Abstract
Biologists have reported on the chemical defences and the phenetic similarity of net-winged beetles (Coleoptera: Lycidae) and their co-mimics. Nevertheless, our knowledge has remained fragmental, and the evolution of mimetic patterns has not been studied in the phylogenetic context. We illustrate the general appearance of ~ 600 lycid species and ~ 200 co-mimics and their distribution. Further, we assemble the phylogeny using the transcriptomic backbone and ~ 570 species. Using phylogenetic information, we closely scrutinise the relationships among aposematically coloured species, the worldwide diversity, and the distribution of aposematic patterns. The emitted visual signals differ in conspicuousness. The uniform coloured dorsum is ancestral and was followed by the evolution of bicoloured forms. The mottled patterns, i.e. fasciate, striate, punctate, and reticulate, originated later in the course of evolution. The highest number of sympatrically occurring patterns was recovered in New Guinea and the Andean mountain ecosystems (the areas of the highest abundance), and in continental South East Asia (an area of moderate abundance but high in phylogenetic diversity). Consequently, a large number of co-existing aposematic patterns in a single region and/or locality is the rule, in contrast with the theoretical prediction, and predators do not face a simple model-like choice but cope with complex mimetic communities. Lycids display an ancestral aposematic signal even though they sympatrically occur with differently coloured unprofitable relatives. We show that the highly conspicuous patterns evolve within communities predominantly formed by less conspicuous Müllerian mimics and, and often only a single species displays a novel pattern. Our work is a forerunner to the detailed research into the aposematic signalling of net-winged beetles.
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Introduction
Defence mechanisms are inextricably linked to biological fitness and among them Müllerian mimicry is one common anti-predatory strategy1,2,3. The evolution of mimicry has been studied from various perspectives, but the considered numbers of interacting species and patterns under consideration have usually been low4,5,6. In order to investigate the evolution of mimicry from a long-term perspective, while simultaneously studying a high number of interacting species, we need to focus on ancient and highly diversified lineages7,8. Unlike the well-studied Heliconius system, some lineages of aposematically coloured insects contain up to several hundred species in a single geographic region and up to a hundred species in a single locality9,10. This means that unpalatable (or more widely unprofitable) aposematically coloured species dynamically interact within complex communities containing multiple patterns, imperfect mimics, as well as Müllerian and Batesian co-mimics. Here, we study the world fauna of net-winged beetles (Coleoptera: Lycidae), an elateroid family with pronounced chemical protection, diverse aposematic colouration, and ~ 4200 species9,11.
Net-winged beetles are a widely distributed family, related to soldier beetles, click beetles, and lampyroid families11,12. Most adults are inactive and sit on leaves under the forest canopy (Figs. 1, 2, 3). Individuals do not activate even when they register a movement very close to their body or when they are gently touched. Only a minority of lycids visit flowers in open-air spaces (Figs. 2P, 3S). The unpalatable lycids use aposematism as a defence strategy and form extensive geographically restricted mimicry rings13,14,15,16,17,18,19,20,21 (Figs. S1–S922). Their anti-predator strategy relies on chemical protection15,22,23. The adults produce a pungent odour as a response to handling, and bitter haemolymph is discharged from ruptures in the elytra and intersegmental membranes in legs and antennae24. Similar bleeding has been reported in related fireflies25,22 (Fig. 4G–I). The jum** spiders have experimentally proved to be effective predators in the case of fireflies55,56, and it is possible that they also prey on net-winged beetles.
