Haug et al. Zoological Letters(2019) RCH ARTICLEOpen AccessA 100-million-year old predator: a fossilneuropteran larva with unusually elongatedmouthpartsJoachim T. Haug1,2* , Patrick Müller3 and Carolin Haug1,2AbstractBackground: Biological diversity is a hot topic in current research, especially its observed decrease in modern times.Investigations of past ecosystems offer additional insights to help better understand the processes underlyingbiodiversity. The Cretaceous period is of special interest in this context, especially with respect to arthropods. Duringthat period, representatives of many modern lineages appeared for the first time, while representatives of more ancientgroups also co-occurred. At the same time, side branches of radiating groups with ‘experimental morphologies’emerged that seemed to go extinct shortly afterwards. However, larval forms, with their morphological diversity, arelargely neglected in such studies, but may provide important insights into morphological and ecological diversity andits changes in the past.Results: We present here a new fossil insectan larva, a larval lacewing, in Cretaceous amber, exhibiting arather unusual, ‘experimental’ morphology. The specimen possesses extremely large (in relation to body size)mandibulo-maxillary piercing stylets. Additionally, the labial palps are very long and are subdivided intonumerous elements, overall appearing antenniform. In other aspects, the larva resembles many otherneuropteran-type larvae.Conclusions: We provide a comparison that includes quantitative aspects of different types of neuropteranlarvae to emphasise the exceptionality of the new larva, and discuss its possible relationships to knownlineages of Neuroptera; possible interpretations are closer relationships to Dilaridae or Osmylidae. In any case,several of the observed characters must have evolved convergently. With this new find, we expand theknown morphological diversity of neuropterans in the Cretaceous fauna.Keywords: Neuroptera, Lacewings, Burmese amber, Cretaceous, Convergent evolutionBackgroundReconstructing changes in past biodiversity has becomean important field of research. In the content of severemodern-day biodiversity losses, we hope to understand,and possibly influence, modern losses by investigatingcomparable processes in the past [1, 2].The Cretaceous period (145–66 mya) has become akind of “hot spot” time period concerning biodiversity,not only because it ended with a dramatic mass extinction that terminated the era of the large dinosaurs.* Correspondence: [email protected] Maximilians University Munich, Biocenter, Großhaderner Str. 2,82152 Planegg, Martinsried, Germany2GeoBio-Center at LMU, Richard-Wagner-Str. 10, 80333 Munich, GermanyFull list of author information is available at the end of the articleThree factors made the Cretaceous an extremely diverseperiod especially among arthropods, i.e. insects, crustaceans, chelicerates, and their relatives, a dominatinggroup of animals in all ecosystems now and in the past:A.) The appearance and diversification of manylineages with nowadays abundant, well-knownrepresentatives, such as ants, bees, termites, orcrabs, but also of less well-known groups, suchas modern-type slipper lobsters [3–9].B.) The survival of older groups, possessingmorphologies still known from Palaeozoic times(ending ca. 252 mya), but being extinct in laterfaunas. This includes, for example, very early The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (, which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication o/1.0/) applies to the data made available in this article, unless otherwise stated.

