Reanalysis and Experimental Evidence Indicate That the Earliest Trace Fossil of a Winged Insect Was a Surface-Skimming Neopteran

ORIGINAL ARTICLE

doi:10.1111/j.1558-5646.2012.01743.x

REANALYSIS AND EXPERIMENTAL EVIDENCE INDICATE THAT THE EARLIEST TRACE FOSSIL OF A WINGED INSECT WAS A SURFACE-SKIMMING NEOPTERAN

James H. Marden1,2 1Department of Biology, Pennsylvania State University, 208 Mueller Lab, University Park, Pennsylvania 16802 2E-mail: [email protected]

Received: April 13, 2012 Accepted: July 3, 2012

A recent description and analysis of an imprint fossil from the Carboniferous concluded that it was made by a mayfly landing in sediment at the edge of water. Here, I reanalyze that trace fossil and supply experimental evidence regarding wing traces and behavior. The thorax of the trace maker lacked structures characteristic of mayflies, but closely matches a modern neopteran insect family (Taeniopterygidae, Plecoptera) little changed from Early Permian fossils. Edges of the folded wings of live Taeniopteryx leave marks on sediment closely matching marks in the trace fossil. Faint marks lateral to and beyond the reach of meso- and metathoracic legs match the location where wings of surface-skimming Taeniopteryx stoneflies lightly touch the sediment when these insects skim onto wet ground at shorelines. Dimensions of the thorax of the trace indicate relatively weak flight ability compared to fossils from the Early Permian, making doubtful the hypothesis that the trace maker was flight capable. Ultimately, this fossil best fits a scenario in which a neopteran insect skimmed across the surface of water, then folded its wings. Surface skimming as a precursor to the evolution of flight in insects is supported by this fossil evidence of skimming behavior in a Carboniferous insect.

KEY WORDS: Carboniferous, ﬂight evolution, fossil, ichnology, Neoptera, paleoichnology, Pterygotes.

Winged insects (Pterygota) diverged and radiated during the This fossil imprint (University of Kansas Natural History Carboniferous (360–300 mya), yet insect fossils from that pe- Museum, SEMC-F97) was made in saturated, water-rippled allu- riod are sparse and consist primarily of wings or wing fragments vial sediment at the edge of fresh water. Ultimately, these sedi- (Grimaldi and Engel 2005). A remarkably detailed full-body im- ments became fine-grained sandstone of the Wamsutta Formation print fossil of a pterygote insect was recently discovered from (Late Carboniferous, Westphalian B-C; 308–314 mya) of south- the Late Carboniferous (Knecht et al. 2011) and provides rare eastern USA, MA. None of this critique challenges the geological information about morphology, habitat, and behavior, but cor- analysis or interpretation of the habitat. A full description of rect interpretation of the imprint features and its maker’s taxo- the fossil and its setting is contained in the original publication nomic identity are critical. Knecht et al. concluded that the imprint (Knecht et al. 2011). was most likely produced by a lineage that gave rise to mayflies (Ephemeropterida). Here, I critique and reinterpret their data and reconstruction, and provide experimental evidence that the trace Materials and Methods fossil contains marks left by the wings of a neopteran insect. The This reinterpretation is based on published images and text in results change conclusions not only about the taxonomy, but also the original report (Knecht et al. 2011), and a high-resolution the behavior of the trace maker. unpublished photograph of the fossil. No features of the fossil

