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ZooKeys 1113: 199–227 (2022)

doi: 10.3897/zookeys.1113.85769 RESEARCH ARTICLE
https://zookeys.pensoft.net Launched to accelerate biodiversity research

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

Jackie Farfán1,2, José Cerdeña1,2, Héctor A. Vargas3,

Gislene L. Gonçalves3,4, Gerardo Lamas5, Gilson R. P. Moreira6

1 PPG Biologia Animal, Departamento de Zoologia, Instituto de Biociências, Universidade Federal do Rio
Grande do Sul, Av. Bento Gonçalves, 9500, Porto Alegre, RS 91501-970, Brazil 2 Museo de Historia Natural,
Universidad Nacional de San Agustín de Arequipa, Av. Alcides Carrión s/n, Arequipa, Peru 3 Departamento de
Recursos Ambientales, Facultad de Ciencias Agronómicas, Universidad de Tarapacá, Casilla 6-D, Arica, Chile
4 Departamento de Genética, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Av. Bento
Gonçalves 9500, Porto Alegre RS, 91501-970, Brazil 5  Departamento de Entomología, Museo de Historia
Natural, Universidad Nacional Mayor de San Marcos, Apartado 14-0434, Lima-14, Peru 6 Departamento
de Zoologia, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500,
Porto Alegre RS, 91501-970, Brazil

Corresponding author: Jackie Farfán ([email protected])

Academic editor: Martin Wiemers  |  Received 26 April 2022  |  Accepted 17 June 2022  |  Published 18 July 2022

https://zoobank.org/C4EB0D82-D142-49EE-A95F-DA97BEBDEBB6

Citation: Farfan 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: 199–227. https://doi.
org/10.3897/zookeys.1113.85769

Abstract
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; Heli-
coniinae) 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

Copyright Jackie Farfán et al. 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.
200 Jackie Farfán et al. / ZooKeys 1113: 199–227 (2022)

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.

Keywords
Chile, Heliconiines, immature stages, Peru, taxonomy

Introduction
The Andes are a ~ 8000 km long mountain belt on western South America, forming
one of the longest mountain ranges on Earth (Montgomery et al. 2001), and harboring
a variety of ecosystems with different environmental characteristics (Myers et al. 2000;
Veblen et al. 2007). The western slopes of the central Andes join the Peru-Chile Pacific
and Atacama deserts from 10 °S in central Peru to 30 °S in northern Chile, where xe-
rophytic shrub land vegetation is predominant between 1,500 and 3,000 m elevation
(Gutiérrez et al. 1998; Montesinos et al. 2012), containing many endemic plants and
animals (Zeballos-Patrón et al. 2001; Arakaki and Cano 2003; Gutiérrez et al. 2019;
Farfán et al. 2020; Málaga et al. 2020). The butterfly fauna in this area is depauper-
ate and poorly known, with very few species recorded among the Papilionidae, Hes-
periidae, Pieridae, and Nymphalidae, including four species of passion-vine butterflies
(Heliconiinae) previously ascribed to Dione Hübner, [1819] and Agraulis Boisduval &
Le Conte, [1835] (Peña and Ugarte 2006; Benyamini et al. 2014; Farfán 2018).
Heliconiinae have been the subject of widespread study in various aspects of biol-
ogy for the last 160 years, comprising more than 700 scientific publications (Bates
1862; Jiggins 2017), highlighting their importance as a model in the study of evolu-
tion (e.g., Mallet and Joron 1999), ecology (e.g., Gilbert 1991; Schluter 2000) and
behavior (Deinert et al. 1994), among others. The phylogenetic relationships of the
members of the Heliconiinae have been analyzed from a morphological (Penz 1999;
Penz and Peggie 2003) and molecular (Brower and Egan 1997; Beltrán et al. 2002,
2007; Kozak et al. 2015) perspective, recognizing four tribes: Acraeini, Heliconiini,
Argynnini and Vagrantini. The Heliconiini included until recently the genera Phila-
ethria Billberg, 1820, Podotricha Michener, 1942, Dryas Hübner, [1807], Dryadula
Michener, 1942, Dione Hübner, [1819], Agraulis Boisduval & Le Conte, [1835], Eue-
ides Hübner, 1816, and Heliconius Kluk, 1780, of which the latter is the most diverse
with ~ 45 species and 200 subspecies (Lamas 2004; Kozak et al. 2015; Zhang et al.
2019). All the heliconiine species have as host plants members of the Passifloraceae s. l.
(Bremer et al. 2009), mainly Passiflora L. (de Castro et al. 2018). There is a large num-
ber of studies related to the coevolution between butterflies and host plants, using the
Passiflora-heliconiine system as a model for the study of coevolution in insects (Ehrlich
and Raven 1964; Gilbert 1972; Benson et al. 1975; de Castro et al. 2018).
Phylogenetic studies have proposed that Agraulis is among the oldest lineages within
the Heliconiini (Brower and Egan 1997; Penz 1999; Beltrán et al. 2007). For a long
time, it was considered as a monotypic genus, with eight subspecies, occupying the larg-
 New species of Dione from xeric western slopes of the Andes 201

