Morphological reassessment of the movable calcar of delphacid planthoppers (Hemiptera: Fulgoromorpha: Delphacidae)

This study presents the morphology of calcar in adult Delphacidae based on representatives of the genera Ugyops Guérin-Meneville, 1834, Notuchus Fennah, 1969 (Ugyopini), Asiraca Latreille, 1798 (Asiracini), Kelisia Fieber, 1866, (Kelisini), Stenocranus Fieber, 1866 (Stenocranini), Chloriona Fieber, 1866, Megadelphax Wagner, 1963, Muellerianella Wagner, 1963, Javesella Fennah, 1963, Conomelus Fieber, 1866, Euconomelus Haupt, 1929, Hyledelphax Vilbaste, 1968, Stiroma Fieber, 1866, Struebingianella Wagner, 1963 and Xanthodelphax Wagner, 1963 (Delphacini). We used SEM electron microscopy, to define seven types of calcar structure (Types 1, 2, 5, 6, 7, 8, and 9) based on combinations of characters including shape, number of teeth and differentiation of sensory structures in species from fifteen genera. Additionally, two other types (Types 3 and 4) were determined based on the calcar descriptions from previous studies. Similarities and differences in calcar structure and function were discussed and emerging relationships between planthopper species and their particular habitats were indicated.

www.nature.com/scientificreports/ spur of Delphacidae-the calcar. It is generally believed that the calcar is used to assist in jumping; however, it is morphologically disparate within delphacid subfamilies. The features of calcar were previously studied under light microscopy, and the results were presented by Metcalfe 62 65 . The reassessment of calcar characters can provide additional data giving insights into the differentiation and morphological disparity of Delphacidae subfamilies; potentially it can indicate adaptation to the structure and surface of their host plants. Additionally, the knowledge of calcar structures will be of great importance for interpreting morphological data available from fossils and tracing evolutionary changes. Therefore, we examined the calcar using SEM to describe new features and possible adaptive characters for moving on different host-plant surfaces.

Material and methods
The studied materials come from the collections of the Upper Silesian Museum in Bytom (USMB) (Notuchus and Ugyops), the collection of the Zoology Research Team, University of Silesia in Katowice (DZUS) (species of several different genera e.g., Asiraca, Kelisia, Chloriona, Megadelphax, Conomelus, Euconomelus, Muellerianella, Hyledelphax, Stiroma, Struebingianella, Xanthodelphax) and the collection of the Laboratory of Terrestrial Invertebrates (LTIB)-State Scientific and Production Amalgamation The Scientific and Practical Center for Bioresources, National Academy of Sciences of Belarus (mainly Stenocranus and Javesella). SEM examinations were conducted in the Laboratory of Scanning Microscopy of the Institute Biology, Biotechmology and Environmental Protection, the University of Silesia in Katowice. The dry material (20 species and 80 specimens) was cleaned in an ultrasonic cleaner for several seconds. Then the specimens were subsequently dehydrated in an ascending ethanol series (30,50,70,80,96, and 100%, for 10 min in each concentration with three 100% ethanol changes) and were air-dried at room temperature for 12 h. The samples were mounted on aluminium stubs with double-sided adhesive carbon tape and sputter-coated in a Pelco SC-6 sputter coater (Ted Pella Inc., Redding, CA, USA) with a thin film of gold. After processing, samples were imaged by the Phenom XL scanning electron microscope. For taking images, an eucentric sample holder was used to allow the sample to freely move, including rotate and tilt, to show all surfaces. The calcar usually protrudes from the tibia, so samples could be rotated and imaged for all elements in detail in SEM.
As previously noted, the metatibial apical movable spur of the Delphacidae, i.e., the calcar, is defined as a special structure usually flattened, foliaceous, and bearing a row of black-tipped teeth on the posterior (trailing) margin or spine-like shaped and not toothed 9 . The historical background of the calcar studies and descriptions are presented in Table S1. Terms of surface sculpturing have been used according to a web available glossary 66 .

