THE WEEVIL ROSTRUM (COLEOPTERA: CURCULIONOIDEA): INTERNAL STRUCTURE AND EVOLUTIONARY TRENDS STEVEN R. DAVIS Division of Invertebrate Zoology American Museum of Natural History BULLETIN OF THE AMERICAN MUSEUM OF NATURAL HISTORY Number 416, 76 pp., 50 figures, 2 tables Issued October 10, 2017 Copyright © American Museum of Natural History 2017 ISSN 0003-0090 CONTENTS Abstract.............................................................................3 Introduction.........................................................................3 Methods ............................................................................3 Taxon Sampling ...................................................................3 Histological Sectioning ............................................................4 Electron Microscopy ..............................................................4 Confocal Microscopy ..............................................................4 Microcomputed Tomography (μCT) .................................................4 Phylogenetic Analysis . .4 Anatomical Terminology ...........................................................7 Results..............................................................................7 General and Internal Anatomy of the Adult Weevil Rostrum ...........................7 Character List ...................................................................20 Phylogeny Utilizing Rostral Characters .............................................26 Phylogenetic Analysis of Rostrum Anatomy..........................................26 Outgroups ....................................................................26 Curculionoidea ................................................................26 Discussion .........................................................................72 Rostrum Structure and Evolution ...................................................72 Phylogenetic Relationships.........................................................73 Acknowledgments...................................................................74 References..........................................................................74 2 ABSTRACT The current classification of weevils has witnessed vast improvements at higher phylogenetic levels, though much remains unstable at the lower levels. In order to develop a more robust mor- phological character system for cladistic analysis of the higher lineages and to gain a comprehensive understanding of the structure of a hallmark feature of weevils, a comparative study was conducted of rostra throughout Curculionoidea. Semithin sections were made of the rostra of 36 exemplar genera representing all seven currently recognized weevil families, as well as 19 of 21 subfamilies within the largest family, Curculionidae, and internal structures were examined for phylogenetically informative characters. While the morphological diversity of rostral forms is impressive, general trends are apparent with respect to life-history traits and modes of feeding. Exploration of internal rostral morphology has yielded valuable but previously unexplored characters that greatly comple- ment the external characters of this structure. Together, these features provide new insight for set- tling current incongruence at the higher levels of classification. Key Words: morphology, histology, phylogeny, adult structure INTRODUCTION of internal rostral morphology provide a valuable complementary set of previously unexplored Weevils (Coleoptera: Curculionoidea) are one characters. Although this study includes rela- of the most diverse family-level groups of extant tively few taxa, it covers a fair range of rostral organisms, with approximately 60,000 described forms and taxonomic divisions, highlighting the species (Anderson, 1995). They are extremely major features apparent in weevil rostrum evolu- important agriculturally and economically, tion and diversification. It will serve as a basis for because they are associated with all major groups continued exploration of rostrum structure of plants and plant parts. The current classifica- throughout weevil subfamilies to determine the tion of weevils, including numerous hypotheses breadth of rostral architecture deviating from the on relationships, is relatively stable at the familial generalized forms determined here. Lastly, this level but remains unstable at the middle to lower work addresses current incongruence at the levels (e.g., subfamilial, tribal; Alonso-Zarazaga higher levels of classification. and Lyal, 1999; Oberprieler et al., 2014; Oberpri- eler, 2014a). The exploration of informative anat- omy has not reached an asymptote for weevils, so METHODS this study was undertaken to deepen our knowl- Taxon Sampling edge of their morphology (see also Davis, 2014, for additional character systems). In order to Semithin sections were produced for all seven develop a more robust morphological character of the currently recognized, extant families system for cladistic analysis of the higher lin- within Curculionoidea, and 19 of 21 subfamilies eages and to gain a comprehensive understand- within Curculionidae (table 2). To adhere to a ing of rostrum structure (for current conservative assessment of current and evolving developmental work), a comparative study was views in the classification, the sampling design conducted of rostrum structure throughout Cur- followed that by Oberprieler et al. (2007, 2014) culionoidea. Semithin sections were made of the for families and and a combination of designs by rostra of exemplar taxa (table 2) representing all Alonso-Zarazaga and Lyal (1999) and Oberpri- seven weevil families, as well as 19 of 21 sub- eler (2014a) for subfamilies (particularly within families within Curculionidae (see methods sec- Curculionidae). To elucidate the variability of tion below), and internal structures were internal rostral morphology, sampling of more examined for useful characters. Although the than one taxon was undertaken when possible, morphological diversity of rostral forms is vast, i.e., within the families Anthribidae, Belidae, general trends in structure are apparent. Features Attelabidae, and Brentidae. Within Curculioni- 3 4 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY NO. 416 dae, sampling of more than one taxon was per- were mounted on SEM stubs using Leit-C-Plast formed for a few subfamilies, including adhesive and an isopropanol-based colloidal Brachycerinae, Dryophthorinae, Entiminae, graphite, and were then coated with gold. Cryptorhynchinae, Scolytinae, Curculioninae, and Molytinae. Of course, it would be of great Confocal Microscopy interest to extend this study to include a more diverse sampling, but the range covered herein is Confocal microscopy was performed on dis- considered adequate to make many well-sup- sected mouthpart components using a Zeiss LSM ported conclusions and hypotheses. 710 and collecting green and red spectra from autofluorescence of the cuticle. Histological Sectioning Microcomputed Tomography (μCT) Many adult taxa were acquired through per- sonal field collecting; several were also sampled Select weevil scans were done from ethanol- from dried and pinned collections. In the former preserved specimens that were subsequently case specimens were collected into 70% ethanol chemically dried using HMDS (hexamethyldisi- and later fixed in 4% paraformaldehyde before lazane) and scanned using a GE Phoenix v-tome- full dehydration in ethanol. In the latter case x μCT; scanning parameters were as follows: specimens were rehydrated by soaking them in voltage 70 kV, current 160 μA, number of images warm water for ~1 hour and then dehydrated in 1200, image acquisition time 333 ms, image aver- ethanol. Although sectioning previously dried ages 5, molybdenum target used. Reconstruction specimens is far from optimal for obtaining his- was done in GE Phoenix datosx 2 reconstruction tological information, sclerotized structures are software. Four separate scans were manually adequately preserved and were sufficient for this stitched in ImageJ, and segmentation done in study. After weevil heads were dehydrated in Volume Graphics Studio MAX v3.0. A rendered 100% ethanol, infiltration of head tissue con- μCT scan movie of the adult head of Rhynchites sisted of approximately 12 hour incubation peri- auratus (same as figures 1–7), showing internal ods through a series of 1:1 then 1:2 ethanol to LR anatomy, is available with this article online White® (an acrylic resin) mixtures. Heads were (https://doi.org/10.5531/sd.sp.27). placed in gelatin capsules and embedded in pure LR White®. Following thermal curing in an oven Phylogenetic Analysis for 24 hours at 60° C, embedded heads were removed from the capsules and sectioned using The morphological character matrix (table 1), a Leica EM UC6 ultratome and diamond knife, consisting of 19 adult rostral characters coded for producing semithin sections ~5–6 μm thick. Sec- 36 taxa, was constructed in WinClada (Nixon, tions were transferred to glass slides, stained in 1999b). Parsimony analyses were performed using toluidine blue, air dried, mounted in Permount™, Nona (Goloboff, 1999), in which the ratchet search and digitally photographed with a Canon EOS-1 algorithm (Nixon, 1999a) was implemented several camera mounted on an Olympus BX51 com- times under different search configurations to max- pound microscope. A z-stack was acquired of imize exploration of tree space and ensure conver- several photomicrographs using the software gence on a stable topology. Successive