est distribution area within the tribe, ranging from the central west of the USA to the
center of Argentina and Chile (Núñez et al. 2022). Recently, Zhang et al. (2019) through
genome-scale phylogenetic analyses suggested that Agraulis is best treated as a subgenus
of Dione. However, later on, Núñez et al. (2022) based on morphometric and molecular
data maintained the two separate genera as valid. They also raised the eight subspecies of
Agraulis to the species level including an undescribed species from the western slope of
the Andes, previously listed as Agraulis n. sp. in the checklist of Neotropical butterflies
(Lamas 2004). The proposal of Zhang et al. (2019) is followed in this study.
This undescribed species has been considered rare, with only seven museum speci-
mens known until 2013. The first one was collected in northern Chile in 1951 at an alti-
tude of 3,000 m, and erroneously identified as A. vanillae (Linnaeus, 1758), as illustrated
in the book Butterflies of Chile (Peña and Ugarte 2006). The additional specimens are
known from central and southern Peru, and northern Chile. During a recent exploration
in the Department of Arequipa, southern Peru, adults of this undescribed species were
observed flying, crossing the road in a sector of a desert hillside with very sparse vegeta-
tion, and laying eggs on the flowers of Malesherbia tenuifolia D. Don (Passifloracae).
Thus, by finding the host plant, and then rearing the immatures, it was possible to obtain
sufficient material to carry out a comparative study at the generic level, where we con-
firmed for all stages that it does not belong to any described species of Dione (Agraulis).
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.

Materials and methods

Immature stages were collected from plants of Malesherbia tenuifolia D. Don (Passiflo-
raceae), 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
(MUSA), and maintained under natural conditions of temperature and humidity. The
eggs were placed until larval hatching. Fresh larvae were transferred to small plastic con-
tainers and fed with M. tenuifolia leaves. Six larvae successfully completed development
to adults, with four males and two females emerging that were deposited in the collection
of the MUSA. Additional field-collected eggs, larvae and pupae were fixed in Dietrich´s
fluid and preserved in 70% ethanol. Also, flying adults were eventually netted near the
host plants. Studies of morphology of the immature stages were conducted at the Labo-
ratório de Morfologia e Comportamento de Insetos (LMCI), Departamento de Zoo-
logia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil.
Adult specimens of related taxa were examined at Museo de Historia Natural,
Universidad Nacional Mayor de San Marcos, Lima, Peru (MUSM), and at MUSA.
Photographs of all relevant type specimens were examined at MUSM; such images are
202 Jackie Farfán et al. / ZooKeys 1113: 199–227 (2022)

also available in Warren et al. (2017). Images taken of male genitalia dissected from the
new species are presented in Suppl. material 1.
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 photo-
graphed 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 da Silva et al. (2006), Beebe et al. (1960), Emsley (1963) and Klots (1970), for
eggs, larvae, pupae, and adults, respectively.
For scanning electron microscope analyses, specimens were dehydrated in a Bal-
Tec 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 ex-
amined and photographed in a XL-30 Philips scanning electron microscope at the
Laboratório Central de Microscopia e Microanálise (LabCEMM) of Pontifícia Univer-
sidade 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 2.
Genomic DNA was extracted from two specimens of the new Dione species (Table 1),
collected in the type locality, following the procedures described in Huanca-Mamani et al.
(2015). A fragment of 650 base pairs of the COI gene was amplified by polymerase chain
reaction (PCR) with the primers LEP-F1 and LEP-R1 (Hebert et al. 2004). PCR ampli-
cons were purified and sequenced by Macrogen (Republic of South Korea) using LEP-F1