The model of Müllerian mimicry is based on the benefit obtained by a protected prey which signals its unpalatability and shares a predation load with its co-mimics1,2,3. Here, predation load is defined as the proportion of individuals removed from co-mimic populations by naïve predators and because of their errors. A single, effective signal should be shared in the order to maximize the benefits of mimicry. To study the processes of signal evolution and effectiveness, we must answer several questions. First, the shared aposematic signal indicates the presence of a selective force, and only rejects a similarity due to common ancestry when it is displayed by unrelated species. Second, we need to evaluate the effectiveness of signalling (the internal and external contrasts and the signalling power of various components of the signal and eventually the various perceptions of the signal by individual predators57,58,59,60,61). Furthermore, the evolution of the signal might be affected by constraints which prevent high levels of similarity (imperfect mimics, relaxed selection62,63,64,65). We also need to consider how many aposematic patterns are present in a region. Multiple patterns in a single place, often within a single aggregation, have been reported9,41,66.
As described above, Lycids are a big group of putatively aposematic, conspicuously signalling species with a world-wide distribution, potentially taking part to several Müllerian mimicry rings. Investigating the diversity of aposematic signalling in ~ 4000 lycids on the scale of zoogeographic regions and over the period of the last hundred million years can provide invaluable information about the origin of aposematism and the evolution of aposematic signals. This work is a starting point for many future experiments focussing on specific local assemblages, but here we focus in discussing the differences between patterns, as well as their phylogenetic origins, i.e. how old are the principal patterns and whether highly conspicuous patterns rapidly evolved from their cryptic relatives or gradually evolved from the aposematically coloured relatives. These factors are also of interest to ecosystem-based studies because phylogenetic structure and signalling plasticity have consequences for the evolution of mimicry in close interactions within each locality.
We assume that the complexity of local rings might partly depend on the regional signalling diversity and on the intensity of migration between neighbouring ecosystems. We thus quantify the diversity of lycid beetle coloration and patterning, focusing on their signal strength in the spectrum visible to humans, relationships, and distribution. Our main aims are to (1) describe colour pattern diversity of lycid beetles; (2) investigate their distribution and evolutionary origins (timing, order of diversification); (3) investigate how alpha-diversity, phylogenetic diversity, and abundance correlate with the number of aposematic patterns occurring in a geographic region.
Methods
Field and museum research
Field observations and voucher sampling were conducted by the majority of the authors from 1989 to 2019 in all zoogeographical regions and additional material was provided by colleagues. The studied specimens are deposited in the authors’ Voucher Collection. The total catching effort cannot be standardized as the number of collected individuals depends on the duration of sampling, the season, weather conditions, and habitat conditions. Therefore, the regional abundance is categorized as follows: (a) low (< 100 individuals collected by a collector and a month, 1–5 species in a place for a fraction of a year); (b) medium (100–300 individual/month; 6–30 species present in ecosystems for about a half of the year), high (> 300 individuals/month, present whole year in up to 100 species in a place). Further data on aposematic patterns were obtained from museum collections (Supplements).
Colour evaluation
Two sets of photographs were used for colour evaluation and estimation of the contrast in the colour spectrum visible to humans. The photographs displaying net-winged beetles in their natural environment were taken by the authors (except Fig. 2L provided by M. J. Eising) using digital cameras under natural light conditions and with automatic colour balance. These photographs are not calibrated. Therefore, their evaluation is limited to the relative contrast within a single snapshot. The further set of photographs consists of calibrated photographs of selected representatives for each aposematic pattern and three plant leaves representing backgrounds (the upper and bottom sides of each leaf). Specimens and leaves were illuminated with three Solux lightbulbs (Tailored Lighting Inc., Rochester, NY) with a colour temperature 4700 K and photographed with a Canon EOS M6 camera equipped with a Canon zoom lens 18–150 mm f/3.5–6.3 IS STM. Three plants were arbitrarily selected as background types for net-winged beetles in order to achieve a range of contrasts: Ficus lyrata Warb. (medium thickness, green upper side, light coloured bottom side), Hoya carnosa (Linnaeus filius) Brown (dark coloured, glossy upper side), and Yucca guatemalensis Baker (light-green leaf).
