Research Article |
Corresponding author: Jackie Farfán ( jjackie4u@gmail.com ) Academic editor: Martin Wiemers
© 2022 Jackie Farfán, José Cerdeña, Héctor A. Vargas, Gislene L. Gonçalves, Gerardo Lamas, Gilson R. P. Moreira.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Farfán J, Cerdeña J, Vargas HA, Gonçalves GL, Lamas G, Moreira GRP (2022) A peculiar new species of Dione (Agraulis) Boisduval & Le Conte (Lepidoptera, Nymphalidae, Heliconiinae) associated with Malesherbia Ruiz & Pavón (Passifloraceae) in xeric western slopes of the Andes. ZooKeys 1113: 199-226. https://doi.org/10.3897/zookeys.1113.85769
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Butterflies associated with xerophytic environments of the Andes have been little studied, and they exhibit high levels of endemism. Herein Dione (Agraulis) dodona Lamas & Farfán, sp. nov. (Nymphalidae; Heliconiinae) is described, distributed on the western slopes of the Andes of Peru and northern Chile, between 800 and 3,000 m elevation. Adults of both sexes, and the immature stages, are described and illustrated based on light and scanning electron microscopy. The immature stages are associated with Malesherbia tenuifolia D. Don (Passifloraceae) found in xeric environments, representing a new record of this genus as a host plant for the subfamily Heliconiinae. Conspicuous morphological differences are presented for all stages at the generic level. Based on a phylogenetic analysis of the COI barcode mitochondrial gene fragment, D. (A.) dodona Lamas & Farfán, sp. nov. is distinguished as an independent lineage within the Agraulis clade of Dione, with ca. 5% difference to congeneric species.
Chile, Heliconiines, immature stages, Peru, taxonomy
The Andes are a ~ 8000 km long mountain belt on western South America, forming one of the longest mountain ranges on Earth (
Heliconiinae have been the subject of widespread study in various aspects of biology for the last 160 years, comprising more than 700 scientific publications (
Phylogenetic studies have proposed that Agraulis is among the oldest lineages within the Heliconiini (
This undescribed species has been considered rare, with only seven museum specimens known until 2013. The first one was collected in northern Chile in 1951 at an altitude of 3,000 m, and erroneously identified as A. vanillae (Linnaeus, 1758), as illustrated in the book Butterflies of Chile (
Thus, in the present paper, the new species is described and illustrated based on the morphology of adults and immature stages. We also present a phylogenetic analysis of mitochondrial (COI) DNA sequences including congeneric species.
Immature stages were collected from plants of Malesherbia tenuifolia D. Don (Passifloraceae), near the village of Pacaychacra (15°50'57"S, 72°38'9"W), Condesuyos Province, 23–24 km SE from the town of Chuquibamba, Department of Arequipa, southern Peru, at 1,800 m elevation. They were brought to the laboratory of Área de Entomología, Museo de Historia Natural de la Universidad Nacional de San Agustin de Arequipa (
Adult specimens of related taxa were examined at Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima, Peru (MUSM), and at
Genitalia dissections were performed using standard techniques, where abdomens of adults were previously soaked in hot 10% KOH solution for 10 min, and dissected parts were stored in glycerol. To study the venation, wings were diaphanized by soaking them in 2% NaClO aqueous solution (bleach), and then dry-mounted.
Morphological observations were performed with the aid of a Zeiss Stemi 305 stereo microscope, and structures selected to be illustrated were previously photographed with a Nikon DS-Ri2 camera through a Nikon SMZ25 stereo microscope at the Laboratório de Sistemática Animal of Universidad Nacional San Agustin de Arequipa (UNSA). Images were assembled and edited in Nikon NIS-Elements and Photoshop version 21.2.0. The descriptive terminology of morphological structures follows
For scanning electron microscope analyses, specimens were dehydrated in a BalTec CPD 030 critical-point dryer, mounted with double-sided tape on metal stubs, and coated with gold in a Quorum Q150R plus sputter coater. They were then examined and photographed in a XL-30 Philips scanning electron microscope at the Laboratório Central de Microscopia e Microanálise (LabCEMM) of Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil. Priority in this case was given to key diagnostic characters that were used to distinguish the new Dione species from congeners; additional scanning electron micrographs are presented for the immature stages in Suppl. material
Genomic DNA was extracted from two specimens of the new Dione species (Table
Specimens used for molecular analyses of Dione (Agraulis) dodona sp. nov. The collection sites (country/locality) and vouchers from which the sequences derived are presented, including the references. Genbank and BOLD identifiers link the record to the databases.
