Structure of the Lepidopteran Proboscis in Relation to Feeding Guild

Home , Cryphia, Eriocraniidae, Proboscis, Proboscis (butterfly), Rhodogastria, Scrobipalpa costella

JOURNAL OF MORPHOLOGY 00:00–00 (2015)

Matthew S. Lehnert,1,2* Charles E. Beard,2 Patrick D. Gerard,3 Konstantin G. Kornev,4 and Peter H. Adler2

1Department of Biological Sciences, Kent State University at Stark, North Canton, Ohio 44720 2Department of Agricultural and Environmental Sciences, Clemson University, Clemson, South Carolina 29634 3Department of Mathematical Sciences, Clemson University, Clemson, South Carolina 29634 4Department of Materials Science and Engineering, Clemson University, Clemson, South Carolina 29634

ABSTRACT Most butterflies and moths (Lepidoptera) (Monaenkova et al., 2012). A pump in the head use modified mouthparts, the proboscis, to acquire flu- then forces the liquid up the food canal to the gut ids. We quantified the proboscis architecture of five (Eberhard and Krenn, 2005; Borrell and Krenn, butterfly species in three families to test the hypothesis 2006; Lee et al., 2014). that proboscis structure relates to feeding guild. We Feeding guilds (i.e., groups of species with simi- used scanning electron microscopy to elucidate the fine structure of the proboscis of both sexes and to quantify lar feeding habits) have long been recognized in dimensions, cuticular patterns, and the shapes and the Lepidoptera and have been associated with sizes of sensilla and dorsal legulae. Sexual dimorphism higher taxa, such as nymphalid subfamilies or was not detected in the proboscis structure of any spe- tribes (Gilbert and Singer, 1975; Krenn et al., cies. A hierarchical clustering analysis of overall pro- 2001). Adult Lepidoptera are conventionally cate- boscis architecture reflected lepidopteran phylogeny, gorized into at least two broad feeding guilds: but did not produce a distinct group of flower visitors flower visitors (nectar feeders) and nonflower visi- or of puddle visitors within the flower visitors. Specific tors (nonnectar feeders), the latter drinking from characters of the proboscis, nonetheless, can indicate wetted surfaces, such as sap flows and rotting flower and nonflower visitors, such as the configuration fruit. Within the flower-visiting guild, the males of of sensilla styloconica, width of the lower branches of dorsal legulae, presence or absence of dorsal legulae at some species routinely drink from damp soil the extreme apex, and degree of proboscis tapering. We (“puddling” sensu Arms et al., 1974). suggest that the overall proboscis architecture of Lepi- Selected features of proboscis architecture have doptera reflects a universal structural organization been associated with adult-feeding habits (Krenn that promotes fluid uptake from droplets and films. On et al. 2001; Monaenkova et al., 2012; Lehnert et al., top of this fundamental structural organization, we 2013; Kwauk et al., 2014; Tsai et al., 2014). Tearing suggest that the diversity of floral structure has and rasping spines and other cuticular modifica- selected for structural adaptations that facilitate entry tions, for example, are on the proboscises of moths of the proboscis into floral tubes. J. Morphol. 000:000– that routinely feed on lachrymal secretions and 000, 2015. VC 2015 Wiley Periodicals, Inc. pierce animal tissue to feed on blood (Banziger,€ KEY WORDS: butterfly; flower visiting; fluid uptake; 1971; Buttiker€ et al., 1996; Hilgartner et al., 2007; mouthparts; nectar feeding; sap feeding Zaspel et al., 2011). Flower-visiting butterflies have darker proboscises (Krenn et al., 2001) and significantly longer proboscises relative to their bodies than do nonflower visitors (Kunte, 2007). Enlarged, INTRODUCTION densely arrayed chemo-mechanoreceptors, that is, Approximately 28% of all fluid-feeding insects are butterflies and moths in the Lepidoptera (Adler and Foottit, 2009). More than 95% of the Contract grant sponsor: National Science Foundation (award number 1354956); Contract grant sponsor: NIH; Grant number: Lepidoptera acquire fluids by means of a tubular 5P02GM103444; Contract grant sponsor: NIFA/USDA. proboscis (Krenn, 1990, 2010). The feeding device consists of two medially concave, elongated maxil- *Correspondence to: Matthew S. Lehnert; 6000 Frank Ave. NW, lary galeae joined dorsally and ventrally by cuticu- North Canton, OH 44720. E-mail: [email protected] lar projections, termed “legulae,” forming a food canal (Eastham and Eassa, 1955; Hepburn, 1971; Received 27 August 2015; Revised 10 October 2015; Krenn, 1997). Spaces between the dorsal legulae Accepted 18 October 2015. facilitate capillary action, supporting the with- Published online 00 Month 2015 in drawal of liquids from pools and porous sub- Wiley Online Library (wileyonlinelibrary.com). strates, such as rotting fruit, into the food canal DOI 10.1002/jmor.20487