Haug et al. Zoological Letters(2019) 5:29offshoots of the evolutionary lineage of mayflies [10]and cockroaches [11].C.) Early side branches of radiating groups with“experimental morphologies” that subsequentlybecame extinct (e.g., Alienoptera: earlyrepresentatives of the lineage towards mantises [12];Tarachoptera: early relatives of caddisflies andbutterflies [13]; Haidomyrmecini: ants distantlyreminiscent of trap-jaw ants [14]).These three factors appear to have led to a very diversefauna in the Cretaceous; very different morphologicaland ecological strategies of different representativeswithin a single group co-occurred within a single fauna[11]. And yet we likely miss larger parts of the period’strue diversity by restricting our view to taxonomic questions, i.e. presence of specific lineages, old ones, newones, side branches etc.What we miss in such an approach is, for example, thediversity of larval forms (for the challenges of the termlarva, see [15]). Larvae are trickier to deal with from thetaxonomic aspect, i.e., to identify them to a narrowersystematic group is often difficult. Yet, especially forHolometabola (such as bees, beetles and butterflies) thelarval phase of their lives appears to have the larger ecological impact, as this phase lasts longer and many individuals do not reach adulthood.In the group Neuroptera, lacewings, spectacular larvalforms are known in modern fauna, such as antlions.Nonetheless, Cretaceous fossils described in recent yearshave demonstrated that 100 million years ago even moreand stranger appearing forms have lived [16–18].Here we report another unusual neuropteran larvafrom 100 million years old Cretaceous Burmese amber(for geological information on Burmese amber, see [19,20]). The larva is challenging to treat in a systematic andtaxonomic context, but exhibits a previously unknownmorphology.Material and methodsMaterialA single larval neuropteran specimen preserved inBurmese amber was investigated (Fig. 1a). The specimenoriginates from the Hukawng Valley, Kachin State,Myanmar and was part of the private collection of one ofthe authors (PM) under the repository number BUB 2943.It is now part of the collections of the Staatliches Museumfür Naturkunde Stuttgart (“Löwentormuseum”) underrepository number SMNS BU-355.The raw amber piece was first cut with a Dremel 3000.Afterwards it was polished with wet sandpaper, first grade200, and then subsequently grade 600, 1000 and 5000.The final polishing was performed with Sidol metal polish.Page 2 of 14DocumentationThe specimen was documented with composite imagingunder different white light conditions, as well as underautofluorescence. The white-light microscopic imageswere recorded with a Keyence VHX-6000 equipped witha 20–2000 objective, either under ring illumination orunder coaxial cross-polarised illumination. Black andwhite background colour was used. To achieve an optimal result, every image was recorded with different exposure times (HDR) [18, 21]. For autofluorescenceimages, a Keyence BZ-9000 was used [22, 23].Each image detail was documented as a stack, with thesingle images of the stack (frames) being recorded in different focal levels in z-axis to overcome limitations indepth of field. The frames of each stack were fused toachieve an entirely sharp image. Several adjacent stackswere recorded in x-y axis to overcome limitations infield of view. All image details were stitched to a finalpanorama image [24, 25].Additionally, based on the stacks the three-dimensionalrelief information was extracted (virtual surface). This information is presented as red-cyan stereo anaglyphs [26].Drawings of the specimen and of comparative materialwere prepared in Adobe Illustrator CS2.MeasurementsDifferent morphological dimensions of the new specimen as well as of different fossil and extant neuropteranlarvae depicted in scientific publications were measured.These measurements include: body length (excludingmandibles), head width, trunk width, mandible length(direct line from proximal joint to distal tip), and labialpalp length (along the outline as it is flexible). From theresulting values ratios were calculated (Table 1), as oftenno scales were available. The ratios were plotted intoscatter plots to illustrate the rough body shape, the relative length of the mandibles, and the relative length ofthe labial palps. The last value (labial palp length) wasonly obtained for groups without lacking (Sisyridae) orvery short (i.e., Myrmeleontiformia) labial palps, approximately corresponding to half of the measuredspecimens.ResultsMorphological descriptionGeneral habitusSmall holometabolan larva, about 2.53 mm long (Figs.1b, c, 2a–c). Body (presumably) organised into 20segments. First body segment (ocular segment) and following five (post-ocular segments 1–5) forming distincthead with sclerotised head capsule. Trunk subdividedinto two functional units. Anterior three trunk segments(post-ocular segments 6–8) sub-similar (thoracic segments) with prominent ventral appendages (thoracic

