Larvae were reared from adults with the following collecting information: “Mossel Bay, South Africa, -34.147305, 22.065792, 9.1.2017, leg. M.J. Kamiński & A.D. Smith”. A total of 20 larvae were reared and examined for this study. Selected specimens were preserved at the entomological collection of the Museum and Institute of Zoology, Polish Academy of Sciences. GenBank accession numbers: MK714051, MK714054, MK714071 (larva).


Differential diagnosis See discussion under Dendarini.


Description TL: 15.0–18.5 mm, HW: 1.3–1.5 mm, PL: 1.2–1.3 mm, PW: 1.6–1.8 mm, PL/mesothoracic length = 1.6; PL/mesothoracic length = 1.2–1.3.


Head Prognathous, distinctly retracted into prothorax; head capsule weakly dorsoventrally flattened; width nearly equal to prothorax; sides rounded; strongly constricted before occipital foramen; yellow with brown spots, same as body segments; punctation absent. Epicranial suture stem length approximately one-third head capsule length; frontal arms sinuate, double branched apically, not reaching epistomal margin. Frons almost smooth, without visible microsculpture, with pair of deep setose points at apex (Fig. 4a). Epicranial plates almost smooth dorsally, without microsculpture; each plate with deep depressions with single long setae in middle and apex; lateral portions sparsely setose (several long setae present); ventral portion of each plate with row of 2–3 long setae along anterior margin near buccal cavity, not confluent with setae on lateral portions of plates (Fig. 4b). Four stemmata present on each epicranial plate, pigmented and well visible. Clypeus trapezoidal, with four deep setose points of which two are located medially while remaining two laterally (Fig. 4a). Labrum transverse, weakly emarginate, medially with pair of long setae, apical margin with row of eight setae. Epipharynx similar as in Eurynotus rudebecki (Fig. 6f). Mandibles asymmetrical, their bifid apices strongly sclerotised and dark; right mandible with triangular premolar tooth. Labium with distinct prementum, mentum and submentum; gula apically well developed, elongated and rectangular; subquadrate prementum, with pair of setae basally and additional one in middle, ligula reduced (Fig. 4c), about 0.2 × of width of first labial segment, labial palps 2-segmented; mentum elongated, hexagonal, with elongated setae distributed in middle; trapezoidal submentum with row of four setae in middle (Fig. 4b). Hypopharyngeal sclerome trilobed, with apical denticle strongly elongated (Fig. 4d). Maxillary palpi 3-segmented, first and third segments of same length, second one about 1.2 longer. Antennae 3-segmented, second antennomere is 3.0 times longer than wide, same length as the first.


Thorax Thoracic tergites yellow with brownish bands in apical part (Fig. 3a). Prothoracic tergum subquadrate, about 2 × length of meso- and 1.4 × metaterga. Meso- and metathoracic tergites wider than long. Thoracic tergites sparsely setose on dorsal surfaces. Mesothoracic spiracle simple, ovate, approximately 1.5 × size of abdominal spiracles; metathoracic spiracle reduced. Thoracic sternites densely covered by microtrichia. Legs. Prothoracic leg noticeably longer, much thicker than meso- and metathoracic ones; prothoracic tarsungulus strongly sclerotized, spade-like; prothoracic trochanter with two stout spines ventroapically; prothoracic femur with ventromedial row of two spines; prothoracic tibia with ventromedial row of three spines (Fig. 4e). Prothoracic tarsungulus about 0.8 × length of tibia; same length as trochanter. Mesotibia with two or three ventromedial spines.

Fig. 3
figure 3

Lateral habitus of five larvae representing the ‘Opatrinoid’ clade: aZadenos mulsanti (Dendarini: Melambiina), bBlapstinus histricus (Opatrini: Blapstinina), cBlapstinus longulus (Opatrini: Blapstinina), dTrichoton sordidum (Opatrini: Blapstinina), and eEurynotus rudebecki (Platynotini: Eurynotina)

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Fig. 4
figure 4

Larval morphology of Zadenos mulsanti: a head in dorsal view, b mouthparts in ventral view, c close-up of ligula, d hypopharyngeal sclerome, e front leg, f pygopods, g pygidium. a9 abdominal segment IX

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Abdomen Abdominal tergites yellow with brownish bands on apex, faintly rugose, without setation. Abdominal sternite I with path of setae basally. Pygidium triangular, urogomphi absent; marginal row of four socketed spines present, arranged as single row around posterior edge; basal row composed of several soft setae (Fig. 4g). Pygopods short, subconical (Fig. 4f).