Results
The present study distinguished nine types of the calcar structure based on the combination of characters of its shape, number of teeth and differentiation of sensory structures. The calcar (clc) (Fig. 1a-f) as a whole is a movable structure, with a membranous connection (ms) at the apex of metatibia, under the metatibial crown (mtt) of a few teeth (apical row of teeth). The sensory structures were identified as sensilla trichoidea (St)-larger setae, hair-like, and sensilla chaetica (Sch)-shorter and stouter setae. Both sensilla belong to a group of the mechanosensilla, which detect most of the tactile sensations perceived by the insect. Sensilla trichoidea (St) are characteristic in a more flexible stem as opposed to sensilla chaetica, which are provided with a stiffer, slightly grooved and acutely terminating stem. Both types of sensilla are embedded in flexible sockets (s), with a flexible membrane (m) surrounding the hair base. The distinguished types of calcar are listed below and in Table 1. www.nature.com/scientificreports/ trinus (Fig. 2d) and Ugyops inermis (Fig. 2e), there are two rows of long sensilla trichoidea (h) and one row of short stout sensilla chaetica (Sch) on the surface. The stout sensilla chaetica resemble sharp teeth. In Notuchus linnavuorii (Fig. 2f), the calcar is more subulate with external (explantar) surface convex and internal (adplantar) surface slightly concave; external (explantar) surface covered with shortened sensilla trichoidea (St). Inthree mentioned Ugyops species, the costate sculpturing of the calcar apex is deeper than that in Asiraca clavicornis.
Type 3: calcar subulate,sparsely dentate. In Vizcaya Muir, 1917, andNeovizcaya Liang, 2002, the calcar is elongated, terete in cross-section, but a distinct row of 8-12 large teeth is present at the adplantar margin (Table S1). Bases of sensilla chaetica are placed above the bases of these teeth. A few sensilla trichoidea are irregularly interspersed between the bases of teeth ( 64 : Fig. 18). of calcar sloping downwards on two sides from a raised external, explantar margin. The ventral adplantar margin with a variable number (more than 8) of large distinct immovable conical teeth (t) (from 9 to 11 teeth, including the apical one), with subbasal, long sensilla trichoidea (St) (Fig. 3a-f). The dorsal adplantar margin is smooth and bent to the ventral side. Adplantar surface of calcar concave, in the form of a wide and shallow groove ( Fig. 4a-f). The size and shape of calcar differ in the studied species.
In Kelisia praecox (Fig. 3a,b) (Kelisiinae) the ventral adplantar margin bears 11 long and thin conical teeth (including the apical tooth) and on the surface of each tooth there is one or two sensilla trichoidea (St). The remainder of calcar surface with a sparse sensilla (St).
In Conomelus anceps (Delphacinae: Delphacini) the ventral adplantar margin bears 9 short and stout teeth (Fig. 3c,d) which are shorter than those in Kelisia praecox. The surface of each tooth possesses one sensilla trichoidea (St). The remainder of calcar surface has a sparse sensilla (St) (Fig. 3c,d).
Type 6: calcar tectiform, densely dentate. The calcar is subtriangular to sickle-shaped in cross-section, dorsal edge less noticeable. The ventral adplantar margin bears more than a dozen small teeth, covered with more or less dense mechanosensilla (St). The adplantar side of calcar lacks sensilla.
In Euconomelus lepidus (Delphacinae: Delphacini) (Fig. 5a,b) the adplantar margin bears about 20 small teeth. The surface of each tooth and remainder of the adplantar slope are covered by abundant mechnosensilla (St), forming a brush hiding the teeth at the adplantar slope of calcar. The calcar adplantar surface bears no sensilla (Fig. 5b).
In Muellerianella brevipennis (Delphacinae: Delphacini) ( Fig. 5c-f the adplantar margin bears 15 small teeth. The surfaces of each tooth and adplantar slope are covered by abundant mechanosensilla (St); however, the tips of the teeth are free of sensilla. These sensilla form a brush on the calcar edge. The inner surface of calcar bears no sensilla (Fig. 5d,f).