VC 2015 WILEY PERIODICALS, INC. 2 M.S. LEHNERT ET AL. sensilla styloconica (Altner and Altner 1986) form a from porous substrates, such as rotting fruit and tree sap brushy tip in nonflower-visiting butterflies (Krenn (Scott, 1986). et al., 2001; Knopp and Krenn, 2003; Petr and Danaus plexippus was received as pupae or adults from Shady Oak Butterfly Farm (Brooker, Florida) or were labora- Stewart, 2004; Krenn, 2010), which takes up fluid tory reared on milkweed (Asclepias spp.). Larvae of P. rapae, more effectively from liquid films (Molleman et al., from Carolina Biological Supply Co. (Burlington, NC) were 2005). We recently discovered that these sensilla reared on artificial diet. Adults of P. glaucus, L. a. astyanax, are hydrophilic, and function like a sponge when and P. interrogationis were captured as adults or reared from 0 0 arranged as a brush (Lehnert et al., 2013), a mech- larvae collected in Clemson, South Carolina (N34839 , W82850 ), from April to September 2011. Voucher specimens were depos- anism further studied by Lee and Lee (2014). In ited in the Clemson University Arthropod Collection. addition, some nonflower-visiting butterflies have a more elliptical proboscis in cross-section, compared Scanning Electron Microscopy with the condition of flower visitors (Lehnert et al., Lepidopteran heads were secured and proboscises straight- 2013). When an elliptical proboscis is dipped into a ened with insect pins on polystyrene foam where they remained liquid, the meniscus rises to a higher level com- through a series of ethanol washes (80%, 95%, 100%; ca. 24 h pared with that of a circular proboscis (Lehnert each) followed by chemical drying in hexamethyldisilazane. et al., 2013; Alimov and Kornev, 2014), which might Specimens were attached to a scanning electron microscope help Lepidoptera to engage more interlegular (SEM) mount with carbon-graphite adhesive tape and sputter- coated with gold or platinum for 1–3 min. A Hitachi TM-3000 spaces for liquid uptake (Kwauk et al., 2014). SEM was used to photograph the dorsum of each proboscis at In accord with the hypothesis that structural 503 magnification, 15 kV, and full vacuum. Selected areas were organization of an organismal device is matched to observed at 1503 or higher magnifications. Composite images its functional demand (Weibel et al., 1991), we of each proboscis were assembled in AdobeVR Photoshop CS2 (Adobe Systems) and used for measurements and illustrations. looked for a testable framework to facilitate the ImageJ software (Rasband, W.S., ImageJ, U. S. National Insti- prediction of general feeding habits (guilds) of but- tutes of Health, Bethesda, Maryland, USA, http://imagej.nih. terflies. Therefore, we asked the following ques- gov/ij/, 1997-2015) was used to acquire measurements. tion: Can visible structural features of the proboscis predict feeding guild? To test the hypoth- Measurements esis that species with similar feeding habits have We used SEM to ensure accuracy of proboscis measurements structurally similar proboscises, we examined 21 and character assessments. Overall, we quantified or catego- proboscis characters for three guilds of butterflies: rized a total of 21 characters for 11–20 (typically 20) specimens of each of the five species (Tables (1–3)). We compared charac- flower visitors, flower-visiting puddlers, and non- ters between sexes for each species [P. glaucus 11 females and flower visitors. To provide a robust analysis of the 9 males for all measured characters, except number of hand- overall proboscis landscape, we built on the study switches (10f, 8m); D. plexippus 10f, 10m; L. a. astyanax 11f, of nymphalid butterflies by Krenn et al. (2001), 9m, except number of handswitches (6f, 6m), widths of Zone 1 and included characters recently associated with (8f, 8m) and Zone 2 (10f, 7m); P. interrogationis 5f, 14m, except for handswitches (3f, 11m), widths of Zone 1 (5f, 13m) and Zone fluid uptake, such as attributes of the dorsal legu- 2 (5f, 13m); P. rapae 8f, 12m, except for handswitches (2f, 5m), lae (Monaenkova et al., 2012; Lehnert et al., and sensilla styloconica stylus length and peg length (6f, 9m)]. 2013), as well as characters with unknown func- Composite SEM images (503 magnification) were used to tions. We also tested the hypothesis that males define areas (i.e., zones) of the proboscis to assist in structural comparisons among species. These structural zones correspond and females differ in the structure of their probos- with well-defined functional zones, that is, the drinking and cises, and predicted differences in the puddling nondrinking regions (Monaenkova et al., 2012; Lehnert et al., group because predominantly males exhibit this 2013). Upper and lower branches of dorsal legulae (Lehnert behavior (Arms et al. 1974). In analyzing the et al., 2013) were present along more than 95% of the length of structural characters, we considered only their each galea of all species, and the width of the upper branch was used to designate structurally defined zones. In ImageJ geometrical features and not their materials software, a straight line was drawn from the base to the tip of properties. a single dorsal legula at the base of the proboscis (Fig. 1). The line was held at constant length and moved distally along the bases of the dorsal legulae until the width of the legulae consis- MATERIAL AND METHODS tently enlarged, signifying the end of one zone and the begin- Species ning of a second zone (cf. Fig. 1b of Lehnert et al., 2013). A third zone was designated on proboscises that lacked dorsal Five species representing three families served as test sub- legulae at the distal tip (Figs. 1 and 2). jects. We selected the monarch butterfly, Danaus plexippus Structural comparisons within and among species included (Linnaeus, 1758) (Nymphalidae), to represent Lepidoptera with total proboscis lengths, zone lengths, percent zone lengths (503 predominantly flower-visiting (i.e., nectar-feeding) habits magnification), galeal widths (measured at the middle of Zones (Brower, 1961); the cabbage butterfly, Pieris rapae (Linnaeus, 1 and 2 and including sensilla styloconica when present, 1503 1758) (Pieridae), and the eastern tiger swallowtail, Papilio magnification), and average widths of upper and lower legular glaucus Linnaeus, 1758 (Papilionidae), to represent flower visi- branches in Zones 1 and 2 (based on five randomly chosen dor- tors with males that also routinely puddle (Arms et al., 1974; sal legulae near the middle of each zone per specimen, 1503 Adler and Pearson, 1982); and the red-spotted purple, Limenitis magnification) (Fig. 1). The medial tips of dorsal legulae were arthemis astyanax (Fabricius, 1775) (Nymphalidae), and the examined in Zone 2 for the presence of a toothlike projection. question mark, Polygonia interrogationis (Fabricius, 1798) The forewing length of each specimen was measured as a possi- (Nymphalidae), to represent nonflower visitors that feed chiefly ble covariate of proboscis length. Zones 1 and 2 each were

