External morphology of the immature stages of Neotropical heliconians: IX. Dione glycera (C. Felder & R. Felder) (Lepidoptera, Nymphalidae, Heliconiinae)

SYSTEMATICS, MORPHOLOGY AND BIOGEOGRAPHY

External morphology of the immature stages of Neotropical heliconians: IX. Dione glycera (C. Felder & R. Felder) (Lepidoptera, Nymphalidae, Heliconiinae)

Héctor A. Vargas^I; Kim R. Barão^II; Darli Massardo^II; Gilson R. P. MoreiraIII,^*

^IDepartamento de Recursos Ambientales, Facultad de Ciencias Agronómicas, Universidad de Tarapacá, Casilla 6-D. Arica, Chile. [e-mail]

^IIPPG Biologia Animal, Departamento de Zoologia, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, 91501–910 Porto Alegre-RS, Brazil. [e-mail]; [e-mail]

^IIIDepartamento de Zoologia, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, 91501–910 Porto Alegre-RS, Brazil

ABSTRACT

External morphology of the immature stages of Neotropical heliconians: IX. Dione glycera (C. Felder & R. Felder) (Lepidoptera, Nymphalidae, Heliconiinae). The biology of the Andean silverspot butterfly Dione glycera (C. Felder & R. Felder, 1861) is still poorly known. This species is restricted to high elevations in the Andes, where the immature stages are found in close association with species of Passiflora belonging to the section Tacsonia (Juss.) Harms, especially P. tripartida var. mollissima (Kunth), which is grown for subsistence by villagers. Herein we describe and illustrate the external features of the egg, larva and pupa of D. glycera, based on light and scanning electron microscopy.

Keywords: Andean silverspot butterfly; banana passion-fruit; Heliconiini; Insecta.

The passion-vine butterflies (Lepidoptera, Nymphalidae, Heliconiini) are well known for their aposematic wing patterns, extensive Müllerian mimicry, and co-speciation with their Passifloraceae host-plants (for reviews, see Benson et al. 1976; Brown 1981; Gilbert 1991). The genus Dione Hübner, [1819] is one of the most basal lineages of passionvine butterflies, and comprises only three species (Lamas 2004): Dione glycera (C. Felder & R. Felder, 1861), Dione juno (Cramer, 1779) and Dione moneta Hübner, [1825]. Of these, only D. glycera shows no differentiation into subspecies throughout its geographic range (Emsley 1963; Lamas 2004), and is closely associated with mountainous landscapes of the Andes, from Venezuela to Argentina (Massardo et al. unpubl. data).

The larvae of D. glycera feed on members of the section Tacsonia (Juss.) Harms of the genus Passiflora L., using preferentially as host plant the banana passion-fruit, Passiflora tripartita (Juss.) Poir. var. mollissima (Kunth). This plant is also native to the Andes (Schwerdtfeger 2004), and is grown from Venezuela to Bolivia, at altitudes above 1,800 m. Its fruits are aromatic, highly appreciated for their pleasant taste and juice (Simirgiotis et al. 2013). Other species of Passiflora, including P. alnifolia, P. cyanea, P. ligularis, P. mixta, P. caerulea and P. edulis, have also been listed as hosts of D. glycera (Beccaloni et al. 2008).

Although the southern limit of D. glycera on the eastern slopes of the Andes is in Argentina, its southern range on the western slopes reaches only to northernmost Chile (Peña & Ugarte 1996). Adjacent to the Atacama Desert, these slopes (Fig. 1) are dominated by arid landscapes (Luebert & Pliscoff 2006), where the presence of D. glycera is generally associated with its main host-plant, P. tripartita var. mollissima. This plant is locally named "tumbo" and is cultivated by villagers for its fruit and fruit juice. The plants are usually grown outdoors on espaliers (Figs. 2, 3), in scattered locations in the valleys, for example at Socoroma, Belén, Codpa and Timar villages in Parinacota Province. Immatures and flying adults have been found in low numbers during the summer on these plants.

Detailed studies describing and illustrating the external morphology of immature stages have been published for D. juno juno (Tavares et al. 2002) and D. moneta moneta (Kaminski et al. 2008). However, knowledge of the immature stages of D. glycera remains restricted to brief descriptions (e.g. Brown 1981; Penz 1999). As part of a series of articles dealing with the morphology of Neotropical heliconians (Antunes et al. 2002; Kaminski et al. 2002, 2008; Tavares et al. 2002; Paim et al. 2004; Silva et al. 2006, 2008; Barão & Moreira 2010), here we describe and illustrate in detail the external morphology of the immature stages of D. glycera, based on light and scanning electron microscopy. We also discuss the characteristics found for D. glycera in relation to congeneric species, and in addition, provide dichotomous keys to identify the species of Dione at the larval and pupal stages.