OPATRINI: Blapstinina


Genus Blapstinus Dejean, 1821


Blapstinus histricus Casey, 1891


Material examined Larvae were reared from adults with the following collecting information: “Canoa Ranch Rest Area, Pima County, Arizona 3-4 Aug 2018 (R.Lumen, K.Kanda)”. More than 100 larvae were reared and examined for this study. Selected specimens were preserved at the entomological collection of the Museum and Institute of Zoology, Polish Academy of Sciences. GenBank accession numbers: MK714042, MK714055, MK714062 (larva), MK714043, MK714056, MK714063 (imago).


Differential diagnosis See diagnosis of Blapstinus longulus.


Description TL: 9.5–12.0 mm, HW: 0.7–0.8 mm, PL: 1.0–1.1 mm, PW: 1.0–1.1 mm, PL/mesothoracic length = 1.3; PL/mesothoracic length = 1.5.


Head Prognathous, distinctly retracted into prothorax; weakly dorsoventrally flattened; width nearly equal to prothorax; sides rounded; strongly constricted before occipital foramen; yellow, with two nearly confluent brown spots covering epicranial plates (Fig. 5a); punctation absent or extremely fine. Epicranial suture stem length approximately one-third head capsule length; frontal arms sinuate, extend to antennal insertion. Frons faintly rugose, with microsculpture. Epicranial plates weakly rugose dorsally, with microsculpture; each plate with deep setose point in middle; lateral portions moderately setose (11–13 setae present); ventral portion of each plate with row of three-to-four long setae along anterior margin near buccal cavity, not confluent with setae on lateral portions of plates. Two stemmata present on each epicranial plate, pigmented and well visible. Clypeus trapezoidal, slightly darker medially, with four deep setose points of which two are located medially and two laterally. Labrum transverse, weakly emarginate, with apical row of elongated setae. Epipharynx anterolateral margins with six spinose setae; four subanterior sensory papillae present, arranged as transverse row subtended by two spinose setae. Mandibles asymmetrical, their bifid apices strongly sclerotised and dark; left mandible with triangular premolar tooth and two distinct apical teeth; right mola reduced, with single apical tooth. Labium with distinct prementum, mentum and submentum; gula well developed, elongated and rectangular; subquadrate prementum bears pair of setae in center, ligula twice shorter narrower than first labial segment, labial palps 2-segmented; mentum elongated, with pair of setae at base and 2 pairs of longer setae lateromedially; trapezoidal submentum bears pair of setae basaly. Hypopharyngeal sclerome pentagonal; tricuspidate, with apical denticle elongated; with deep depression in middle. Maxillary palpi 3-segmented, first and third segments of same length, second one about 1.2 longer. Antennae 3-segmented, second antennomere is 2.0 times longer than wide and about 2 times longer than first (Fig. 5a).

Fig. 5
figure 5

Morphology of the analysed larvae representing Blapstinina: a–cBlapstinus histricus, d–gBlapstinus longulus, h–kTrichoton sordidum. a, e, i Head and thoracic segments in dorsal view, h tip o head in dorsal view, d mouthparts in ventral view, abdominal segments in (b, c, f, k, j) dorsal and (g) lateral views. a1–a9 Abdominal segments I–IX; t1–t3 thoracic segments I–III

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Thorax Thoracic tergites yellow, covered with brown spots (Figs. 3b,5a). Prothoracic tergum subquadrate, about 2 × length of meso- or metaterga; dorsally covered with single brown spot. Meso- and metathoracic tergites wider than long, apically each with brown band. Brown spots of thoracic segments narrowly interrupted medially. Thoracic tergites sparsely setose on dorsal surfaces. Lateral margin of protergum with eight to ten long setae in anterior part. Mesothoracic spiracle simple, ovate, approximately 1.5 × size of abdominal spiracles; reduced metathoracic spiracle visible. Legs. Prothoracic leg slightly longer, thicker than meso- and metathoracic legs; prothoracic tarsungulus sclerotized; prothoracic trochanter with two stout spines ventroapically; prothoracic femur and tibia with ventromedial row of two spines. Prothoracic tarsungulus about 0.5 × length of femur and tibia; about 0.7 × length of trochanter. Mesotibia with two ventromedial spines.


Abdomen Abdominal tergites yellow, faintly rugose, with sparse elongate seate laterally. Abdominal sternite I with narrow brown band in apical part. Abdominal laterotergites with lateral margins indistinctly pigmented. Abdominal segment VIII with row of four setae basally, entirely covered by brownish spot. Pygidium rounded apically; with wide brown band (Fig. 5b); suddenly upturned to apex, urogomphi absent; marginal row of 12–16 socketed spines present, arranged as single row around posterior two-thirds to one half of segment (Fig. 5c); basal row of eight soft setae located outside of brown spot. Pygopods short, subconical.