Journal of Morphology TABLE 1. Forewing and proboscis measurements (means 6 SE) for ﬁve butterﬂy species Zone length (mm) (Percent total proboscis length) Galea width (lm) (n) Flower Forewing Proboscis Species visitor n length (mm) length (mm) Zone 1 Zone 2 Zone 3 Zone 1 Zone 2 P. glaucus Yes (p) 20 56.2 6 0.95a 17.95 6 0.40a 15.29 6 0.35a 2.65 6 0.08a 0.01 6 0.0b 229.78 6 6.05b 113.87 6 3.86b (85.2%)D (14.7%)B (0.1%)A P. rapae Yes (p) 20 24.55 6 0.35e 9.46 6 0.16c 8.75 6 0.16d 0.70 6 0.02d 0.02 6 0.0a 136.62 6 2.86d 53.61 6 2.34c (92.5%)A (7.5%)E (<0.1%)A D. plexippus Yes 20 49.65 6 0.74b 14.39 6 0.26b 13.06 6 0.24b 1.31 6 0.04c 0.01 6 0.0b 241.91 6 4.39b 106.83 6 2.75b (90.8%)B (9.1%)D (0.1%)A L. a. astyanax No 20 42.45 6 0.82c 13.17 6 0.18c 10.74 6 0.17c 2.43 6 0.04b NA 297.81 6 9.54a 201.39 6 6.38a (81.5%)E (18.5%)A (16) (17) PROBOSCIS BUTTERFLY OF STRUCTURE P. interrogationis No 19 31.21 6 0.39d 12.77 6 0.26c 11.33 6 0.23c 1.44 6 0.04c NA 201.53 6 4.69c 114.06 6 2.82b (88.7%)C (11.3%)C (18) (18)

Lowercase and uppercase letters indicate signiﬁcant differences (P < 0.05) within columns, as determined using Fisher’s LSD test. p, Puddling by males; NA, Zone 3 does not exist. Sample size (n) is given in parentheses only when it differs from the sample size presented in the n column.

TABLE 2. Measurements (means 6 SE) and characteristics of dorsal legulae for ﬁve butterﬂy species

Dorsal legulae width (mm) Upper Lower Upper No. of Dorsal legulae Origin of branch branch branch handswitches arrangement dorsal Toothlike Species n (Zone 1) (Zone 1) (Zone 2) in Zone 1 (n) (Zone 2) legulae* projections P. glaucus 20 37.91 6 0.98a 46.68 6 1.02c 71.7 6 2.23b 7.17 6 1.5b (18) Not overlapping 3 Absent P. rapae 20 13.7 6 0.47b 45.46 6 1.46c 26.03 6 1.03e 105.86 6 27.96a (7) Overlapping 3 Absent

ora fMorphology of Journal D. plexippus 20 7.56 6 0.58d 43.02 6 1.26c 45.81 6 1.23d 0.15 6 0.08b Not overlapping 2 Absent L. a. astyanax 20 11.05 6 0.56c 77.22 6 2.88a 81.18 6 1.88a 2.92 6 1.06b (12) Overlapping 2 Present P. interrogationis 19 10.2 6 0.32c 60.17 6 2.13b 60.04 6 1.89c 2.86 6 0.98b (14) Overlapping 1 Present

*Arising from base of lower branch (1) or galeal wall (2), or well-developed along proboscis length, except in Zone 3 (3). Lowercase letters indicate signiﬁcant differences (P < 0.05) within columns, as determined using Fisher’s LSD test. Sample size (n) is given in parentheses only when it differs from the sample size presented in the n column. 3 4 M.S. LEHNERT ET AL.

) divided into three sections of equal length and the presence of n sensilla was determined in the middle section of each zone if

0.66a they were within 20 mm (basiconica, styloconica) or 50 mm (tri- 0.24b

6 chodea) of the base of the dorsal legulae. The average widths of 0.18c (15) 6

6 five randomly chosen sensilla styloconica were determined for each specimen (stylus and peg separately). The galeal surface sculpture was observed at 503 and higher magnifications in Zone 1. The number of switches in handedness of the dorsal legulae was determined throughout Zone 1 at 1503 magnification. ) Peg length ( n

llum styloconicum, whereas Statistics 2.46a 20.95 1.93b 8.76 6 6 Analysis of covariance was used to compare species with 0.17c (15) 5.01

6 regard to proboscis length, adjusting for forewing length as a proxy for body size (Miller, 1977). Analysis of variance was used to test for significant differences (P < 0.05) in proboscis lengths, zone lengths and widths, percent zone lengths, legular widths, handswitches, and stylus and peg lengths of sensilla styloconica between sexes within and among species. Signifi- cant differences in means among species were compared using Fisher’s least significant difference (LSD) test. An agglomerative hierarchical clustering algorithm using average linkage distance (Johnson and Wichern, 2007) was employed to simultaneously evaluate the means of 13 proboscis characters for the five species. A dendrogram depicting the results of the hierarchical cluster analysis was produced, cali- brated from 1.0 to 1.5, representing a matrix of distances. Characters used for analysis included proboscis length, percent

SE) in Zone 2 on proboscises of ﬁve butterﬂy species length of Zone 2, number of handswitches in Zone 1, width of 6

column. galea in Zones 1 and 2, percentage change in width between

n Zones 1 and 2, width of the lower and upper branches of dorsal legulae in Zone 1, percentage of lower branch of dorsal legulae covered by upper branch in Zone 1, width of upper branch of dorsal legulae in Zone 2, dorsal legular arrangement, presence/ absence of a toothlike projection at medial tips of dorsal legu-

) Arrangement Distribution Stylus length ( lae, and presence/absence of styloconica sensilla. Nonordinal n categorical variables and variables based on measured structures that were absent in certain species were omitted from the clustering analysis. All statistical analyses were conducted in SAS version 9.3.

