Highly Specialized Cretaceous Beetle Parasitoids (Ripiphoridae) Identified

Systematic Entomology (2019), 44, 396–407 DOI: 10.1111/syen.12331

Highly specialized Cretaceous beetle parasitoids (Ripiphoridae) identiﬁed with optimized visualization of microstructures

JAN BATELKA1, JAKUB PROKOP1 , HANS POHL2,MINGBAI3, WEIWEI ZHANG4 andROLF G. BEUTEL2

1Department of Zoology, Faculty of Science, Charles University, Praha 2, Czech Republic, 2Institut für Zoologie und Evolutionsforschung, FSU Jena, Jena, Germany, 3Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing, China and 4Unaffiliated, Chongqing, P. R. China

Abstract. Extremely miniaturized longipedes insects (body length c. 0.3 mm) embedded in two pieces of Cretaceous amber from Myanmar are described and interpreted. Using inverted fluorescence and light microscopy for detailed analysis of microstructures, the inclusions were identified as primary larvae of the beetle family Ripiphoridae, subfamily Ripidiinae. While the structure of thoracic and abdominal segments including appendages corresponds well with the groundplan known in recent members of Ripidiinae, a curved prosternal ridge with prominent spines (each c.5𝜇m), the reduced condition of stemmata and antennae and the lack of sharp mandibles are unique features within the entire family, apparently apomorphies of the longipedes larvae. A sinuate prosternal edge with a dense row of spines (prosternoctenidium) might be homologous with ‘head ctenidia’ in some previously described miniaturized conicocephalate larvae, but further investigation is needed. The morphological differences between the head of longipedes larvae and extant Ripidiinae are interpreted as adaptations to different groups of hosts and life strategies. Palaeoethology of the longipedes larvae is briefly discussed. In addition, the systematic placement of conicocephalate larvae from Canadian, Myan- mar and Russian Cretaceous ambers, already interpreted by various authors as primary instars within Coleopterida (assigned to either Strepsiptera or to the coleopteran Tene- brionoidea: Ripiphoridae), is discussed.

Introduction data is particularly difficult in fossil larvae, where the exoskele- ton is often too thin and structures too small to be well preserved The evolution and age of origin of extant beetle lineages are cur- and/or observed. Even in cases of very detailed descriptions of rently being investigated and discussed intensively (Hunt et al., larvae of Coleopterida preserved as compression fossils, < 40% 2007; McKenna et al., 2015; Toussaint et al., 2017). Amber of characters used for data matrices including extant terminals inclusions and compressed fossils of Mesozoic species with phy- became available (see, e.g., Pohl, 2009; Fikácekˇ et al., 2014). logenetically important characters could be an important source Cretaceous larvae are of particular interest as their morphol- of calibration data (Szwedo & Nel, 2015), even though results in ogy could shed light on the early evolutionary history of the palaeoentomology are facing certain constraints (Nel & Prokop, respective clades. Larvae discovered in various World amber 2009). Fossil taxa are only useful for systematic and phyloge- deposits have been primary larvae generally attributed to either netic purposes when the description is detailed enough to be used the parasitic Strepsiptera (Grimaldi et al., 2005; Kathirithamby for comprehensive differential diagnosis or for a data matrix also et al., 2017) or to parasitoid beetles of the family Ripiphori- including extant taxa (e.g. Huang et al., 2016). Obtaining such dae with first instars of similar habitus (Grimaldi et al., 2005; Beutel et al., 2016). However, none of the descriptions of these Cretaceous larvae provided so far allows a precise analysis of Correspondence: Jakub Prokop, Department of Zoology, Faculty of Science, Charles University, Vinicnáˇ 7, 128 43 Praha 2, Czech Republic. phylogenetically important details and a subsequent confidential E-mail: [email protected] systematic placement.

