Immatures of the New World treehopper tribe Amastrini (Hemiptera, Membracidae, Smiliinae) with a key to genera

Abstract The immatures stages of 8 of the 11 genera (Amastris Stål, Bajulata Ball, Erosne Stål, Harmonides Kirkaldy, Idioderma Van Duzee, Neotynelia Creão-Duarte & Sakakibara, Tynelia Stål, and Vanduzea Goding) of the tribe Amastrini are described for the first time along with brief diagnoses of Membracidae and the subfamily Smiliinae. A key to genera and notes on biology are provided. Multiple species of most genera are illustrated. Based on its distinct nymphal morphology, Vanduzea laeta nolina Ball is elevated to specific rank as Vanduzea nolina stat. n., and Bajulata, despite the superficial similarity of its adults to those of Vanduzea, is confirmed as warranting generic rank based on its unique nymphal morphology. Colombia is a new country record for Tynelia.


Introduction
Treehoppers (Membracidae, Aetalionidae, and Melizoderidae) are well known for the expanded, often extravagantly developed pronotum common to adults of nearly all of the more than 400 genera and 3,000 species (McKamey 1998). What is less well known is that the adult pronotum is usually displayed, in miniature form, in the last, fifth instar. In addition to this nascent enlarged pronotum, immatures are often covered with various arrangements of large spinelike structures (scoli) on the head, all thoracic segments, and the abdomen that are usually absent in the adults. Indeed, except for the nascent pronotum, treehopper immatures show a vast array of structures that to a large extent have evolved independently of the adult forms. Despite this wealth of potential diagnostic features, there has been no previous identification guide to genera of immature treehoppers.
Perhaps two of the earliest accounts of treehopper immatures were Scheller's (1783Scheller's ( -1794 and Fairmaire's (1846, Pl. 3 fig. 17 of Centrotus cornutus [Linnaeus]). The former were cited by Buckton (1902), who also refers to "the Dutch paper read in 1868 before the Ent. Soc. of the Netherlands which treats the metamorphosis of these insects," and reproduces images in larger form of three species. Buckton's (1902) crude illustrations of several Neotropical species are also added, including that of a laterally compressed Cymbomorpha Stål nymph misidentified as Membracis continua Walker, which has laterally compressed adults, but not laterally compressed nymphs; Fowler (1894) also alludes to this "Membracis" nymph. Both references were apparently based on Westwood's misidentification of a nymph in the Hope Collection at Oxford.
Moreover, the first accounts of any merit on New World treehopper immatures were by Funkhouser (1917), which treated and illustrated the life histories of treehoppers of the Cayuga Lake Basin in New York, and Haviland (1925), who provided brief descriptions and illustrations of the nymphs and egg masses of various species found in the Republic of Guyana. Further works are by Richter (1941aRichter ( , b, 1942aRichter ( , b, 1947Richter ( , 1955 in Colombia; Quisenberry et al. (1978) in the United States; Strümpel (1986), who described eggs and nymphs of Havilandia spiralis (Haviland); Flock and Gill (1987), who illustrated and briefly described the nymph of Parantonae Fowler in Arizona; Pratt and Wood (1992) on the Enchenopa binotata (Say) species complex; Miyazaki and Buzzi (1985) on Membracis; Creão-Duarte and Sakakibara (1987), who described the immatures of Kronides Kirkaldy; Dietrich and Deitz (1991) on Aconophorini;and McKamey and Deitz (1996) on Hoplophorionini. Beyond other isolated descriptions, the features of immatures of New World taxa had received little attention until 11 characters of 56 genera (2 Amastrini genera) were included in the first phylogenetic estimate of membracid phylogeny (Dietrich et al. 2001). More recently, Godoy et al. (2006) provided images of immatures of many Neotropical genera, and Strümpel and Strümpel (2014) illustrated nymphs of a few Enchenopa and Enchophyllum species. Lencioni-Neto and Sakakibara (2013) described the immature of Alcmeone Stål (Darninae). The immatures of the unusual Antillean endemic genera Antillotolania Ramos and Deiroderes Ramos have also been described (McKamey and Brodbeck 2013). Regardless, formal descriptions or even illustrations of New World taxa are restricted to very few genera, and keys are nonexistant with the exception of Quisenberry et al. (1978) and Pratt and Wood (1992), severely hindering identification.
Immatures of Old world treehoppers, consisting of most of Centrotinae (Membracidae) and Darthula Kirkaldy (Aetalionidae) have received even less attention (Capener 1962(Capener , 1968Ananthasubramanian 1978Ananthasubramanian , 1982Ananthasubramanian and Ghosh 1982;Ahmad andPerveen 1984, Yuan andChou 2002). Despite having many variable features, they are morphologically less diverse than New World forms and beyond the scope of this study. No other subfamilies occur in both hemispheres. This is the first installment describing the nymphs of New World treehopper genera, with examples of multiple species of some, egg masses of some, and other biological information. This contribution covers the tribe Amastrini, a predominantly Neotropical tribe, and includes 8 of the 11 genera (immatures of Aurimastris Evangelista & Sakakibara, Hygris Stål, and Lallemandia Funkhouser are unknown).