MATERIAL AND METHODS

Eggs and larvae were collected from leaves of tumbo in Socoroma Village, Parinacota Province on the western slopes of the northern Chilean Andes, at ca. 3,000 m elevation, from January 2009 to July 2013 (Fig. 1). They were brought to the entomology laboratory of the Facultad de Ciencias Agronómicas, Universidad de Tarapacá, Arica, and placed in plastic bottles, with leaves of the host plant added when necessary, and reared at room temperature. Some specimens of each stage and instar were fixed in Dietrich's fluid and preserved in 70% ethanol for subsequent observation. Additional specimens were reared until the adult stage in order to confirm the taxonomic identification. The immature specimens were deposited in the collection of the Laboratório de Morfologia e Comportamento de Insetos (LMCI), Departamento de Zoologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil, under accession number LMCI 79. Adult vouchers were deposited in the Colección Entomológica de La Universidad de Tarapacá (IDEA), Arica, Chile.

Aspects of the general morphology of the immature stages were analyzed from material that was either fixed or embedded in glycerin jelly. Head capsules were hydrated, cleared in a 10% potassium hydroxide solution (KOH), and slidemounted in glycerin jelly. Drawings were made from fixed specimens, using a reticulated ocular attached to a Leica® M125 stereomicroscope. The tegumentary ultrastructure was studied at the Centro de Microscopia Eletrônica of UFRGS. For the analyses, the specimens were dehydrated in a Baltec® CPD030 critical-point dryer, mounted with double-sided tape on metal stubs, and coated with gold in a Bal-tec® SCD050 sputter coater. Specimens were examined and photographed in a JEOL® JSM5800 scanning electron microscope.

To distinguish larval instars, the greatest width of the head capsule in frontal view was measured with a micrometer scale mounted in the ocular of the stereomicroscope. By using the least-squares method, the resulting data were adjusted to the power function, y = ae^bx, and following the procedure described by Snedecor & Cochran (1980).

For the egg, we use the nomenclature employed by Dell'Erba et al. (2005). Larval body areas are labeled according to Peterson (1962). For the primary chaetotaxy and crochet of prolegs, we follow Stehr (1987) with the modification proposed by Duarte et al. (2005) for the CD group of the head. The setae of the paraproct and anal prolegs are labeled according to Kitching (1984). To describe the scoli, we follow Beebe et al. (1960), and for the nomenclature of the pupa, we follow Mosher (1916).

RESULTS AND DISCUSSION

Egg (Figs. 4, ^8–12). Sub-spherical; flat base abruptly narrowed near apex, with a slightly depressed area at apex. Dimensions (mean ± standard error; n = 8): diameter = 1.06 ± 0.03 mm; height = 1.38 ± 0.01 mm. Light yellow when recently deposited, with brown and whitish spots subsequently (Fig. 4). Chorion ornamented (^{Figs. 8–12}) with vertical (Vr) and horizontal (Hr) smooth carinae, their number varying from 14–16 and 18–23, respectively. Vertical carinae about three times as thick as horizontal carinae. All vertical carinae start at base, but some do not reach the apex. Intersections of vertical and horizontal carinae delimit cells, with broadly rounded internal angles and smooth surface; the lower cells (Lc) (^{Fig. 12}) are mostly rectangular, with the horizontal margin about 2.5 times as long as the vertical margin; the upper cells (Uc) (^{Fig. 11}) are pentagonal. Micropylar region at chorion apex (^{Fig. 9}) slightly rugose, ornamented with two strata of polygonal cells peripherally forming the annulus, and one stratum of oval cells centrally forming the rosette in the center of which are the micropyles. Aeropyles (^{Fig. 10}) as small circular pores at intersections of vertical and horizontal carinae.

The gross morphology, color variation and ornamentation of the egg of D. glycera fit the pattern previously reported for the eggs of D. juno juno (Tavares et al. 2002; Dell'Erba et al. 2005) and D. moneta moneta (Dell'Erba et al. 2005; Kaminski et al. 2008). These aspects resemble the eggs of Agraulis vanillae maculosa (Stichel, [1908]) (Dell'Erba et al. 2005; Silva et al. 2006). However, in the lower cells of the chorion of the three species of Dione the length of the horizontal margin is more than twice that of the vertical margin, whereas in the lower cells of A. vanillae maculosa the length of the horizontal margin is less than twice that of the vertical one (Dell'Erba et al. 2005; Silva et al. 2006). Thus, the lower cells of Dione are comparatively broader, whereas in A. vanillae maculosa the lower cells are higher. Furthermore, some of the vertical carinae in the three species of Dione are well defined until near the micropylar region, slightly projected distally, encircling the micropylar region; whereas the vertical carinae of A. vanillae maculosa change slightly in orientation along the distal third, and are not projected distally (Dell'Erba et al. 2005; Silva et al. 2006).