In this study we describe minute longipedes insects from marked in Fig. 1D), ‘nematoceran’ fly with unclear affinity mid-Cretaceous amber from Myanmar, similar to a morphotype (two), and brachyceran species with unclear affinity (two). with extremely prolonged tibio-tarsi reported from New Jersey Turonian amber by Grimaldi et al. (2005). Using inverted fluorescence and light microscopy, we revealed hitherto overlooked Optical devices microstructures important for the systematic placement of these Burmese and also North American fossils. The results allowed The specimens from sample SMNS BU-60 were visual- us to place these beetle larvae confidentially to subfamilial level, ized under an Olympus IX81 inverted fluorescence microscope although they possess some structures not yet observed in their with UIS2 objective lenses and equipped with an ORCA-AG extant relatives, and simultaneously lack some features typical monochromatic 12-bit charge coupled device camera (Ham- for the respective clade. matsu, Japan). The mirror images were pseudocoloured (red for Cy3, blue for DAPI and green for fluorescence) and super- imposed with cell^r software (Olympus Soft Imaging Solu- Materials and methods tions, Japan). Images from the inverse microscope are mirrored. For this scope, we used the imagej (64-bit) software (Rasband, Material W.S., ImageJ, U.S. National Institutes of Health, Bethesda, MD, U.S.A.; http://imagej.nih.gov/ij/, 1997–2015). Two pieces of Burmese amber of longipedes primary larvae The specimens embedded in SMNS BU-60 were also observed were at our disposal, with 15 and 46 specimens, respectively. under an Olympus BX40 microscope and habitus photographs Both examined pieces came from deposits in the Hukawng were taken with an attached Canon D550 digital camera (Canon Valley of Myanmar. The age is estimated as c. 99 Ma (earliest Inc., Japan) in RAW format. Original photographs were pro- Cenomanian) (Shi et al., 2012). cessed using Adobe photoshop cs4 (Adobe System Incor- The syninclusion with 15 larvae, examined by the Prague porated, San Jose, CA, U.S.A.), and for some images the team, is stored in the Staatliches Museum für Naturkunde, focus-stacking software zerene stacker (Zerene Systems, Stuttgart, Germany (hereinafter coded as SMNS). Its precise Richland, WA, U.S.A.) was used to produce final photographs. mining locality is unknown. Because of different states of The specimens in the IZAS amber piece were observed under a preservation of each particular larva, a description based on Keyence VHX-2000 digital microscope (Keyence Deutschland single selected specimen would be inappropriate. Owing to the GmbH, Germany). extremely small size of the individuals, some structures are easily overlooked or they can only be identified with certainty in one or a few of them by using different optical techniques (see later). Therefore, all specimens were numbered (see Fig. 1A) Comparative taxonomy and terminology and coded ‘SMNS’. The particular number(s) used in the description or plates indicates the specific specimen(s) in which For differential diagnosis and comparative analysis of char- a character has been observed or documented. Some specimens acters of the longipedes larvae, we chose descriptions of the are preserved in the dorsal view, and others in the ventral view. following primary larvae: Ripidius quadriceps Abelle de Per- Specimens SMNS_9, SMNS_12, and SMNS_15 are interpreted rin, described by Besuchet (1956); Blattivorus inquirendus as visible in the ventral view. The amber piece SMNS BU-60 (Silvestri), described by Silvestri (1906); generic synonymy measures 19.4 × 20.2 × 8.0 mm. Remnants of a fly specimen of Rhyzostylops Silvestri and Blattivorus Chobaut is after (Diptera: Phoridae gen. et sp. indet.) (Fig. 1B) and one small Batelka (2009) (both Ripidiinae); Macrosiagon ferruginea larva of a possible Elateroidea (Coleoptera) (Fig. 1C) are also (Fabricius) is by Grandi (1936); Macrosiagon bimaculata embedded with the larvae. (Fabricius) is by Chobaut (1906); Macrosiagon cruenta (Ger- The syninclusion with 46 larvae (accession number mar) and Ripiphorus smithi Linsley et MacSwain are by BU-002386) (see Fig. 1D), examined by the Jena team, is Linsley et al. (1952); Ripiphorus arabiafelix caboverdianus integrated into the collection of the Institute of Zoology, Chi- Batelka et Straka is by Batelka & Straka (2011); Metoecus nese Academy of Sciences (Beijing, P. R. China) (hereinafter paradoxus (Linnaeus) is by Švácha (1994) (all Ripiphori- coded IZAS) (Beijing, P. R. China). It comes from a mining nae); and Pelecotoma fennica (Paykull) is by Švácha (1994) locality at Noije Bum (near Tanai Village, 26∘21′33.41′′N, (Pelecotominae). With respect to the morphology of these 96∘43′11.88′′E) (e.g. Cruickshank & Ko, 2003; Grimaldi et al., listed genera, we stayed with the respective reference, unless 2002). The IZAS amber piece measures 26 × 22 × 10 mm. otherwise stated. Fossil conicocephalate larva from Taimyr Syninclusions along with the longipedes larvae comprise the (Kathirithamby et al., 2017), Myanmar (Beutel et al., 2016) following groups of arthropods: Arachnida (nine specimens) and Manitoba (Grimaldi et al., 2005) were compared with the (one marked in Fig. 1D), orthopteran nymph (one), Psocodea present longipedes larvae in available structural details. Larvae (one), Sternorrhyncha (one), apocritan Hymenoptera (two), of Meloidae are discussed based on studies of Pinto et al. Berothidae (Neuroptera) (one) (Fig. 1D), primary larva of (1996), Bologna & Pinto (2001) and Bologna et al. (2002). Strepsiptera (one) (Pohl et al., 2018), Elateridae (1) (Fig. 1D), Data on the morphology of Strepsiptera larvae are taken from Polyphaga with unclear affinity (two) (both Coleoptera) (one Pohl (2000).

