How uniform are snakefly larvae?
Extant snakefly larvae are largely rather uniform, and many fossil forms greatly resemble such modern-day forms. Nonetheless, some prior observations reported Cretaceous larvae that deviated at least in certain aspects from this seemingly stereotypical morphology. Specimen 75 described by Engel (2002) is certainly unusual compared to modern forms, possessing a rather slender, elongate head not seen in modern larvae, and quite prominent antennae, while in modern forms, the antennae are usually rather short. Another specimen from Burmese amber like specimen 75 is specimen 87, reported by Haug et al. (2020a), which possesses even more prominent and larger antennae. Haug et al. (2020a) established various metrics for characterizing the exceptional nature of these two individuals, more conclusively demonstrating that such Cretaceous morphologies lacked analogs among modern snakeflies.
Our significantly expanded data set further emphasizes the notion that during the Cretaceous snakefly larvae occupied a greatly expanded range of morphospace. These Cretaceous taxa exhibited a range of morphological diversity (≈ disparity) that has subsequently contracted, likely in connection with the loss of species diversity and the extinction of unique forms, perhaps also linked to biologies and life histories no longer represented among raphidiopteran diversity today. Interestingly, although the aforementioned specimens 75 and 87 are here further demonstrated to be clearly set apart from modern-day forms (Engel 2002; Haug et al. 2020a), they are only a subset of an even wider morphological variety. Among the new larvae reported there are those that are even more exceptional, significantly expanding the overall morphospace occupied (Figs. 29c, d, 30c, d).
Changes over time
Our results demonstrate that larval forms in the Cretaceous exhibit a much larger morphological diversity (≈ disparity) relative to more modern forms. While this is most apparent in the length of the antennae and the head shape, it is also apparent in other factors. However, some of the variation may be explained by different preservational aspects of the thorax and certainly the abdomen in several of the fossil larvae. Nonetheless, the analysis restricted to the more heavily sclerotised elements, i.e., head capsule and antennae, provides a rather similar perspective.
Specimens from the Eocene show a much more restricted morphospace, while remaining slightly greater than that of modern larvae. This is especially interesting as the sub-data-set for both fossil groups is significantly smaller than the sub-data-set of the modern forms, suggesting that sample-size corrections are not necessary (refer to Haug et al. 2020b).
It is apparent that over evolutionary time the overall body shape of snakefly larvae did not change significantly, i.e., these larvae seem to have always been quite slender. By contrast, the variability in relative proportions of the head shape and antennae have changed significantly during the last 100 million years. Indeed, the considerable morphological diversity observed during the Cretaceous decreased significantly by at least the Eocene and then continued to retract through to the present day. It is possible that this is reflective of differing sensory requirements and different modes of predation among these larvae. Certainly, some developmental requirements changed considerably during this expanse of time as all of the known fossil snakefly larvae occur in palaeo-climates inconsistent with the necessary exposure to extended freezing to bring on pupation that is otherwise so characteristic of our modern fauna (Gruppe et al. 2020). Accordingly, the expectation that there were similar or even related changes in diet, predation specialization and behaviours, and ecology seems well founded. In fact, the loss of diversity was perhaps brought about by the loss of species as former groups, such as Baissopteridae and Mesoraphidiidae, were eventually replaced by a restricted group among this diversity, namely the group Neoraphidioptera (Liu et al. 2014; Engel et al. 2018). This restriction would have been both in terms of phylogenetic diversity as well as morphological diversity, and perhaps also in ecology, behaviour, and physiology.
Evolutionary mechanisms
Evolution acts via subtle changes and processes. Heterochrony describes a set of slight evolutionary changes to developmental timing. We might therefore assume that heterochrony might have played a role in forming the morphology of snakefly larvae. Haug et al. (2020a) suggested that the large antennae in specimen 87 might represent a specialisation and that small antennae (as seen in modern-day larvae) might be ancestral, but the significantly larger data set available with this study suggests otherwise. In fact, comparing the ratios for relative antennal length among Cretaceous, Eocene, and extant larvae indicates a clear trend to less variability, but also progressively shorter antennae. This suggests that ancestrally the antennae of snakefly larvae were longer and that the shorter antennae seen in modern larvae is the derived condition, at least relative to the raphidiomorphans. Naturally, this implies that the phylogenetic distribution for this trait is that a number of early consecutive-diverging snakefly lineages had longer larval antennae relative to more derived and younger monophyletic groups. Unfortunately, we do not know the phylogenetic relationships among the fossils included in our study, not even allowing identification to higher groups (e.g., family). Moreover, larvae remain unknown for the diversity of Jurassic species, and particularly those of the extinct group Priscaenigmatomorpha (Engel 2002), and these could alter our conclusions once such material is discovered and put into a proper phylogenetic context with the present material. Nonetheless, even in the absence of a phylogeny based on larval characters, the general distribution of antennal lengths and their changes through time remain consistent with the pattern and character polarity outlined herein.