Images were converted from a Canon raw format (CR2) to a 48-bit tiff with dcraw program (https://www.dechifro.org/dcraw/) in linear intensity scale, calibrated with a IT8.7 calibration target (R110112, www.coloraid.de) using a CoCa program with implemented Argyll Colour Management System (www.argyllcms.com), and finally converted to 24-bit sRGB IEC61966-2.1 colour space using perceptual rendering intent. The colour space characteristics of the bright and dark coloured elytral areas were quantified in the CIE La*b* absolute colour space (International Commission on Illumination, CIE 1976). i.e., the colour spectrum visible to humans. The colour samples were taken five times from an area of 31 × 31 pixels at various points of measured body parts in Photoshop 13.0. The average colour values were computed and the colour distance DeltaE La*b* CIE 76 was counted as the Euclidian distance between colour positions in the 3D CIE La*b* colour space. The processing of photographs follows the methods used by the authors of an earlier study41. The DeltaE distances were counted between beetle dorsum and background (both, bright and dark coloured body parts versus each of the six leaf backgrounds, i.e. the upper and bottom side of each leaf, the external contrast is further designated as EC-DeltaE/Upper, EC-DeltaE/Bottom) and distance between colours displayed on the dorsum, i.e. internal contrast, is designated as IC-DeltaE67.
Additionally, the collection and voucher specimens were photographed so we could document the diversity of aposematic signalling, the variability of aposematic patterns, and intraspecific variability. These illustrations were taken using a Canon EOS 700D digital camera attached to an Olympus SZX-16 binocular microscope, using two StoneMaster LED light panels (temperature 5700 K) and a white reflective panel. The white balance was custom set with a white target panel. The photographs were taken at various focus levels and the final photographs were assembled using Helicon Focus v.7.6.1.
We mapped the geographic distribution of aposematic patterns through the examination of sequenced individuals, and we additionally, evaluated specimens in collections and searched the relevant literature. Nineteen categories were used for descriptions of patterns; aberrant patterns were described individually as non-categorized. We considered the uniform colouration displayed on the dorsal side of the body, the different colours displayed on the pronotum and the elytra, and a group of patterns with two colours displayed on the elytra. Additional patterns include striate, punctate, tricoloured, and reticulate types, and several unique patterns, e.g. green, green/black, black/amethyst; see Figs. S1–S922, Table S1). Further, we noted the morphological modifications of elytra connected with the strength of the signal—the posteriorly widened elytra and globuliform elytra (Figs. 2L, 3A).
Molecular data, phylogenetic analyses, and zoogeography
The phylogenetic investigation is based on earlier published cox1, nadh5, and rrnL mtDNA, SSU and d2 loop of LSU rRNA, and the transcriptomic and genomic data11,24,68. No data were specifically produced for this. We assembled a matrix of 751 lycid terminals and Iberobaenia minuta, a sister to the Lycidae69, as an outgroup (Table S2). The length invariable protein-coding cox1 and nadh5 sequences were aligned using TRANSALIGN70 and the rRNA fragments using default parameters of MAFFT v.7.271. The individual gene alignments were concatenated into a supermatrix using FASconCAT-G v.1.0472. The substitution models were identified using ModelFinder73. All models and partitions are listed in Table S3. We estimated maximum likelihood (ML) trees using IQ-Tree v.1.6.174 and the option-g allowing us to constrain the deep topology. The bootstrap support (BS) was obtained by applying UFboot75 with 5000 replicates. To obtain a tree with deep splits supported by the phylogenomic analysis, the relative position was fixed for 23 terminals whose relationships have been inferred from ~ 4200 orthologs (representatives of all subfamilies and 22 of 30 recognized tribes, Fig. S923). The positions of other terminals, including the tribes for which transcriptomic data are still unavailable, were unconstrained. In such a way, the relationships among most tribes are based on transcriptomics and the shallow splits on rRNA and mtDNA fragments.
The complete dataset could not be reliably analysed using BEAST 1.8.17,10.