Haug et al. Zoological Letters(2019) 5:29Page 3 of 14Fig. 1 Overview and details of the new neuropteran larva (SMNS BU-355) with large stylets. a–d. Composite white-light micrograph. a. Overviewimage of the amber piece. b–d. Ring illumination, reflective. b. Overview of specimen in ventral view (white background). c. Overview of specimen indorsal view (black background) d. Close up of head in ventral view (black background). e, f. Composite auto-fluorescence micrographs; dorsal view onhead. e. Native. f. Structures highlighted by colour-markings; stemmata in orange, subdivision of antennae in blue and cyan. g, h. Composite whitelight micrographs. g. Ventral region of head (white background); labial palp subdivision highlighted by colour-markings. h. Posterior trunk (abdomen)under coaxial cross-polarised light, providing more contrast for recognising folds and setae. Colour in background is side effect of polarisation.Abbreviations: 1–22 number of elements; ab abdomen; at antenna (antennula in neutral arthropod terminology); hc head capsule; lp labialpalp (maxillary palp in neutral arthropod terminology); sp. scale-like pattern; sy mandibulo-maxillary stylet; th thoraxappendages). Posterior eleven trunk segments withoutsuch appendages (Figs. 1h, 2g). Body without appendagesabout three times as long as maximum width. Maximumwidth at about 30% along the anterior-posterior axis.Head regionHead capsule of sub-trapezoidal shape in dorsal (or ventral) view, dorso-ventrally flattened (Figs. 1d–g, 2d, e).Anterior edge of head capsule almost straight, about0.46 mm wide, central third slightly drawn out anteriorlyinto a flat triangle. Posterior edge almost straight; onlyabout 60% as wide as anterior edge, about 0.28 mm.Length of head capsule about as long as width of posterior edge. Lateral edges slightly bulging, not straight. Dorsal surface of head capsule covered with scale-likeornament, at least 35 loosely organised rows of suchscale-like ornaments from one lateral edge to the other(Fig. 2d). Slight impression of a Y-shaped moult suture(mainly visible under fluorescence; Figs. 1e, 2d).Ocular segment recognisable by larval eyes (stemmata). Stemmata located on a slight protrusion arisingfar anteriorly laterally from the head capsule (Fig. 1f). Atleast five individual stemmata per side. Stemmata largerthan scale-like ornaments; diameter about as wide asthree scale-like ornaments.Post-ocular segment 1 recognisable by a pair of appendages, the antennae (antennulae in neutral arthropodterminology). Antennae arising from small hump far laterally from the anterior-dorsal region of the head capsule (Fig. 1f). Diameter at the base slightly larger thandiameter of stemma. Antenna long, elongate, about threetimes as long as the length of the head capsule,

Haug et al. Zoological Letters(2019) 5:29Page 4 of 14Table 1 Ratios of measured dimensions of different neuropteran larvae for scatter plots in Fig. 3GroupSpeciesLarvalinstarNew fossil phidaeAscaloptynxfurciger1AscalaphidaeHaploglenius sp.AscalaphidaeLibelloides sp.AscalaphidaePuer lulodesmexicanaAscalaphidaeOccurrence body length /trunk widthhead width /trunk widthmandible length /body lengthlabial palp /body .250.001[27]4extant2.681.060.590.00Ululodes myia S.27 opidae–3Source Fig. ae Coniopteryx sp.3[29]C-173extant3.100.480.070.10Coniopterygidae niopterygidae ilaridaeNallachius extant10.750.330.040.03HemerobiidaeHemerobius rces clara1[38]23extant––0.120.14MantispidaeDitaxis na[40]15extant3.260.490.120.003

Haug et al. Zoological Letters(2019) 5:29Page 5 of 14Table 1 Ratios of measured dimensions of different neuropteran larvae for scatter plots in Fig. 3 aceaNemopteridaeLarvalinstarSource Fig. insourceOccurrence body length /trunk widthhead width /trunk widthmandible length /body lengthlabial palp /body iese dae?Rophalis relicta[42]07.21 tant9.730.550.080.07Nevrorthidae–[42]07.18 s[35]S.26 idae–1[42]07.04 otidae –1Psychopsidae–SisyridaeSisyra dalii2[45]64extant3.520.390.190.00SisyridaeSisyra dalii3[45]67extant2.810.300.280.00SisyridaeSisyra fuscata[35]S.26 u.r.extant2.630.370.380.00SisyridaeSisyra isyra isyra syra syra Gextant3.260.290.340.00subdivided into 22 articles. Proximal article about 1.5times as long as wide (diameter). Article 2 slightly moreslender and significantly longer, about three times aslong as preceding element. Article 3 shorter, about 50%of preceding article, slightly more slender. Article 4 resembling article 3, only very slightly more slender.