Species | Country/Locality | Voucher | Genbank/BOLD Accession code | Reference |
---|---|---|---|---|
Dione (Agraulis) dodona sp. nov. | Peru / Arequipa | J151 | OM925454/BIGLE001-22 | This study |
Dione (Agraulis) dodona sp. nov. | Peru / Arequipa | 459 | OM925453 /BIGLE002-22 | This study |
Dione (Agraulis) incarnata | Costa Rica / Guanacaste | 00-SRNP-16229 | GU333737 / MHACG518-04 |
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Dione (Agraulis) forbesi | Peru | G3 | MZ229712.1 |
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Dione (Agraulis) insularis | Dominican Republic | NW152-16 | GQ864730 |
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Dione (Agraulis) lucina | Ecuador | LEP-58352 | MZ229704.1 |
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Dione (Agraulis) maculosa | Argentina/ Entre Ríos | MACN-Bar-Lep-ct 01616 | MF545390 / LEPAR178-11 |
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Dione (Agraulis) vanillae | Ecuador | LEP-55200 | MZ229702.1 |
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Dione (Dione) glycera | - | BMC17102 | MN306819.1 |
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Dione (Dione) juno | Peru/ San Martín | 8727 | KP074744.1 |
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Dione (Dione) moneta | Argentina/ Salta | MACN-Bar-Lep-ct 07589 | MZ335918.1/LNOA484-16 | NCBI deposit |
Abbreviations for the museum collections and institutions from which specimens were examined are:
IDEA Colección Entomológica de la Universidad de Tarapacá, Arica, Chile;
MUSM Museo de Historia Natural, Universidad Nacional Mayor San Marcos, Lima, Peru.
Dione vanillae:
Agraulis vanillae:
Agraulis vanillae forbesi: Herrera,1972: 73.
Agraulis
[n. sp.]:
Agraulis
sp. n.:
Agraulis
sp.:
Peru, Arequipa, Pacaychacra [15°54'S, 72°33'W], 1500 m.
Holotype
♂, Peru, Arequipa, Pacaychacra, 15°54'S, 72°33'W, 1500 m, reared from eggs collected on Malesherbia tenuifolia (Passifloraceae), 24.VII.2019, J. Farfán leg. deposited in MUSM. Paratypes (25♂, 17♀): Peru. Lima: 1♂, San Bartolomé, 1600 m, [11°55'S, 76°31'W], 21.iii.[19]81, P. Hocking (MUSM); 1♀, Cocachacra, 1450 m, [11°55'S, 76°32'W], 6.x.[19]83, P. Hocking [MUSM-ENT 008630] (MUSM); 1♀, Río Rímac, Chaute, 2350 m, 11°56'S, 76°30'W, 12.v.2012, P. Hocking (MUSM). Arequipa: 2♀, 7 km E Cháparra, 1450m, [15°41'S, 73°49'W], 14.iv.[19]88, G. Lamas [MUSM-ENT 008631, 008632] (MUSM); 1♂, entre Majes y Chuquibamba, 15°55'S, 72°33'W, 1500 m, 24.iv.2017, G. Lamas (MUSM); 1♂ same data as holotype [
Immature stages preserved in 70% ethanol, with the same data as the holotype, collected on Malesherbia tenuifolia (Passifloraceae) with dates VI.2018, X.2019 and IV.2021, were deposited in
Dione (Agraulis) dodona sp. nov. can be easily distinguished from its congeners by the wing pattern, presenting a black postdiscal spot between M3-A1 veins on the dorsal hindwing that is absent in all other species, and, also, by presenting a divided or partially divided silver spot in the discal cell on the ventral hindwing, always undivided in other species of Dione (Agraulis). In the male genitalia, the valvae have a rounded termen, without denticles, and the distal portion of the crista is narrow and straight. In other species, the termen is sub-triangular and shows denticles on the margin, and the distal apex of the crista is transversally enlarged. Also, the juxta has its upper edge slightly split in dodona, which is widely open in other species. The aedeagus is straight in lateral view without cornuti in dodona, up-curved near distal end with cornuti in other species. The female genitalia possess evenly wide signa in the proximal portion, composed of robust spines, unlike other species that have smaller spines; the proximal apex of signa is narrower and progressively enlarges distally.