Blapstinus longulus LeConte, 1851


Material examined Larvae were reared from adults with the following collecting information: “Sunset Point Rest Area, Yavapai County, Arizona 25 July, 2018 (R. Lumen, E. Lumen, A.L. Smith)”. More than 100 larvae were reared and examined for this study. Selected specimens were preserved at the entomological collection of the Museum and Institute of Zoology, Polish Academy of Sciences. GenBank accession numbers: MK714045, MK714058, MK714065 (larva), MK714044, MK714057, MK714064 (imago).


Differential diagnosis Larvae of Blapstinus longulus can be easily separated from those of B. histricus by different color patterns of the dorsal surface of the body, i.e., head with two separate brown spots constricted to dorso-median parts of the epicranial plates (head with a single large spot covering epicranial plates and basal part of frons in B. histricus) (Fig. 5a, e); prothoracic tergum with two separate brown spots on the sides (the whole dorsal surface covered by the spot in B. histricus) (Fig. 5a, e); pygidium without a brown spot on the ventral side (spot present in B. histricus) (Fig. 5c, f, g). Moreover, the larvae of those two Blapstinus species can be separated by different length of dorsal spines of pygidium (short, not longer than the gap between first and second spine in B. longulus; elongate, longer than the gap between first and second spine in B. histricus) (Fig. 5c, f).


Description TL: 10–13.1 mm, HW: 0.9–1.0 mm, PL: 1.1–1.2 mm, PW: 1.0–1.1 mm, PL/mesothoracic length = 1.3; PL/mesothoracic length = 1.5.


Head Prognathous, distinctly retracted into prothorax; weakly dorsoventrally flattened; width nearly equal to prothorax; sides rounded; strongly constricted before occipital foramen; yellow, with two brown spots each in center of epicranial plates (Fig. 5e); punctation absent or extremely fine. Epicranial suture stem length approximately one-third head capsule length; frontal arms sinuate, extend to antennal insertion. Frons faintly rugose, with microsculpture. Epicranial plates weakly rugose dorsally, with microsculpture; each plate with deep setose area in middle; lateral portions moderately setose (11–13 setae present); ventral portion of each plate with row of two to three long setae along anterior margin near buccal cavity, not confluent with setae on lateral portions of plates. Two stemmata present on each epicranial plate, pigmented and well visible. Clypeus trapezoidal, slightly darker medially, with four deep setose areas of which two are located medially and others laterally. Labrum transverse, weakly emarginate, without noticeable setation. Epipharynx anterolateral margins with six spinose setae; four subanterior sensory papillae present, arranged as transverse row subtended by two spinose setae. Mandibles asymmetrical, their bifid apices strongly sclerotised and dark; left mandible with triangular premolar tooth and two distinct apical teeth; right mola reduced, with single apical tooth. Labium with distinct prementum, mentum and submentum; gula well developed (Fig. 5d), elongate, rectangular; subquadrate prementum bears pair of setae in center, ligula twice shorter, narrower than first labial segment, labial palps 2-segmented; mentum elongated, with pair of setae at base and 2 pairs of longer setae lateromedially; submentum trapezoidal with pair of setae basally. Hypopharyngeal sclerome pentagonal; tricuspidate, with apical denticle elongated; with deep depression in middle. Maxillary palpi 3-segmented, first and third segments of same length, second one about 1.2 longer. Antennae 3-segmented, second antennomere is 4.0 times longer than wide and about 1.2 times longer than first.


Thorax Thoracic tergites yellow (Fig. 3c). Prothoracic tergum subquadrate, about 2 × length of meso- or metaterga; with two brown spots laterally (Fig. 5e). Meso- and metathoracic tergites wider than long, apically each with brown band. Thoracic tergites sparsely setose on dorsal surfaces. Lateral margin of protergum with eight to ten long setae in anterior part. Mesothoracic spiracle simple, ovate, approximately 1.5 × size of abdominal spiracles; reduced metathoracic spiracle visible. Legs. Prothoracic leg slightly longer and thicker than meso- and metathoracic legs; prothoracic tarsungulus strongly sclerotized; prothoracic trochanter with two stout spines ventroapically; prothoracic femur and tibia with ventromedial row of two spines. Prothoracic tarsungulus about 0.5 × length of femur and tibia; about 0.7 × length of trochanter. Mesotibia with two ventromedial spines.