Fig. 1. Amber arthropod syninclusions. (A–C) Specimen SMNS BU-60, Staatliches Museum für Naturkunde, Stuttgart, Germany: (A) longipedes larvae – each specimen numbered; (B) Diptera: Phoridae gen. et sp. indet.; (C) Coleoptera: Elateroidea (?) – larva. (D) IZAS specimen, Institute of Zoology, Chinese Academy of Sciences and the Three Gorges Entomological Museum, Chongqing, China; white arrows indicate the position of longipedes larvae; Ar, Arachnida family indet.; CEl, Coleoptera: Elateridae gen. et sp. indet.; CPo, Coleoptera: Polyphaga family indet.; NBe, Neuroptera: Berothidae gen. et sp. indet. Scale bars: (A, D) 1 mm, (B) 500 𝜇m, (C) 100 𝜇m. [Colour figure can be viewed at wileyonlinelibrary.com].

In this study we introduce the term ‘longipedes’ larvae for the et al., 2005; Beutel et al., 2016; Kathirithamby et al., 2017), newly described first instars from Cretaceous Burmese amber thus avoiding the undesirable usage of two parallel binominal and similar New Jersey long-legged larvae, referring to their systems for larvae and adults, without the option of synonymiza- extremely prolonged tibio-tarsus. This character discerns them tion. The only disadvantage of this approach is that such organ- readily from all other known extant and fossil first instars of par- isms without formal description are usually neglected by authors asitic beetle larvae. The proposed term constitutes complemen- of taxonomic catalogues. tary labelling to the term ‘conicocephalate’ larvae introduced for another Cretaceous larval morphotype by Kathirithamby Results et al. (2017). Both terms allow the immediate orientation of researchers and serve as replacements for scientific binomens Taxonomy for fossil larvae, as used in some other cases (e.g. Pohl, 2009; Fikácekˇ et al., 2014). We do not follow the designation of fossil Family Ripiphoridae Gemminger larvae by binomenal names, instead we follow the practice Subfamily Ripidiinae Gerstaecker of authors who dealt with these instars previously (Grimaldi Ripidiinae gen. et sp. indet.

AB C

GHI

Fig. 2. Micrographs of longipedes larvae habitus preserved in Burmite. (A–E, G) SMNS BU-60: (A) SMNS_2-6; (B) SMNS_1; (C) SMNS_11; (D) SMNS_12; (E) SMNS_15; (G) SMNS_13. (F, H, I) IZAS specimens; lps, one long preapical seta; ls, long setae; mp, maxillary palpus; pct, prosternoctenidium. Scale bars: 100 𝜇m. [Colour figure can be viewed at wileyonlinelibrary.com].

Habitus. Extremely small, c. 0.3 mm long excluding ter- or slightly longer, or shorter. Meso- and metathorax and abdom- minal setae. Well sclerotized, with smooth cuticle. Minute inal segments I–V parallel-sided. Legs extremely elongated, finger-shaped sensilla with ring-shaped base inserted close to with flagellate tibio-tarsus and very thin distal part. Posterior midline on pro- and mesothorax, two pairs on the former, and abdominal segments VI–X narrowing strongly over short dis- tance towards rounded apex, with conspicuous concentration of one pair on the latter. Pair of long bristle-like setae inserted 13–17 long setae. on posterior edge of metanotum and pair of very similar setae Head. Parabolic shape, with evenly rounded anterior margin close to lateral edges. Abdominal segments connected by soft and evenly widening posteriorly, with greatest width at hind membranous cuticle, resulting in varying length of abdomen, margin. Moderately sized compared with length of postcephalic depending on preservation as long as thorax and head combined, body, c.20𝜇m from anterior to posterolateral margin in dorsal