C 2012 The Author. 1 Evolution JAMES H. MARDEN

were considered beyond what was presented in the original re- particularly their consideration of Plecoptera, which they incor- port. New data were provided by experiments performed using rectly described as “not known or predicted to extend deep into the live Taeniopteryx sp. (possibly T. burksi, nebulosa, or another of Carboniferous.” The earliest fossil stonefly is from the a group of highly similar species) collected as adults from Bald Carboniferous (Bethoux et al. 2011) and a diverse group of fam- Eagle Creek, Centre County, PA in February and March of 2011 ilies are represented in Early Permian fossils (reviewed in Illies and 2012. These stoneflies were used to create shallow impres- 1965), hence indicating radiation prior to the Permian and dis- sions in fine-grained kaolinite sediment, and to film their behavior proving the claim (Grimaldi and Engel 2005) that Plecoptera is skimming across a water surface onto rippled, saturated sediment. not a particularly old order of insects. Knecht et al. referred to Impressions were made by allowing live stoneflies to walk freely “basal” and “primitive” Plecoptera without citing a source, and over wet kaolinite, or by gluing a pin to the dorsal thorax of a used assumptions about basal plecopteran morphology not trace- stonefly and pressing it lightly into kaolinite to the approximate able to a rooted phylogeny or to fossil Plecoptera. By this method, depth of the trace fossil. Photos of stoneflies and of experimental they excluded Plecoptera based on the trace’s contiguous rather impressions were taken with a digital camera attached to a stere- than broadly separated leg bases (coxae), a prothorax not wider omicroscope. Video sequences were filmed at normal speed using than other body segments, and absence of relatively short forelegs. a Sony TRV9 camera and at 240 frames per second using a Casio As I show below, these exclusions of Plecoptera are baseless. Exilim EX-ZR100 camera. Plecoptera fossils from the Early Permian (Illies 1965) in- clude Paleotaeniopteryx (Fig. 1D). Modern members of this family (Taeniopteryx; Taeniopterigidae) have ventral morphology Results strikingly similar to the trace fossil (Fig. 1A, B), closely matching A first step toward taxonomic identification of the SEMC-F97 the overall body shape, posture, position of the leg bases, relative trace maker requires consideration of the head and terminus of breadth of the prothorax, and ventral sclerites of all thorax seg- the abdomen, locations of key features for distinguishing orders ments. Notably, the trace fossil does not possess the ventral thorax of insects. Mayflies and dragonflies (Odonata) have tiny anten- morphology of mayflies (Fig. 1C, showing Siphlonurus, thought nae unlikely to leave a discernable trace, whereas early Neoptera to have the most primitive morphology of modern Ephemeroptera; typically had long, segmented antennae. In this trace fossil, only Matsuda 1956), which have a mesothoracic furcasternum (F2) the ventral rear of the head left an impression, and anterior to the that is large and posterior to the mesothoracic leg bases, and a fu- head is a disturbance obscuring the sediment surface. Regardless, sion of basisternum 3 and furcasternum 3 (B3+F3). All modern live insects tend to hold their antennae above the ground in a po- Ephemeroptera have an enlarged and strongly convex F2 sclerite, sition unlikely to leave marks. Knecht et al. concluded that long the ventral attachment point of the subalar–sternal muscles, di- antennae were absent, but the fossil provides no clear evidence rect wing depressors absent in Neoptera (Kluge 1994). Lack of for either presence or absence of long antennae. ephemeropteran F2 and B3+F3 morphology in the imprint fossil Mayflies typically have three filaments at the terminus of indicates that its maker was not a mayfly, or that the sternal mor- the abdomen whereas insects such as stoneflies (Plecoptera) have phology and arrangement of flight muscles in modern mayflies only two. In the trace fossil, Knecht et al. concluded that “two diverged radically subsequent to the time of this imprint fossil. narrow impressions lie in curved projection from the terminus of Flight ability of all types of insects is a function of the final tapered segment, representing the drag marks of some the mass of the flight muscles relative to total body weight of the terminal structures, either both cerci or one cercus and the (Marden 1987). Flight muscles occupy most of the interior volume median caudal filament.” This unambiguous description of two of the meso and metathorax, and in situations in which it is not impressions extending from the abdomen terminus contrasts with possible to dissect and weigh the body segments, relative dimen- their Figure 2C legend, which claimed that “basal articulations of sions of these thoracic segments and the abdomen provide a metric the three structures are discernible along the margin of the last seg- strongly correlated with flight ability (Srygley and Chai 1990). In ment.” How two impressions in the primary description became the SEMC-F97 trace fossil, the width of the meso and metatho- three basal articulations in the figure legend is not clear, but lack- rax does not exceed that of the abdomen (both 3.8 mm; Knecht ing definitive evidence of three filaments, the abdomen terminus et al. 2011). Even in Taeniopteryx, a marginally flight-capable provides no reason to identify the trace maker as a mayfly. insect (Marden and Kramer 1994), the thorax is broader than Much of the remaining taxonomic interpretation in Knecht the abdomen (Fig. 1A; ratio = 1.26). Across a taxonomically et al. was based on a process of elimination. Paleodictyoptera broad sample of early fossil pterygotes (Early Permian; Table were eliminated by the absence of a ventrally folded rostrum, S1, N = 14), the highest ratio of thorax to abdomen breadth which would have been clearly visible in this well-resolved im- (1.59) occurs in a mayfly, Protereisma, whereas only one species pression. Other eliminations of taxa were less well justified, has a ratio less than one. Modern insects in which the thorax is

