Description of larval morphology and phylogenetic relationships of Heterotemna tenuicornis (Silphidae)

In total 48 larval specimens of H. tenuicornis were obtained and analysed. We identified 30 larvae of the first instar, 14 of the second instar and 4 of the third instar. Two larvae and one adult specimen of H. tenuicornis were used for molecular phylogenetic placement of the genus within the subfamily Silphinae. The phylogenetic tree was obtained using Bayesian analysis from the concatenated partial 16S (434 bp) and COI (609 bp) sequences (Fig. 1).

Species identification based on genetic distances

The calculated p-distances between concatenated sequences of 16S and COI of larval and adult specimens of H. tenuicornis were between 0.0029 and 0.0078 (the mean calculated p-distance within Heterotemna specimens was 0.01). Conversely, the distance between different species of Silpha was shown to be higher (mean calculated p-distance within the Silpha species was 0.08), thus the larval specimens were confirmed as belonging to the same species as the adult specimen, H. tenuicornis (SM1).

Phylogenetic analyses

The Bayesian analysis (posterior probability 99), maximum parsimony bootstrap (84) and maximum likelihood bootstrap (93) strongly supported a clade of the genera Silpha, Heterotemna, Ablattaria and Phosphuga, suggesting close relationships of these genera with Heterotemna inside the genus Silpha, which makes the genus Silpha paraphyletic. The position of H. tenuicornis as a sister lineage to S. tristis Illiger, 1798 was strongly supported by the Bayesian analysis (97) but not strongly supported by the other analyses. The results confirmed the monophyly of the genera Thanatophilus Leach, 1815, Necrodes Leach, 1815, and Oiceoptoma Leach, 1815 within the subfamily Silphinae (Fig. 1).

Morphometry

The two commonly used measurements for instar identification, head width and width of protergum , are applicable in the case of H. tenuicornis (Fig. 2c, d) as these two measurements do not overlap between the instars and show significant differences. More specifically, the following measurements were very different between instars; head width (F statistic = 231 on 2, df = 45, p value < 2.2e−16), protergal width (F statistic = 4.109 on 2, df = 45, p value < 2.2e−16). Significant difference was also observed in the length of the first segment of the maxillary palpus (F statistic = 9.181 on 2, df = 44 , p value < 0.0004653), all three antennomeres (AI F statistic = 112.3 on 2, df = 45, p value < 2.2e−16, AII F statistic = 143.2 on 2, df = 45, p value < 2.2e−16, AIII F statistic = 69.19 on 2, df = 45, p value 1.868e−14) and both urogomphal segments (UI F statistic = 95.25 on 2, df = 45 , p value < 2.2e−16, UII F statistic = 4.109 on 2, df = 45, p value 0.02296). In the case of the urogomphal segments, we observed isometric growth in the first segment but not in the second segment (SM4).

Larval morphology

Diagnosis

Body teardrop-shaped (widest at front), distinctly dorsoventrally flattened, dark brown with lighter paratergites on thorax and abdominal segments I–VIII (Figs. 3a, d, f, 4c), with two lighter subcircular spots on protergum (Fig. 3a, d). Short median unsclerotized line extending beyond epicranial stem anteriorly (Fig. 6e). Epipharynx anterolaterally with two pairs of sensory pegs on its heavily sclerotized margin (Fig. 7b, lsp). Ventral epicranial ridges present, extending past the posterior edge of the hypostomal ridge (Fig. 4d). Antenna very slender and elongated (Figs. 4a, 5a). Antennomere II with large, round and flattened sensorium, bearing several sclerotized pores surrounded by narrow, sclerotized ring (Fig. 6f), externally with a wide unsclerotized area. More apically, antennomere II with three additional smaller sensilla (Figs. 4a, 5a, 6f). Anterior margin of protergum medially with narrow emargination (Fig. 8i). Rudimentary spiraculum present on metasternum (Fig. 3b). Paratergites of meso- and metathorax and abdominal segments I–VIII resembling the shape of pig ears, constricted posteriorly between tergite and paratergite, with apex pointed posteriorly (Figs. 3a, 4c, g). Ventrite II on abdomen entire, not subdivided into three sclerites (Fig. 3b).