Haug et al. Zoological Letters(2019) 5:29Page 6 of 14Fig. 2 Overview and details of the new neuropteran larva (SMNS BU-355) with large stylets (continued). a, b, d, g, composite auto-fluorescencemicrographs; c, e, stereo red-cyan-anaglyphs based on virtual surface reconstruction, colour-inverted; use red-cyan-glasses to view. a, overview ofspecimen in ventral view. b, overview of specimen in dorsal view. c, overview of specimen in dorsal view. d, close-up on surface of head andcervix, dorsal view. e, close-up on head, ventral view. f, composite white-light micrograph with ring illumination, reflective, white background;close-up on thorax with walking appendages; note long tibiae. g, close-up on abdomen; note setae on surface. Abbreviations: cx coxa; fe femur; ta tarsus; ti tibia; tr trochanterArticle 5 to article 21 of sub-similar morphology. Article5 about 50% of length of article 4, following articlesslightly varying in size, in general, decreasing in lengthand also slightly in diameter. Distal article (22) abouttwo times as long as preceding article, distally rounded.Post-ocular segment 2 (intercalary segment) not externally recognisable.Post-ocular segment 3 recognisable by a pair of appendages, the mandibles. Mandibles very long, elongate;about 50% of the main body proper length (Fig. 1b, d).Very gentle S-curvature in dorsal view slightly curvingoutside (laterally) proximally and inside (medially) distally. In most regions, about as wide as two stemmata,wider at the base. Strongly tapering at the tip, providinga sharp, syringe-like impression. A thin line along themandible indicates a not directly observable groove atthe functional ventral side (posterior side; Figs. 1d, 2e).Post-ocular segment 4 recognisable by a pair of appendages, the maxillae (maxillulae in neutral arthropodterminology). Closely associated with the mandibles,forming mandible–maxilla complex, a functional stylet,about 1.20 mm long (Fig. 1d). Maxilla wider at base andthis wider part reaching further distally than that ofmandible; exact length not observable, most likely similar in length to mandible.Post-ocular segment 5 recognisable by a pair ofappendages, the labium, representing a functional coupledpair of originally individual appendages (maxillae or“second” maxillae in neutral arthropod terminology).Proximal region difficult to discern, based on the insertionof the distal parts, elongate, reaching to the anterior edgeof the head. The distal parts, palps (endopods, “telopods”)are prominently developed. They are very elongate, andeach is subdivided into at least 16 elements (Fig. 1g).

Haug et al. Zoological Letters(2019) 5:29Proximal element about as long as wide. Next distal region(counted as element 2) elongate curved, possibly eitheroriginally subdivided into 8–10 elements (based on lengthcomparison) or to be subdivided during ontogeny. Element 3 longer than proximal element, hence slightly longerthan wide. Following elements (4–15) all sub-similar toelement 3. Distal element (16) about as long as fourfurther proximal elements, tapering distally.Trunk regionTransition of head to trunk with a distinct collar-like sclerite, cervix (Fig. 1b, g). Anterior edge slightly wider than posterior edge of head capsule, first widening and thentapering posteriorly, posterior edge about as wide as anterior edge, widest region in the middle. Length about 30% ofwidth of anterior edge. The middle with a more or less distinct abaxial crest. Surface with an indication of scale-likeornament, yet much less distinct than on the head capsule.All trunk segments with distinct dorsal and ventralsurfaces, set off from anterior and posterior structuresby distinct folds. Not distinctly sclerotised into easily detectable tergite and sternite. First three trunk segmentswith sub-similar morphology, forming a functional unit,thorax.Post-ocular segment 6 (trunk segment 1), prothorax,wider than cervix at anterior edge, narrowing towardsthe mid region, but widening further posteriorly. Anterior edge about as wide as maximum width of head capsule, posterior edge wider than head capsule. Lengthabout 50% of width of anterior edge. Surface with scalelike ornament similar to pattern of head capsule, moreapparent towards the lateral. A single seta on each sideanterior-laterally (Fig. 2b, d). Ventrally, close to the posterior edge a pair of prominent walking appendages,foreleg (thoracopod, Fig. 2f).Foreleg composed of five individual elements. Mostproximal element (coxa, most likely basipod in neutralarthropod terminology) ring-like in ventral view, tapering distally. More distal elements (endopod, “telopod”)significantly