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Figure 1. Comparison of thorax ventral morphology in stoneflies and mayflies with the SEMC-F97 trace fossil (Knecht et al. 2011). (A, B) Ventral morphology of an extant neopteran insect (Plecoptera, Taeniopteryx) and the trace fossil (scale bars: A, 5 mm; B, 10 mm). Highlighted are sternal sclerites of Taeniopteryx, which, with minimal changes in size (primarily width) and relative position, match the fossil. Poststernum 1, basisternum 3, and furcasternum 3 are clearly defined in the fossil. (C) Diagram (adapted from Matsuda 1956) of the ventral pterothorax of the mayfly Siphlonurus (Ephemeroptera). Note how the mayfly furcasternum 2 (F2) is enlarged and posterior to the mesothoracic leg bases (L2), and that basisternum 3 and furcasternum 3 are fused (B3+F3). This ventral morphology is a shared derived feature in Ephemeroptera, in which F2 provides the ventral attachment point of the subalar–sternal muscles, direct wing depressors absent in Neoptera (Kluge 1994). (D, E) Forewing of Paleotaeniopteryx (top, scale bar 1 mm; adapted from Illies 1965) and modern Taeniopteryx (lower, scale bar 3 mm; adapted from Bethoux´ 2005). This lineage has undergone little change in wing design over 260 million years. not wider than the abdomen tend to be small and wind-blown (e.g., Thysanoptera), have abdomens swollen with eggs (e.g., temporarily flightless female mantids), or have an abdomen mass reduced by dorsoventral flattening (e.g., Hemiptera, many Coleoptera), large abdominal air sacs, or a narrow waist and terminus (many Hymenoptera). Among insects sharing the general- ized cylindrical body design and habitat of the SEMC-F97 trace fossil, a thorax not wider than the abdomen occurs in flightless Allocapnia stoneflies (Plecoptera) that sail across the surface of water until they encounter shorelines (Fig. 2; Marden and Kramer Figure 2. Side view of an Allocapnia stonefly using its wings to 1995). sail across water. Note the weakly developed thorax, not wider The canonical difference between paleopteran (including than the abdomen. This species has small flight muscles and cannot Ephemeroptera) and neopteran insects is the ability of Neoptera fly (Marden and Kramer 1995). to fold their wings parallel to and just above the long axis and Taeniopteryx; Fig. 3A) or slant downward (e.g., Megaloptera: of their body (a trait independently evolved in the extinct Di- Sialis) along the sides of the body, with the leading edges lightly aphanopterodea; Kukalova-Peck and Brauckmann 1990). In many touching the ground surface when the insect is at rest. Marks Neoptera, the folded wings curve around (e.g., Plecoptera: Leuctra likely to have been left by the edges of wings held in this fashion

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Figure 3. Evidence for marks made by the edges of folded neopteran wings in the SEMC-F97 trace fossil. (A) Ventral view of a live Taeniopteryx stonefly. Arrows show the fore and hind wing leading edges, which sit flush against the ground when the wings are folded. Inset shows close-up of the paired wing edges. (B) Wing edge marks (at arrowheads) beside the abdomen of the SEMC-F97 trace fossil (Knecht et al. 2011). (C, D) Traces of wing edges of Taeniopteryx stoneflies in experimental impressions in kaolinite. (E) Close-up of the two fine parallel marks (at arrowheads) beside the abdomen of the SEMC-F97 trace fossil. Scale bars: A, 5 mm (inset 0.1 mm); B, 10 mm; C, 5 mm; D, 0.2 mm; E, 0.4 mm. are present in the SEMC-F97 trace fossil but were not interpreted the edges of the overlapping fore and hind wings contacted the as such by Knecht et al. The marks in question comprise pairs of sediment surface, strikingly similar to the marks present in the closely spaced lines running parallel to and equidistant from the trace fossil (Fig. 3C, D). Distance between these paired lines was sides of the body (Fig. 3B, E). Knecht et al. attributed these marks 0.1 mm in Taeniopteryx (Fig. 3D) compared to 0.18 mm in the to scratches left by spines projecting from the posterior femora trace fossil (Fig. 3E), consistent with the twofold difference in that dragged as the animal came to rest in wet mud while moving body length. Across experimental replicates, marks left by edges in a posterior-to-anterior direction. However, this path of motion of folded Taeniopteryx wings were variable in their presence, is contradicted by their conclusion that the insect moved laterally, absence, and number at all locations along the body, including as indicated by faint marks beyond the farthest reach of the fully between the meso- and metathoracic legs, caused by variations in extended legs (the linear green arrows in Knecht et al.’s Fig. 3). the lateral tilt of the body, irregularities of the sediment surface, Clearly the animal could not have moved simultaneously in two and the movement and wing posture of individual insects. different directions, nor could these marks have been made by leg Additional faint marks on the sediment surface lateral to the motions after the insect had settled, as such traces would follow right legs of the trace fossil provide additional evidence regarding arcs rather than lines parallel to the body axis. the behavior of the trace maker. As described by Knecht et al., To test the hypothesis that folded neopteran wings can leave these marks are outside the reach of the limb impressions of all marks on the sediment surface, I experimented with live insects. three right legs. Their description however conflicts with their When Taeniopteryx stoneflies walked across a fine wet kaolinite reconstruction (reproduced here in Fig. 5), which shows marks sediment, they left wing edge traces adjacent and parallel to the beyond the reach of only the meso and metathoracic legs, but abdomen (Fig. 4). Similarly, resting Taeniopteryx stoneflies with not the forelegs. These distal marks are considerably shallower folded wings (Fig. 3A), pressed lightly into kaolinite to a depth and less distinct than the deeper and more proximal impressions similar to the SEMC-F97 trace fossil, left parallel lines where clearly made by legs. Given the setting of the trace fossil, these