Table 1. 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 Núñez et al. 2022
Dione (Agraulis) forbesi Peru G3 MZ229712.1 Núñez et al. 2022
Dione (Agraulis) insularis Dominican Republic NW152-16 GQ864730 Núñez et al. 2022
Dione (Agraulis) lucina Ecuador LEP-58352 MZ229704.1 Núñez et al. 2022
Dione (Agraulis) maculosa Argentina/ Entre Ríos MACN-Bar-Lep- MF545390 / LEPAR178-11 Lavinia et al. 2017
ct 01616
Dione (Agraulis) vanillae Ecuador LEP-55200 MZ229702.1 Núñez et al. 2022
Dione (Dione) glycera - BMC17102 MN306819.1 Marín et al. 2021
Dione (Dione) juno Peru/ San Martín 8727 KP074744.1 Kozak et al. 2015
Dione (Dione) moneta Argentina/ Salta MACN-Bar-Lep- MZ335918.1/LNOA484-16 NCBI deposit
ct 07589
 New species of Dione from xeric western slopes of the Andes 203

primer. Sequences obtained in this study were deposited in GenBank and BOLD databas-
es (Table 1). The phylogenetic status of the new Dione species was explored by combining
our sequences with COI data of six congeners (D. (A.) incarnata Riley, 1926, D. (A.)
forbesi Michener, 1942, D. (A.) insularis (Maynard, 1889), D. (A.) lucina (C. Felder & R.
Felder, 1862), D. (A.) maculosa Stichel, [1908], and D. (A.) vanillae (Linnaeus, 1758)) ob-
tained from Núñez et al. (2022). In addition, three species of Dione (Dione), juno (Cramer,
1779), glycera (C. Felder & R. Felder, 1861) and moneta Hübner, [1825] were included in
the analysis (Table 1). The COI-tree was inferred by using the Maximum Likelihood (ML)
method and General Time Reversible model (Nei and Kumar 2000), with heuristic search
obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of
pairwise distances, with 500 bootstrap replications. Analysis was conducted in MEGA X
(Kumar et al. 2018). Genetic distances between species of subgenera Agraulis and Dione
were quantified using the Kimura 2-parameter model in MEGA.
Abbreviations for the museum collections and institutions from which specimens
were examined are:

LMCI Laboratório de Morfologia e Comportamento de Insetos, Universidade

Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil;
MHNS Museo Nacional de Historia Natural de Chile, Santiago de Chile, Chile;
IDEA Colección Entomológica de la Universidad de Tarapacá, Arica, Chile;
MUSA Museo de Historia Natural, Universidad Nacional de San Agustín de Areq-
uipa, Arequipa, Peru;
MUSM Museo de Historia Natural, Universidad Nacional Mayor San Marcos,
Lima, Peru.

Taxonomic account
Dione (Agraulis) dodona Lamas & Farfán, sp. nov.
https://zoobank.org/687E38BC-19C6-48F0-9B93-EE7B9A7C40A5
Figs 1–10

Dione vanillae: Peña 1951: 262.

Agraulis vanillae: Ureta 1963: 114–115; Pérez-D’Angello 1970: 6; Etcheverry 1970:
95; Peña and Ugarte 2006: 313, figs; Benyamini et al. 2014: 18.
Agraulis vanillae forbesi: Herrera,1972: 73.
Agraulis [n. sp.]: Lamas 2004: 264.
Agraulis sp. n.: Farfán 2018: 367.
Agraulis sp.: Núñez et al. 2022: 152–178.

Type locality. Peru, Arequipa, Pacaychacra [15°54'S, 72°33'W], 1500 m.

Type material. 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
204 Jackie Farfán et al. / ZooKeys 1113: 199–227 (2022)

Figure 1. 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.