Type 8: calcar densely denticulate with bristly mechanosensilla. This type seems to be further modified than Types 4 and 5, triangular to sickle-shaped in cross-section, with the ventral adplantar margin elongated teeth, and rows of densely packed mechanosensilla and with apex diminutive.
In Stenocranus fuscovittatus (Stenocraninae) (Fig. 10a-f), the calcar adplantar margin presents a row of stout teeth (14), but in Stenocranus major (Fig. 11a-d) the adplantar margin bears 20 teeth. Specific features of this type includes deep separation of each tooth on the adplantar margin and location of the brush of sensilla trichoidea along with the teeth. Type 9: calcar tectiform toothless (edentate). Here the calcar is flattened, sickle-like in cross-section, but the explantar (dorsad) edge is obsolete; the edplantar edge is toothless and covered with several rows of sensilla trichoidea. www.nature.com/scientificreports/ In Stiroma affinis (Fig. 12a,b,d) and Hyledelphax elegantulus (Fig. 12c) (Delphacinae: Delphacini) the calcar is toothless (edentate). Its edge is covered with several rows of sensilla trichoidea. The proximal and median portions ofcalcar are significantly wider than the distal, tapering portion.

Discussion
Although a charismatic feature of Delphacidae, the calcar has not been previously the subject of detailed comparative and morpho-functional studies. The delphacid tibial calcar has been widely used in taxonomic treatments of members of this family from Muir 19 71 and Liang 64 . However, since then, no further attention has been directed to studies of calcar structure. The calcar is present also in the nymphs, since the 1 st instar; however it is different in size, shape and armature from those in the imagines 25,63,70,72,73 . A preliminary attempt to trace evolutionary changes of the calcar was presented by Muir 19   www.nature.com/scientificreports/ A general tendency involves a change from a subulate calcar to a tectiform one (Table 1), which is variously armed and flattened. These opinions could be confirmed by analysing the data in recent phylogenetic studies of Delphacidae 4,5 and our results. In the present study, in SEM, the calcar differs between various tribes of Asiracinae. Type 1 calcar present in Asiracini (Asiracinae) seems to be the most plesiomorphic condition, movable, an awl-shaped and long spine, not different except size and movability from apical teeth. A slightly modified Type 1calcar, more quadrangular in cross-section is mentioned in the genera Notuchoides Donaldson, 1988 and Kiambrama Donaldson, 1988 74 . The same type of calcar is observed in other Asiracini genera Copicerus Swartz, 1802 and Elaphodelphax Fennah, 1949 27,75 .
A little more modified type, subular and with sensilla chaetica and sensilla trichoidea organized in rows (Type 2) is present in Ugyopini (Asiracinae). Within this tribe, in species of the genus Ugyops the calcar is elongate, subular and angulate with sensilla chaetica and sensilla trichoidea arranged in rows. Within this model variabilities are observed in particular species: a row of the sensilla chaetica and two rows of long sensilla trichoidea in U. nemestrinus and U. inermis as well as three rows of short and stout sensilla chaetica in U. taranis. In other known species of Ugyops (Table S1), the calcar presents the same model (Type 2). However, the calcar in   47 and similar to that in Ugyopini of Dominican amber (Fig. 13). Solórzano-Kraemer 49 briefly described another fossil from Miocene Mexican amber, placed in the genus Eucanyra Crawford, 1914 (synonym of Ugyops). The calcar in this fossil also seems to represent Type 2.
In the Vizcayinae (genera Vizcaya Muir, 1917 and Neovizcaya Liang, 2002) the calcar appears to represent Type 3-it is subulate, round in cross section with a row of teeth on the adplantar side ( 28 67 ). Species of these genera inhabiting Hawaii and Marquesas seem to be more related to trees and shrubs of various plant families, mostly dicotyledonous, probably using a greater diversity of microhabitats available 85,86 and having adapted to them. Asche 10 postulated that this type of calcar evolved independently, possibly to enhance walking on particular surfaces (e.g., on woody substrates). Interestingly, 'alohine' calcars were mentioned also in Burnilia (Plesiodelphacinae) 84 but earlier Asche 83 described the calcar in Plesiodelphacinae as "kelisioid" rather than "alohinid". An "alohinoid" calcar was also reported for the genus Sparnia Stål, 1862 (Delphacini), by Asche & Emeljanov 87 .