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the shore, the stoneflies often continued wing-powered skimming across a short distance of saturated sediment, and continued flapping their wings until they were unable to proceed farther, only then folding their wings and beginning to move their legs (see Sup- porting Information Video S1 online). This behavior is a good fit for the setting and events evidenced in the trace fossil. Also clear in this video is that the stoneflies folded their wings in a raised position above their dorsum, with no portion of the wings touching the ground during the wing folding maneuver. Only when the wings were fully folded and parallel to the body axis were they lowered to a point where the wing edges contacted the ground. Hence, no arced traces made by fold wings, rotating from the wing bases, are expected or observed in the trace fossil. Consid- ered at a population level, surface-skimming insects repeatedly arrive at shorelines onto soft sediment, consistently recreating the conditions necessary to create the SEMC-F97 trace fossil.

Figure 4. Wing trace made by a Taeniopteryx stonefly walking on wet kaolinite. Walking direction was from bottom to top. Angled Discussion lateral marks were made by the legs; a broad band was left by the Knecht et al. concluded that the maker of the trace fossil flew and abdomen (right edge at arrow). The line parallel to the abdomen landed at a shoreline location and rejected the hypothesis that the was left by the edges of the folded wings. insect skimmed across the water surface. The only evidence to support the flying hypothesis is absence of a walking track leading to the body impression, but insects commonly disembark from floating objects, jump from rocks or stems above the ground, or skim across water without leaving marks. The body impression in the sediment was not likely to have been caused by impact after flight, because the lightweight bodies of small insects do not dis- place by impact a substantial volume of heavy wet sediment held together by surface tension. A more physically realistic scenario is that the maker of the trace skimmed across the wet sediment Figure 5. Comparison of striations distal to the legs in the SEMC- surface, leaving minimal marks, until it encountered a patch of F97 trace fossil with location of wingtips of a surface skimming sticky, possibly organic-laden mud in which it became temporar- stonefly. Left: Interpretation of Knecht et al. (2011) requiring lat- ily stuck. Struggling and adhesion, as opposed to impact, were eral movement of the entire body to leave striations beyond the most likely the forces responsible for creating the impression. reach of the meso- and metathoracic legs while also moving for- ward to leave the linear marks parallel to the abdomen (i.e., two How the insect extracted itself and departed without leaving an contradictory paths). Right: Dorsal view of a Taeniopteryx stonefly exit track is unclear, but insects commonly respond to obstacles skimming on the surface of water or wet sediment (see Support- or impedance by reaching upward and lifting themselves onto ing Information Video S1 online). Tips of the fore and hind wings overhanging objects. contact the surface at locations consistent with the distal lateral Knecht et al.’s rejection of surface-skimming as a plausible marks in the trace fossil. behavior for creation of the imprint was based on a set of false arguments rather than evidence from the fossil. Surface- distal lateral marks could have been made by beating wings of skimming locomotion by adult aquatic insects was first described an insect like Taeniopteryx as it skimmed across the surface of in Plecoptera (Marden and Kramer 1994), accompanied by water and came ashore onto saturated sediment. A photo of a mechanical, physiological, and morphological data indicating skimming Taeniopteryx superimposed onto Knecht et al.’s recon- that skimming could have been an evolutionary forerunner to struction (Fig. 5) shows that the tips of the fore and hind wings, at powered flight. Almost immediately, the flight-from-skimming maximum downstroke, align with these distal lateral marks. Fur- hypothesis was challenged on the grounds that the phylogenetic thermore, it is easy to recreate this scenario experimentally with placement of skimming indicated evolutionary novelty (Will Taeniopteryx skimming across water onto mud. Upon reaching 1995). However, no other taxa or fossils had yet been examined