Terminology Studies of the lepidopteran proboscis have produced a mix of functional, structural, and positional terminology (Eastham and Eassa, 1955; Hepburn, 1971; Kingsolver and Daniel, 1979; Krenn, 1990, 1998; Walters et al., 1998; Krenn and Muhlberger,€ 2002; Krenn and Kristensen, 2004; Petr and Stewart, 2004; Zaspel et al., 2011; Monaenkova et al., 2012; Lehnert et al., 2013). We, therefore, explicitly deﬁne the terms that we use. Dorsal legula (pl., legulae) (Figs. 1–12)—cuticular exten-

) Styloconica Basiconica ( sion(s) along the dorsal midline of the proboscis. Each dorsal n legula consists of an upper and a lower branch. The lower branches of opposing galeae overlap in the nondrinking region Presence (P) or absence (A*) of sensillaof the Styloconica proboscis, but are more widely spaced in the drinking region (Lehnert et al., 2013) where they overlap little or not at all. The upper branches are enlarged and more widely spaced Trichodea ( in the drinking region and can have a toothlike projection at the medial tip. The upper branches are either well developed along the length of the proboscis or appear to originate from

n other structures in the nondrinking region, such as bumps at 2020 P P A* P P P NA Sparse Scattered NA 5.32 NA NA 2020 P P A* P P P NA Dense Uniform NA 117.57 NA NA 19 P (14) P P (11) Dense Uniform 67.28 the bases of the lower branches of dorsal legulae or as small,

TABLE 3. Arrangement of sensilla trichodea, basiconica, and styloconica (means triangular spines on the galeal wall adjacent to the lower branch. Longitudinal groove (Figs. (5 and 12))—trough of variable ) is given in parentheses only when it differs from the sample size presented in the

n width that runs lengthwise along the dorsum of each galea for part of the proboscis length, usually next to a longitudinal ridge that separates the trough and dorsal legulae. The longitudinal

Species groove is not present in all species. Microbumps (Fig. 12)—minute tubercles of various shapes P. glaucus P. rapae D. plexippus L. a. astyanax P. interrogationis *Chemosensilla are reduced ina size stylus and shorter do thanNA, its not Not peg meet applicable. indicated ourSample a classiﬁcation size sensillum criteria ( basiconicum. for sensilla styloconica; a stylus longer than its peg indicated a sensi on the galeal surface. The distribution pattern of microbumps

Journal of Morphology STRUCTURE OF BUTTERFLY PROBOSCIS 5

Fig. 1. Schematic of the proboscis of P. glaucus, with scanning electron micrographs (insets A–E), showing measurements of linear features. (A) Upper (ub) and lower (lb) branches of dorsal legulae were measured from their bases to the distal tip at the midpoint of Zone 1 (indicated by red box on illustration). (B) The transition from Zone 1 to Zone 2 (indicated by black line) is based on dorsal legular (dl) widths (Monaenkova et al., 2012). Sensilla trichodea (st) on the galea (ga) were counted if they were within 50 mmofthe dorsal legulae. (C) The presence of Zone 3 was based on absence of dorsal legulae; the boundary of Zones 2 and 3 is indicated by a black line. Sensilla basiconica (sb) were recorded if they were within 20 mm of the dorsal legulae. (D) Midpoint of Zone 1 indicating where galeal width was measured (red line). (E) Galeal width also was measured at the midpoint of Zone 2. Scale as in Figure 2. can change along the length and width of the proboscis, but lap those on the right galea. Switches in handedness can occur tends to be expressed as a series of microbumps perpendicular multiple times along the proboscis. to the proboscis midline. Sensillum styloconicum (pl., sensilla styloconica)(Figs.(3and Microvalley (Fig. 12)—gap between neighboring microbumps 6), and 8211)—sensory organ with a stylus longer than its peg. within a series of microbumps. Sensilla styloconica can be sparse (more than one sensillum Macrovalley (Fig. 12)—trough between each series of micro- apart) or dense (less than one sensillum apart), and their distri- bumps. Macrovalleys are typically wider and deeper than bution can be uniform (in a single row) or scattered (not in a sin- microvalleys. gle row). Various criteria are used to distinguish sensilla Proboscis handedness (Fig. 5)—arrangement determined by styloconica from sensilla basiconica (Petr and Stewart, 2004; which galea has dorsal legulae overlapping the dorsal legulae Bauder et al., 2013; Faucheux, 2013), which with the presence of of the adjacent galea. A proboscis is considered left-handed, for potentially intermediate forms of sensilla styloconica (Paulus instance, if the dorsal legulae on the organism’s left galea over- and Krenn, 1996), confound classiﬁcation of sensory structures.

Journal of Morphology 6 M.S. LEHNERT ET AL.

Fig. 2. Butterflies and their proboscises (distal region to right). The top two proboscises indicate the relative zone lengths of nonflower visitors (P. interrogationis and L. a. astyanax), followed by the proboscis of the flower visitor, D. plexippus. The two bottom proboscises represent flower visitors with male puddling habits (P. glaucus and P. ra pa e). The black bars indicate 1 mm.

We adopted the terminology and criteria used by Petr and Stew- spaced upper branches of dorsal legulae than those in Zone 1. art (2004), and use an operational classification scheme based This zone is hydrophilic (Lehnert et al., 2013). solely on the size of the stylus in relation to the peg. Zone 3 (Figs. 1–3)—distalmost region of the proboscis that Sensillum basiconicum (pl., sensilla basiconica) (Figs. (3 and lacks dorsal legulae; it is not present in all species. This zone is 6), and 9)—sensory organ with a stylus shorter than its peg. hydrophilic and, hence, a functional subset of Zone 2, the drink- Sensillum trichodeum (pl., sensilla trichodea) (Figs. (6 and ing region (Lehnert et al., 2013). 9), and 12)—hair-like or spine-like sensory organ without a stylus or peg. Sensilla trichodea are treated here as synonymous with sensilla cheatica (Xue and Hua, 2014), although Faucheux RESULTS (2013) suggested functional differences. Sexual Dimorphism Zone 1 (Figs. 1–426, and 12)—nondrinking region of the proboscis that extends from the junction of the proboscis with the No significant sexual dimorphism was detected head to where the upper branches of dorsal legulae begin to for any variable of any species (df 5 4, P > 0.05). enlarge. This zone is hydrophobic (Lehnert et al., 2013). Zone 2 (Figs. 124, 628, and (10 and 11))—drinking region of Proboscis Length the proboscis that extends from where the upper branches of dorsal legulae begin to widen to where dorsal legulae are no Proboscis lengths differed significantly among longer present. Zone 2 is characterized by larger, more widely species (F 5 136.41; df 5 4, 94; P < 0.0001) and