Fig. 3. Line drawings of longipedes larva habitus (reconstructions): (A) dorsal view; (B) ventral view. ca, caput; fg, foregut (narrow pharynx and oesophagus); fs, finger-shaped sensilla; ls, long setae; md, mandible; mp, maxillary palpus; tr, trochanter; pn, pronotum; ps, prosternum; pct, prosternoctenidium; mss, mesosternum; msn, mesonotum; mts, metasternum; mtn, metanotum; t I–VIII, tergite I–VIII; s I–VIII, sternite I–VIII. Scale bar: c. 100 𝜇m. view. Slightly less than 30 𝜇m wide posteriorly. Stemmata or of the maxillary palpus, with very short portion projecting eye spots absent. Transverse frontoclypeal strengthening ridge beyond the edge of the head (SMNS_12, Fig. 4H). and dorsal ecdysial sutures (frontal sutures and coronal suture) Thorax. Prothorax almost as long as meso- and metatho- not visible, apparently absent. Posteriorly directed minute pro- rax combined. Pronotum well developed, distinctly widening jection present at about anterior third of lateral cephalic mar- posteriorly, with rounded posterolateral corners. Distal part gin. Posterodorsal margin slightly retracted, overlapped by dis- of foregut discernible, obliterating on posterior third of seg- tinctly convex anterior pronotal margin. Scale- or lobe-like pos- ment (Figs 3A; SMNS_5, Fig. 4C). Anterior margin slightly terolateral extensions of head capsule absent. Posteroventral convex, very slightly broader than posterior cephalic margin. cephalic margin adjacent with anterior prosternal edge. Tento- Distinct lateral edge absent. Four finger-shaped sensilla with rial grooves not recognizable, but massive posterior tentorial distinctly wider ring-shaped base arranged bisinuate close to base visible through semitransparent cuticle of posterior head anterior margin of pronotum. Row of another similar four reg- capsule. Labrum not recognizable as a separate structure, appar- ularly spaced finger-shaped sensilla placed close to posterior ently fused with clypeofrons. Head capsule with clearly visi- pronotal margin (SMNS_13, Fig. 5). One pair of the same type ble straight and narrow foregut (consisted of mouth, pharynx of sensilla located laterally at midlength of pronotal margin, and oesophagus) (see Bésuchet 1956: 101 “Tube digestif formé with short portion projecting beyond lateral edge (SMNS_12, d’un œsophage étroit, assez long, traversant tout le thorax … ”, Fig. 4H). Four bristle-like setae regularly placed along pos- “fig. 27”; see also Figs 3A, 4C). Antennae also not recognizable. terior margin of pronotum, each next to the finger-shaped Mandibles movable, protruding in some specimens (SMNS_2, sensilla. Sternal region with strongly curved sinuate edge Fig. 4B; SMNS_15, Fig. 4E), triangular, with straight inner mar- with dense row of c. 25–30 apically rounded, prominent gin. Apices not exceeding beyond margin of head and not spines (c.5𝜇m), which are referred to as prosternoctenid- overlapping each other when closed (SMNS_5, Fig. 4C). Max- ium in the following (SMNS_13, Fig. 2G; SMNS_12, Fig. 4A). illary palpus long, bristle-like (SMNS_2, Fig. 4B; SMNS_5, Prosternal plate absent and posteriorly directed, fixed spinulae Fig. 4D; SMNS_13, Fig. 4F; SMNS_6, Fig. 4G), with only one on posteroventral segmental margin also missing. Prominent, segment visible and basal segments or proximal maxillary ele- elongate, cone-shaped procoxae inserted posterolaterally, very ments unrecognizable (SMNS_5, Fig. 4D). One pair of minute distinctly protruding beyond lateral pronotal margin. Trochanter finger-shaped sensilla present dorsally just posteriad of the level small and only visible as indistinctly delimited element, but

ABC

D E F

Fig. 4. Micrographs of head structures of longipedes larvae. (A) Head and pronotum with indication of prosternoctenidium SMNS_12 in fluorescence; (B) head with maxillary palpus SMNS_2; (C) head with narrow pharynx and oesophagus, SMNS_5; (D) head with indication of maxillary palpus SMNS_5; (E) head with indication of mandibles SMNS_15; (F) head with indication of maxillary palpi SMNS_13; (G) head with marked posterior corners (arrowheads) and maxillary palpus SMNS_6; (H) head and pronotum with finger-shaped sensilla SMNS_12. fg, foregut (narrow pharynx and oesophagus); fs, finger-shaped sensilla; md, mandible; mp, maxillary palpus; pm, posterior margin of head; pct, prosternoctenidium. Scale bars:(A–D, F–H) 50𝜇m; (E) 10 𝜇m. [Colour figure can be viewed at wileyonlinelibrary.com]. present and articulated with distal procoxal articulatory areas than prothorax, with almost exactly parallel lateral margins. by slightly narrowed proximal part; slightly widening distally, Anterior mesonotal corner overlapped by broadly rounded pos- broadly connected with femur; oblique separating line indis- terolateral pronotal edge; posterior corners narrowly rounded, tinct, devoid of any setae (SMNS_13, Fig. 6A). Femur strongly almost acute. Mesonotum simple, undivided, without median elongated (c. 0.1 mm), slender, strongly narrowed basally, with ecdysial line; row of four regularly spaced finger-shaped sen- very slightly convex hind margin, and one long preapical silla placed close to posterior mesonotal margin (SMNS_13, seta (SMNS_13, Figs 2G, 6A; SMNS_11, Fig. 2C; SMNS_12, Fig. 5). Four bristle-like setae regularly placed along posterior Fig. 2D). Tibio-tarsus extremely elongated (c. 225 𝜇m), about margin of mesonotum, each next to the finger-shaped sensilla. 3 × longer than femur, very thin, strongly tapering distally. Distal Sternal region simple, without recognizable surface structures; half of tibio-tarsus with at least two extremely small, semi-erect, rows of spinules absent. Middle legs very similar to forelegs but laterally directed spines (SMNS_13, Fig. 6E). Pretarsus termi- even more elongated, total length 0.36 mm. Mesocoxa similar in nated by minute pad. Mesothorax distinctly shorter and narrower shape and size to procoxa, trochanter more robust, preapical seta