Abdomen Abdominal tergites yellow, faintly rugose, with sparse elongate seate laterally. Abdominal sternite I with narrow brown band in apical part. Abdominal laterotergites with lateral margins indistinctly pigmented. Abdominal segment VIII with row of four setae basally and brownish spot in middle. Pygidium triangular and with brown spot in dorsal view (sometimes brown spot is absent—Fig. 5f), gradually upturned to apex, urogomphi absent; marginal row of 12-16 socketed spines present, arranged as single row around posterior two-thirds to one half of segment; basal row of eight soft setae located outside of brown spot (Fig. 5f, g). Pygopods short, subconical (Fig. 5g).


GenusTrichoton Hope, 1841


Type species: Trichoton cayennense Hope, 1841 (by original designation).


= Epilasium Curtis, 1844 syn. by Mulsant and Rey (1853)


Type species: Epilasium rotundatum Curtis, 1844 (by monotypy).


= Bycrea Pascoe, 1868 syn. nov.


Type species: Bycrea villosa Pascoe, 1868 (by monotypy).


Justification for new generic synonymy Comparison of the larval morphology of T. sordidum and Bycrea villosa (see Steiner 2004) revealed that both those species share unique clypeal and labral setation (Fig. 5h) and similar pigmentation of the body segments—especially the head (Fig. 5i). Moreover, newly analysed molecular data suggest a close phylogenetic affiliation of Trichoton and Bycrea. Both genera form a separate clade within Blapstinina (Fig. 2). Analysis of imaginal morphology and available references show Trichoton is only defined by the lack of presumably autapomorphic features of Bycrea (i.e. absence of strongly elongated spine on protibia, weakly developed (not widened) basal protarsomeres, mesotibia without median tooth on the outer side (all three features concern male morphology), protibia not triangular, antennomeres 7–11 not transverse, maxillary palp more triangular). On the other hand, both entities share many key features, i.e. curved protibiae, distinctly bisinuate base of pronotum and triangular scutellum (see Davis 1976), glabrous (not covered with setae) basal fcorners of the fifth abdominal ventrite (presently observed feature, Fig. 1b). According to Aalbu and Triplehorn (1985) both genera can be separated by differing head structure (eyes not fully divided in Bycrea; fully divided into dorsal and ventral portions in Trichoton). However, this statement is false. The eyes of Bycrea villosa are fully divided, just as in all Trichoton and other Blapstinina (for details see Iwan and Kaminski 2016).

To sum up, all of the analysed data sets (larval and imaginal morphology, molecular data) suggest a close phylogenetic relation between Trichoton and Bycrea. As a result, Bycrea is hereby interpreted as a synonym of Trichoton. In the light of gathered data, Bycrea could not be interpreted as a separate genus-group taxon close to Trichoton because of the lack of morphological synapomorphies supporting monophyly of Trichoton in such a phylogenetic scenario. The apomorphic morphology of Trichoton villosum comb. nov. is likely due to this species’ myrmecophilous affiliation(s) (Rojas 1988).

Label data of the studied adult Trichoton specimens used for reference are presented in Electronic Supplementary Material Appendix 1.


Note In 2001, Ferrer & Moraguès introduced Marcuzzichoton as a new subgenus of Trichoton. However, the authors did not designate a type species for the new genus-group name, as required by Articles 13.3 and 67.4.1 of the ICZN (1999), and therefore the new name was considered unavailable. After this mistake was pointed out, the same authors published a short paper to rectify the situation (Ferrer and Moragues 2002). In this second paper, they selected Opatrum occidentale Berg as the type species of the new genus-group name they proposed in 2001. However, a subsequent designation of a type species is not allowed by the ICZN (1999) for the generic names proposed after 1930. Therefore this action did not make Marcuzzichoton available. Furthermore, by referring to “Marcuzzichoton Ferrer & Moraguès, 2001” in their second paper, they failed to fulfil Article 16.1 of the the ICZN (1999). As a result, Marcuzzichoton Ferrer and Moraguès is unavailable in both of their papers.


Trichoton sordidum (LeConte, 1851)


Material examined Larvae were reared from adults with the following collecting information: “Boulder Recreation Area, Maricopa County, Arizona Nov. 23rd 2017”. A total of three larvae were reared. However, two of them represent earlier instar larvae. Therefore, the following description was made based on a single, not dissected, specimen. GenBank accession numbers: MK714048, MK714061, MK714068 (imago).