Detailed description

Body Instar III. Mean total body length: 16.65 mm ± 0.815 mm. Teardrop-shaped, fusiform to onisciform larvae, narrowing towards both ends, widest at metathorax (Fig. 3a–g), dorsoventrally distinctly flattened (Fig. 3c). Terga well-sclerotized and sparsely and regularly covered with short, recumbent setae (Fig. 4b, c). Dorsal surface dull, sparsely granulate, with regular isodiametric microsculpture (Fig. 4b). Paratergites of meso- and metathorax and abdominal segments I–VIII wide, resembling the shape of pig ears, constricted posteriorly between tergite and paratergite, with apex pointed posteriorly (Figs. 3a, 4c, g). Abdominal ventrites more glossy, without distinct microsculpture, posterior margin of ventrites with row of long, semierect setae (Fig. 4e). Dark brown pigmentation present on thoracic and abdominal sclerites, as well as on bases of paratergites. Remaining area of paratergites and lateral edges of protergum ochre (Fig. 4c, g). Laterally, tergites with darker brown spots and shallow depressions, arranged in two, slightly irregular, longitudinal rows; medially, dorsum with pale ecdysial line, reaching posteriorly to abdominal tergite IV. Additionally, pair of lighter subcircular translucent spots present on the frontal medial area of protergum, duller and not clearly delimited (Fig. 3a, b). Instar II. Pair of lighter subcircular translucent spots on protergum not clearly delimited (Fig. 3f–g). Mean total body length: 15.55 mm ± 1.16 mm. Instar I. Two lighter subcircular spots present on protergum more apparent, well-delimited (Fig. 3d–e). Mean total body length: 13.02 mm ± 0.77 mm.

Head capsule Instar III. Prognathous. Head capsule covered dorsally and laterally by regular, densely arranged erected setation (Fig. 6a–c). Longest setae present on anterior and anterolateral part of frons (Fig. 6d–e). Cranium with isodiametric microsculpture, latero-posteriorly with paler spots (Fig. 6g). Epicranial stem (= coronal suture) present (Fig. 6a, es), V-shaped frontal arms (= frontal sutures) passing into U-shaped base in one third of their length (Fig. 6e, fa). Short median desclerotized line extending beyond epicranial stem (Fig. 6e, ml). Six stemmata on both sides of the head organized into two groups – four forming sub-rhomboid pattern placed dorsally behind antennal socket (Fig. 6e), and two ventro-laterally behind the antennal socket (Figs. 5a, 6c). Frontoclypeal suture absent, rudiments present only laterally as distinctly sclerotized, transverse, extension of anterior tentorial pits (Fig. 6d, atp). Clypeus trapezoidal; anterior margin distinctly sclerotized, widely emarginate and only narrowly desclerotized medially (Fig. 6b, d). Epipharynx, with the exception of medial part, covered by densely arranged microtrichia oriented posteriorly and medially (Fig. 7b). Anterolaterally, the heavily sclerotized epipharyngeal margin bearing two pairs of lateral sensory pegs (Fig. 7b, lsp). Anterior part emarginate, at its lateral angles with 1 pair of median sensory pegs (Fig. 7b, msp). Anteriorly, on first porous area, are two pairs of large sensilla (Fig. 7b, fpa). More posteriorly, secondary porous area consisting of two pairs of small sensilla (Fig. 7b, spa). More posteriorly, near base, with broad, weakly arched parabolic row of 18 pores (or cibarial plates), bounded posteriorly on each side by group of 5 pores (quinqueporous area) (Fig. 7b, qpa). Two pairs of bilobate pegs located more laterally (Fig. 7b, bp). Hypopharynx membranous, with transverse hypopharyngeal bracon. Tentorium consisting of pair of sclerotized anterior arms, laterally narrowly extended by fine hyaline lobes in their basal 2/3, before the dorsal arms connecting to them; hyaline dorsal arms connected to frons near the beginning of the U-shaped base of the frontal arms; and sclerotized posterior arms connected to broad posterior tentorial bridge. Ventral epicranial ridges present, extending past the posterior edge of hypostomal ridge (Fig. 4d). Hypostomal rods absent. Gular region very short, with gular sutures converging anteriorly. Head size: HW 2.842 mm ± 0.077 mm. Instar II. Head size: HW 2.327 mm ± 0.075 mm. Instar I. Head size: HW 1.927 mm ± 0.045 mm.

Mandibles (Fig. 8a–d) Instar III. Symmetrical (Fig. 6b), simple without mola or prostheca. Apical tooth longer than sub-apical tooth, apex regularly bent dorsally in inner lateral view (Fig. 8b). Two stout setae present dorsally and dorso-laterally on mandibular base; additional short, stout seta present dorsally in the mid-length of the mandible. Inner margin of both apical and subapical teeth finely serrate (Fig. 8d). Instar II and Instar I same as Instar III.