Fig. 5. Distribution of finger-shaped sensilla marked by arrowheads on thorax of longipedes larva SMNS_13, fluorescent probe – DAPI mode (381–392 nm). Scale bar: 100 𝜇m. on femur not visible, probably missing (SMNS_13, Fig. 6A). Distal half of tibio-tarsus with three extremely small semi-erect spines arranged in more or less regular row, oriented to same side as ultimate pretarsal pad (SMNS_2, Fig. 6D). Extremely small pretarsal structures recognizable as minute pad, with one possible pretarsal segment (unguitractor?) (SMNS_2, Fig. 6D; SMNS_13, Fig. 6E). Metathorax similar in shape to mesothorax, lacking finger-shaped sensilla on metanotum, but with two pairs of fairly long bristle-like setae on posterolateral region (SMNS_13, Fig. 5; SMNS_11, Fig. 2C). Metanotum slightly shorter than mesonotum, with more acute posterolateral angles. Hind legs very similar to middle legs (SMNS_15, Fig. 6B, C). Abdomen. Ten-segmented (SMNS_11, Fig. 2C) (c. 125 𝜇m depending on preservation). Segments I–V parallel-sided, with more or less straight lateral margin. Segments I and II shorter than III and IV, all four lacking setae. Segment V with four long setae arranged in two pairs inserted close to its posterior Fig. 6. Micrographs of structures on legs of longipedes larvae: (A) margin. Segment VI also with two pairs of setae on rounded trochanters in pro-, meso- and metathoracic legs SMNS_13; (B) spines on metathoracic leg SMNS_15; (C) spines and pulvillus on metathoracic posterolateral corner (only one visible on right side) and a pair leg SMNS_15; (D) spines and pulvillus on meso- and metathoracic of setae close to the midline. Terminal segments form a rather legs SMNS_2; (E) spines and pulvillus on prothoracic leg, metathoracic short and compact, laterally and posteriorly rounded unit; sharp leg without discernible spines SMNS_13. Lps, long preapical seta lateral edges mark borders between segments VII, VIII and IX; on profemur; tr, trochanter, white arrowheads indicate tibio-tarsal seven long setae inserted on tergal surfaces. Segment X conical, spines, black arrowhead points to pulvillus. Scale bars: (A) 50 𝜇m; without any projection or sucker. (B–E) 10 𝜇m.

Discussion into tarsomeres, and the abdomen definitely lacks an 11th segment. A prognathous and wedge-shaped head and a distinctly Systematic placement developed pronotal shield very likely belong to the groundplan of Coleopterida, and the presence of this feature sug- As in the case of a recently described immature insect from gests that the larvae described here belong to this clade. The Burmese amber (Beutel et al., 2016), the specimens under con- shape and composition of the legs, the absence of three stout sideration here are obviously holometabolous larvae. External bristles on the ventral side of the coxae, and the lack of an wing buds are completely absent and also compound eyes and 11th abdominal segment with a jumping apparatus formed by ocelli, the tarsus is part of a tibio-tarsus and not subdivided strongly developed bristles exclude it from Strepsiptera. The

Fig. 7. Line drawings of details in recent Ripidiinae. (A, B, G) Blattivorus (Rhyzostylops) inquirendus Silvestri, 1906: (A) head and pronotum dorsally; (B) head and prosternum ventrally; (G) metathoracic leg. (C–F) Ripidius quadriceps Besuchet, 1956: (C) head dorsally; (D) pronotum and mesonotum; (E) prosternum and mesosternum; (F) metathoracic leg. an, antenna; fs, finger-shaped sensilla; md, mandible; mp, maxillary palpus; bs, bristle-like setae; st, stemata; tr, trochanter. (A, B, G) Redrawn from Besuchet, 1956; (C–F) redrawn from Silvestri, 1906. Not to scale. loss of the antennae is a character shared with this order. How- Falin et Engel, which are similar to some extant members of ever, this is apparently a result of convergence, probably linked the tribe Ripidiini, and the genera Paleoripiphorus Perrichot, with endoparasitism (Misof et al., 2014). The characters dis- Nel, et Néraudeau, (French amber) (Batelka et al., 2016b, cussed below indicate that the longipedes larvae belong to the 2018) and Protoripidius Cai, Yin et Huang (Burmese amber) tenebrionoid family Ripiphoridae of the coleopteran suborder of uncertain generic affinities. It will probably be impossible Polyphaga. in the near future to assign the longipedes larvae to one of Within Ripiphoridae, the subfamily Ripidiinae is the most these genera or to another, yet undescribed genus. However, diversified clade. With 22 (or 23) genera, it represents almost the longipedes larvae from New Jersey (Grimaldi et al., 2005) half of 46 currently valid extant and extinct genera (see File provide evidence that Ripidiinae was widely distributed in S1). Moreover, Ripidiinae displays remarkable morphological the Cretaceous period, including also the North American and sexual variability among genera (Lawrence et al., 2010), landmass, far away from the French and Myanmar Cretaceous and its representatives are quite common in the fossil record forests. (Batelka et al., 2016b, 2018). However, of this known diversity, only descriptions of primary larvae of Blattivorus and Ripidius are available for comparison. Both genera differ in chaetotaxy, Interpretation of structural features and differential diagnosis but simultaneously they share many characters unknown in more or less superficially similar larvae of Ripiphorinae, Meloidae 1) Grimaldi et al., (2005: fig. 7) interpreted stiff, bristle-like and Strepsiptera, and also missing in conicocephalate larvae (see setae on the head of their longipedes larvae from New Jersey as later). Considering the fragmentary knowledge of the diversity antennae. Our results reveal that these setae, which are probably of ripidiine immature stages, it is not surprising that longipedes not antennae, are inserted close to the base of the mandibles, larvae show several ‘unexpected’ autapomorphic features in hidden below the clypeolabral region in the frontal half of addition to features shared with Ripidiinae. This includes, the head. Considering Ripiphoridae as a potential candidate for instance, the absence of antennae and eyes, prolonged for a placement of these larvae, we may readily exclude tibio-tarsi, and the presence of prosternoctenidium. Ripidiinae Pelecotominae (albeit known from Burmese amber; Batelka are represented in Cretaceous Burmese amber by the genera et al., 2016b). Even though the larval antennae in Pelecotominae Amberocula Batelka, Engel et Prokop and Cretaceoripidius are also inserted close to the mandibular base, their shape and

