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) embed- ded in two pieces of Cretaceous amber from Myanmar are described and interpreted. Using inverted fluorescence and light microscopy for detailed analysis of microstruc- tures, the inclusions were identified as primary larvae of the beetle family Ripiphoridae, subfamily Ripidiinae. While the structure of thoracic and abdominal segments includ- ing appendages corresponds well with the groundplan known in recent members of Ripidiinae, a curved prosternal ridge with prominent spines (each c.5m), 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, Vinicnᡠ7, 128 43 Praha 2, Czech Republic. phylogenetically important details and a subsequent confidential E-mail: [email protected] systematic placement. 396 © 2018 The Royal Entomological Society Cretaceous primary larvae of Ripiphoridae 397 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 fluo- rescence 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