First instar larva (Figs. 5, ^13–15, ^23–36). Head (Figs. 5, ^13–14, ^23–29). Blackish, with setae smooth, variable in size, with apex widened in P2 and L1 (^{Figs. 13–14}, ²⁹) and pointed in the remaining cephalic setae (^{Figs. 13–14}, ²⁸); frontoclypeus triangular; frontoclypeal suture not differentiated; anteclypeus membranous, as a straight transverse stripe between frontoclypeus and labrum; antennal pocket lateral to mouthparts (^{Figs. 23}, ²⁵), membranous, with many round protuberances; a group of six stemmata (^{Figs. 13}, ¹⁴, ²⁴) dorsal to each antennal pocket; stemmata 1–5 in semicircle, stemma 6 slightly posterior to an imaginary line from stemmata 1 to 5. Antenna tri-segmented (^{Fig. 25}); first segment ring-like, short, partially hidden by antennal pocket; second segment cylindrical, diameter about two-thirds that of first segment, four sensilla at apex; third segment cylindrical, short and narrow, about half length and diameter of second segment, also with four sensilla at apex.

Mouthparts of chewing type (^{Figs. 13}, ¹⁴, ²³). Labrum (^{Figs. 13}, ²³) bilobed, with distal margin broadly cleft at middle, six pairs of short, hair-like setae on external surface; mandible with five teeth on distal margin, two setae on external surface; maxilla (^{Fig. 27}) with palpus and galea well differentiated; palpus with eight sensilla at apex and one on medial surface; galea with seven sensilla; labium (^{Fig. 26}) with pair of bi-segmented palpi, each with one sensillum at apex, first segment about six times longer than second segment; spinneret postero-ventral to labial palpi, about 2.5 times the length of a labial palpus, with orifice opening at apex and a pair of small setae near the base.

Thorax and abdomen mostly reddish brown, with creamwhite spots and a faint yellowish-brown dorsal stripe; prothoracic dorsal shield, anal shield, pinnacles, setae, thoracic legs and lateral plates of prolegs black; setae smooth, variable in size, with apex either acute or widened (^{Figs. 15}, ³⁰, ³¹). Prothoracic dorsal shield rectangular with rounded angles, anterior and posterior margins with slight cleft at middle. Anal shield trapezoidal, with broadly rounded angles and posterior margin broadly convex. Spiracles (^{Figs. 15}, ³⁶) laterally on prothorax and A1–A8, circular, with peritrema elevated; prothoracic spiracle posterior to pinnacle of lateral group of setae; abdominal spiracles between lateral and sub-dorsal groups of setae; diameter of prothoracic spiracle similar to that of A8 and slightly larger than the remaining A1–A7. Two well-developed legs (^{Fig. 32}), ventrally on each thoracic segment; coxa whitish brown, with a black transverse stripe interrupted laterally; trochanter a narrow transverse stripe between coxa and femur; femur, tibia, tarsus and tarsal claw black; tarsal claw slightly curved, with a plain, round enlargement at base and sharp apex. Two well-developed prolegs (^{Fig. 33}) ventrally on A3–A6 and A10, each with a blackish lateral plate, and provided with 13–14 crochets at apex; crochets of A3–A6 in uniordinal, uniserial circle, those of A10 in semicircle. Integument mostly smooth, ventral area, prolegs and area surrounding the anus provided with conical microtrichia (^{Fig. 34}), those surrounding the anus with longer apex (^{Fig. 35}).

Chaetotaxy. Head (^{Figs. 13}, ¹⁴). 17 pairs of setae, 4 pairs of microsetae and 11 pairs of pores. Adfrontal (AF) group; bisetose; AF2 near dorsal angle of frontoclypeus; AF1 at about dorsal third of adfrontal suture; AFa pore closer to AF2 than to AF1. Anterior (A) group: trisetose; A1 dorsal to antennal pocket; A2 dorsomedial to A1; A3 dorsolateral to A2; Aa pore ventrolateral to A3. Cephalo-dorsal (CD) group: trisetose; CD1, CD2 and CD3 microsetae, almost in straight line posterior to P2; pore CDa posteroventral to CD1. Clypeal (C) group: bisetose; both setae near ventral margin of frontoclypeus; C1 closer to intersection of ventral margin of frontoclypeus and adfrontal suture; C2 medial to C1. Frontal (F) group: unisetose; F1 slightly displaced toward ventral half of frontoclypeus. Lateral (L) group: unisetose; L1 about midway between P2 and S2; La pore posteroventral to L1. Microgenal (MG) group: unisetose, MG1 a microseta, posteroventral to S3; MGa pore anterodorsal to MG1. Posterodorsal (P) group: bisetose; P1 and P2 forming an imaginary line almost parallel to epicranial suture; P2 posterodorsal to P1; pore Pb anteroventral to P2; pore Pa ventral to P1. Stemmatal (S) group: trisetose; S1 at about midpoint between stemmata 1 and 5; S2 posterior to stemma 1; S3 posteroventral to S2; pore Sa ventral to S2; pore Sb between stemmata 3 and 4. Substemmatal (SS) group: trisetose; SS2 between stemmata 5 and 6; SS1 near antennal pocket; SS3 between SS1 and SS2; pore SSb between SS3 and SS2; pore SSa not found.