© 2018 The Royal Entomological Society, Systematic Entomology, 44, 396–407 404 J. Batelka et al. the general configuration of the body are very different from metanotum, all conicocephalate larvae have a ratio 0.7–0.9:1, the invasive first instars of Ripiphorinae and Ripidiinae, which all Ripidiinae have a ratio 1:1, longipedes larvae have a ratio show the greatest similarity to the Cretaceous longipedes larvae. 1.2–1.4:1, and all Ripiphorinae have a ratio 1.5–2:1. In the In Ripiphorinae and Ripidiinae, the antennae are always placed length of head and thoracic segments, the longipedes larvae are close to the posterior corner of head capsule, which is in contrast thus intermediate between Ripidiinae and Ripiphorinae. How- to the apparent loss of the antenna in the longipedes larvae. ever, in Ripiphorinae, the head is always much longer than the In conicocephalate larvae from Canada and Russia, neither pronotum. In this character, longipedes larvae are more similar antennae nor palpi are described. In Burmese conicocephalate to Blattivorus. larva, there is one visible seta in a similar position to that 5) The presence of two types of setae in longipedes lar- in longipedes larvae, probably erroneously interpreted as an vae, short finger-shaped sensilla and long bristle-like setae, is antenna by Beutel et al. (2016). one of the most important diagnostic characters in compari- The observed condition of the maxillary palpi is also in son to other potentially related parasitic larvae. In first instars contrast to possible relationships to Ripiphorinae, in which of Meloidae, Ripiphorinae, conicocephalate larvae and Strep- these appendages are always much shorter than the antennae, siptera, only bristle-like setae are known. In contrast, two types with a short and cylindrical ultimate palpomere. By contrast, of setae occur in Blattivorus and Ripidius.InBlattivorus,spi- the terminal maxillary palpomere of Blattivorus and Ripidius nis and setis are reported. Spinis are thick setae (sensilla) with (Ripidiinae) is almost identical to the equivalent in longipedes a rounded base, distributed on the head and thoracic segments larvae, albeit in Ripidiinae, in contrast to longipedes larvae, the only, while setis are long bristle-like setae distributed ventrally basal palpomeres are visible. on the head and thorax segments and also on both sides of all 2) The length of the tibio-tarsi in relation to the femora may abdominal segments. In Ripidius, similar structures have been also be relevant for the systematic placement of longipedes described as spinules and soies. The former are thick sensilla larvae. In Ripidius the tibio-tarsi are about 1.5× as long as distributed dorsally on the head and thorax, and ventrally on all femora, and in Blattivorus about 2× as long (Ripidiinae). In Rip- thoracic segments and the first three segments of the abdomen. iphorinae, the tibio-tarsus is always shorter than the respective Soies are bristle-like setae distributed over the entire body sur- femur (0.9× in Macrosiagon ferrugineum;inRipiphorus smithi face both dorsally and ventrally. We interpret the rows of spinis and R. arabiafelix caboverdianus the femora is also described as of Blattivorus and spinules of Ripidius as a possible autapomor- ‘little’ or ‘slightly’ longer than the corresponding tibio-tarsus). phy of Ripidiinae and homologous with rows of finger-shaped In the length of the tibio-tarsus (about 3× as long as femora), sensilla of longipedes larvae (Fig 7A–E). For completeness, we longipedes larvae are more similar to larvae of Ripidiinae than note that a pair of similar sensilla are also present on the proster- to those of Ripiphorinae. In conicocephalate larvae the legs are Macrosiagon Metoecus quite different in their basic configuration and cannot be directly nal plate of and (Ripiphorinae). How- compared with those of Ripiphoridae and longipedes larvae. The ever, their homology with those of Ripidiinae cannot be con- tibiae and tarsal segment(s) are still distinguishable as separate firmed; the function of these rows of sensilla in Ripidiinae is segments, at least in the Canadian and Russian specimens. unknown. Their similarity with sensilla on antennae and max- 3) Another relevant character is the row of extremely small illary palpi of other larvae of Coleoptera (e.g. Pelecotominae; semi-erect spines on the distal half of the tibio-tarsus, which is see Švácha, 1994) suggests that they might serve a chemosen- oriented towards the same side as the pretarsal pad, and one sory function as they search for their hosts (see Palaeoethology spine with the opposite orientation. A similar row of spines later). is present on the tibio-tarsus of Ripidius (Besuchet, 1956: fig. 17) and Blattivorus (Silvestri, 1906: fig. 15), of the same size, span and orientation (see Fig 7F, G). By contrast, the setation is either much more complex in Ripiphorinae (Ripiphorus)or Diagnostic overview absent (Macrosiagon). In Ripidius, two pretarsal claws with a large pad are present, while in Blattivorus two small pretarsal Longipedes larvae share several characteristic features claws with a small pad are reported. In Ripiphorinae, pretarsal with extant Ripidiinae: (i) long, bristle-like terminal segment claws are absent and a large pretarsal inflatable pad (bladder) is of maxillary palpi; (ii) proportions and shape of femora, always present (apomorphy for the subfamily). trochanter and tibio-tarsus; (iii) regular row of semi-erect spines 4) Due to the telescopically connected abdominal segments, on tibio-tarsus; (iv) proportions of head and prothorax (with the length of the abdomen is too variable among specimens to Blattivorus); (v) presence of both finger-shaped sensilla and be measured and compared with other body segments. However, bristle-like setae on head, and thorax; (vi) trochanter devoid of relative ratio measurements can be obtained for the head and seta otherwise present in Pelecotominae and Ripiphorinae (see thoracic segments. The head is as long as the pronotum in conic- later). Longipedes larvae differ from those of Ripidiinae in the ocephalate larvae from Canada and Taimyr, and also in Ripidius. following characteristics: (i) missing pretarsal claws; (ii) more It is distinctly shorter in conicocephalate larva from Myanmar, in prolonged tibio-tarsus; (iii) possible absence of antennae and longipedes larvae and Blattivorus. In all Ripiphorinae, the head stemmata with cornea lenses (eyes in primary larvae of extant is distinctly longer than the pronotum (1.2–1.6×). Considering Ripidiinae are placed laterally, so their visibility in flattened the combined length of head + pronotum versus mesonotum + longipedes larvae is less likely).