Within the Kelisiinae (genera Anakelisia Wagner, 1963 and Kelisia), the calcar represents Type 5, with a subtriangular cross-section, a slightly concave adplantar surface and a distinct row of long, conical teeth on the adplantar margin. As observed in Kelisia praecox and Anakelisia fasciata (Kirschbaum, 1868), each tooth bears one or two mechanosensilla. The remainder of calcar surface bears sparse mechanosensilla.
In the Stenocraninae, at least these with known detailed structures of calcar, it is Type 8. This model is observed in Stenocranus major (Kirschbaum, 1868) [89][90][91] . It is not clearly evident from available sources, but Type 8 is probably present also in the genus Preterkelisia Yang, 1989 92,93 . Almost nothing is known on the calcar in the Stenocraninae genus Proterosydne, except it, is "… solid, elongate, narrow, with 8 spines" ( 94 : 131), suggesting it could represent Type 5 or Type 8.
Within the representatives of subfamily Delphacinae the calcar diversity is the largest, with Types 5, 6, 7 and 9 distributed among various taxa. In the Saccharosydnini various types are reported, e.g., various species of the genus Saccharosydne Kirkaldy, 1907 present    The highest variability of calcar is known among Delphacini, e.g., Type 9 is present in Paranectopia Ding et Tian, 1981 originally described in Tropidocephalini, but moved to Delphacini 5 . Type 9 is also known in the genera Achorotile Fieber, 1866, Astatometopon Campodonico, 2017, Eurybregma Scott, 1875, Hyledelphax Vilbaste, 1968, Nataliana Muir, 1926, Stiroma Fieber, 1866 27 . Type 5 is present e.g., in the genera Conomelus Fieber, 1866, Onidodelphax Yang, 1989, and species Formodelphax formodus Yang, 1989 93 . The calcar attributed to Type 6 is present e.g., in genera Euconomelus Haupt, 1929, Isodelphax Fennah, 1963, Javesella Fennah, 1963, Laodelphax Fennah, 1963  www.nature.com/scientificreports/ The exact function of calcar and its mode in Delphacidae remains unresolved. The reasons for the disparity of calcar structures are not well recognized. It is generally believed that the calcar is used to assist in jumping; however, observations and experiments have not confirmed it. The recent observations of Delphacidae jumping mechanisms focused on the thorax, its musculature and base of legs with no attention to the role or function of calcar at the process 103,104 . With the presence of several types of sensory hairs, the calcar seems to be involved in the process of jumping, but its variability could be related to the surfaces from which the jump is taken. It seems plausible that this diversity is somewhat related to the structure and properties of the surface on which planthopper is living. Delphacids are relatively host-specific, and most mainland species (92% of records) attack monocots; dicot feeding dominates (82% of records) only on oceanic islands 3,10,67 . The Asiracinae Ugyopini recorded on dicotyledones mainly feed on woody dicots (most probably secondarily) and on monocotyledon Arecaceae. The family Arecaceae comprises 240 genera and approximately 2700 species predominantly concentrated in tropical and subtropical regions 105,106 , with fossil record reaching Late Cretaceous 107 . Type 2 calcar, as in Ugyopini, could be, on the one hand, a conservative model and, on the other, an expression of adaptation and a long co-evolutionary history with their host plants. Type 1 calcar, is present in Asiracini; for these planthoppers, www.nature.com/scientificreports/ most monocot records is related to Cyperaceae, but some are recorded on dicots and even ferns (this is a definitively secondary adaptation to host plant). Cyperaceae crown groups appeared in the Late Cretaceous-Early Paleogene, but their diversification took place at the end of the Eocene 108,109 . Host plants of Vizcayinae remain unknown; Kelisiinae seem to be strictly associated with Cyperacae and Juncaceae, plants of extraordinary ecological importance, occupying a broad range of habitats from rain forests to tundra, as components of open habitats including many types of wetlands, temperate and tropical grasslands and savannas, especially in moist sites, or more shaded ones as understory of forests 110,111 . The evolutionary shifts in the history of these plants took place at the terminal Eocene-Early Oligocene, during a global cooling period and in the Miocene, during