Journal of Morphology STRUCTURE OF BUTTERFLY PROBOSCIS 7

Fig. 3. Scanning electron micrographs indicating the presence or absence of Zone 3 on butterfly proboscises. A and B show the dorsal and lateral view of Zone 3 on a galea of P. rapae, respectively. Zone 3 is characterized by a proboscis tip (tp) that lacks dorsal legulae (dl). A also indicates the sensilla styloconica (ss) and basiconica (bs), and ventral legulae (vl) on proboscises of P. rapae. C and D represent the tips of proboscises of nonflower visitors, L. a. astyanax (C) and P. interrogationis (D). C shows the toothlike projection that extends from the medial tip of the dorsal legulae. The nonflower visitors have dorsal legulae extended to the tip and, therefore, lack Zone 3. B shows that Zone 3 extends beyond the food canal (fc). were longest in P. glaucus (Table 1). Forewing than did other flower visitors (F 5 164.61; df 5 2, length differed among species (F 5 345.51; df 5 4, 57; P < 0.0001; Table 1). 94; P < 0.0001), and was not a significant covariate The galeal widths in Zone 1 (F 5 106.57; df 5 4, of proboscis length in P. rapae or P. interrogatio- 89; P < 0.0001) and Zone 2 (F 5 189.82; df 5 4, 90; nis, but was in the other species. P < 0.0001) differed significantly among species, with L. a. astyanax having the widest Zones 1 and 2 (Table 1). Measurements in the middle of Zones Zone Dimensions 1 and 2 indicated that the proboscis tapered dis- Zone 1 occupied more than 80% of the total pro- tally in all species, with the greatest reduction of boscis length for all species (Table 1; Fig. 2). Zone width (>50%) in D. plexippus, P. glaucus, and P. lengths differed significantly among species (Zone rapae. Polygonia interrogationis had a 43.4% 1, F 5 108.17; df 5 4, 94; P < 0.0001), as did the reduction in width and L. a. astyanax a 32.4% percent of proboscis length represented by each reduction, when including sensilla styloconica. zone (Zone 1, F 5 280.20; df 5 4, 94; P < 0.0001; Table 1). The percent of the proboscis length desig- Dorsal Legulae—Upper Branches (Zone 1) nated as Zone 2 was greatest in L. a. astyanax The upper branches of the dorsal legulae varied and shortest in P. rapae. Pieris rapae and P. glau- in width, shape, and point of origin among species cus had the shortest and longest average proboscis (Table 2, Fig. 4). Widths of the upper branches in lengths, respectively, and did not have similar pro- Zone 1 differed significantly among species portions of the proboscis represented by each zone (F 5 392.80; df 5 4, 94; P < 0.0001) and were widest (Table 1; Fig. 2). Zone 3 was absent in nonflower in P. glaucus and P. rapae.InP. glaucus, they visitors (Fig. 3). The proboscis of P. rapae had a were nearly as wide as the lower branches significantly greater percentage length for Zone 3 (Fig. 4A). The upper branches of dorsal legulae in

Journal of Morphology 8 M.S. LEHNERT ET AL. or pentagonal and nearly ﬂush with the ridge of the galeal surface.

Dorsal Legulae—Lower Branches (Zone 1) The lower branches of dorsal legulae in Zone 1 were pointed in the Nymphalidae and spatula shaped in P. rapae, but were partially covered by the upper branches in P. glaucus (Figs. 4 and 5). The number of handswitches differed signiﬁcantly among species (F 5 32.62; df 5 4, 66; P < 0.0001). Handswitches occurred along Zone 1 an average of <7 times in all species except P. rapae, which had signiﬁcantly more handswitches, producing a zipper-like appearance (Table 2; Fig. 5B). Hand- switches in P. glaucus uniquely occured among the upper branches of dorsal legulae (Fig. 5A).

Dorsal Legulae—Widths All species exhibited a structural change of the upper branches of dorsal legulae, demarcating Zones 1 and 2 (Table 2; Fig. 6); however, the changes in overall dorsal legular widths between these zones differed among species, regardless of whether the upper or the lower branches were wider. Dorsal legulae of P. glaucus were approximately 50% wider in Zone 2 than in Zone 1, were of similar width between zones in the Nymphali- dae, and decreased in width in P. rapae (Table 2). In P. rapae, the upper branches of dorsal legulae approximately doubled in width from Zone 1 to Zone 2; however, the lower branches decreased in width in Zone 2, resulting in a reduction in overall dorsal legular width. The upper and lower branches of P. rapae formed a groove between them in Zone 2 (Fig. 7B).

Dorsal Legulae—Shapes of Upper Branches in Zone 2 The dorsal legulae in Zone 2 of L. a. astyanax, P. Fig. 4. Scanning electron micrographs of the origin and transi- interrogationis, and P. rapae overlapped in tion of the upper branches of dorsal legulae. A shows enlarged upper branches (ub) of dorsal legulae in Zone 1 of proboscises of shingle-like fashion (Table 2, Fig. 7), resulting in P. glaucus, which enlarge and demarcate Zones 1 and 2 (inset). interlegular spacing that was not completely visi- B and C show smaller upper branches of dorsal legulae in Zone ble from the dorsum (Fig. 8). The dorsal legulae of 1ofL. a. astyanax and P. interrogationis, respectively. The lower D. plexippus and P. glaucus were positioned side- branches (lb) of dorsal legulae are apparent in Zone 1, whereas the upper branches of dorsal legulae in L. a. astyanax (shown in by-side and were nearly ﬂat, with the enlarged B) appear as triangular projections from the galeal wall. The interlegular spaces visible from the dorsum (Fig. upper branches on proboscises of P. interrogationis appear as 8). Polygonia interrogationis and L. a. astyanax small bumps at the base of the lower branches of dorsal legulae had a toothlike projection on the tip of each dorsal in Zone 1. All species have upper branches of dorsal legulae enlarged, demarcating Zones 1 and 2 (insets). legula (Fig. 3; Table 2). The upper branches of L. a. astyanax were serrated distally (Figs. 7 and 8).