A conspicuous and derived feature shared by longipedes larvae basal taxa and in distinctly derived taxa of Ripiphoridae and and the larva described by Beutel et al. (2016) is the pres- Meloidae. Larval trochanters certainly belong to the groundplan ence of an anteroventral prosternoctenidium – in the longi- of Tenebrionoidea, with only some exceptions in Mordellidae pedes larvae, a sinutate prosternal ridge or line densely set with (Beutel & Friedrich, 2005). In contrast, all three hitherto known flattened, prominent spines. This was considered as a ventral conicocephalate larvae are described as lacking or not having cephalic structure in Beutel et al. (2016), probably a misinter- recognizable trochanters. Kathirithamby et al. (2017) stated that pretation corrected here. Ventral cephalic structures overlapping trochanters were absent for their larva from Taimyr; Beutel et al. the prosternum are absent from all known beetle larvae. Com- (2016) noted trochanters (of forelegs) were not recognizable, bined with a pronotum overlapping the head capsule on the dor- e.g. on middle and hind legs for their specimen from Myanmar; sum, this would completely immobilize the head. Moreover, this and in the case of the Canadian larva of Grimaldi et al., (2005) would imply the presence of an extremely widened gula or sub- trochanters are neither mentioned nor depicted. mentum (or hypostomal bridge) with a conspicuous armature, a The new observation presented here shows that, in the case condition unknown in holometabolous larvae. of the longipedes larvae trochanters, although small and incon- Certain characters of longipedes larvae from New Jersey spicuous they are similar to those depicted in Besuchet (1956: (Grimaldi et al., 2005) have to be re-examined and compared fig. 17), even though they are slightly shorter (see Fig. 6A). Itis carefully. In specimens from this locality, the ventral ctenidium conceivable that the border separating the trochanter from the has been considered as a part of the posteroventral pronotal femur might became more and more obsolete as an effect of edge. The serration is described (and illustrated) as very ‘fine’. If size reduction, rendering it difficult or impossible to identify the this suggests homology with the serration on the posteroventral trochanter. A complete reduction in the conicocephalate larvae prosternal edge of the Burmese longipedes larvae, the different described earlier is very unlikely, considering the apparent high size of the teeth may be a diagnostic character on the species movability of the leg. In any case, a careful re-examination of the level. material with optimized techniques is desirable and will probably facilitate a reliable placement.