Furthermore, the immature stages of Dione (Agraulis) dodona sp. nov. show differences with the available data compared to other Agraulis species (
Adult. (Figs
Adults of Dione (Agraulis) dodona sp. nov. A, B male, holotype C, D female paratype E, F details of hindwing pattern color (indicated by blue squares in A and D respectively), open arrow points black spot on Cu1-Cu2 cell, and close arrow on silver discal spot. Left: dorsal view; right: ventral view. Scale bars: 2 cm.
Adult morphology of Dione (Agraulis) dodona sp. nov. A head, male, lateral view B wing venation, male C detail of forewing venation, female (red setae points differences with male) D, E distal portion of prothoracic tarsi under scanning electron microscopy, male in ventral view D female in lateral view E distal tarsomere indicated by open arrow in E. Scale bars: 1 mm (A); 5 mm (B); 50 µm (D); 250 µm (E).
Male genitalia
: Rounded and subtriangular valvae occupying most of the genital capsule, being wide anteriorly and narrowest in the apex, with rounded pointed apex with hairs on ventral margin (Fig.
Male genitalia of Dione (Agraulis) dodona sp. nov. A general, mesal view B distal portion of valve crista under scanning electron microscopy (pointed with open arrow in C) C right valve D termen of valve under scanning electron microscopy (pointed with closed arrow in C) E aedeagus, lateral F juxta, posterior. Scale bars: 1mm (A); 250 µm (B); 500 µm (C, E, F); 200 µm (D).
Female: Wingspan 50–52 mm, forewing length 26–27 mm. Very similar to male, but paler, with the most prominent dorsal marks and spots mainly in the forewing apex, where it presents a faint stain between the veins R1 to M1, absent in males and with a paler background than the rest of the wing (Fig.
Female genitalia
: Eighth segment narrow. Posterior apophysis ~ 1/2 the length of the papilla anales (Fig.
Egg
(Figs
Sub spherical, flat base slightly narrowed near apex. Yellow when recently laid (Fig.
Immature stages of Dione (Agraulis) dodona sp. nov. A freshly-laid egg, lateral view B egg just prior to hatching, lateral C first instar, latero-dorsal D, E fifth instar, head in detail (anterior view) and general aspect (lateral), respectively F, G, H pupa in ventral, dorsal and lateral views, respectively. Scale bars: 400 µm (A); 1 mm (C); 5 mm (E); 4 mm (H).
(Figs
Larval chaetotaxy of Dione (Agraulis) dodona sp. nov. A, B head capsule, frontal and lateral view respectively C prothoracic dorsal shield, dorsal D first instar, lateral E fifth instar, lateral. A, anterior seta; AF, adfrontal seta; C, clypeal seta; D, dorsal seta; F, frontal seta; L, lateral seta; P, postero-dorsal seta; S, stemmatal seta; SS, sub-stemmatal seta; PL, seta of proleg cylindrical section of tenth abdominal segment; PP, paraproctal seta; Sc, cephalic scolus; SD, subdorsal seta; Sl, subspiracularscolus; Sn, anal scolus; So, dorsal scolus; Sp, supraspiracularscolus; SV, subventral seta; XD, prothoracic seta. Scale bars: 200 µm (A); 100 µm (B, C); 500 µm (D); 3 mm (E).
From the second instar on, the head is black with thorax and abdomen yellow with two bands running along the subdorsal and subspiracular area. Three chromatic patterns were observed, mainly in the fifth instar, one of these patterns (brownish) is characterized by the head, legs and black scoli; thorax and abdomen yellow with brown band in the subdorsal and subspiracular area, with a thin dorsal brown line, the head has a pattern of white spots located on the frontoclypeous, and with the brown labrum, lateral plates of prolegs black (Fig.
