Life habits, hox genes, and affinities of a 311 million-year-old holometabolan larva
© Haug et al. 2015
Received: 26 March 2015
Accepted: 3 July 2015
Published: 29 September 2015
The Erratum to this article has been published in BMC Evolutionary Biology 2016 16:169
Holometabolous insects are the most diverse, speciose and ubiquitous group of multicellular organisms in terrestrial and freshwater ecosystems. The enormous evolutionary and ecological success of Holometabola has been attributed to their unique postembryonic life phases in which nonreproductive and wingless larvae differ significantly in morphology and life habits from their reproductive and mostly winged adults, separated by a resting stage, the pupa. Little is known of the evolutionary developmental mechanisms that produced the holometabolous larval condition and their Paleozoic origin based on fossils and phylogeny.
We provide a detailed anatomic description of a 311 million-year-old specimen, the oldest known holometabolous larva, from the Mazon Creek deposits of Illinois, U.S.A. The head is ovoidal, downwardly oriented, broadly attached to the anterior thorax, and bears possible simple eyes and antennae with insertions encircled by molting sutures; other sutures are present but often indistinct. Mouthparts are generalized, consisting of five recognizable segments: a clypeo-labral complex, mandibles, possible hypopharynx, a maxilla bearing indistinct palp-like appendages, and labium. Distinctive mandibles are robust, triangular, and dicondylic. The thorax is delineated into three, nonoverlapping regions of distinctive surface texture, each with legs of seven elements, the terminal-most bearing paired claws. The abdomen has ten segments deployed in register with overlapping tergites; the penultimate segment bears a paired, cercus-like structure. The anterior eight segments bear clawless leglets more diminutive than the thoracic legs in length and cross-sectional diameter, and inserted more ventrolaterally than ventrally on the abdominal sidewall.
Srokalarva berthei occurred in an evolutionary developmental context likely responsible for the early macroevolutionary success of holometabolous insects. Srokalarva berthei bore head and prothoracic structures, leglet series on successive abdominal segments – in addition to comparable features on a second taxon eight million-years-younger – that indicates Hox-gene regulation of segmental and appendage patterning among earliest Holometabola. Srokalarva berthei body features suggest a caterpillar-like body plan and head structures indicating herbivory consistent with known, contemporaneous insect feeding damage on seed plants. Taxonomic resolution places Srokalarva berthei as an extinct lineage, apparently possessing features closer to neuropteroid than other holometabolous lineages.
We provide additional documentation and a formal description of Srokalarva berthei morphology in the Additional file 1, where important structural details on a tagma-by-tagma and segment-by-segment basis are listed and described. Relevant details regarding the taxonomic affiliation of this specimen with particular early-appearing lineages of Holometabola also are provided.
The head and mouthparts
The head is less well-preserved than previously thought. In the original reconstruction, head structures [6, 14] were divided into substructures such as cephalic capsule sclerites, antennal articles and palpal segments. Under certain angles of illumination weak subdivisions of these structures are apparent, but other angles of illumination reveal alternative demarcations. Neither surface texture or ornamentation, nor color provides a clear guide for the presence of cephalic sutures (Additional file 1). It is likely that the cephalic capsule indeed was subdivided, yet their exact delimitation remains indeterminate. Fundamental head structures are evident, such as the antennae, clypeus and its surrounding sutures, some primary segments, and the mouthparts and their subelements.