Microstructures do matter Palaeoethology

A controversial topic is the systematic position of Cretaceous All longipedes larvae from New Jersey and Myanmar are pre- conicocephalate larvae from Myanmar, Canada and Russia. served as syninclusions from three, 15 and 46 specimens per They are attributed either to Strepsiptera (Grimaldi et al., 2005, piece of amber. No eggshells are preserved with them, so these Kathirithamby et al., 2017) or to Ripiphoridae (Beutel et al., clusters do not represent freshly hatched larvae, but rather fully 2016). Arguments for each conclusion will not be repeated developed primary larvae, waiting for their host. Simultane- here, but we will rather direct the attention to one neglected ously, there are no plant fragments to which the larvae may be character, to underline the inherent problems of the systematic associated with [such as, for example, in extant Ripiphorinae placement of these extinct immature insects. In Ripiphoridae, (Batelka & Straka, 2011; Batelka, 2011)]. We may thus con- primary larvae of three subfamilies are known: Pelecotominae clude, that larvae were entrapped in resin when they were wait- (Pelecotoma), Ripidiinae (Ripidius,andBlattivorus) and Rip- ing assembled on a tree producing resin flow. However, by the iphorinae (all included genera). In the first-instar larva of Pele- shape of their head and mandibles, and the absence of antennae cotoma [with a basal position within Ripiphoridae based on and eyes, they differ markedly from larvae of their extant Ripidi- type of larva (Švácha, 1994) and molecular data (Batelka et al., inae relatives. This may indicate that hosts of longipedes larvae 2016a)], the following characteristics of the leg are known: were not roaches, which are the hosts of extant Ripidiini. trochanter unequal, reduced laterally, very long medially, dis- Larvae of extant Ripidiinae are invasive but nonphoretic. They tinctly divided in two, distal portion with a moderately long attack their future host directly (roach nymph), and no other medial seta (Švácha, 1994: fig. 12). In Ripidius, the trochanters organism is used as vector by the first instars (Besuchet, 1956). are described as ‘très petits, à peine plus longs que larges’ Assuming the lack of eyes and mechanoreceptive antennae in (Besuchet, 1956: 100) and in Blattivorus as ‘trochantere bre- the longipedes larvae, the question arises as to how these larvae vissimo’ (Silvestri, 1906: 320) (for trochanters of longipedes were able to find their host. Some kind of cooperation within larvae, see earlier description) (Fig 7F, G). All known larvae larval clusters, known, for example, in invasive and phoretic of Ripiphorinae also have well-developed trochanters with a triungulins of the beetle family Meloidae (Saul-Gershenz & very long seta: Metoecus (Švácha, 1994: fig. 49), Macrosiagon Millar, 2006; Vereecken & Mahé, 2007), is conceivable and (Grandi, 1936: fig. VIII; Chobaut, 1906: 271), and Ripipho- should be a topic for future research on Ripidiinae. rus (Linsley et al., 1952: 294; Batelka & Straka, 2011: 57). Trochanters are also distinguishable in primary larvae of either Meloidae such as basal Eletica (Pinto et al., 1996) or in highly Supporting Information derived triungulins of Stenodera (Bologna et al., 2002). Additional supporting information may be found online in Trochanters are well developed in all extant and fossil par- the Supporting Information section at the end of the article. asitic larvae similar to Cretaceous conicocephalate larvae. Moreover, they are present both in phylogenetically relatively File S1. Generic list of Ripiphoridae.

Acknowledgements Bologna, M.A. & Pinto, J.D. (2001) Phylogenetic studies of the Meloidae (Coleoptera), with emphasis on the evolution of phoresy. JB and JP are much obliged to Arnold Staniczek and Karin Systematic Entomology, 26, 33–72. Wolf-Schwenninger (both Staatliches Museum für Naturkunde, Bologna, M.A., Giulio, A.D. & Pinto, J.D. (2002) Review of the genus Stuttgart, Germany) for loan of the amber piece with syninclu- Stenodera with a description of the first instar larva of S. puncti- collis European Journal of Entomology 99 sion of longipedes larvae from Myanmar under their care. The (Coleoptera: Meloidae). , , 299–313. authors also cordially thank Ondrejˇ Šebesta (Charles University) Chobaut, A. (1906) Communications. Le triongulinide du Macrosiagon for his help with examination setup on the Olympus IX81. tricuspidata Lepech (Col.). 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