Net-winged beetles are a relatively ancient lineage and, presumably, they separated from the Iberobaeniidae ~ 177 mya69 (Figs. 5, 6, 7). Our results show the early origin of Dexorinae as a sister to other lycids (Figs. 5, 6). Without information on their chemical protection and ecology, the species-poor Dexorinae can possibly be left out as a potentially non-aposematic group110,111 (Figs. S383, S384). The representatives of other lycid lineages are unpalatable and most are brightly coloured, and we assume that the origin of aposematically signalling in lycids can be set, at the latest, to ~ 125 mya when the radiation of major, highly diverse lycid lineages started (Fig. 7A, Fig. S925).
We prove that similar aposematic patterns evolved repeatedly in unrelated lineages although weak phylogenetic conservatism was identified (δ-statistics values 2.13–5.90). The indicated level of phylogenetic conservativism must be interpreted cautiously because the colouration is at least partly linked with the geographical origin and large clades from a single area are present in the tree (Fig. S918). Therefore, the δ-statistics value of 5.9 for Metriorrhynchini might also be affected by the geographical structure of the dataset. Conversely, colour conservativism is highly probable in the Dictyopterini and Taphini. Both lineages seldom signal their unpalatability via their bicoloured elytra, even in the ecosystems where the highly conspicuous forms that use such signals dominate (Figs. S6, S176). Our results indicate that the most recent common ancestor of Dictyopterini and Taphini was bright red, and the lineage is ancient (the earliest split in the clade ~ 92 mya). The early origin of aposematism in this clade is supported by the recently discovered mid-Cretaceous net-winged beetles, which are morphologically similar to the extant bright red Erotinae79,80.
Further, we suggest the gradual evolution of aposematic signalling. The reconstruction recovers simple, relatively low-contrast, uniformly coloured patterns in an initial phase of lycid diversification (Fig. 7, Fig. S926). We avoid the estimation of the colour tone for the earliest lycids as the shifts between the yellow and dark coloured forms are controlled by the same set of genes, and therefore, they are simple and common112,113 and the estimation would be prone to error. We hypothesize subsequent origins of further aposematic patterns such as the contrast between the differently coloured pronotum and the elytra, bicoloured elytra, and various characteristically mottled forms41 (Fig. 7, Fig. S926). All unique, uncommon patterns such as punctate, metallic blue, red/blue, green, reticulate, and tricoloured are only present in terminal lineages, mostly in a few separate species. Therefore, we cannot date them with confidence and must surmise their recent origins (Fig. S929).
We assume that the initial dependence on external contrast is supplemented by an internal contrast whose effectiveness should be similar under various ambient conditions. Later, the signalling is enhanced by more complex patterns combining different colours in a characteristic arrangement. We propose that various other conspicuous patterns continuously evolve and persist in net-winged beetles’ rings, regardless of the continuing dominant presence of less conspicuous ancestral forms. A low number of aberrantly coloured aposematics is a disadvantage, so the origin and persistence of new patterns have been discussed by several authors114,115,116,117,118.
Gregariousness was proposed as the trait which supported the origin of new highly conspicuous signals114,115. Conspicuous new patterns are often displayed by a single species with low abundance in a very restricted area (Figs. S1–S922). Therefore, they cannot assemble their own aggregations and we found them as members of aggregations predominantly formed by other species, which regularly contained several aposematic patterns9,40,41. Potentially, all brightly coloured individuals in multi-species aggregations40,41 can be collectively perceived as unprofitable or they may emit a smell, even when they are not disturbed30,36,119,120. In such a way, participation in an aggregation can enhance the survival of visually aberrant aposematics. Detailed information about these factors is unavailable for the present study which considers several thousand species from all zoogeographical regions and theoretically should have taken into account a similar number of predators97, as well as the impact of the presence of imperfect Müllerian and Batesian mimics from other groups of insects120. Nevertheless, our results show that a wide spectrum of aposematic signals can evolve within a single lineage of beetles (Table S8), numerous patterns are present within a single area (Table S7), differently coloured unprofitable prey co-exist in close interaction, and low contrast patterns, transitional colouration, and imperfect mimics regularly occur alongside highly conspicuous forms (Figs. S1–922).