Photographs of adults of all membracid genera are available online (Deitz and Wallace 2010).

Materials and methods
Some of the species examined are new species but their nymphal descriptions below are not intended to constitute a description recognized by the International Code of Zoological Zomenclature (International Commission on Zoological Nomenclature 1999).
Late instars of Membracidae are usually sturdy enough to maintain their form when dried, so pinned specimens were used to determine characters. The only characater that would likely be affected is the length of abdominal segment IX relative to other body parts, due to contraction. Nevertheless, this effect should be roughly equivalent in all pinned specimens.
Because some form of parental care or at least aggregation of nymphs is widespread among treehoppers, for many subfamilies it is easy to associate adults, nymphs, and eggs. In the case of solitary taxa, repeated adult-nymph-host association, rearing, and in a few cases the extrapolation of the miniature pronotom have been used to associate adults and nymphs. Generally, treehopper immatures will be identifiable to genus, and sometimes to species, so the genus is described but multiple species, if known and distinctive, are also shown. Characteristics such as oviposition style, parental care, nymphal aggregation, and ant-attendance are usually uniform within tribes. Host plants and location of feeding are also reported here as far as known.
Most vouchers are deposited in the National Museum of Natural History in Washington, D.C. (USNM) in cabinet drawers designated "McKamey et al. Membracidae Immatures Vouchers," except those of Tynelia (see below). Each voucher also has the label "Immatures Project Voucher, McKamey et al. 2015" and the species name or, if unidentified, its species number. In most cases, additional nymphs were examined, but only those placed into the separate, synoptic collection are listed in Material examined.
Images were captured with a Microvision system and Cartograph 8.0.6 automontage software and adjusted in Adobe Photoshop. Scanning Electron Micrographs taken with A Philips XL-30 SEM using a gaseous phase on uncoated specimens.

Morphology, characters, and terminology
The features used by (Dietrich et al. 2001) in their first phylogenetic estimate are used, except the protrusions on abdominal tergum II, which are difficult to determine in many specimens and not critical to differentiate nymphs. Also, because many states occur on the ventrolateral margins of the abdomen exist, our definition of "lamellae" has been refined, such that the nymph of Tolania Stål figured in the above paper (their Fig. 8b) is no longer considered as having lamellae but rather as having a row of enlarged chalazae. Dietrich et al. (2001) used 11 nymphal characters and 28 character states; and for many taxa the nymphs were unknown. Presently the current authors have 76 nymphal characters and 322 character states with multiple genera of every New World membracid tribe except Centronodini and the monobasic tribes, which are also represented. Because new characters are expected to be discovered as more higher taxa are surveyed, and it is desirable to keep characters grouped by body sections, listing the characters and character states in each paper would complicate comparative studies and use of the character matrix in future phylogenetic studies. The character descriptions and data matrix are therefore, instead, posted online on the Systematc Entomology Laboratory website (https://www.ars.usda. gov/Main/docs.htm?docid=25448). Because many of the character states have never been observed before, some of the terms used here are likewise novel or require elaboration.
The morphology of Amastrini and other Smiliinae are illustrated in Figs 1-11. As in adults, the pronotum of nymphs consists of a premetopidium (basally) and a postmetopidium (posteriorly), separated by a metopidial sulcus, which is usually marked by lateral callosties (see below) or by a transverse indentation. In contrast to the adults, all three of these structures provide useful characters in Smiliinae, and the pre-and postemetodidium bear scoli, or not, as if they were separate segments. Chalazae consist of a base, which can be tuberculate or stalked, and a seta, which can be needle-or hairlike (Fig.  2), subcylindrical and capitate (Fig. 3), or paleate (Fig. 4). Chalazae can cover the entire body, be restricted to certain areas, or be entirely absent, and enlarged chalazae can occur in various places as well, There are sometimes up to 3 longitudinal rows of enlarged chalazae or even scoli present on the abdomen, which sometimes extend onto the mesoand metanotum. On abdomial tergum IX, chalazae may be in paired longitudinal rows, irregularly arranged, or completely absent preapicaly and at or very near the apex. The larger, spinelike scoli (Figs 1, 9-11) may occur on every segment (Fig. 1), on none ( Fig.  13), or some combination, often bear chalazae (Figs 9-11), and are almost always paired in the Amastrini genera currently represented and most other membracids. The placement of scoli, their basal and distal direction, and sizes relative to other scoli and lengths compared to their basal widths are all character states useful in distinguishing genera and, in some cases, species within genera. Flock and Gill (1987) illustrated scoli similar Figures 1-11. Structure and character states of Smiliinae. 1 Neotynelia nigra, with principal structures of an membracid nymph labelled 2-4 Needlelike, subcylindrical, and paleate setae (in Bajulata) of chalazae with turbuculate bases, respectively 5-6 Length of first tarsomere relative to second, distinctly shorter than, or subequal in length to, respectively 7-8 Quadrinarea sp., Length of pro-and mesothoracic first tarsomere relative to length of metathoracic first tarsomere 9-11 Tuberculate chalazae on scoli (9-10) and stalked chalazae (11).