(Figs
The successive instars can be accurately distinguished by the width of the head capsule, because they do not overlap (Table
Mean and standard error (SE), interval of variation (IV), and growth rates (GR) of head capsule width in larval instars of Dione (Agraulis) dodona sp. nov. reared on Malesherbia tenuifolia D. Don.
Instar | N | Head capsule width (mm) | ||
---|---|---|---|---|
Mean ± SE | IV | GR | ||
I | 19 | 0.50 ± 0.01 | 0.46–0.56 | - |
II | 12 | 0.72 ± 0.01 | 0.58–0.83 | 1.44 |
III | 8 | 1.07 ± 0.03 | 0.95–1.17 | 1.48 |
IV | 8 | 1.69 ± 0.04 | 1.56–1.87 | 1.52 |
V | 5 | 2.50 ± 0.05 | 2.44–2.70 | 1.48 |
Pupa
(Figs
Immature stages of Dione (Agraulis) dodona sp. nov. under scanning electron microscopy A egg, lateral view B first instar, upper portion of fifth abdominal segment, lateral C fifth instar, prothoracic plate, postero-dorsal (protuberance pointed by close arrow) D, E, F pupal head (dorsal view), mesothoracic meso-dorsal crest (lateral), and latero-dorsal tubercle of third abdominal segment (lateral), respectively. Scale bars: 500 µm (A, F); 100 µm (B); 250 µm (C); 1.5 mm (E, D).
The specific epithet is based on the locality of Dodona (Greece); it was a city-sanctuary in ancient Greece, where there was an oracle in which Dione was venerated as the (temporary) wife of Zeus, until she was replaced by Hera. Thus, the new species is named “dodona” to continue the classical Greek tradition.
Adults of Dione (Agraulis) dodona sp. nov. are known from distinct populations, located in central and southern Peru, and northern Chile, on the western slopes of the Andes. In Peru, it has been found in the Departments of Lima (1,400–2,400 m elevation), Arequipa (between 800 and 2,600 m elevation), Moquegua (1,800–2,100 m elevation), and Tacna (1,800 m elevation). In Chile, two specimens were collected in 1951 and 1968 from two localities in the Tarapacá Region between 2,300 and 3,000 m elevation, and recently other two specimens were collected from Arica Region (1,580 m elevation).
Malesherbia tenuifolia D. Don (Passifloraceae) is the only host plant known for the immature stages of Dione (Agraulis) dodona sp. nov. This species was described originally from northern Chile. It is distributed between 19–21 °S in Chile (
Life history of Dione (Agraulis) dodona sp. nov. A general view of type locality, Pacaychacra valley, Arequipa, Peru (blue close arrow points to larval hostplant) B host plant, Malesherbia tenuifolia Don C female laying eggs (close arrow point) on flowers of host plant D eggs (pointed by setae) on flower E detail of flower showing damage (entrance) by first instar larva (open arrow). Scale bar: 5 mm.
Adults of Dione (Agraulis) dodona sp. nov. were only observed on sunny days in the type locality, beginning to fly around 08:00 a.m., quickly disappearing when weather conditions became cloudy. They usually fly close to the ground, up to 1–2 m high. Females were seen alighting on the host plant. Territorial behavior, courtship and mating behavior were not observed. Adults were seen feeding on flowers of other plants growing around Malesherbia hosts. The species is multivoltine in the population of the type locality, flying all year round. From three to seven individuals were usually observed in a typical sunny day (4–5 h of observation), most of which were males. This species is sympatric with Dione (Agraulis) forbesi, but they do not fly in the same habitat; forbesi was observed in areas with higher humidity and vegetation, compared to dodona, which was found only in xerophytic areas associated with the host plant (Fig.
Dione (Agraulis) dodona sp. nov. was recovered as an independent lineage within the Agraulis clade of the COI-tree (Fig.