more slender than coxa, overall tubeshaped. Proximal endopod element (trochanter) short,curved outward, about as long as diameter of coxa,about two times as long as wide. Endopod element 2(femur) more than three times as long as trochanter;diameter similar to that of trochanter. Endopod element3 (tibia) very elongate and even more slender. Slightlylonger than trochanter and femur combined, only abouthalf the diameter of the trochanter; in anterior view verygently curved outwards. Distal endopod element (element 4, tarsus) not further subdivided. Shorter than trochanter, diameter similar to that of tibia. Distallycarrying a pair of hook-shaped claws slightly longer thanthe diameter of the tarsus.Page 7 of 14Post-ocular segment 7, mesothorax, wider than prothorax at anterior edge, widening posteriorly. Lateraledges with three distinct humps each. Lateral regions ofdorsal side covered with scale-like ornament. Close toanterior edge is a row of about eight (estimated) smallerhumps each carrying a seta (Fig. 2b). Closer to posterioredge is a row of about six such humps. Indistinct foldssubdivide the dorsal region of the segment into roughlythree regions. Ventrally, close to the posterior edge is apair of prominent walking appendages, midleg, subsimilar to that of the prothorax (Fig. 2f).Post-ocular segment 8 more difficult to observe, as thetrunk is slightly distorted here. Appears slightly narrower than preceding segment, very slightly taperingposteriorly. Longer in anterior-posterior axis. Observablesurface ornamentation resembling that of preceding segment, including scale-like ornament, lateral and dorsalhumps with setae and folds. Ventrally close to the posterior edge is a pair of prominent walking appendages,hindleg, sub-similar to that of the prothorax, but slightlymore elongated (Fig. 2f).Post-ocular segments 9–19 (trunk segments 4–14)sub-similar, forming a functional unit, abdomen (Figs.1h, 2g; Insecta-type abdomen not comparable to thatof other euarthropods); only 10 segments externallyvisible; most likely segments 10 11 not differentiablefrom each other. Abdomen in anterior region slightlynarrower than posterior edge of thorax, strongly tapering posteriorly, each segment is narrower than thenext anterior segment; abdominal segment 1 is fivetimes as wide as terminal segment (10 11). Abdomen further differentiated. Post-ocular segments 9–15(abdominal segments 1–7) more similar to each other.Ventral region of abdomen largely obscured bybubble.Post-ocular segment 9 (abdominal segment 1) shorterthan metathorax, only about 30% of the length (anteriorposterior length). Segment indistinctly subdivided intothree regions by abaxial folds (Fig. 2b). Each of the threeregions with small humps of which most bear setae. Exactnumber difficult to discern, estimated 12 in anterior region, six in middle region, four in posterior region.Postero-laterally the segment bears a hump on each sidefrom which socketed setae arise (exact number difficult,most likely two longer ones and one shorter one withoutdistinct socket).Post-ocular segment 10 (abdominal segment 2) subsimilar to preceding segment. Due to being slightly narrower fewer humps (approx. 11, 6, 4). In addition to thepostero-lateral hump a smaller less distinct hump anterolaterally (on each side) with two (?) setae without distinctsockets.Post-ocular segment 11 (abdominal segment 3) subsimilar to preceding segment. Due to being slightly

Haug et al. Zoological Letters(2019) 5:29narrower fewer humps (approx. 10, 6, 4). Postero-lateralhump more distinct, almost cone-shaped.Post-ocular segment 12 (abdominal segment 4) subsimilar to preceding segment, due to being slightlynarrower fewer humps (approx. 9, 4, 4).Post-ocular segment 13 (abdominal segment 5) subsimilar to preceding segment, due to being slightlynarrower fewer humps (approx. 8, 4, 2).Post-ocular segment 14 (abdominal segment 6) subsimilar to preceding segment, due to being slightly narrower fewer humps (approx. 7, 2, 2).Post-ocular segment 15 (abdominal segment 7) subsimilar to preceding segment, due to being slightlynarrower fewer humps (approx. 