to those of Fig. 11 and referred to them as "spinose tubercles," which is insufficient to account for the morphological variation among all membracids bearing scoli with chalazae. Instead of scoli, but in the same places, there may occur paired enlarged chalazae or paired clusters of them (Fig. 41). As noted by Dietrich et al. (2001), the enlarged chalazae (or chalazal clusters) in the same placement of scoli suggest that the enlarged chalazae are homologous. In addition to the form and arrangement of chalazae on the tibiae, an important feature of the legs is the length of the metathoracic first tarsomere relative to the first tarsomere of the more anterior legs (Fig. 7 vs. 8) and to the metathoracic second tarsomere (Figs 5,6). There are also smooth callosities sometimes present on the head, pronotum, or mesonotum that are diagnostic.
Characters are described from the overal body form, the head, all thoracic segments including legs and forewing wingpads, and segments III-IX of the abdomen with special emphasis on segment IX. Because new characters will undoubtedly be discovered as we explore other taxa, each of the above major regions is assigned a number and the characters are indicated by that number and a letter in the character descriptions posted online (see above). Many of these character states are related. For example, if scoli are present on the pronotum, they often occur on all thoracic and abdominal segments. There are enough exceptions, however, that separate characters are warranted for each segment. Abdominal segment X, which is the first anal segment, is sometimes long and sclerotized, but because it is often retracted into segment IX, is not included among the characters.

Membracidae Rafinesque
Nymphs of Membracidae can be distinguished from all other Auchenorrhyncha, including the treehopper families Aetalionidae and Melizoderidae, by a ventrally fused abdominal segment IX, which thereby forms a tube through which the anal segments (X and XI) can be exerted by the nymph in defense or to proffer exudate to attendant ants, melaponine bees, or Parachartergus Ihering vespid wasps. Ants include the opportunistic genera Azteca, Camponotus, Crematogaster, and Ectatomma, which sometimes build vegetative enclosures around membracids, and which also collect from extrafloral nectaries and sometimes consume membracids (SHM observations, Haviland 1925). No nymphs of any of the treehopper families jump, but this is a feature shared with some leafhoppers (e.g., Eurymelinae and Macropsis) and some planthoppers (e.g., Tettigometridae).
Eggs of treehoppers are either laid in masses of 40-70 eggs, inserted singly or in groups of 2-5 into the tissue or not, and covered, uncovered, or enveloped by a white or brown secretion (whose characteristics have not yet been investigated) in usually distinct patterns. In Membracinae there is sometimes auxillary deposits by the female of waxlike or clear and sticky material above, below, or around the egg mass. Oviposition surfaces include crossvein leaf surfaces, leaf midribs, petioles, tendrils, twigs and thicker stems. In most genera of Hoplophorionini (Membracinae), additional incisions are made, prior to eclosion, through which the nymphs feed.
The descriptions below and in other installments are for the fifth instar, but most features apply equally to earlier instars. In younger instars some features are more pronounced, including an increased length of the scoli relative to their basal widths and to the overall body size, an increased the length of the abdominal segment IX relative to the rest of the body, and less development of the pronotum and wing pad.

Amastrini Goding
Note. Deitz (1975) characterized the adults of the tribe and listed eight genera in Amastrini: Amastris Stål, Bajulata Ball, Erosne Stål, Harmonides Kirkaldy, Idioderma Van Duzee, Lallemandia Funkhouser, Tynelia Stål, and Vanduzea Goding. Hygris Stål was later referred to Amastrini (Sakakibara 1998) and the genera Neotynelia Creão-Duarte & Sakakibara (2000) and Aurimastris Evangelista & Sakakibara (2007) were later described. The nymphs of Lallemandia, Aurimastris and Hygris are unknown. Below we provide a description of the immatures of the tribe and the other genera, and their character states are posetd online (see Methods). Members of Amastrini are subsocialso, in contrast with solitary taxa such as Darninae, the association of the immatures with adults is straightforward (Fig. 65).