Thorax (^{Fig. 15}). Prothorax: 10 pairs of setae (D1, D2, XD2, SD1, SD2, L1, L2, SV1, SV2, V1); D and XD groups on the prothoracic dorsal shield; D1 and XD2 near anterior margin, with XD2 displaced toward anterolateral angle of shield and D1 toward the middle; D2 near posterior margin of shield; pore XDc between XD2 and D2. SD group bisetose, with both setae on a pinnacle ventral to dorsal shield. L group bisetose, with both setae on a pinnacle anterior to spiracle. SV group bisetose, with both setae on a pinnacle posteroventral to L group. Ventral group unisetose, posteromedial to prothoracic coxa. Meso- and metathorax: eight pairs of setae (D1, D2, SD1, SD2, L1, SV1, SV2, V1); D1 and D2 on separate pinnacles, with D1 dorsal and slightly anterior to D2; SD group on a single pinnacle, ventral and slightly anterior to D2; L1 on a ventral pinnacle and slightly anterior to SD group; SV group on a single pinnacle ventral to L1; V1 posteromedial to mesothoracic coxa.

Abdomen (^{Fig. 15}). A1: seven pairs of setae (D1, D2, SD1, L1, L2, SV1, V1); D1 and D2 on separate pinnacles, with D1 anterodorsal to D2; SD1 on a pinnacle anteroventral to D2 and dorsal to spiracle; L group on separate pinnacles, with L1 posterodorsal to L2; SV1 on a small pinnacle between L group and V1. A2: eight pairs of setae (D1, D2, SD1, L1, L2, SV1, SV2, V1), with spatial distribution similar to preceding segment, but SV group on two small separate pinnacles. A3–A6: eight pairs of setae (D1, D2, SD1, L1, L2, SV1, SV2, V1), with distribution similar to preceding segment, but SV group and V1 on the respective prolegs; SV group on a broad lateral plate, and V1 on the medial surface. A7, A8: six pairs of setae (D1, D2, SD1, L1, L2, SV1), with distribution of setae similar to A1, but V1 absent. A9: five pairs of setae (D1, D2, SD1, L1, PL1, PL2, PL3, PL4, PL5, V1); D and SD groups on anal SV1); pinnacles of right and left D1 medially fused; D1, D2, shield; PP1 and SP1 on small pinnacles on posterior surface SD1 and L1 almost forming a straight line; SV1 posteroventral of proleg; PL1, PL2, PL3 and PL4 on lateral plate of proleg; to L1. A10: 12 pairs of setae (D1, D2, SD1, SD2, PP1, SP1, PL5 and V1 on medial surface of proleg.

The gross morphology and chaetotaxy of the first instar of D. glycera fit the general pattern for Heliconiini (Antunes et al. 2002; Kaminski et al. 2002, 2008; Tavares et al. 2002; Paim et al. 2004; Silva et al. 2006, 2008; Barão & Moreira 2010). However, a conspicuous variation among the first instars is related to the shape of the apex of some primary setae of the head, thorax and abdomen, which is generally widened, as in D. glycera (this study), D. moneta moneta (Kaminski et al. 2008), A. vanillae maculosa (Silva et al. 2006), Heliconius erato phyllis (Fabricius, 1775) (Kaminski et al. 2002), Dryadula phaetusa (Linnaeus, 1758) (Silva et al. 2008), Dryas iulia alcionea (Cramer, 1779) (Paim et al. 2004), and Philaethria wernickei (Röber, 1906) (Barão & Moreira 2010), but it is acute in Eueides isabella dianasa (Hübner, [1806]), and 3–4 tipped in D. juno juno (Tavares et al. 2002).

The distribution of the setae with widened apex on the head capsule enables us to separate D. glycera from D. moneta: setae P2 and L1 have widened apices in D. glycera, whereas in D. moneta setae P1 and P2 have this shape (Kaminski et al. 2008).