global warming, then cooling periods, resulting in rapid diversification 111,112 . Therefore it could be assumed that Kelisiinae retaining Type 5 of calcar shifted to these host plants simultaneously with or after their diversification and spreading. In Stenocraninae, the calcar of Type 8 is present, which could be related to adaptations to a broader array of host plants. Stenocraninae seem to be strongly associated with monocotyledons in modern fauna, with a clear dominance of Poaceae over Cyperaceae 3,90 . In Delphacinae, the variability of types of calcar is the highest (Table1); while in vast majority they feed on various Poaceae, shifts to other monocots or dicotyledons are known 3,4 . Also, modifications of calcar were reported in Delphacini taxa associated with water plants (e.g., waterlily), e.g. Megamelus davisi Van Duzee, 1897 with an exceptionally large, thin and leaflike calcar with about 20 small teeth, but allowing to place it to Type 7 [113][114][115][116] . The shape of the calcar probably facilitates jumping from the water surface (personal communication, Ch. Bartlett). Delphacini seem to have experienced several host shifts but do not present a co-evolutionary pattern; representatives of this lineage are clearly ecological opportunists 4 . Therefore the variability and disparity of calcar and its pattern in this group is homeoplasous, not giving strong phylogenetic signal, but could be a good tool to understand the ecological history of the Delphacini and their temporal shifts and adaptations to host plants. Increased diversification within Delphacini may reflect a shift to grass-feeding, and host shifts within Poaceae, perhaps from grasses with C3-C4 photosynthetic pathways 4 , and different anatomical features of C3 and C4 grasses 117 . It must be noted that host plant associated diversification within Delphacidae was mediated by co-evolutionary relationships with endosymbiotic bacteria and fungi 118,119 ; therefore, the natural selection and adaptation of these planthoppers took place at various planes. The calcar is the most significant character defining Delphacidae but its evolutionary origin is not fully resolved. Basing on its postembryonic development, it is supposed that the calcar is derived from one of lateral apical teeth of the hind-tibia as present in Cixiidae 29,63,120 . Modern Cixiidae, as well as most of the known fossils, present some variability in the armature of metatibial apex; however, the elongate outer tooth of the external group often stands out, and the two next teeth of the same group are shorter; these three teeth form a medial   (Fig. 13). The other Miocene taxon: Amagua fortis Cockerell, 1924 from Late Eocene/Early Oligocene of Amgu (Amagu) River, Sikhote-Alin, Russia is a  Cockerell, 1921 should be assigned to Delphacidae and the original materials must be revised; Chattian, Oligocene fossil named 'Delphax' rhenana Statz, 1950 does not seem to represent Delphacidae 48,50,52-54 . The calcar from fossil resins is not different from the one present in modern Ugyopini. The taphonomic potential of Delphacidae for fossilisation as adpressions seems not to be high due to various extrinsic and intrinsic factors 125,126 ; therefore, the inclusions in amber and other fossil resins remain an invaluable source of information.

Conclusion
The calcar of Delphacidae is their unique synapomorphy, defining the family as a whole. Its structure is highly variable, but the use and estimation of its phylogenetic and classification values seem challenging, as detailed knowledge of theof calcar structure and its function remains limited. On the other hand, the variability and disparity of calcar and its patterns observed in Delphacidae could be a good tool to understand the ecological history of these insects and their temporal shifts and adaptations to host plants. Being a charismatic character of the Delphacidae, the calcar still poses a number of problems to be addressed, e.g., its evolutionary origin, the developmental ways of formation, factors influencing its disparity, even the exact function. Here, we presented the first attempt to systematize calcar models and structures and evaluate its potential in morphological, evolutionary and ecological studies. We have also justified the need for restudying this morphological structure.   www.nature.com/scientificreports/