Sensilla Zone 1 of the three Nymphalidae were not well developed. The upper branch of a dorsal legula of Sensilla basiconica and trichodea were present P. interrogationis in Zone 1 was a small bump at on all proboscises (Fig. 9). Sensilla styloconica, the base of the lower branch, whereas the upper however, were found only on proboscises of L. a. branch of L. a. astyanax was a small, triangular astyanax, P. interrogationis, and P. rapae (Figs. 9 protrusion on the adjacent galeal wall (Fig. 4B,C). and 10). The short chemosensilla in Zones 2 and 3 The upper branch of D. plexippus was rectangular on proboscises of P. glaucus and D. plexippus were

Journal of Morphology STRUCTURE OF BUTTERFLY PROBOSCIS 9

Fig. 5. Scanning electron micrographs showing handswitches of dorsal legulae in Zone 1. (A) Handswitches (hs) on proboscises of P. glaucus occur with the upper branches of dorsal legulae (dl). Handswitches in other species occur solely with the lower branches, shown here with proboscises of P. ra pa e (B)andL. a. astyanax (C). Proboscises of P. rapae have a large number of handswitches that appear zipper-like. A galea (ga) is shown as a reference in each image. The longitudinal groove (lg) on the proboscis of P. glaucus appears near the dorsal legulae. classiﬁed as sensilla basiconica (according to crite- (Fig. 12). These patterns also changed along the ria outlined in our Terminology section above). proboscis length and width within species. Papilio The sensilla styloconica were dense and uniformly glaucus, D. plexippus, and P. interrogationis had distributed in rows in L. a. astyanax and P. inter- similar microbump patterns on the lateral surface rogationis, giving Zone 2 an overall brush-like of the galeae, which consisted of rows of circular appearance (Fig. 11, Table 3), but were sparse and microbumps, with microvalleys interspersed by scattered in P. rapae. The stylus and peg lengths macrovalleys. The rows of circular microbumps of sensilla styloconica differed signiﬁcantly among continued medially to the longitudinal groove near species (F 5 768.92; df 5 2; P < 0.0001 and the dorsal legulae of D. plexippus, but transitioned F 5 334.35; df 5 2; P < 0.0001, respectively) and to a zigzag pattern in P. glaucus and were spine- were of different shapes (Fig. 10, Table 3). like in P. interrogationis. The microbump pattern of L. a. astyanax produced a wrinkled appearance, which became spine-like near the dorsal legulae Microbumps, Microvalleys, and Macrovalleys (Fig. 12). In P. rapae, it consisted of crosswise Each species had unique shapes and patterns ridges with a series of spike-like microbumps and of microbumps and micro- and macrovalleys broad macrovalleys (Fig. 5B) that transitioned into

Fig. 6. Scanning electron micrographs showing the boundary of Zones 1 and 2. All proboscises have upper branches (ub) of dorsal legulae that abruptly enlarge, indicating the transition (tr) from Zone 1 to 2. The lower branches (lb) of dorsal legulae are prevalent throughout Zone 1 in all species except P. glaucus, where they are hidden by the upper branches. Zone demarcation was observed in proboscises with relatively smooth galeae (ga) along the proboscis length (e.g., D. plexippus, A) and proboscises with sensilla styloconica (ss) (e.g., L. a. astyanax, B). Sensilla trichodea (ts) are on the galeal wall, but are more abundant in Zone 2.

Journal of Morphology 10 M.S. LEHNERT ET AL.

Fig. 7. Scanning electron micrographs of the enlarged dorsal legulae in Zone 2. Upper branches (ub) of dorsal legulae of all species are wider in Zone 2 than in Zone 1; however, the arrangement and shape of the dorsal legulae differs among species. Limenitis a. astyanax (A)andP. rapae (B) have dorsal legulae that overlap shingle-like in Zone 2. The lower branches (lb) of dorsal legulae in L. a. astyanax appear as large triangular projections, whereas the upper branches are serrated. Upper and lower branches of dorsal legulae of P. rapae fuse together, forming a channel-like groove. bumps in Zone 2. The microbump patterns of L. a. P. interrogationis for approximately 10% of the pro- astyanax and P. interrogationis in Zone 2 were boscis length, but was absent in the other species. obscured by sensilla styloconica (Fig. 11). Grouping of Species Based on Proboscis Structure Longitudinal Grooves A dendrogram (Fig. 13) produced from the hier- The proboscises of L. a. astyanax and P. inter- archical clustering analysis of 13 proboscis charac- rogationis had a wide longitudinal groove near the ters showed that the nonflower visitors L. a. ridge that ran nearly the length of Zone 1 into astyanax and P. interrogationis clustered together. Zone 2. The longitudinal groove also was present The flower visitor D. plexippus clustered with in D. plexippus and P. glaucus, but narrower, and these two species, and the flower visitor and pud- was absent in P. rapae. A single row of hair-like dler P. rapae, in turn, clustered with this group of setae (Fig. 2) occupied the longitudinal groove of three species. Papilio glaucus was the most dis- each galea near the base of the proboscis of tant from the other groups.

Fig. 8. Scanning electron micrographs showing the dorsal legular arrangement and interlegular spaces. Nonﬂower visitors have dorsal legulae (dl) in Zone 2 that overlap (L. a. astyanax with sensilla styloconica [ss] shown in image A), whereas ﬂower visitors have dorsal legulae that do not overlap (P. gl au cu s shown in B). Differences in dorsal legular arrangement are coupled with differences in the arrangement of the corresponding interlegular spaces (il).

Journal of Morphology STRUCTURE OF BUTTERFLY PROBOSCIS 11

Fig. 9. Scanning electron micrographs of sensilla on proboscises. Sensilla trichodea (ts) are on the galeae (ga) of all species (L. a. astyanax shown in A). Sensilla styloconica (ss) are on nonﬂower visitors and P. rapae; however, they are denser and more uniform in distribution in Zone 2 on proboscises of nonﬂower visitors (sensilla styloconica of L. a. astyanax shown in B). Sensilla basiconica (bs) are present on all species (D. plexippus shown in C).