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 [LMCI 357–51] (LMCI); 1♂ same data as holotype (MUSA); 1♂, 2♀,
same data as holotype but with date 15.XII.2020 (MUSA); 1♂ same data as holotype
 New species of Dione from xeric western slopes of the Andes 205

Figure 2. Adult morphology of Dione (Agraulis) dodona sp. nov. A head, male, lateral view B wing vena-
tion, male C detail of forewing venation, female (red setae points differences with male) D, E distal por-
tion 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).
206 Jackie Farfán et al. / ZooKeys 1113: 199–227 (2022)

Figure 3. 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).

but with date 24.vii.2021 (MUSA); 4♂ Aplao, Valle Majes, 15°53'46"S, 72°28'03"W,
800 m, 02.vi.2013, Leg. J. Cerdeña / M. Delgado (MUSA); 1♂, Condesuyos,
Pacaychacra,15°54'59.2"S, 72°33'01.5"W, 1500 m, 02.IX.2020, Leg. Jose Cerdeña
 New species of Dione from xeric western slopes of the Andes 207

Figure 4. Female genitalia of Dione (Agraulis) dodona sp. nov. A external view, lateral B signum C stink
club under scanning electron microscopy, specialized scales indicated by closed arrows. Asterisk indicated
dorsal glands. Scale bars: 1 mm (A); 500 µm (B); 200 µm (C).

(MUSA); 1♂, same data, but 13.XII.2020 (MUSA); 1♀, same data, but 24.VII.2021,
Jackie Farfán (MUSA); 1♀, same data, but 12.XI.2019, [LMCI 357–52] (LMCI);
3♂, Yura, 2 Km SW Yura viejo, 16°13'19"S, 71°42'23"W, 2600 m 21.IV.2022, Leg.
Jackie Farfán (MUSA). Moquegua: 6♂, 1♀, Torata, 170630/705036 [17°06'30"S,
70°50'36"W], 2090m, 26.VII.2021, Leg. Jackie Farfán (MUSA); 1♂, 1♀, same
data [LMCI 357–53, LMCI 357–54] (LMCI); 4♀, La Capilla, 13 Km S Puquina,
164450/711054 [16°44'50"S, 71°10'54"W], 1800 m, 28.XII.2013, Leg. J. Farfán /
J. Cerdeña (MUSA); 1♀, Omate, 3 Km SW Omate, 16°41'15.27"S, 70°59'13.73"W,
2000 m, 27.X.2017, Leg. Robert Cornejo (MUSA). Tacna: 1♂, Chululuni, 17°22'02"S,
70°28'24"W, 1800 m, 18.XII.2020, Leg. Jackie Farfán (MUSA). CHILE. Arica: 1♂,
Las Peñas, 18°33'08"S, 69°46'03"W, 1580 m, 02.XII.2020, H. A. Vargas leg. [IDEA-
LEPI–2022–008] (IDEA); 1♀, same data, but 10.XI.2020; [IDEA-LEPI–2022–007]
(IDEA). Tarapacá: 1♀, Iquique, Parca, 3000 m, [20°01'S, 69°01'W], ix/x.[19]51, L.
E. Peña (MUSM); 1♂, Q[uebrada] de Guatacondo, “Cauquenisca” [= Cautenicsa],
2300 m, [20°56'S, 69°01'W], 26-X-1968, P. Millas (MHNS).
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
208 Jackie Farfán et al. / ZooKeys 1113: 199–227 (2022)

Figure 5. 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).

IV.2021, were deposited in LMCI, under accession numbers 357–31 (14 eggs), 357–33,
357–40, 357–44 (20 larvae), 357–46, 357–47, 357–48, 357–49, 357–50 (5 pupae).
Diagnosis. 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
 New species of Dione from xeric western slopes of the Andes 209

Figure 6. 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 seg-
ment; 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).

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
210 Jackie Farfán et al. / ZooKeys 1113: 199–227 (2022)

Figure 7. Variation in coloration patterns among fifth-instar of Dione (Agraulis) dodona sp. nov., shown
schematically for the head capsule and fourth abdominal segment, respectively, in frontal and lateral views
A brownish B grayish C reddish.