Naturally, shorter appendages can be interpreted as paedomorphic, i.e., retained earlier developmental morphologies in later stadia. This may be achieved by the later onset of development (post-displacement), a shorter developmental time (progenesis), or simply slower development (neoteny) of the antennae (Webster and Zelditch 2005). With the available data, we cannot differentiate between these possibilities, and reconstructed larval sequences would be necessary for making such a differentiation. For the moment, it seems likely that some of the fossil larvae may represent different stages of a single species (e.g., Figs. 16 and 21d, e), and therefore there should be the potential for exploring such an analysis in the future. In particular, several specimens trapped in a single piece of amber should have a greater likelihood of being conspecific (e.g., Figs. 18, 19, 25, 26). In addition, fossil larvae with a more extreme morphology may be good candidates for identifying conspecifics in other pieces (e.g., Figs. 16 and 21d, e). For example, it is tantalizing to consider those larvae with elongate heads (e.g., specimen 75) with species such as Rhynchobaissoptera hui (Lu et al. 2020), although as we note below there is some degree of disconnect in certain lineages between larval head shape and that of the corresponding adult.
It would be fascinating and perhaps revealing to expand the comparison to include pupae and adults. It may be possible that this type of heterochronic shift of the antenna only affects larval stages, while the morphology of the adult remains unchanged (cf. Haug et al. 2016; Haug 2020a, b). Modern-day pupae have clearly longer antennae than modern-day larvae, but the antennae are differently structured, with many small antennomeres, while the long antennae of the fossil larvae have only a few but often long antennomeres.
Likewise, the varying head shapes of the fossil larvae would be interesting to set in a framework including adults. Modern pupae apparently have already a longer head and a relatively shorter thorax (the latter is also observed in the retraction of the thorax when forming the pre-pupa; Fig. 2b, c; see also Haug et al. 2020c for a comparable process in lacewings). It could follow then that the higher variability in fossil larvae is the result of differential timing of the point from which the elongation of the head capsule begins. Alternatively, it is possible that in the past snakefly adults had a similarly greater head-shape variability, a matter which should be addressed in future analyses.
It has not escaped our notice that head shape, where we observe so much variation and changes in larvae, is correlated in adults with differences between the two major modern lineages (families) of Raphidioptera (Neoraphidioptera). Among the traits that differentiate the two lineages is an overall difference in the shape of the head capsule posteriorly: those of Inocellidae are rather robust and broad posteriorly, with broadly rounded posterolateral angles, while those of Raphidiidae taper more gently caudally. In the Eocene fauna, there is at least one group that intermingles features of these two groups in terms of such head shape, specifically the group Electrinocelliinae (Engel 1995). Despite these differences, the head capsules of larvae of these two groups are more similar, and all are quite robust and broad posteriorly, at least relative to that observed in adults of Raphidiidae. This may be an indication that the broader head morphology (in both adults and larvae) is plesiomorphic, at least for this group, with that observed in Raphidiidae representing a specialized novel character state. Alternatively, there may be some degree of decoupling between larval and adult head morphology; perhaps not surprising given the dramatic developmental rearrangement between the larval and pupal/adult stadia. Nonetheless, such matters could be more fully explored in detail by the potential future expanded analyses outlined here.
It would also be beneficial to discover fossil snakefly pupae for expanding the comparisons initiated herein. As noted, snakefly pupae are quite active with considerable shared resemblance to the adult. In many other holometabolan lineages, the pupa is quiescent and differs more starkly from the adult (e.g., Beutel et al. 2014; Saltin et al. 2016). It remains unclear whether these two aspects are ancestral or derived for the broader group. Fossil pupae could provide an important insight into this aspect of neuropteridan, and perhaps greater holometabolan, evolution and would certainly allow us to enrich our comparisons.
While our data are still limited for answering such evolutionary or palaeo-evo-devo questions and despite the difficulties of placing fossil larvae in robust phylogenetic estimates (but see Badano et al. 2018), we can already extract some signal indicating paedomorphosis in the evolution of modern-day snakefly larvae.