Subsequent instars. Color pattern is variable starting in the second instar. Three chromatic patterns were observed in the fifth instar, with a large number of intermediate forms (Figs. 6, ^17–22). One of these patterns (^{Figs. 21}, ²²) is characterized by the head, thorax, prolegs, scoli and verrucae mostly pinkish-yellowish brown; head with a blackish area associated with the stemmata, mandibles dark orange; thorax and abdomen with a broad whitish-cream lateral stripe, dorsal scoli on a longitudinal light-gray stripe with an irregular yellowish-orange spot at the base of each scolus and some similar, scattered spots; an irregular yellowish-orange spot at base of the remaining scoli, enclosed by an irregular light-gray spot; some scattered small dark-gray spots; scoli darkening distally.Another pattern (^{Figs. 17}, ¹⁸) has the head, legs, prolegs, scoli and verrucae black; thorax and abdomen with a broad whitish-cream lateral stripe; dorsal scoli on a yellow longitudinal stripe with an irregular orange spot at the base of each scolus and some similar, scattered spots; an irregular orange spot at the base of the remaining scoli, enclosed by an irregular yellow spot; lateral plates of the prolegs black or dark yellowish brown. In a third, intermediate pattern (^{Figs. 19}, ²⁰), two pairs of black stripes present frontally on the orange head, and the body is bluish. There is substantial variation in the corresponding tones of blue and also in the size of the black stripes on the head.

The chaetotaxy also changes strikingly starting from the second instar (^{Fig. 16}). The primary setae are replaced by secondary setae (Figs. 37, 38, 41), scoli (Fig. 39) and verrucae (Fig. 40). The head (Fig. 37) is smooth and covered by a large number of hair-like setae which vary in length; and bears two short stout scoli dorsally (Figs. 16–19), namely the cephalic scoli, which are provided with several short, hairlike setae. The thorax and abdomen have the integument covered by conical, striated microtrichia (Figs. 41, 42) and scattered hair-like setae (Fig. 41); prothoracic dorsal shield (Fig. 38) bears a number of short hair-like setae and two pairs of stout spine-like setae on small dorsal conical projections; elliptical spiracles (Fig. 43) with slightly elevated peritrema laterally on prothorax and abdominal segments A1–A8, those of prothorax and A8 slightly larger than the remaining spiracles; prolegs (Fig. 44) with lateral plates covered by several hair-like setae, crochets (Fig. 45) in uniserial and multiordinal arrangement. Thirty pairs of thoracic and abdominal scoli, which are elongated conical tegumentary outgrowths, provided with some hair-like setae on the surface, one of which, typically the longest one, is placed at the apex; 11 dorsal pairs (T1–T2 and A1–A9); ten supraspiracular pairs (T1–T2 and A1–A8), with those of the meso- and metathorax anteriorly displaced; eight lateral pairs (A1–A8), and one anal pair (A10). Thirteen pairs of thoracic and abdominal verrucae; three pairs on prothorax, one between dorsal shield and spiracle, which is provided with a spine-like seta, another greatly reduced pair anterior to spiracle, and another pair between spiracle and coxa; two pairs on meso and metathorax, one posterior to supraspiracular scolus and another dorsal to coxa; one pair on A1–A2 and A7–A8, which is ventral to lateral scolus; and two pairs on A9, one near the dorsal scolus, and another on the lateroventral face of the segment.

As in the first instar, the gross morphology of the subsequent instars of D. glycera fits the general pattern described for Heliconiini (Antunes et al. 2002; Kaminski et al. 2002, 2008; Tavares et al. 2002; Paim et al. 2004; Silva et al. 2006, 2008; Barão & Moreira 2010). However, the length of the cephalic scoli of D. glycera is less than half the height of the head capsule, appearing as a short, stout projection. This pattern is found with even greater reduction in D. juno juno (Tavares et al. 2002); meanwhile in the other congener D. moneta moneta (Kaminski et al. 2008) these structures are elongated, as in the remaining heliconians whose immature stages have been studied (Antunes et al. 2002; Kaminski et al. 2002; Paim et al. 2004; Silva et al. 2006, 2008; Barão & Moreira 2010).

The distribution pattern of the verrucae resembles that of D. moneta moneta (Kaminski et al. 2008). However, the spinelike seta on the verruca between the prothoracic dorsal plate and the prothoracic spiracle in D. glycera is absent in D. moneta moneta. This seta has not been reported for any other heliconian for which the early stages are described. Furthermore, the verruca anterior to the prothoracic spiracle and that on the lateroventral face of A9 are less developed in D. glycera than in D. moneta moneta.

Another interesting aspect of the larvae of D. glycera is the striking variation in the color pattern among individuals (Figs. 17–22). Intraspecific variation in the color pattern of heliconiines has been also recorded for the congeneric D. moneta moneta (Kaminski et al. 2008) and for A. vanillae maculosa (Silva et al. 2006).