As a result, the interactions of multi-species and multi-pattern assemblages with various local predators are possibly very complex96. For example, the predators’ learning may be affected by the complexity of local mimetic rings, and the fluctuations in their composition. Conversely, the effectiveness of the aposematic signalling will reciprocally depend on the dynamically changing spectrum of predators, both in time and space. At least some predators should be able to enhance the avoidance of aposematic prey by social learning121,122, but such a factor is limited to birds and cannot be expected in various invertebrates, which are putatively important predators of small-bodied lycids.
Strictly speaking, a pattern displayed by a single species is not a case of mimicry. But, if such a species is unpalatable, emits an aposematic signal, and occurs sympatrically with other unprofitable prey, it contributes to the predators’ perception of the whole community, and over the course of time the single-species pattern might be adverged as a model by co-mimics. Therefore, we propose a potential role for the multi-pattern environment in the evolution of new patterns117. Under such conditions, prior experience with the unprofitability of less conspicuous prey produces a positive bias for avoidance learning of more conspicuous defence signals123. The learning process needs high frequency encounters and possibly that is one of the reasons why New Guinea and the Colombian-Ecuadorian Andes, where lycids are highly diverse and common, are apparent hot-spots of aposematic signal diversity (Fig. 8, Fig. S1–S922). The observed persistence of multi-pattern communities may also be supported by phylogenetic conservativism, high levels of migration between local mimetic assemblages, the diversity of predators, and microhabitat diversity in mountain ecosystems, but the evaluation of these factors will need differently designed research.
Aposematic patterns and diversity
We assume that there are several potential predictors of the diversity of aposematic signalling in a region: phylogenetic diversity, alpha-diversity, and abundance. Further factors include the ecosystem diversity62,124,125 and genetic constraints126 on which we do not have sufficient information. The evaluation of considered factors depends on the availability of data. Although we can reliably record the number of tribes occurring in a region (as an indicator of phylogenetic diversity), we currently do not have data on the phylogenetic structures of net-winged beetle communities. Similarly, our estimation of alpha-diversity depends on the number of species already described11 and these are only a part of the real diversity44,84. Additionally, we dependent on an approximate estimation of abundance in various ecosystems (see “Methods”).
The highest phylogenetic diversity was identified in the Oriental region (the Greater Sundas and Indo-Burma, 18 and 17 tribes, respectively) and in Eastern Asia (15 tribes; Fig. 8A). Nevertheless, the number of aposematic patterns is moderate in these regions (6–10 patterns, usually under five patterns in a location; Fig. 8C, Table S7). We only identified some endemic aberrant patterns in the high mountains of the Greater Sundas41,44. In contrast with the low phylogenetic diversity, the highest numbers of patterns were recorded in New Guinea (only four tribes, with Metriorrhynchini predominant, but 22 recorded patterns, Tables S7, S8) and the northern Andes (five tribes recorded, three of them dominant; 14 patterns, Table S7). The low correlation between phylogenetic diversity and the number of aposematic patterns is shown in Fig. S924 (R2 = 0.057).
We can consider alpha-taxonomic diversity in separate regions (Table S7, Fig. 8B, Fig. S924). If we compare the number of species and the number of patterns, we identify an expected positive slope, but with a low fit of the model (R2 = 0.23), although the interpretation is limited by the present state of the biodiversity inventory84,85,127,128. If we consider the real number of species identified in New Guinea (~ 1300 spp., unpublished data), the R2 value doubles. Although we can keep the working hypothesis that the number of aposematic patterns and the number of species are positively correlated, the evidence is ambiguous and only future studies of a high number of mimetic communities where individuals are in real contact can produce reliable data.