DISCUSSION boscis architecture, however, does permit grouping Does Overall Proboscis Landscape Reflect of the nonflower visitors. Feeding Habits? The absence of a distinct grouping of flower visi- Although adult Lepidoptera typically feed oppor- tors, based on an overall set of proboscis characters, does not mean that a flower-visiting species tunistically, they often are grouped as flower visi- cannot be categorized on the basis of selected tors and nonflower visitors (Gilbert and Singer, structural features. Our study taxa and many 1975; Krenn et al., 2001; Knopp and Krenn, 2003; examples in the literature (Krenn et al., 2001; Lehnert et al., 2013). Our data, however, do not Knopp and Krenn, 2003; Molleman et al., 2005; support the hypothesis that external proboscis Lehnert et al., 2013) can be categorized as flower architecture, based on an overall set of proboscis visitors or nonflower visitors. How, then, can our characters, indicates a distinct guild of flower visi- clustering analysis be reconciled with the ability tors. The smooth proboscis of D. plexippus, for to recognize a flower visitor on the basis of visible instance, groups with the brushy proboscises of structural characters of its proboscis? The answer P. interrogationis and L. a. astyanax. Overall pro-

Fig. 10. Structures measured and shapes of sensilla styloconica. Sensilla styloconica consist of a peg and stylus (A). The length of the peg (pe) and stylus (st) and distance between sensilla styloconica and dorsal legulae (dl) differ among L. a. astyanax (B), P. in ter - rogationis (C), and P. rapae (D). Sensilla styloconica are dense and ﬂattened in nonﬂower visitors (L. a. astyanax, P. interrogationis), but scattered and rounded on the proboscis of P. rapae.

Journal of Morphology 12 M.S. LEHNERT ET AL.

Fig. 11. Scanning electron micrographs showing differences in overall appearance of Zone 2 between butterflies of different feeding habits. Zone 2 of flower visitors, such as D. plexippus (A), have sensilla basiconica (bs) and nonoverlapping dorsal legulae (dl). Non- flower visitors, such as L. a. astyanax (B), have enlarged sensilla styloconica (ss) and overlapping dorsal legulae. lies in the overlay of structural modifications for for fluid uptake are present in the oldest extant specialized feeding habits on the generalized struc- haustellate Lepidoptera, such as the Eriocraniidae tural arrangement of all lepidopteran proboscises. (Krenn and Kristensen, 2000; Monaenkova et al., The fundamental organization of all lepidopteran 2012) and, therefore, date to more than 100 mya proboscises—a tapered, porous tube with a rough (Grimaldi and Engel, 2005). Lepidoptera that feed galeal surface often bearing slender cuticular pro- from flowers must be able to acquire fluids not jections well depicted by Krenn and Kristensen only from pools, but also from droplets and films (2000)—reflects adaptations for capillary action in the corolla when nectar is in limited supply and the acquisition of liquid from surface films (Monaenkova et al., 2012). and droplets, including those in floral corollas that With the diversification of flowering plants, the often provide only a trace of nectar (Monaenkova early Lepidoptera bearing fibrous proboscises et al., 2012; Lehnert et al., 2013; Kwauk et al., would have been structurally positioned to exploit 2014). These fundamental structural adaptations new food sources such as flowers with nectar.

Fig. 12. Scanning electron micrographs showing proboscis surface patterns and topography. A shows the longitudinal groove (lg) on proboscises of L. a. astyanax near the dorsal legulae, with lower branches (lb) and triangular upper branches (ub). Sensilla trichodea (ts) and basiconica (bs) are in or near the longitudinal grooves. The microbump (mb) pattern on proboscises of L. a. astyanax changes from a bumpy texture near the dorsal legulae to rows of bumps on ridges interspersed by macrovalleys. The proboscises of D. plexippus (B) have longitudinal grooves that are narrower than those of L. a. astyanax and microbumps with microvalleys (mi) and macrovalleys.

Journal of Morphology STRUCTURE OF BUTTERFLY PROBOSCIS 13

Fig. 13. Dendrogram depicting agglomerative hierarchical clustering with average linkage distance of means of 13 proboscis characters for ﬁve species of butterﬂies. Although the dendrogram implies no ancestral or derived relationships and is not equivalent to a cladogram, it nonetheless mirrors the topology of current phylogenetic relationships (e.g., Regier et al., 2013).

Subsequent modifications of the proboscis would addition, puddling behavior, although often have adapted flower-visiting lepidopterans to reported as a male-specific behavior, has been increased foraging efficiency from tubular flowers. reported in females of P. glaucus and P. canaden- Proboscis characters unique to flower visitors, sis (Scriber, 1987, 2002). Hence, the apparent such as a reduced sensillar brush (Krenn et al., absence of sexual dimorphism and lack of struc- 2001) and tapering, are probably related to flower tural indicators in the proboscis of puddling spe- entry. On the basis of these characters alone, a cies is not unexpected. Nonetheless, the possibility species can be categorized as predominantly a of finer structural differences, such as the interle- flower visitor or a nonflower visitor. Most proboscis gular spacing, between males and females cannot characters in our analysis probably are related to be excluded (Kwauk et al., 2014). the entry of fluid into the proboscis of all haustel- Selection pressures exerted by diverse floral late Lepidoptera (Monaenkova et al., 2012; Leh- structure (Soltis et al., 2009; Tiple et al., 2009) nert et al., 2013); therefore, the suite of characters probably have resulted in the diversity of proboscis for acquiring fluids from droplets and wetted structure among flower visitors. The best-known surfaces swamps out other characters that aid flo- relationship between flower and proboscis struc- ral entry, which are relevant only to flower visi- ture is the corresponding length of the floral tube tors. Although not indicative of feeding guilds, the and the proboscis (Kunte, 2007; Krenn, 2010; set of proboscis characters used in our analysis Arditti et al., 2012; Bauder et al., 2015); however, produces a dendrogram with a topology that other patterns can be found. The Papilionidae and reflects the evolutionary relationships of the five Nymphalidae, for example, which feed on nectar species (Regier et al., 2013; Kawahara and Brein- from similar (e.g., larger) flowers (Tiple et al., holt, 2014), affirming the relevance of proboscis 2009), have similar dorsal legular shapes that dif- structural characters in lepidopteran classification fer from those of pierids. Future studies could (Kristensen, 1984; Krenn and Kristensen, 2004; examine a potential relationship between proboscis Kristensen et al., 2007). structure and nectar composition and viscosity. The two flower-visiting species with puddle- visiting males, P. glaucus and P. rapae, did not Which Proboscis Characters Are Indicators cluster together, nor did males differ from females of Feeding Habits? for any of the 21 evaluated proboscis features. Lepidoptera that feed from porous substrates, Puddling species use the same structural features such as decaying fruits, have a brushy proboscis involving uniform principles of fluid acquisition formed of dense rows of elongated sensilla stylo- from wetted surfaces that are present in all haus- conica (Knopp and Krenn, 2003; Molleman et al., tellate Lepidoptera (Monaenkova et al., 2012). In 2005). Species without a brushy proboscis, by