Differential diagnosis Larvae of Trichoton sordidum can be easily separated from those of the above described Blapstinus species by the differing setation of clypeus (row of eight spinose setae on anterior margin of clypeus; lack of spinose setae in both Blapstinus species), labrum (row of four spinose setae on anterior margin of labrum in T. sordidum; lack of spinose setae in both Blapstinus species), mandibles (pair of spinose setae present laterally on apical part in T. sordidum; lack of spinose setae in both Blapstinus species) and pygidium (24 spinose setae irregularly arranged on margin in T. sordidum; 12–16 spinose spines arranged as single row around posterior two-thirds to one half of pygidium in both Blapstinus species) (Fig. 5c, f, k). Moreover, the larvae of T. sordidum are also characterised by having prothoracic tibia with ventromedial row of three spines (two spines in both Blapstinus species). The clypeal and labral setation of Trichoton villosum comb. nov. is similar to that observed for Trichoton sordidum (see above). Both Trichoton species can be easily told apart by differing spot patterns of the abdominal terga (segments IV–VII with brownish bands in the apex in T. sordidum; without brownish bands in T. villosum (see Steiner 2004).


Description TL: 8.5 mm, HW: 1.0 mm, PL: 1.0 mm, PW: 1.5 mm, PL/mesothoracic length = 2.0; PL/mesothoracic length = 1.3.


Head Prognathous, distinctly retracted into prothorax; weakly dorsoventrally flattened; narrower than prothorax; sides rounded; strongly constricted before occipital foramen; yellow, single brown spot covering epicranial plates and two narrow brown bands directed anteriorly (visible on frons) (Fig. 5i); punctation absent or extremely fine. Epicranial suture stem length approximately one-third head capsule length; frontal arms sinuate, extend to antennal insertion. Frons faintly rugose, with microsculpture. Epicranial plates weakly rugose dorsally, with microsculpture; each plate with deep setose area in middle; dorsal and lateral portions densely setose; ventral portion of each plate with row of four long setae along anterior margin near buccal cavity, not confluent with setae on lateral portions of plates. Two stemmata present on each epicranial plate, pigmented and well visible. Clypeus trapezoidal, yellow without darker spots, with row of eight spinose setae in apical portion. Labrum transverse, weakly emarginate, with apical row of four spinose setae (Fig. 5h). Epipharynx not dissected. Mandibles asymmetrical; pair of spinose setae present laterally on apical part. Labium with distinct prementum, mentum and submentum; gula well developed, elongated and rectangular; subquadrate prementum bears pair of setae in center, ligula half as long, narrower than first labial segment, labial palps 2-segmented; mentum elongated, with pair of setae at base and 2 pairs of longer setae lateromedially; submentum trapezoidal with pair of setae basally. Hypopharyngeal sclerome not dissected. Maxillary palpi 3-segmented, first and third segments of same length, second one about 1.2 longer. Antennae 3-segmented, second antennomere is 2.0 times longer than wide and about 2 times longer than first.


Thorax Thoracic tergites yellow, but covered with brownish spots (in some cases confluent) (Figs. 3d,5i). Prothoracic tergum transverse, about 2 × length of mesoterga and 1.5 × length of metaterga. Brown spots of thoracic segments interrupted medially (Fig. 5i). Thoracic tergites denselly setose on dorsal and lateral surfaces. Mesothoracic spiracle simple, ovate, approximately 1.5 × size of abdominal spiracles; metathoracic spiracle not visible. Legs. Prothoracic leg slightly longer, thicker than meso- and metathoracic legs; prothoracic tarsungulus strongly sclerotized; prothoracic trochanter with two stout spines ventroapically; prothoracic femur with ventromedial row of two spines; prothoracic tibia with ventromedial row of three spines. Prothoracic tarsungulus about 0.7 length of trochanter, femur and tibia. Mesotibia with two ventromedial spines.


Abdomen Abdominal tergites covered with brownish spots, especially noticeable on segments IV–VIII (Fig. 5j) and covered with long and dense setae. Pygidium triangular and with wide brown band, suddenly upturned to apex, urogomphi absent; marginal row of 24 socketed spines present, not arranged as single row; basal row of eight soft setae located outside of brown spot (Fig. 5j, k). Pygopods short, subconical.

PLATYNOTINI: Eurynotina


Genus Eurynotus Kirby, 1819


Eurynotus rudebecki Koch, 1955


Material examined Larvae were reared from adults with the following collecting information: “Mossel Bay, South Africa, -34.147305, 22.065792, 9.1.2017, leg. M.J. Kamiński & A.D. Smith”. A total of 48 larvae were reared and examined for this study. Selected specimens were preserved at the entomological collection of the Museum and Institute of Zoology, Polish Academy of Sciences. GenBank accession numbers: MK714050, MK714053, MK714070 (larva), MK714049, MK714052, MK714069 (imago).


Differential diagnosis See discussion under Platynotini.