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 dif-
ferences with the available data compared to other Agraulis species (Beebe et al. 1960;
Brown 1981; da Silva et al. 2006). In the egg, the number of horizontal carinae almost
doubles the number described for Dione (Agraulis) maculosa [cited as ‘A. vanillae´]
(11–13 vs. 17–19), the egg being taller in Dione (Agraulis) dodona sp. nov. In relation
to the larval stage, in the first instar, the main difference is the size of the D2 setae in
the abdominal segments, being very small in maculosa and insularis (1/4 the length of
D1) (Beebe et al. 1960; da Silva et al. 2006), but reaching more than half the length
of D1 in Dione (Agraulis) dodona sp. nov. (Fig. 9B). In the fifth instar, the head scoli
 New species of Dione from xeric western slopes of the Andes 211

Figure 8. Pupa of Dione (Agraulis) dodona sp. nov. under A ventral B dorsal, and C lateral view, respec-
tively. A, abdominal segment; T, thoracic segment. Scale bar: 4 mm.

in maculosa are well developed, whereas Dione (Agraulis) dodona sp. nov. bears short
stout scoli (Figs 5D, 6E). Furthermore, the prothoracic dorsal plate in Dione (Agraulis)
dodona sp. nov. has spine-like setae on top of enlarged conical projection (Fig. 9C),
unlike maculosa, which has a simple seta bearing on a small projection (da Silva et al.
2006). In the pupa, the main differences are related to the head protuberances, which
are small in Dione (Agraulis) dodona sp. nov. (Fig. 9D), more conspicuous, and as long
as half the length of head in maculosa; also, the meso-dorsal crest is less pronounced
in Dione (Agraulis) dodona sp. nov. (Fig. 9E), in maculosa the dorsal margins are more
enlarged; protuberances on the abdominal segments do not occupy the entire length of
the segment in Dione (Agraulis) dodona sp. nov. (Fig. 9F), in contrast to maculosa where
protuberances fill all the length of the third abdominal segment.
Description. Adult. (Figs. 1–4). Male: Wingspan 44–52 mm (holotype 50 mm).
Head: antennae approximately two thirds of the forewing in length, black with the
tip of club orange (Fig. 2A), with 36 antennomers, 11 of which define the club. Pal-
pus elongated, approximately twice the size of the head, with a dark brown dorsal
color with light orange hairs, in ventral view white covered by white and orange hairs
212 Jackie Farfán et al. / ZooKeys 1113: 199–227 (2022)

Figure 9. 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, pro-
thoracic 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).
 New species of Dione from xeric western slopes of the Andes 213

(Fig. 2A). Thorax: Generally brown. Body dorsally black with brown and orange hairs,
ventrally covered by white and light orange scales, legs dorsally light orange with white
and orange hairs at the base, ventrally white. Forewing length 25–28 mm (holotype:
27 mm), hindwing length 18–20 mm. Wing venation as described in Michener (1942)
(Fig. 2B). Wing color pattern typical for the Agraulis clade except a black postdiscal
spot between veins M3-A1 on dorsal hindwing and a silver spot located in the discal
cell on ventral hindwing divided or partially divided (Fig. 1E, AF). Abdomen: dorsally
brown with orange hairs, ventrally covered by white scales.
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. 3D), costa with eversible pouch on its inner surface,
bearing a median-ventral crista (Fig. 3C); crista narrow, turbinate-shaped, with apex
protruded to dorsal margin of valve, and with little spines on surface (Fig. 3B), with
wider prolongation on the proximal ventral surface connecting with saccus (Fig. 3A).
Saccus short, with anterior process curved upwards and thinner apex. Tegumen long
and wide, in dorsal view the basal portion of the uncus is wider, narrowing towards the
tip, ending in a narrow apical process. Gnathos present, well developed, short tongue-
shaped slightly up curved in lateral view (Fig. 3A), vinculum slim and proximally
incurved with dorsal projection. Juxta wide in ventral view with a pointed anterior
portion and a widened posterior portion as the shape of two slightly divided lobules
(Fig. 3F). Aedeagus straight in lateral view, ~ 1/2 the length of the genital capsule,
evenly wide, sclerotized, without cornuti (Fig. 3E).
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. 1C, D). Abdomen with stink-
clubs attached to a lateral fold, dorsally on posterior margin of the eighth sternum,
densely covered with elongated, either single or bifid, specialized scales (Fig. 3C).
Female genitalia: Eighth segment narrow. Posterior apophysis ~ 1/2 the length of
the papilla anales (Fig. 3A). Two signa slightly arched with the proximal tip near the
ductus bursae, formed by four or five rows of wide spines (Fig. 3B).
Immature stages. Egg (Figs 5A, B, 9A, 2S)
Sub spherical, flat base slightly narrowed near apex. Yellow when recently laid
(Fig. 5A), reddish brown with a whitish band subsequently, and showing larva by
transparency close to hatching (Fig. 5B). Size (mean ± standard error): diameter -
0.92 ± 0.03 mm; height -1.13 ± 0.02 mm (n = 10). The chorion is adorned with 19–
20 vertical and 16 or 17 horizontal carinae of smooth surfaces, which when intersect
delimit cells (Fig. 9A). In the lower part of the egg, the vertical carinae are generally
twice as wide as the horizontal ones and some are interrupted at one or two cells before
the micropylar region. In the upper third, they have similar widths. Aeropiles scattered
in the intersection of carinae, and similar in shape to those of “Agraulis vanillae”, as
interpreted by da Silva et al. (2006). Micropylar region is surrounded by a rosette-like
sculpture of the chorion.
214 Jackie Farfán et al. / ZooKeys 1113: 199–227 (2022)