Journal of Morphology 14 M.S. LEHNERT ET AL. default, typically are recognized as flower visitors, demophon (Nymphalidae) (Krenn, 2010), and V. although scattered sensilla styloconica can be cardui (Nymphalidae) (Kwauk et al., 2014). present (Krenn et al., 2001; Petr and Stewart, No evidence suggests that legular hand switches 2004). Nonflower visitors have unique proboscis or microbump patterns indicate feeding guilds. characters in addition to a dense sensillar brush, Hand switches, especially the zipper-like hand such as dorsal legulae extended to the tip of the switches of P. rapae, might increase proboscis flex- proboscis and wider, lower branches of dorsal legu- ibility or minimize galeal separation during feed- lae in the proximal region (Zone 1). For flower vis- ing (Lehnert et al. 2014). Zipper-like hand itors, the opposite conditions hold. The presence of switches also are found in P. icarus (Lycaenidae) Zone 3 in the flower visitors might have adaptive (Krenn et al., 2005). Microbumps and valleys value in facilitating the proboscis to enter narrow might facilitate channeling of fluids to regions of floral corollas, but requires further study. Probos- the proboscis where uptake can occur (Lehnert cis structure of nonflower visitors might provide et al., 2013) and aid proboscis flexibility while increased hydrophilic surface area and capillarity feeding (Krenn et al., 2005) and coiling (Krenn, for fluid uptake from porous substrates (Lehnert 1990; Krenn et al., 2005; Lehnert et al. 2015). The et al., 2013). presence of surface sculpturing on the proboscis The dense rows of sensilla styloconica and dor- might represent a general character for feeding sal legulae that extend to the apex, might hinder from droplets and films; pool feeding would not the proboscis from entering floral tubes by increas- require external channeling of fluid. ing friction or drag in the corolla. Serrations on the dorsal legulae of L. a. astyanax could impede CONCLUSIONS floral entry, but might assist in fluid uptake by The proboscis of haustellate Lepidoptera is fun- scraping surfaces of rotting fruit. All proboscises damentally adapted for fluid uptake from droplets that we studied taper distally, especially in flower and surface films. Its overall structure does not visitors, which could enhance floral entry and unequivocally permit species to be assigned to access to nectar (Krenn et al., 2001), particularly flower-visiting versus nonflower-visiting groups or in combination with a distalmost region of the pro- to a puddle-visiting group. Specific characters of boscis (Zone 3) free of dorsal legulae. the proboscis, however, such as the presence or absence of a dense sensillar brush, allow most spe- Which Proboscis Characters Are Poor cies to be assigned to a feeding guild. Characters Indicators of Feeding Habits? related to floral entry, such as increased tapering A longer Zone 2 has been associated with of the proboscis, characterize flower visitors. nonflower-visiting nymphalids (Krenn et al., We suggest that the diversity of proboscis struc- 2001). Although a longer Zone 2 might increase ture among flower visitors reflects the diversity of the surface area of the proboscis applied to wetted floral structure. Flower-visiting nymphalids surfaces, such as rotting fruit, (Knopp and Krenn, exhibit greater structural variation of the probos- 2003; Molleman et al., 2005; Lehnert et al., 2013), cis than do nonflower-visiting nymphalids (Krenn the nonflower visitors in our study do not have a et al., 2001). Proboscis length, in general, varies proportionally longer Zone 2 than the other more among the flower-visiting Lepidoptera than butterflies. it does among nonflower visitors (Kunte, 2007). On We showed that changes in the width of the the contrary, feeding from wetted surfaces, such as upper and lower branches along the proboscis, rotting fruit, involves less structural variation, which are associated with the structural distinc- owing to greater uniformity of the porous nature tiveness of the drinking region (Lehnert et al., of the substrates. Thus, no structural indicators 2013; Kramer et al., 2015), are species specific and for puddling species or between genders in species that only the upper branches of dorsal legulae with puddling males were apparent. The adaptive enlarge from Zone 1 to Zone 2 in our studied spe- value of other structural attributes of the probos- cies. The structural origins of the upper branches cis, such as the zipper-like handedness of dorsal of dorsal legulae in Zone 2 differ among species, legulae, require further exploration. but their consistent presence across species suggests functional importance and similar selection ACKNOWLEDGMENTS pressures. The grooves between the upper and We thank the Clemson University Electron lower branches of the dorsal legulae of P. rapae Microscopy Laboratory for assistance with scan- might help channel fluids to the food canal. ning electron microscopy, Ms. Bennie M. Saylor for Grooves in dorsal legulae with overlapping photographs of the adult butterflies, Angela H. arrangements are found in many species including Newman for illustrations of the proboscises, and Cryphia muralis (Noctuidae), Scrobipalpa costella Prof. Harald W. Krenn for insightful comments on (Gelechiidae) (Faucheux, 2013), Archaeoprepona the manuscript. This is Technical Contribution No.

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