Instar identification. The first instar is strikingly different from the other stages in the absence of scoli, and also in the color pattern. After the first molt, identification based on color pattern or morphology is difficult, because the instars do not show stable differences in these attributes. However, the successive instars can be accurately distinguished by the width of the head capsule, because the widths measured in successive instars did not overlap (Table I). The following exponential growth equation was adjusted for the five instars (y = 0.652e^0.198x; n = 32; r² = 0.975; p < 0.05). Thus, the growth pattern of the head capsule follows the Brooks-Dyar rule (Daly 1985). The mean growth ratio among instars was 1.56, similar to ratios previously reported for other Neotropical heliconians (Antunes et al. 2002; Kaminski et al. 2002, 2008; Tavares et al. 2002; Paim et al. 2004; Silva et al. 2006, 2008; Barão & Moreira 2010).

Pupa (Figs. 7, ^46–65). Variable in coloration throughout ontogeny, with mixture of light gray, dark brown, pinkish brown, cream white and black (Fig. 7). Abdominal segments A1 and A2 with pair of silvery blotches dorsally. Length (mean ± standard error; n = 5) = 19.55 ± 0.34 mm.

Head with pair of short, angled cephalic projections (Figs. 46, ⁴⁸, ⁵¹); epicranial suture absent; eyes (^{Fig. 49}) with sculptured region near antenna, bearing few short hair-like setae (^{Fig. 50}), and another smooth region near front; labrum as a slight, short longitudinal stripe between the mandibles; maxilla with well-developed galeae, along midline of ventral surface, anteriorly delimited by labrum and mandibles, slightly surpassing posterior margin of A4; antennae arising dorsally on head, projected ventrally to apex of maxilla, with many smooth, round tubercles on surface (^{Fig. 52}).

Thorax with the three segments exposed. Prothorax as a small hexagonal plate in dorsal view, with anterior and posterior margins broadly excavated, with a pair of lateral tubercles (^{Figs. 47}, ⁵³). Mesothorax broadly expanded laterally along anterior half, with well-defined basilar tubercles (Fig. 57) and a longitudinal, meso-dorsal crest (^{Figs. 47}, 54) that is greatly developed, broadly rounded, between a pair of round lateral tubercles; two parallel rows, termed submarginal and postmedian, with three tubercles each near apex of wing (Fig. 58, 59); mesothoracic spiracle (Fig. 61) opening laterally at anterior margin of segment, as a simple cleft and with microtrichia. Metathorax as a narrow plate with anterior margin broadly excavated, with pair of lateral tubercles, hindwings as straight stripes between forewings and abdominal segments.

Abdomen with segments A1–A4 partially hidden by wings; with pair of lateral tubercles on A1–A8, which are little developed on A1–A2 (Fig. 55), most developed on A3, and decreasing in size posteriorly; one meso-dorsal tubercle on A5–A7 (Fig. 56); one supraspiracular tubercle (Fig. 60) on A3–A4; pair of ventral tubercles (Fig. 63) on A5–A6; spiracles of A1 and A2 hidden and partially hidden, respectively, by forewings, and spiracles of A3–A7 elliptical (Fig. 62) with a well-developed filter apparatus; spiracles of A8 greatly reduced. Cremaster (Fig. 64) quadrate, with truncate apex, and a large number of short, curved hooks (Fig. 65).

The pupal morphology of D. glycera fits the pattern recognized by Mosher (1916) for Nymphalidae, characterized by the absence of an epicranial suture, and by the prothoracic and mesothoracic legs extended anteroventrally, reaching the posterior margin of the eye.

The cephalic projections of the pupa of D. glycera are short, slightly surpassing the anterior margin of the head. This pattern is also found in the pupae of the other two species of Dione (Tavares et al. 2002; Kaminski et al. 2008). The cephalic projections are more developed in A. vanillae maculosa (Silva et al. 2006); they substantially exceed the anterior margin of the head, and are as long as ca. half of the head.

Further remarks. This is the first detailed study dealing with the external morphology of all the early stages of D. glycera. As similar studies are available for the other two species of the genus (Tavares et al. 2002; Kaminski et al. 2008), we compared the egg, first-instar larva, fifth-instar larva, and pupa of the three species of Dione in order to test ter group Agraulis (Penz 1999; Massardo et al. unpubl. data). the diagnostic value of the morphology of the early stages. However, as mentioned above, we could not find stable dif-The egg morphology easily separates Dione from the sis-ferences to distinguish among the species of Dione at this stage. On the other hand, field observations suggested that females of D. glycera generally lay eggs individually on the leaves of the host plant, usually members of Tacsonia (Juss.) Harms within the subgenus Passiflora L. Rarely more than one egg is deposited per leaf. This egg-laying behavior is strikingly different from that reported for the other two species of the genus. Clusters of 60–140 eggs have been mentioned for D. juno (Dell'Erba et al. 2005), and clusters of up to 20 eggs have been recorded for D. moneta (Dell'Erba et al. 2005; Kaminski et al. 2008). Occasionally the eggs can also be deposited individually in this species (Brown 1944; Brown 1981; DeVries 1987), which may make it difficult to distinguish them from eggs of D. glycera by means of this characteristic. Dione moneta and D. glycera are sister species, but the former occurs at much lower elevations, and the larvae are specialized feeders not on species of the subgenus Passiflora L., but on Decaloba (DC.) Rchb. (Massardo et al. unpubl. data).