6, 2, 2).Post-ocular segment 16 (abdominal segment 8) withless distinct surface, appearing rough, but no apparenthumps or similar. A single abaxial fold separates an anterior and a posterior region of the segment. Comparably to more anterior segments with two humps on eachside, both almost cone-shaped, with a single socketedseta each.Post-ocular segment 17 (abdominal segment 9) narrower, simple, no humps or folds. Laterally with twolonger socketed setae, and one unsocketed shorter seta.Terminal trunk element (most likely representingconjoined post-ocular segments 18 19, i.e. abdominalsegments 10 11) simple terminally rounded, with fivesetae on each side.Results of scatter plotsConcerning the general body shape, the new larva clusters among many other neuropteran larvae (Fig. 3a).However, concerning relative mandibular length the newlarva clusters above the majority of the investigatedneuropteran larvae, hence having relatively longer mandibles, but there are still other larvae clustering in thePage 8 of 14same area, all of them being first instar larvae (Fig. 3b,Table 1). When comparing the relative length of mandibles and labial palps, the new larva is clearly differentfrom all other investigated neuropteran larvae (also ofthose in which labial palps are not lacking (Sisyridae) orvery short (Myrmeleontiformia)). It does not only haverelatively longer mandibles, but especially much longerlabial palps (Fig. 3c).DiscussionPhylogenetic interpretation: neuropteran relationshipsThe small larva can easily be identified as a neuropteran.The soft-appearing outer cuticle with three pairs ofwalking appendages, distinct head capsule and especiallythe stemmata (few simple eyes) immediately support aningroup position within Holometabola. The prominentmandibles, apparently interconnected with parts of themaxilla, in a far anterior position (prognathous positionof mouthparts), as well as a distinct collar-like connection between head and trunk (cervix) are autapomorphies of Neuroptera [41].While an ingroup position of Neuroptera is easily supported by the observable characters, further-reachingconclusions are, as discussed in the following, more challenging. The ancestral state for the mouthparts of larvaein Neuroptera is a specialised mandible-maxilla complex,with mandibles and maxillae forming a pair of piercingsucking structures or stylets, hence mandibulo-maxillarystylets [41, 46, 47]. To use these effectively, i.e., for piercing possible prey, a certain force is necessary, and furthermore an equal counterforce. Many neuropteranstherefore have curved mandible-maxilla complexes thatact against each other. In this way, force and counterforceare produced with the same mechanism; in fact a comparable mechanical solution is present in the like-wisepiercing-injection type mouthparts of labidognathanFig. 3 Scatter plots of ratios of measured dimensions of different neuropteran larvae (see Table 1). a, head width:trunk width ratio vs. body length:trunkwidth ratio; this plot describes the general body shape. b, mandible length:body length ratio vs. body length:trunk width ratio; this plot illustrates therelative length of the mandibles compared to the body dimensions. c, labial palp length: body length ratio vs. mandible length: body length ratio; this plotshows the relative lengths of mandibles and labial palps; note the eccentric position of the new larva

Haug et al. Zoological Letters(2019) 5:29spider or epimorphan centipedes. Yet, of course, here verydifferent appendages have formed these counteractingmouthparts; the spider chelicerae correspond to theantennae of the neuropteran, the maxillipeds of the centipedes to the first walking appendages. Still this arrangement of counteraction orientation appears very effective.The exact relationships of neuropterans are still a matterof debate [48]; review in [47]. The more traditional viewinterpreted Nevrorthidae (a species-poor group with relicdistribution) as sister group to all other neuropterans. Theremaining neuropterans were often further subdividedinto Hemerobiiformia and Myrmeleontiformia. Alternatively, Myrmeleontiformia was interpreted as being nestedinside Hemerobiiformia, the two branches Sisyridae andOsmylidae branching off before the split of Myrmeleontiformia and the rest of the neuropteran groups. Thesupposed sister group to Neuroptera, i.e., Megaloptera,and also the neuropteran in-groups Nevrorthidae andSisyridae have fully aquatic larvae. Many larvae ofOsmylidae can be considered as semi-aquatic. Based onthis character distribution many authors have reconstructed the evolutionary history of Neuroptera with ancestrally aquatic larvae [42].Yet, other phylogenetic reconstructions have drawn adistinctly different picture [49–51]. Here Coniopterygidae(in older reconstruction an ingroup of Hemerobiiformia)is the sister group to the remaining neuropterans.Nevrothidae, Sisyrid

ferent focal levels in z-axis to overcome limitations in depth of field. The frames of each stack were fused to achieve an entirely sharp image. Several adjacent stacks were recorded in x-y axis to overcome limitations in field of view. All image