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for presence/absence of the trait, and hence a phylogenetic homologous across aquatic insect orders (Grimaldi and Engel analysis was premature at that time. Subsequent studies revealed 2005) refers to filamentous and other types of nonflappable gills that surface-skimming locomotion is widespread across modern that occur in various body locations, but these derived structures Plecoptera families (Marden et al. 2000; Marden and Thomas are irrelevant to the homology of the segmental flapping gills 2003), appears to be an ancestral trait within the order (Thomas et thought to have given rise to wings. al. 2000), and occurs sporadically in modern mayflies (Ruffieux In conclusion, the original description of the SEMC-F97 et al. 1998; Marden et al. 2000) and damselflies (Samways 1996). trace fossil and interpretations in that report do not withstand a Wing structures important for aerial flight became more elaborate critical analysis. Rather than providing evidence of the landing and diverse during the radiation of modern stoneflies (Thomas position of a flying mayfly, this imprint fossil was more likely et al. 2000), consistent with weaker flight in early Plecoptera made by a weakly flying or flight-incapable neopteran insect. and the weakly developed thorax of the SEMC-F97 trace fossil. The setting in wet rippled shoreline sediment, along with a weakly Similarly, wings and thoraces of fossil mayflies (Carboniferous developed thorax and marks where wingtips of a skimmer would and Permian) indicate reduced flight ability compared to modern have repeatedly struck the ground, indicate that the trace maker species (Wootton and Kukalova-Peck 2000). These results agree may have skimmed across water onto wet ground before becoming with a model in which flight originated on water, a setting in stuck and folding its wings into a resting position. Such behaviors which the body weight is supported and only a small amount are readily observable in certain extant insects like Taeniopteryx of thrust is required for locomotion, followed by a transition to and are a predicted transitional stage during the evolution of three- three-dimensional flight requiring a more elaborate flight motor dimensional flight in pterygote insects (Marden and Kramer 1994; and wings. Developmental studies have found support for the Marden 2008). Ultimately, this trace fossil provides evidence that wings-from-gills hypothesis for wing origins (Cohen et al. 1993; surface-skimming locomotion has a deep history in neopteran Averof and Cohen 1997; Franch-Marro et al. 2006), and molec- insects and perhaps insects in general, thereby strengthening the ular phylogenetics indicate that hexapods evolved from aquatic, likelihood that skimming across water was an intermediate stage crustacean ancestors (Regier et al. 2010). Although the proximal in the evolution of flight. antecedents of pterygote insects were fully terrestrial (e.g., Protura, Archaeognatha, Zygentoma), each of these groups may ACKNOWLEDGMENTS have lost gills early in their divergence from an aquatic stem group C. Marone generously donated the kaolinite used for making imprints. that retained gills and an aquatic habit all the way through to the J. Benner shared a high-resolution photograph of the fossil. R. Schilder emergence of pterygotes. Clearly, evidence supporting a possible helped with videos of surface-skimming and he, T. Carlo, and two anony- aquatic and surface-skimming origin for insect flight has grown mous reviewers provided helpful comments on the manuscript. This work was supported by NSF IOS-0950416. considerably since the original publication of the hypothesis, yet inexplicably Knecht et al. cited the premature and unrooted 1995 phylogenetic analysis by Will as authoritative and concluded that LITERATURE CITED “support for the skimming hypothesis is entirely ad hoc.” Averof, M., and S. M. Cohen. 1997. Evolutionary origin of insect wings from Knecht et al. extended their arguments against the skimming ancestral gills. Nature 385:627–630. Bethoux,´ O. 2005. Wing venation pattern of Plecoptera (Insecta: Neoptera). hypothesis by stating there is no evidence for aquatic forms in Illiesia 1:52–81. any life stage of early Odonata, Ephemeroptera, and Plecoptera. Bethoux,´ O., Y. Cui, B. Kondratieff, B. Stark, and D. Ren. 2011. At last, a This is a curious stance because flappable abdominal gills, com- Pennsylvanian stem-stonefly (Plecoptera) discovered. BMC Evol. Biol. monly thought to be serial homologs of wings (Kukalova-Peck 11:248. 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Supporting Information The following supporting information is available for this article:

Table S1. Maximum breadth and body length dimensions of fossil insects from the Early Permian (the Elmo fauna). Video S1. Stoneflies skimming onto shore and continuing to flap while on soft sediment. Video S2. Flappable articulated segmental gills: a shared ancestral trait in pterygote insects.

Supporting Information may be found in the online version of this article.

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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