Snakefly diversity
It seems generally accepted that snakeflies were more diverse in the past (Engel 1995: 187; Aspöck 2002: 35, 36; Aspöck and Aspöck 2007: 478–480, 2009: 53; Liu et al. 2016; Engel et al. 2018). The taxonomic view on this aspect is difficult. In few cases does pure species counting as a basis for taxonomic comparison between time periods provide a clear view. The group Dinosauria is a notable example. While most people believe their maximal diversity ended at the Cretaceous-Paleogene transition, the more than 10,000 extant bird species clearly outnumber formally described fossil species by a factor of 10, suggesting that dinosaurian diversity (as represented by their ingroup Aves) experienced its greatest diversification during the Cenozoic! Only a few lineages prominently show a large number of fossil species against a significantly smaller extant number (notable examples among insects include Mastotermitidae, Megalyridae, Scolebythidae: Grimaldi and Engel 2005). Given that there are about 248 extant and only 100 extinct species of Raphidioptera (Liu et al. 2016; Engel et al. 2018; Lu et al. 2020), the raw number of species does not alone support the conclusion of declining diversity. Furthermore, the documentation of declining diversity is not a mere comparison of the modern fauna versus the cumulative total of all species that existed prior to the present (although most often researchers rely solely on a lumping of all past faunas). Instead, it is more properly an assertion that in progressively younger faunas each has fewer and fewer species, or at least an overall trend in such a direction. From this perspective, the picture is even murkier as all of the known fossil faunas for Raphidioptera are a mere fraction of the modern diversity, and there are no sufficient data to establish such a trend.
Accordingly, where is the taxonomic signal for a higher diversity in the past? In recent history, neuropterists have rightly considered the number of extinct species described to be a mere fraction of the diversity during any given moment in the past and therefore concluded that, when lumping all palaeofaunas into one, the 100 or so fossil species are reflective of a far greater total diversity. Under such a scenario, if the 100 represented even a tenth of the actual fauna over that period, then certainly the numbers dwarf that of the modern fauna. Nonetheless, data are insufficient to apply such an extrapolation to any given palaeofauna as the amount of material is scant and conclusions on high diversity in any one fauna become increasingly speculative. Thus, aside from such gross estimations, higher ranks have been used as rough proxies for diversity. Extant snakeflies have been classified into two higher groups, traditionally recognized as families, the fossil diversity into six, and this gives an impression of more diversity (Engel 1995, 2002; Liu et al. 2016). Naturally, higher groups are to some degree subjective and reflective of the degree to which the individual taxonomist circumscribes the observed diversity. Two independent taxonomists recognizing the same species and monophyletic groups could arrive at radically different numbers of higher taxa solely based on how broadly or narrowly each circumscribed the groups. Each opinion is, ideally, founded in valid morphological observations among the living and fossil diversity, and ideally organized within a phylogenetic framework. Nonetheless, the raw numbers are somewhat artificial. For example, if each of the groups now ranked as subfamilies of Neoraphidioptera was elevated to familial rank (resulting in Electrinocelliidae, Succinoraphidiidae, Raphidiidae, and Inocelliidae), then right away the apparent change in diversity has diminished when comparing the Cenozoic and Mesozoic raphidiopteran faunas, and yet the morphological and phylogenetic observations remain unchanged. This would become even more dramatic if in-groups in Raphidiidae and Inocelliidae were similarly accorded such a rank. If a higher group would be identified as paraphyletic, then the validity of the distinction would only be further eroded in either direction in terms of changing diversity. Ranks are a tool for orientation in a greater phylogenetic outline and serve only as the most coarse and imprecise of qualitative proxies for diversity.
The approach presented here provides an alternative proxy, and although it has its own conceptual limitations, it at least offers some quantitative framework in which to observe changes in the absence of absolute (or even raw) species numbers from each time period. If morphological diversity is a proxy for species diversity in Raphidioptera, then the trend quantified herein is consistent with the traditional hypothesis of increased past diversity and that modern snakeflies are relict. Of course, morphological diversity outlining a large morphospace may be achieved by a few isolated and extremely divergent species rather than a greater number and variety (diversity) of species. Nonetheless, it is a more quantifiable and direct proxy for diversity than categorial ranks.