Description TL: 29.0–32.0 mm, HW: 2.1–2.2 mm, PL: 1.9–2.2 mm, PW: 2.4–2.7 mm, PL/mesothoracic length = 1.5–2.5; PL/mesothoracic length = 1.2–1.4.


Head Prognathous, distinctly retracted into prothorax; head capsule weakly dorsoventrally flattened; slightly narrower than prothorax (in earlier stages HW = PW); sides rounded; strongly constricted before occipital foramen; color dark brown, same as body segments (Fig. 3e); punctation absent. Epicranial suture stem length approximately one-third head capsule length; frontal arms sinuate, double branched apically, not reaching epistomal margin (Fig. 6a). Frons faintly rugose, with visible microsculpture, with pair of deep setose points at apex (Fig. 6a). Epicranial plates weakly rugose dorsally, with microsculpture; each plate with deep setose points in middle and apex (Fig. 6a); lateral and ventral portions densely setose (several long setae present). Five stemmata in two rows (transverse ophthalmic spots) present on each epicranial plate, pigmented and well visible. First row composed on three separated ocelli situated just before epicranial margin near antennal insertion; second row with two partly fused stemmata. Clypeus trapezoidal, with four deep setose points of which two are located medially while remaining two laterally (Fig. 6b). Labrum transverse, weakly emarginate, basally with pair of long setae, apical margin with row of eight setae. Epipharynx as on Fig. 6f. Mandibles asymmetrical, apical tooth of right mandible well developed, covering apical part of the tooth of left mandible, their bifid apices and molar part strongly sclerotised and dark; outer margin roundly extended outwards (visible from dorsal view), with distinct emargination in middle of the outer ridge (Fig. 6b). Labium with distinct prementum, mentum and submentum; gula apically well developed (Fig. 6c), elongated and trapezoidal; subquadrate prementum, setation variable, ligula of same width as first labial segment (Fig. 6d), labial palps 2-segmented; mentum elongated, hexagonal, with elongated setae distributal at base and sides; trapezoidal submentum with irregular setation. Hypopharyngeal sclerome pentagonal; tricuspidate, with apical denticle slightly elongated (Fig. 6e). Maxillary palpi 3-segmented, first and third segments of same length, second one about 1.2 longer. Antennae 3-segmented, second antennomere is 2.0 times longer than wide, same length as first one (Fig. 6c).

Fig. 6
figure 6

Larval morphology of Eurynotus rudebecki: a, b head in dorsal view, c mouthparts in ventral view, d close-up of ligula, e hypopharyngeal sclerome, f epipharynx, g front leg, h mesothorax in ventral view, i pygidium

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Thorax Thoracic tergites dark brown (Fig. 3e). Prothoracic tergum subquadrate, about 2 × length of meso- and 1.4 × metaterga. Meso- and metathoracic tergites wider than long). Thoracic tergites sparsely setose on dorsal surfaces. Mesothoracic spiracle simple, ovate (ca. 2.4 × as long as wide) (Fig. 6h), approximately 1.5 × size of abdominal spiracles; reduced metathoracic spiracle visible. Thoracic sternites densely covered by microtrichia (Fig. 6h). Legs. Prothoracic leg noticeably longer, much thicker than meso- and metathoracic legs; prothoracic tarsungulus strongly sclerotized, with visible suture at base on ventral side; prothoracic trochanter with two stout spines ventroapically; prothoracic femur with ventromedial row of four spines; prothoracic tibia with ventromedial row of two or three spines (Fig. 6g). Prothoracic tarsungulus about 0.8 × length of tibia; same length as trochanter. Mesotibia with two ventromedial spines.


Abdomen Abdominal tergites dark brown, faintly rugose, with sparse elongate seate laterally. Abdominal sternite I with patch of setae basally. Pygidium triangular, distinctly narrowing apically, urogomphi absent; marginal row of four socketed spines present at 6/7 length from base (in earlier stages pygidium more ovate at apex, spines located at 4/5 length from base), arranged as single row around posterior edge (distance between spines equals spine length); basal row composed of four soft setae (Fig. 6i). Pygopods short, subconical.