Nonetheless, we found clear differences for the first- and fifth-instar larvae and for the pupal stage, which allow their prompt identification, and which are summarized in the following dichotomous keys:

Key to first-instar larvae of Dione

1.

Dorsal setae of thorax and abdomen with widened apex ... 2

1'.

Dorsal setae of thorax and abdomen 3–4 tipped ... D. juno

2.

Head seta P2 with widened apex and L1 with acute apex.. ... D. moneta

2'. Head seta P2 with acute apex and L1 with widened apex ... D. glycera

Key to fifth-instar larvae of Dione

1.

Prothoracic dorsal shield without scoli ... 2

1'.

Prothoracic dorsal shield with pair of scoli ... D. juno

2.

Cephalic scoli longer than half width of head ... D. moneta

2'. Cephalic scoli shorter than half width of head ... D. glycera

Key to pupae of Dione

1.

One pair of golden spots dorsally on A1–A2 ... 2

1'.

Dorsal surface of A1–A2 without silver or golden spots.. ... D. juno

2.

Cephalic projection broadly round and expanded laterally ... D. moneta

2'. Cephalic projection angled, not expanded laterally ... D. glycera

ACKNOWLEDGEMENTS

We are grateful to the staff members of the Centro de Microscopia Eletrônica of UFRGS for the use of facilities and for their assistance in scanning electron microscopy analyses. Thanks are also due Janet W. Reid for editing the text. The financial support for this study came in part from a CAPES Doctoral Fellowship granted to D. Massardo. The work was also supported by CNPq, Brazil (Project 490124/ 2010-0, PROSUL – 08/2010, and project number 309676/ 2011-8), granted to G.R.P. Moreira.

Received 11 September 2013;