The most prominent mouthpart element is a large, triangular mandible, identifiable by shape and position. The mandible is ill defined and the inner surface apparently has partly collapsed while the outer border has remained intact. Our interpretation of the mandible is a more massive structure than provided in the initial account . The mandible under reflected light from above (Fig. 2a, b) or at other angles of incidence (Fig. 2c, d) would suggest a surface bounded by a proximal border, indicating a less massive structure. However, at different light angles (Fig. 2e, f), the proximal border appears positioned further dorsal and the general shape is more slender. The expanded size of the mandible is corroborated by stereomicroscopic imaging (Fig. 2g, h). Above the dorsal mandible margin is a structure more challenging to interpret. This structure is broad, lobate, well-sclerotized and originally was interpreted as an eye, possibly compound. This structure is prominently upraised (Fig. 2), but with no indication it contains ocular features, and appears to have been partly compressed under the mandible. These features suggest that it is related to the mouthparts; the most plausible interpretation is a hypopharynx. A less likely possibility is that it represents a large, projecting condyle of the opposite mandible. (Although it appears the specimen lacks compound eyes, a cluster of miniscule, circular structures may be stemmata (Fig. 1a–f), but their identity is ambiguous.) The two serial structures that are posterior to the mandible (Fig. 2g, h) likely represent head segmental regions with ventral appendages. Based on structure and position, they are interpreted as the maxilla and labium, as originally described. A suture separating these two, posterior, segmental regions from the rest of the head capsule was not observed.
The thorax and legs
The abdomen and leglets
The abdominal appendages differ in certain aspects from the original interpretation. The abdominal appendages are inserted more dorsad along the abdominal sidewall, or pleurite, than formerly recognized, and are partially covered by the body. This indicates longer appendages than originally reconstructed, but they are somewhat shorter than the thoracic ones. None of these appendages preserve unequivocally a distal claw. As the exact subdivision of these appendages is difficult to discern, there appears to be two broader proximal elements and up to five smaller, distally tapering ones. The abdominal appendages therefore are composed of more elements and are more markedly gracile than the thoracic ones.
The presence of fully developed abdominal leglets in Srokalarva berthei (Fig. 6a), was followed ca. 8 million-years later by more diminutive leglets and the absence of the anteriormost (A1) and posteriormost (A8) leglets in the abdominal series of Metabolarva bella (Fig. 6b). This latter condition indicates that activation of the Ubx/Abd-A/Abd-B repressor genes may have occurred within basalmost holometabolans, even though these genes were latently present in their ancestors . These relatively simple patterns likely were superseded by more complex regulatory networks involving Abdominal-B (abd-B) genes and complicated by a series of activator and repressor factors that bound with regulatory proteins to express or repress appendage development [18, 26]. Evidence for this includes extant basal lineages of Coleoptera and Hymenoptera, which have more complex abdominal leglet development and distribution patterns [27, 28] than those of Srokalarva berthei or Metabolarva bella.
A campodeiform body facies and life habits has been favored as the ancestral endopterygote larva, characterized by an active, predaceous, dorsoventrally flattened larva with forwardly-directed mouthparts. Recently, a hypothesis that the ancestral endopterygote larva was eruciform has gained broader acceptance, typified by a cylindrical, sluggish, herbivorous, caterpillar-like larva with downwardly-directed mouthparts. The morphology of Srokalarva berthei and Metabolarva bella  supports the eruciform hypothesis, although such an attribution may represent an oversimplification [29, 30]. The eight million-year-older Srokalarva berthei has features that hint at a campodeiform habitus, such as limited dorsoventral flattening (Additional file 1: Figure S3) and laterally extended legs (Fig. 4b). Most features, including a caterpillar form, presence of abdominal leglets typically used for anchoring to vegetation, and downwardly directed mouthparts featuring broadly attached, herbivore mandibles , indicate an eruciform larva. Plant damage from an unknown external feeder on contemporaneous medullosan foliage of Macroneuropteris scheuchzeri  would be expected from a Srokalarva berthei larva. Additionally, a legless eruciform larva similar to Srokalarva berthei could have been responsible for creating primitive, elongate pith galls in the frond rachises of marattialean Psaronius ferns a few million years later .