Genetic distance (%) between Dione (Agraulis) dodona sp. nov. and its congeners. Analysis used 650 base pairs sequences of the Cytochrome oxidase subunit I gene under the Kimura 2-parameter model. Specimens included in the analysis are presented in Table
Taxa | 1. | 2. | 3. | 4. | 5. | 6. | 7. | 8. |
---|---|---|---|---|---|---|---|---|
1. Dione (Agraulis) dodona sp. nov. | - | |||||||
2. Dione (Agraulis) forbesi | 5.8 | - | ||||||
3. Dione (Agraulis) lucina | 4.6 | 3.9 | - | |||||
4. Dione (Agraulis) vanillae | 5.3 | 4.4 | 2.2 | - | ||||
5. Dione (Agraulis) incarnata | 5.1 | 4.6 | 3.4 | 3.4 | - | |||
6. Dione (Agraulis) insularis | 4.6 | 3.9 | 1.9 | 1.9 | 3.2 | - | ||
7. Dione (Agraulis) maculosa | 4.4 | 3.4 | 1.1 | 1.7 | 2.7 | 1.1 | - | |
8. Dione (Dione) spp. | 9.5 | 8.9 | 8.6 | 8.6 | 9.3 | 9.4 | 8.8 | - |
Phylogenetic status of Dione (Agraulis) dodona sp. nov. based on 650 bp-sequences of the Cytochrome oxidase subunit I gene. The consensus tree was inferred with the Maximum Likelihood method and General Time Reversible model, with 500 bootstrap replicates. The branch support (bootstrap) is shown next to the nodes.
The identification key to adults of Agraulis provided by
Thus, results presented herein show clearly that Dione (Agraulis) dodona sp. nov. is distinct from its congeneric species at all development levels. On the other hand, particularly in the larval stage, our study also showed that it shares some characters with species of the Dione clade; in the first instar, the D2 setae are well developed accordingly in the latter; in subsequent instars, the cephalic scoli are reduced similar with Dione moneta, and the prothoracic plate with enlarged conical protuberances bears setae. In fact, such traits had been used up to now to separate genera of heliconiines at the larval stage (
Females of Dione (Agraulis) dodona sp. nov. lay eggs predominantly on flowers, where first instar feed on the internal parts. There is no documentation of another species of Heliconiinae that oviposits preferentially on flowers or larvae feeding on them (
Finally, it is important to mention that, historically, Dione (Agraulis) dodona sp. nov. was erroneously cited for decades as “Agraulis vanillae” in Chile, based on two specimens collected between 1950 and 1970 in the Tarapacá region, northern Chile.
We are grateful to Leandro Menezes Baum from Laboratório Central de Microscopia e Microanálise (LabCEMM) of Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil, for helping with scanning electron microscopy analyses. Thanks also to Dr. Evaristo López (Museo de Historia Natural, Universidad Nacional San Agustín, Arequipa, Perú [
Figure S1
Data type: Image.
Explanation note: Male genitalia of Dione (Agraulis) species congeners to Dione (Agraulis) dodona sp. nov. (A) D. (A) galapagensis; (B) D. (A.) lucina; (C) D. (A.) maculosa; (D) D. (A.) vanillae; (E) D. (A.) forbesi; (F) D. (A.) insularis; (G) D. (A.) incarnata incarnata; (H) D. (A.) incarnata nigrior. From right to left column: General, mesal view, aedeagus removed; termen of valve, mesal; juxta, ventral; aedeagus, lateral. Scale bars: 1mm, 0.25mm, 0.25mm, 0.5mm, respectively.
Figure S2
Data type: Image.
Explanation note: Scanning electron micrographs of Dione (Agraulis) dodona sp. nov. immature stages: (A-C) Egg; (D-J) First instar; (K-P) Fifth instar. (A) micropylar region; (B) aeropyle; (C) upper cells; (D) stemmatal region, lateral; (E) antenna, antero-lateral; (F) spinneret, lateral; (G) metathoracic leg; (H) apex of setae of abdominal segment, lateral; (I) proleg with exposed crochets; (J) last abdominal segment, posterior; (K) stemmatal region, dorso-frontal; (L) short chalaza-like setae; (M) proleg of six abdominal segments; (N) seventh abdominal segment, latero-dorsal; (O) spiracle; (P) corresponding spiracle in detail. Mp, micropyles; Ac, aeropyle; Uc, upper cell. Scale bars: 50 µm (A, E, F, I, L, Q); 10 µm (B); 150 µm (C, P); 100 µm (D, G, J); 25 µm (H, M); 250 µm (K); 0,5 mm (N); 1mm (O).