Antenna (Figs. 4a, 5a, 6f) Instar III. Trimerous and fully sclerotized, inserted in membranous socket on lateral distal margin of genal region. Sensorium of AII placed on outer lateral area of its distal end, consisting of round, flattened bulb covered with several large pores, and belted by sclerotized ring and three small sensilla arranged in triangle adjacent posteriorly to the sensorium (Fig. 6f). All three antennomeres densely covered by stout setae across the surface (Fig. 5a). Apex of AIII with three articulated sensilla and one elongate, slender seta (as on Fig. 5b). Antennomere proportions: first two segments slightly shorter than third segment (Fig. 4a); AI 1.213 mm ± 0.138 mm, AII 0.912 mm ± 0.009 mm, and AIII 0.989 mm ± 0.164 mm. Instar II. Antennomere proportions: AI (1.214 mm ± 0.215 mm) slightly longer than AII (1.031 mm ± 0.15 mm) and AIII (1.093 mm ± 0.117 mm). Instar I. Antennomere proportions: AI (0.713 mm ± 0.083 mm) and AII (0.07 mm ± 0.041 mm) of similar length and AIII on average slightly longer (0.824 mm ± 0.06 mm).

Maxilla (Fig. 7c–e) Instar III. Attached closely laterally to labium. Cardo broad (Fig. 7e, cd), separated in ventral view into two parts by narrow, heavily sclerotized, longitudinal apodeme (joined anteriorly to inner margin of stipes). Base of cardo with one large seta placed on outer ventral margin and two smaller setae, one placed dorsally and another ventrally. Stipes elongate, subrectangular; ventral side regularly sclerotized, with more distinctly sclerotized apodeme on inner margin, joining cardo with inner base of lacinia (Fig. 7e, st). Ventral surface with group of several short setae and one large seta in the middle of the length. Outer lateral margin with another small seta and additional single large seta, placed more anteriorly. Dorsal surface only narrowly sclerotized along outer margin, with irregular row of short setae and pores (Fig. 7d, st). Most of dorsal surface weakly sclerotized. Lacinia and galea basally fused, separated only apically (Fig. 7d–e). Sclerotized basal fused part ca. 1.4 as long as wide. Distinct cuticular spines present on inner lateral and dorsal base of lacinia; base in dorsal view with two distinctly sclerotized, slightly dorsally elevated regions (Fig. 7d). Fused basal part on outer margin in ventral view with two large, laterally pointing setae, and several small setae and pores. Another two large setae present at base. Free apical part of lacinia distinctly sclerotized, with elongate longitudinal apodeme in ventral view. Inner margin with heavily sclerotized lobes, with 9–11 large, stout teeth. Lacinia elongate apically, apex shorter than galea. Galea with a compact, relatively small brush of setae (Fig. 7d, e, ga). Maxillary palpus trimerous, short basal palpifer present (Fig. 7c, mpf). Third segment cylindrical, apically with group of sensilla. Proportions of maxillary palpomeres: MPI 0.248 mm ± 0.009 mm, MPII 0.277 mm ± 0.057 mm, MPIII 0.468 mm ± 0.026 mm. Instar II: Basal fused part of lacinia and galea shorter and wider, sclerotized part only slightly longer than wide. Galea with large brush of setae. Proportions of maxillary palpomeres: MPI 0.189 mm ± 0.032 mm, MPII 0.245 mm ± 0.024 mm, MPIII 0.396 mm ± 0.087 mm. Instar I: Basal fused part of lacinia and galea short and wide, sclerotized part as long as wide. Galea with large brush of setae. Proportions of maxillary palpomeres: MPI 0.158 mm ± 0.041 mm, MPII 0.199 mm ± 0.029 mm, MPIII 0.384 mm ± 0.067 mm.

Labium (Fig. 8e–g) Instar III. Submentum present ventrally at base of maxillo-labial complex aslarge, broadly trapezoidal sclerite, posterolateral corners extending to base of cardo, and to transversely oriented posterior tentorial pits (Fig. 4d). Anterior part weakly sclerotized, distinctly separated from mentum. Surface postero-laterally with two pairs of large setae, surface irregularly covered by several additional short setae. Mentum wide, sclerotization extended also laterally, with two pairs of very large setae positioned laterally, surrounded by additional several short setae and pores (Fig. 8e, mt). Prementum transverse, narrowing anteriad, irregularly sclerotized. Laterally with one pair of large setae, medially with several small setae (Fig. 8e, pm). Ligula bilobed, heart-shaped, sclerotized laterally and basally, each lobe along the sagittal plane covered dorsally by numerous longitudinal lines of fine short setation and dense bulbous projections apically and centrally between the two lobes (as on Fig. 8f, li). Labial palpus bimerous; basally with longitudinal, laterally sclerotized palpifer (as on Fig. 8f, lpf). Basal palpomere club-shaped, laterally with four and ventrally with two short setae, distal palpomere conical, with several very short setae and several sensilla laterally and apically. Instar II. Mentum ventrally with one pair of very long and one pair of slightly shorter setae laterally (Fig. 8g). Instar I. Setae on mentum as in instar II.