Discussion

Larval morphology has been proven to be an important data source for inferring phylogenetic relations among major groups of darkling beetles (Skopin 1962, 1964; Watt 1974). Because of their different lifestyles, adults and immature stages have separate evolutionary histories (Corbet 1962; Matthews et al. 2010). Theoretically, this should enable cross-testing of phylogenetic hypotheses obtained based on adult and immature data. However, in most cases, it is unclear how specific selective pressures acting on the larval stage might influence their usefulness for phylogenetic studies. In the case of opatrinoid beetles, it was proven that at least part of the currently known morphological variability was derived from ecological factors, especially in desert living (Schulze 1969) or myrmecophilous taxa (see Trichoton). This is likely the main reason why despite having a relatively comprehensive coverage of larval descriptions across the known subtribal diversity (Table 1), no features concerning immature stages were able to be used to define any family-group taxa within the ‘Opatrinoid’ clade (Iwan and Kamiński 2016; Kamiński et al. 2019). The present study revealed a great variability of larval morphology among the major opatrinoid subclades recovered by Kamiński et al. (2019). However, larval features seem to be extremely informative at the generic level in some cases (see below).

Further descriptive studies should focus primarily on representatives of unstudied genera and species with outstanding ecology (e.g., ultrapsammophilous taxa). In addition, further larval descriptions may also have utilitarian uses, as larvae of many species representing the ‘Opatrinoid’ clade are common pests (Table 2).

Table 2 Selected, non-exhaustive list of species of Opatrines and their hosts with associated publication(s)
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Dendarini. The description presented here for Zadenos mulsanti is the first comprehensive contribution to our knowledge of larval morphology within Melambiina. The other available publications are rather accidental (Table 1); thus, only general conclusions can be made here.

Both representatives of Melambiina share the same setation pattern of pygidium, i.e., four distinct spines present on the anterior edge (Fig. 4g). This feature is also shared by Dendarus punctatus from Dendarina (Medvedev 1968; Skopin 1978; Cherney 2005). However, it cannot be treated as a common feature of all Dendarini since other known larvae (i.e., Heliopates and Phylan) possess eight spines instead of four (Skopin 1978). According to the phylogeny presented by Kamiński et al. (2019), Heliopates is paraphyletic in relation with Phylan. Therefore, the above-mentioned spine arrangement might be diagnostic for the Heliopates + Phylan clade. However, more descriptions are required to fully test this hypothesis.

Larvae of Z. mulsanti can be easily distinguished from those of D. punctatus by the different arrangement of spines on front legs (Fig. 2a). In Zadenos, the trochanter and femur are each equipped with a pair of spines, while there are three spines are present on the tibia (Fig. 4e). Contrarily, in Dendarus, the following pattern is observed: trochanter with four spines, femur with six, and tibia with two (Cherney 2005). The other available descriptions concerning Dendarini are incomplete or ambiguous, preventing further comparisons.


Opatrini. The analysed material revealed a great level of morphological variability within the studied Blapstinina larvae (Fig. 5). At this point, it is impossible to propose any diagnostic characters concerning this developmental stage which define the subtribe. However, it can be noted that the front legs of Blapstinina larvae are only slightly enlarged in relation with other known cases representing the Opatrinoid clade, with the exception of Stizopina (Schulze 1963). On the other hand, the larval morphology proved to be extremely informative at the generic level (see above under Trichoton).

A similar pattern was revealed for the most intensively studied subtribes of Opatrini [i.e., Opatrina and Stizopina (Table 1)]. For example, the spine pattern of the edge of pygidium varies significantly in Opatrina (eight spines arranged on a single row in some Penthicinus species; up to 22 in Melanestes; ~ 20 distributed on margins in middle of pygidium in Scleropatrum) (Dai et al. 2000; Jia et al. 2014; Jinxia and Youzhi 2000; Li et al. 2013; Ren et al. 2000; Yu et al. 2000a, b, c; Yu and Ren 1994a, b, Zhang and Yu 2004; Cherney 2005). In Stizopina, this variability is even greater (Fig. 2). The strongest modifications within this subtribe were observed for the highly specialised, psammophilous genera, such as Psammogaster. In this case, no apical spines on pygidium are present, while the other known taxa have up to 18 spines (in different arrangements) (Schulze 1963). On the other hand, all known Stizopina larvae do not have ocelli, which seems to be unique across the ‘Opatrinoid’ clade (Schulze 1963).

An additional observation of interest is that despite the high variability in pygidium morphology, no Opatrini species were reported to possess four distinct apical spines—a commonly observed feature within other closely related tribes (Figs. 4g,6i). This also holds true for Amphidorini (Wade and Boving 1921; Smith et al. 2014), a tribe recovered sister to the ‘Opatrinoid’ clade (Kamiński et al. 2019).

The available contributions for other subtribes (Ammobiina and Heterotarsina) are extremely fragmentary, with the few represented taxa reflecting little of the diversity of the group(s), inhibiting further analysis (Table 1).