accepted 27 February 2014

• Antunes, F.A., Menezes, A.O. Jr., Tavares, M. & Moreira, G.R.P. 2002. Morfologia externa dos estágios imaturos de heliconíneos neotropicais: I. Eueides isabella dianasa (Hübner, 1816). Revista Brasileira de Entomologia 46: 601610.
• Barão, K.R. & Moreira, G.R.P. 2010. External morphology of the immature stages of Neotropical heliconians: VIII. Philaethria wernickei (Röber) (Lepidoptera, Nymphalidae, Heliconiinae). Revista Brasileira de Entomologia 54: 406418.
• Beccaloni, G.W., Viloria, Á.L., Hall S.K. & Robinson, G.S. 2008. Catalogue of the hostplants of the Neotropical butterflies Monografías Tercer Milenio, vol. 8. Zaragoza, Sociedad Entomológica Aragonesa, 403 p.
• Beebe, W., Crane, J. & Fleming, H. 1960. A comparison of eggs, larvae and pupae in fourteen species of Heliconiine butterflies from Trinidad, W. I. Zoologica 45: 111154.
• Benson, W.W., Brown, K.S. Jr. & Gilbert, L.E. 1976. Coevolution of plants and herbivores: passion vine butterflies. Evolution 29: 659680.
• Brown, F.M. 1944. The egg, larva and chrysalis of Dione moneta Hübner. Bulletin of the Brooklyn Entomological Society 39: 132134.
• Brown, K.S. Jr. 1981. The biology of Heliconius and related genera. Annual Review of Entomology 26: 427456.
• Daly, H.V. 1985. Insect morphometrics. Annual Review of Entomology 30: 415438.
• Dell'Erba, R., Kaminski, L.A. & Moreira, G.R.P. 2005. O estágio de ovo dos Heliconiini (Lepidoptera, Nymphalidae) do Rio Grande do Sul, Brasil. Iheringia, Série Zoologia, 95: 2946.
• DeVries, P.J. 1987. The butterflies of Costa Rica and their natural history. Volume I: Papilionidae, Pieridae, Nymphalidae Princeton, Princeton University, 327 p.
• Duarte, M., Robbins, R.K. & Mielke, O.H.H. 2005. Immature stages of Calycopis caulonia (Hewitson, 1877) (Lepidoptera, Lycaenidae, Techlinae, Eumaeini), with notes on rearing detritivorous hairstreaks on artificial diet. Zootaxa 1063: 131.
• Emsley, M. 1963. A morphological study of imagine Heliconiinae (Lepidoptera: Nymphalidae) with a consideration of the evolutionary relationships within the group. Zoologica 48: 85129.
• Gilbert, L.E. 1991. Biodiversity of a Central American Heliconius community: Pattern, process, and problems, p. 403427. In: Price, P.W., Lewinsohn, T.M., Fernandes, G.W. & Benson, W.W. (eds.). Plantanimal interactions: Evolutionary ecology in tropical and temperate regions New York, John Wiley & Sons, 639p.
• Kaminski, L.A., Dell'Erba, R. & Moreira, G.R.P. 2008. Morfologia externa dos estágios imaturos de heliconíneos neotropicais: VI. Dione moneta moneta Hübner (Lepidoptera, Nymphalidae, Heliconiinae). Revista Brasileira de Entomologia 52: 1323.
• Kaminski, L.A., Tavares, M., Ferro, V.G. & Moreira, G.R.P. 2002. Morfologia externa dos estágios imaturos de heliconíneos neotropicais: III. Heliconius erato phyllis (Fabricius) (Lepidoptera: Nymphalidae: Heliconiinae). Revista Brasileira de Zoologia 19: 977993.
• Kitching, I.J. 1984. The use of larval chaetotaxy in butterfly systematics, with special reference to the Danaini (Lepidoptera: Nymphalidae). Systematic Entomology 9: 4961.
• Lamas, G. 2004. Checklist: Part 4A. Hesperioidea Papilionoidea, p. 261274. In: Heppner, J.B. (ed.). Atlas of Neotropical Lepidoptera Volume 5A. Gainesville, Association for Tropical Lepidoptera, Scientific Publishers, xxxvi+439 p.
• Luebert, F. & Pliscoff, P. 2006. Sinopsis bioclimática y vegetacional de Chile. Santiago, Editorial Universitaria, 316 p.
• Mosher, E. 1916. A classification of the Lepidoptera based on characters of the pupa. Bulletin of the Illinois State Laboratory of Natural History 12: 1165.
• Paim, A.C., Kaminski, L.A. & Moreira, G.R.P. 2004. Morfologia externa dos estágios imaturos de heliconíneos neotropicais. IV. Dryas iulia alcionea (Lepidoptera: Nymphalidae: Heliconiinae). Iheringia, Série Zoologia, 94: 2535.
• Penz, C.M. 1999. Higher level phylogeny for passion-vine butterflies (Nymphalidae, Heliconiinae) based on early stage and adult morphology. Zoological Journal of the Linnean Society 127: 277344.
• Peña, L.E. & Ugarte, A.J. 1996. Las mariposas de Chile. The butterflies of Chile. Santiago, Editorial Universitaria, 359 p.
• Peterson, A. 1962. Larvae of insects.An introduction to Nearctic species. Part I Lepidoptera and plant infesting Hymenoptera Ann Arbor, Edwards Brothers Inc., 315 p.
• Schwerdtfeger, M. 2004. Passiflowers of the Andes, p.7577. In: Ulmer, T. & MacDougal, J.M. (eds.) Passiflora: Passionflowers of the World Portland, Timber Press, 432 p.
• Silva, D.S., Dell'Erba, R., Kaminski, L.A. & Moreira, G.R.P. 2006. Morfologia externa dos estágios imaturos de heliconíneos neotropicais: V. Agraulis vanillae maculosa (Lepidoptera, Nymphalidae, Heliconiinae). Iheringia, Série Zoologia, 96: 219228.
• Silva, D.S., Kaminski, L.A., Dell'Erba, R. & Moreira, G.R.P. 2008. Morfologia externa dos estágios imaturos de heliconíneos neotropicais: VII. Dryadula phaetusa (Linnaeus) (Lepidoptera, Nymphalidae, Heliconiinae). Revista Brasileira de Entomologia 52: 500509.
• Simirgiotis, M.J., Schmeda-Hirschmann, G., Bórquez, J. & Kennelly, E.J. 2013. The Passiflora tripartita (Banana Passion) Fruit: A Source of bioactive flavonoid C-glycosides isolated by HSCCC and characterized by HPLCDADESI/MS/MS. Molecules 18: 16721692.
• Snedecor, G.W. & Cochran, W.G. 1980. Statistical methods Ames, Iowa State University, 507 p.
• Stehr, F.W. 1987. Order Lepidoptera, p. 288305. In: Stehr, F.W. (ed.). Immature insects Vol. I, Dubuque, Kendall/Hunt Publishing Company, 975 p.
• Tavares, M., Kaminski, L.A. & Moreira, G.R.P. 2002. Morfologia externa dos estágios imaturos de heliconíneos neotropicais: II. Dione juno juno (Cramer) (Lepidoptera: Nymphalidae: Heliconiinae). Revista Brasileira de Zoologia 19: 961976.

 

Publication Dates

• Publication in this collection
  14 July 2014
• Date of issue
  June 2014

History

• Accepted
  27 Feb 2014
• Received
  11 Sept 2013