Thorax (Fig. 3a, b) Instar III. Three-segmented. Protergum sub-semicircular (Figs. 3a, 8i). N1W 8.633 mm ± 0.283 mm, protergum widest at base with narrow emargination at medial part of anterior margin (Fig. 8i). Pair of subcircular lighter patches present anteriorly, only vaguely delimited (Fig. 3a). Mesotergum wider than protergum, metatergum being the widest part of the body (Fig. 3a). Paratergites of meso- and metatergum distinctly longer than paratergites on abdomen (Fig. 3a, b), both segments also distinctly more robust than abdominal segments (Fig. 3a). Venter of prothorax composed of prosternum, subdivided into three semi-sclerotized areas; lateral ones small; medial area largest. Ventrolateral areas of pro-, meso-, and metathorax composed of episternum and epimeron with short pleural suture in between them and well-sclerotized pre- and postcoxale. Meso- and metasternum subdivided by transverse fold into membranous basisternum and sternellum; basisterna on both segments medially with well sclerotized areas, sagittally divided into two plates (Fig. 3b). Laterotergites poorly sclerotized, with mesothoracic spiraculum opened, its inner part densely covered with multi-branched filtration hairs (Fig. 5f). Rudimentary spiraculum also present on laterotergites of metasternum (Fig. 3b, rs). Instar II: N1W 6.909 mm ± 0.340 mm. Instar I: Pair of subcircular lighter patches well-delimited (Fig. 3d). N1W 5.713 mm ± 0.237 mm.

Legs (Fig. 5c) Pentamerous, including pretarsus, relatively elongated (femur ca. 4 times as long as wide); regularly covered with two types of setae—short and thin and long and stout (Figs. 5d, 6i). Surface with regular, isodiametric microsculpture (Figs. 5d, 6i). Femur ventrally covered with additional row of longer setae (Fig. 5c). Tibiotarsus covered regularly with longer, stout setae (Fig. 5d). Tarsal claw with pair of opposed setae (Fig. 5d).

Abdomen (Figs. 3a, b, 4b, c, e–g, 5e, 6h): Instar III Ten-segmented. Tergites regularly narrowing posteriad, length of segments slightly increasing towards abdominal apex (Fig. 3a). Paratergites I–IX covered with minute setae, antero-lateral margin with four larger setae, posterior apex with single, large seta (Figs. 4f, 6h). Anterior part ventrally with distinctly sclerotized spiraculum, opened postero-laterally (Fig. 8h). Ventrite I reduced, present only medially, lateral portions largely unsclerotized. Ventrite II entire, similar to ventrites III–IX, not broken into three sclerites (Fig. 3b). Tergite IX subrectangular, with reduced paratergites, with two posteriorly oriented apical setae and with well-developed pair of two segmented urogomphi (Figs. 4g, 5e). Basal segment of urogomphi densely covered by recumbent setae; only scarce setae present ventrally on distal segment (Figs. 4h, 5e). Segment X subcylindrical, holding pygopod with six eversible lobes, covered by fine spines (Figs. 5e, 7a). Distal edge covered with row of posteriorly oriented setae (Fig. 4g). First (basal) urogomphal segment (URI 1.006 mm ± 0.065 mm) widest at its base, narrowing posteriorly, shorter than segment X (Fig. 4h). Second segment conical, narrowing towards its end (URII 0.234 mm ± 0.019 mm) with short stout seta (US 0.143 mm ± 0.013 mm) (Figs. 4h, 5e). Instar II: First segment of urogomphi longer than segment X (Fig. 4i), URI 0.803 mm ± 0.051 mm, URII 0.188 mm ± 0.033 mm and US 0.152 mm ± 0.021 mm. Instar I: First segment of urogomphi longer than segment X (Fig. 4j), URI 0.621 mm ± 0.064 mm, URII 0.208 mm ± 0.037 mm and US 0.29 mm ± 0.044 mm.

Identification key to instars of Heterotemna tenuicornis

1. 1.

   Pair of lighter subcircular translucent spots on frontal medial area of protergum more distinct, well-delimited (Fig. 3d, e). Head width 1.83–2.01 mm. Protergum width 5.24–6.19 mm. … First instar

2. –

   Pair of lighter subcircular translucent spots present on frontal medial area of protergum, duller and not clearly delimited (Fig. 3a, b, c, e). Head width 2.19–2.91 mm. Protergum width 6.38–9.11 mm².

3. 2.

   Head width 2.19–2.43 mm. Protergum width 6.38–7.47 mm. First segment of urogomphi longer than segment X (Fig. 4i). … Second instar

4. –

   Head width 2.72–2.91 mm. Protergum width 8.39–9.11 mm. First segment of urogomphi shorter than segment X (Fig. 4h). … Third instar

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