Abundance is another factor, although it is potentially linked with alpha-diversity. Currently we do not have hard data to evaluate the abundance of net-winged beetles in various regions and, preferably, individual mimicry communities should be the subject of analyses rather than regions, as in this study. An estimation can only be based on typical collecting success in various regions, and then we could see that regions where net-winged beetles are most common also have the highest diversity of aposematic signals, i.e. New Guinea, the northern Andes, and South East Asian mountains such as Sumatran volcanoes and Mt. Kinabalu in Borneo.
Conclusion
The main goals of our study were to assemble information on the aposematic patterns of > 4000 net-winged beetles, investigate the link between patterns and phylogenetic relationships, and identify the factors affecting aposematic signalling.
We have documented numerous aposematic signals (Figs. 1, 2, 3, 4, Figs. S4–S922). The patterns displaying internal contrast and the complex arrangement of differently coloured patches evolved from simple uniformly coloured forms (Fig. 7). Further, we show that the most conspicuous patterns evolved in limited areas from their aposematic relatives and are displayed by a limited number of species. Despite conspicuousness and putative high level of protection, they sympatrically occur with their less conspicuous ancestral forms, and the local faunas regularly contain a relatively high number of imperfect mimics and transitional forms (Figs. S1–S922). As a result, predator avoidance learning is not a simple choice between cryptic and aposematic prey, but each of many predators has to discriminate between cryptic prey and the whole spectrum of aposematic net-winged beetles in a ring which also contains Batesian and Müllerian co-mimics9.
We assume that regional signalling diversity will be taken in account when specific lycid mimetic complexes are studied in the future. We found the highest number of aposematic patterns in the areas with very high alpha-diversity and abundance but low phylogenetic diversity (New Guinea, the northern Andes), as well as in areas with moderate abundance but high phylogenetic diversity (South East Asia as the whole). The relative importance of these factors needs further study limited to a strictly defined community of interacting mimics and predators. The lycid colour patterns are so diverse in some regions that continuous migration could affect convergence to a common signal. Although lycids species often occur in multi-species communities, it is possible that some of them face a different spectrum of predators and so their patterns are a compromise, which indicates the presence of more complex conditions than the traditionally considered simple prey/predator pairs.
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Acknowledgements
The Czech Science Foundation is acknowledged for the funding of the project (GACR #14942S). The numerous photographs of the longhorn beetles mimicking the Lycidae were generously provided by Luboš Dembický (Brno) who is cordially acknowledged for his invaluable help. We are also very grateful to J. McClarin (Cosanga), M. J. Eising (Utrecht), A. Coache (La Brillanne) for approval to publish their photographs of net-winged beetles and their co-mimics. T. Rulkens, B. Dupont, and Encyclopaedia of Life generously released their photographs under the CC-BY licence. We thank O. Konvička (Zlin) for access to his private collection. We are very grateful to numerous colleagues who provided specimens for our study and to D. M. Richardson for proof reading of the manuscript.
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L.B., M.M., D.K., and T.Y. conceived the idea of this study. M.M., D.K., M.M., M.B., Y.L., R.B., and L.B. performed the field research, M.M., D.K., M.M., M.B., Y.L., R.B., and L.B. performed the laboratory work; J.K. and L.B. took calibrated photographs and analysed colour data, M.M. and DK analysed phylogenetic data, L.B. and M.M. performed the literature search, L.B., M.M. and Y.L. prepared illustrations, M.M. and L.B. lead the manuscript writing. All co-authors commented on the drafts of the manuscript and gave the final approval for the publication.
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Motyka, M., Kusy, D., Masek, M. et al. Conspicuousness, phylogenetic structure, and origins of Müllerian mimicry in 4000 lycid beetles from all zoogeographic regions. Sci Rep 11, 5961 (2021). https://doi.org/10.1038/s41598-021-85567-x
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DOI: https://doi.org/10.1038/s41598-021-85567-x
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