Pedinini. Although no immature stages of this tribe are described here, the comparison of available larval descriptions with the newly introduced phylogenetic context resulted in some interesting conclusions. One of the main problems encountered by Kamiński et al. (2019) concerned the phylogenetic relationship between Helopinina and Leichenina. In one scenario, the latter was recovered sister to all other Pedinini, while in the second, it was nested within Helopinina (sister to the genus Drosochrus). Based on adult morphology, Kamiński et al. (2019) favored the first solution. As a result, the three following subtribes were designated: Helopinina, Leichenina, and Pedinina. The larval descriptions for all those entities presently exist (Table 1).

Comparative analysis revealed that Helopinina is the most variable subtribe in terms of larval morphology. It is best visualised in the modifications of the setation arrangement of the pygidium (Schulze 1968). Contrary to Leichenina and Pedinina, no known Helopinina larvae possess four apical spines (Schulze 1969). Moreover, the larvae of Leichenum are clearly characterised by a unique, within Pedinini, structure of the clypeus and labrum (St. George 1930; Cherney 2005; Dunford and Steiner 2007). In conclusion, no larval features directly linking Helopinina and Leichenina were found during the present study. This supports the classification concept proposed by Kaminski et al. (2019). For detailed analysis of the larval morphology within Helopinina, see Schulze (1968).

Finally, the setation of larval forelegs seems to be relatively stable within Pedinini. The majority of known species are characterised by having a pair of spines on each of the following segments: trochanter, femur, and tibia (St. George 1930, Keleinikova 1966; Medvedev 1968; Schulze 1968; Cherney 2005; Dunford and Steiner 2007). The only known exceptions concern a few species of Micrantereus species, which posses a single spine on the protibia (Schulze 1968). This spine arrangement (2–2–2) is not shared by the most closely related tribes, i.e., Dendarini and Platynotini (e.g., Figs. 4e,6g). However, it was observed within several species representing Opatrini (Dai et al. 2000; Jia et al. 2014; Jinxia and Youzhi 2000; Li et al. 2013; Ren et al. 2000; Yu et al. 2000a, b, c; Yu and Ren 1994a, b; Zhang and Yu 2004, Cherney 2005).


Platynotini. Because Schulze (1969) did not attach any descriptions for the Heteropsectropus larva illustrated in her paper, and Tschinkel’s (1978) contribution concerned the first instar larvae, this paper is first to provide data which can be used for comparative studies on higher phylogenetic levels. The available reference knowledge on Platynotina is presented by Iwan (1995), Iwan and Bečvář (2000) and Iwan and Schimrosczyk (2008).

The morphology of E. rudebecki larvae is highly consistent with that of the platynotoid and trigonopoid Platynotina (for definitions, see Iwan 2002, and Kamiński 2015), i.e., Alaetrinus, Anchophthalmus, Bantodemus, Glyptopteryx, Opatrinus, and Zophodes (Table 1). This seems to also hold true for the undescribed larvae of Heteropsectropus (Schulze 1969); however, the material concerning this species was not realised here. The above-mentioned affiliation is supported by the following features: clypeus with two pairs of setae (Fig. 6a, b), labrum with a median pair of setae and eight apical setae (Fig. 6a, b), and pygidium with four apical spines (Fig. 6i). However, these characteristics are likely plesiomorphic for the Dendarini + Pedinini + Platynotini clade, as they were also recovered in Pedinina and Melambiina (Fig. 4a). It was also observed by Schulze (1969) that larvae of platynotoid Platynotina resemble those of Scaurini to an astonishing extent.

The larval morphology of other Platynotina lineages is extremely variable (Schulze 1962, 1964, 1978; Iwan and Bečvář 2000). No key features seem to be shared with the presently described larvae of E. rudebecki. However, this species can be easily distinguished from the other known Platynotini larvae by having lateral extensions of mandibles (Fig. 6b).


Economic importance. The number of species with currently known larvae within the ‘Opatrinoid’ clade is surprisingly low, especially when economic factors are taken into account. A brief literature search performed here indicates that larvae representing this group have been noted as pests of various plant species. The ‘Opatrinoid’ clade is represented globally, with infestations reported across several tribes and subtribes from such distant countries as Australia, Turkmenistan, The United States of America, and Zimbabwe (Table 2). On the other hand, several tenebrionid species (including representatives of ‘Opatrinoid’ clade) were observed to feed on common weed species (Ogloblin and Kolobova 1927), i.e., Chenopodium album L., Convolvulus arvensis L., or Polygonum aviculare L. As these insects are cosmopolitan in distribution and impact a wide array of crops, greater research effort into their larval stages (rearing, descriptions, life history, etc.) is needed and warranted to curb negative and potentially enhance positive effects of their presence.