Within Holometabola, and based on the original description, Srokalarva berthei previously has been interpreted as an antliophoran . Yet, the presence of supposed suture lines on the posterior head does not be support such an attribution. By contrast, the comparably long and well-developed abdominal appendages on eight segments do have a distinct comparison among extant larvae: the aquatic larvae of Megaloptera possess comparable appendages. Nevertheless, other aspects of the morphology of Srokalarva berthei are not compatible with a megalopteran identity. For example, the head morphology is rather unspecialized, while megalopterans have a prognathous head with long mandibles and rather short antennae. Still, the presence of eight abdominal appendages may be an autapomorphic specialization of Srokalarva berthei and not necessarily an ancestral feature of Holometabola. The presence of serial abdominal appendages could indicate that Srokalarva berthei is closely related to Megaloptera, but branched from this lineage [9, 10] and evolved a variety of features indicating specialized herbivory typical of a “primitive”, eruciform caterpillar , known from younger deposits .
Based on our morphological description of this specimen, the earliest holometabolous larvae were thick-bodied, cylindrical, inactive, herbivorous, and possessed a downwardly oriented head and mouthparts that are representative of eruciform larvae. This condition differs significantly from the alternative hypothesis of the earliest holometabolous larvae as thin-bodied, flattened, highly active, predatory forms with forwardly-directed head and mouthparts typical of campodeiform larvae.
The developmental biology of early holometabolous larvae of the Late Carboniferous soon involved homeotic gene regulation of abdominal leglets through repression of the Distalless gene by the Abdominal-A and Ultrabithorax genes. Activation of this regulatory mode may have happened during the Late Pennsylvanian, between the demise of Srokalarva berthei and the appearance of Metabolarva bella. This early experimentation in abdominal appendage developmental regulation generated the beginnings of abdominal appendage diversity seen today in the larvae of major holometabolous lineages.
Based on their morphologies, the diet of Srokalarva berthei and Metabolarva bella likely were herbivorous, as external foliage feeders. This inference is consistent with insect feeding damage occurring on a several seed-plant taxa in contemporaneous deposits.
The taxonomic affinities of Srokalarva berthei preferentially lies with the neuropteroid branch rather than the antliophoran branch of the Holometabola. This basal bifurcation of the Holometabola probably predates its separation into the major lineages of today.
The single known specimen is preserved as part and counterpart and required special imaging methods for viewing and documentation. A broad habitus view of the entire specimen was provided by combining separate images into a composite form. Each component image was taken from separate focal planes that subsequently were integrated by CombineZM® from a series of images. These different images then were stitched with Adobe Photoshop® CS 3 software under cross-polarized light with a Canon Rebel T3i camera equipped with a MP-E 65 mm lens and a MT 24 EX Canon Twin Flash. For comparison of the specimen to earlier studies that used standard light-microscopy, fully reflected vertical light as well as directed side light from different angles was used. In order to document specimen relief, two techniques were applied. The first approach was stereo-imaging that employed the Canon Rebel T3i camera. A second procedure involved virtual surface reconstruction resulting from stacks of images recorded on a Zeiss Axiophot microscope attached to a Skopetech DCM 510 camera. Cross-polarized light was provided by multiple, optical-fiber light sources. Virtual surfaces were calculated in Image Analyzer® software.
Our study did not involve the use any human participants or animals.
We thank Finnegan Marsh for configuration of the figures and drafting Figure 6. Marie K. Hörnig (Zoological Institute and Museum, Ernst Moritz Arndt University, Greifswald, Germany) assisted with the imaging of the specimen. Torsten Wappler (Steinmann Institute, Bonn University) provided Figure 5. JTH has been funded by the Alexander von Humboldt Foundation with a Feodor Lynen return fellowship. CH has been supported by the German Academic Exchange Service (DAAD) with a return fellowship. JTH and CH thank Derek E.G. Briggs (Department of Geology and Geophysics, Yale University), Steffen Harzsch (Zoological Institute and Museum, Ernst Moritz Arndt University, Greifswald, Germany) and J. Matthias Starck (Department of Biology II, Ludwig-Maximilians University, Munich, Germany) for their support. We thank all people programming open source software. This is contribution 301 of the Evolution of Terrestrial Ecosystems consortium at the National Museum of Natural History, in Washington, D.C.
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