Our analysis permits us a view into larval diversity. Naturally, this poses some challenges as fossil larvae most often cannot be reliably identified to species or associated with species previously circumscribed on the basis of adults (but see a rare case in Batelka et al. 2021). Yet, in many holometabolans, the larvae represent the longest portion of the life cycle and therefore by some measures the most critical for avoiding harm and obtaining sustenance. Therefore the approach presented here may support earlier, taxonomy-based findings, but perhaps provides a more reliable perspective concerning the overall ecological role of the animals involved.
Quite intuitively, for a group assumed to have declining diversity, we found a trend toward declining morphological disparity. That is the opposite of that observed for ants where Cretaceous ants were less diverse and followed by significant diversification, and correspondingly exhibit a narrower morphospace relative to their more diverse modern counterparts (Barden and Grimaldi 2016). Nonetheless, in ants the Cretaceous morphospace overlaps considerably with modern ant morphospace (occupying about 10% of modern ant space). In the present case of snakefly larvae, the extant morphospace (PC1 and PC2) is only 23% of the size of the Cretaceous morphospace. The overlap is even smaller, only 7% of the Cretaceous morphospace is shared with modern snakefly larvae morphospace. Thus, in ants as diversity increased, more and more morphospace was explored, while in snakeflies, a once-broad swath of Cretaceous morphospace was eroded away as specific groups or specialized niches occupying portions of snakefly morphospace became extinct.
A broader view on Neuropterida
The snakeflies are sister to the monophyletic group Eidoneuroptera (= Megaloptera + Neuroptera) (Engel et al. 2018; Winterton et al. 2018). Megaloptera comprises the dobsonflies, fishflies, and alderflies, the larval forms of which are all aquatic predators. The fossil record of these larvae is quite scarce (Baranov et al. 2022), not easily facilitating a comparison of larval diversity or disparity. Neuroptera includes many different types of lacewings, and lacewing larvae are better represented in the fossil record (Pérez-de la Fuente et al. 2020). From a qualitative perspective, it is quite clear that in the past, there were many larval types of lacewings that are no longer present today (Pérez-de la Fuente et al. 2012b, 2016; Wang et al. 2016; Liu et al. 2016, 2018; Badano et al. 2018; Haug et al. 2019a, b, c), but at the same time, many quite modern forms also occurred (e.g., Wang et al. 2016; Haug et al. 2018; Pérez-de la Fuente et al. 2020), collectively providing for a larger overall diversity (including morphological diversity; see discussion in Haug et al. 2019b). Quantitative aspects also demonstrated that some larvae were already quite modern (Herrera-Flórez et al. 2020), while others have a morphology unparalleled in the modern fauna (Haug et al. 2019b, 2020b, c, d). Applying morphometric approaches (although based on outlines instead of measurements) revealed that in two lineages of Neuroptera, Psychopsidae (silky lacewings) and Nymphidae (split-footed lacewings), the larval diversity decreased significantly after the Cretaceous (Haug et al. 2020b, 2022). For long-necked antlions, i.e., the larvae of Crocinae (thread-winged lacewings), it remains partly unclear owing to challenges with preservation whether the diversity actually decreased; nonetheless, we also see a loss of certain larval morphologies (Haug et al. 2021).
The data obtained here for snakefly larvae reveal a similar trend, specifically that diversity of larval forms was significantly greater 100 million years ago. The loss of morphological diversity also indicates a loss of ecological diversity or ecological functions. As (almost) all modern larvae of Neuropterida (Raphidioptera, Megaloptera, and Neuroptera) are predators, we can conclude that the same was true for their many fossil species. Such a conclusion is well founded given that many of the predatory tools used by modern larvae are present in the fossil forms (e.g., scythe-like mandibles, piercing mandibles, etc.). A loss of diversity would mean a loss of predators, and while many were certainly generalist predators like many modern species, others were assuredly specialised for a particular type of prey, as is also observed in their modern neuropteridan counterparts. Thus, some aspects of the overall loss might reflect co-extinction, whereby specific types of preys went extinct and resulted in the loss of their specialist predators. For the moment, it remains unknown what preys were victimized by fossil snakeflies, but these could have included animals of the groups Acari (mites), Auchenorrhyncha (e.g., treehoppers, leafhoppers), Sternorrhyncha (e.g., whiteflies, greenflies, scale insects), and other preys often taken by modern snakeflies and certainly well represented in many of the palaeofaunas from which our snakefly material originated.
It has been asserted that Neuropterida is an ancient and relict group of Holometabola. Their larvae appear to attest to such an evolutionary pattern, at least during the last 100 million years.