Nymphal description. Overal body. Cross-section subtriangluar; chalazae dense on thorax and abdomen, obvious throughout body (except sparse in Harmonides reticulata and some Neotynelia); no parts of body covered with waxlike substance; overall body in dorsal view elongate. Head. Dorsal or anterior rounded protuberances absent (except present in some Neotynelia); chalazal bases tuberculate; compound eye surface setae present (except absent in Idioderma and some Neotynelia); no enlarged chalazae between eyes; enlarged chalazae in front of ventral margin of eye absent (except present in Amastris exigua); enlarged chalazae adjacent to central or dorsal margin of eye absent (except present in Harmonides and Vanduzea laeta); frons not extending over central margin of eye (exception: Bajulata bajula). Prothorax. Dorsal and lateral suprahumeral horn buds absent; pronotal lateral margin simple (except emarginate in Bajulata); postmetopidium without elevation and carination that is absent in adult; metopidial sulcus not incised, continuous with adjacent surfaces above and below it; posterior extension distally narrowly convex or acute. Mesothorax. Anterior basal side of scoli, if present, without cluster of enlarged chalazae. Legs. Prothoracic tibia form simple (except foliaceus in Bajulata); metathoracic tarsal length subequal to pro-and mesothoracic tarsal length; all first tarsomeres distinctly shorter than second tarsomeres. Abdomen. Terga III-IV dorsally with paired apically acute scoli (except none in Idioderma, a single middorsal projection in Bajulata, and paired apically rounded or blunt scoli in Amastris elevata); terga V-VI dorsally with paired apically acute scoli (except paired enlarged chalazae in Idioderma, single middorsal projection in Bajulata, paired apically rounded or blunt scoli in A. elevata); tergum VII with spaired apically acute scoli dorsally (except single middorsal projection in Bajulata, and paired apically rounded or blunt scoli in A. elevata); tergum VIII with paired apically acute scoli dorsally (except none in Neotynelia sp. 1, single middorsal projection in Bajulata, and paired apically rounded or blunt scoli in A. elevata); lamellae absent; scoli bearing tuberculate chalazae (expect scoli absent in A. exigua). Segment IX. Distal half in cross-section usually subtriangular; preapically covered with irregularly arranged chalazae (except paired row of enlarged chalazae in some Neotynelia); fused portion of segment IX distal to unfused portion; unfused portion distally not bifurcate.
Discussion. Although each genus of Amastrini can be distinguished within the tribe, it is difficult to neatly circumscribe Amastrini. Immatures of most amastrine genera have paired, straight scoli on abdominal terga III-VIII, some also have them on the head, or thoracic nota, or the head and all thoracic nota. The genus Bajulata Ball is the most divergent, in having a single middorsal process on each abdominal segment, a unique condition among Smiliinae. In contrast to other Amastrini, Bajulata also has paleate setae on the head and thorax.
Material examined. Distribution. Brazil and Peru northward to the United States. Biology. As far as known, all Amastris are subsocial, with the female parent sitting atop her uncovered egg mass, which are inserted into stems of the host, and tending her nymphs after hatching (Fig. 65). Nymphal aggregations are almost always antattended. Ant specimens pinned under vouchers include Camponotus, Crematogaster, and Azteca.
Discussion. Broomfield (1976) revised this large genus, but many species remain undescribed, including some of the specimens examined here. Nymphs of the genus Amastris Stål are difficult to characterize because some species bear no distinguishing enlarged chalazae or scoli or other features and thus resemble some taxa of other Smiliinae tribes and even other subfamilies.
Distribution. United States.
Distribution. Brazil to northern South America. Discussion. At present, the only feature found to distinguish Erosne nymphs from Amastris is the slightly further posterior extension of the pronotum in Erosne, which is undoudtedly coupled with the more extensive pronotum in the adults. The Erosne species examined is new. [52][53] Diagnosis. Head and pronotum without scoli; meso-and metanota and terga III-VIII each with short paired scoli; body evenly covered with chalazae with long setae.
Biology. Similar to other Amastrini genera, Vanduzea aggregate on their host plants and some species, for example Vanduzea arquata (Funkhouser, 1915), are tended by Formica ants (Funkhouser 1915, Fritz 1983, Crocroft 2003. These treehoppers feed on herbaceous and woody dicot hosts, such as Albizia julibrisson, Citrus sp., Melilotus alba, Bidens alba, Eupetorium capillofolium, Lespedeza sp., Quercus sp., and Robinia pseudoacacia Yonke 1973c, Dietrich et al. 1999). Vanduzea is a common and widespread genus in the Nearctic, Neotropics, West Indies, and has been introduced to the Hawaiian Islands . SHM has observed them in Hawaii tended by ants, which are also introduced.
Discussion. The nymph of Vanduzea laeta has unequal but not posteriorly increasing or decreasing sizes of abdominal scoli with greatly elongate scoli on terga IV and V (Fig. 36). In contrast, V. laeta nolina has abdominal scoli that are subequal in length and all short (Fig. 37), so the latter is here elevated to specific status.