Figure 10. 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.
 New species of Dione from xeric western slopes of the Andes 215

First instar. (Figs 5C, 6A–D, 9B, S2). Length (mean ± standard error;
n = 6) = 3.56 ± 0.34 mm. Head, prothoracic dorsal shield, anal shield, pinnacles, legs
and lateral plates of prolegs blackish; thorax and abdomen mostly creamy white, slight-
ly translucent, reddish brown dorsally on A1–5 (Fig. 5C). Prothoracic dorsal shield
trapezoidal with rounded angles, posterior margin with cleft at middle. Chaetotaxy as
shown in Fig. 6A–D; SV group unisetose on T2–3; D2 ~ 1/2 the length of D1; and
spiracles laterally on prothorax and A1–8, circular, with peritrema elevated (Fig. 9B).
Subsequent instars. 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 pat-
terns (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. 7A); the second colored
pattern (greyish) with head and black setae, thorax and abdomen with bands in the
subdorsal and subspiracular area gray, the base of prolegs gray too, head with pattern
of cream coloration with a larger area than the previous one and the gray labrum, with
a triangular spot into the frontoclypeous, lateral plates of prolegs cream (Fig. 7B); the
third pattern (reddish) is similar to second pattern but the color of the bands in abdo-
men, base of scoli and the head is more reddish (Fig. 7C). However, the predominant
pattern observed in the field was the brownish one (Fig. 5D, E).
Fifth instar. (Figs 5C, D, 6E, S2) Length (mean ± standard error;
n  =  5)  =  25.45  ±  1.78 mm. Head blackish, covered by a large number of hair-like
setae and short chalaza-like setae, which vary in length, and bears two short stout scoli
dorsally (Fig. 5D). The thorax has the integument covered by conical, striated micro-
trichia, prominent on coxa of legs and latero-ventral face of prothorax; abdomen with
cuticular sculpturing composed of irregular ribs, except the last segment ventrally on
posterior face of anal proleg with conspicuous microtrichia; prothoracic dorsal shield
bears a number of short chalaza-like setae and two pairs of stout spine-like setae on
dorsal conical projections; elliptical spiracles with pronounced peritrema laterally on
prothorax and abdominal segments A1–8, those of prothorax and A8 slightly larger
than the remaining ones; prolegs with lateral plates covered by several hair-like and
chalaza-like setae, crochets in uniserial and multiordinal arrangement. Thirty pairs of
thoracic and abdominal scoli, which are elongated conical, integumentary outgrowths,
provided with some short chalaza-like setae on the surface, one of which, typically the
longest one, is placed at the apex; 11 dorsal pairs (T2–3 and A1–9); ten supra-spiracular
pairs (T2–3 and A1–8), with those of the meso- and metathorax anteriorly displaced;
eight lateral pairs (A1–8), and one anal pair (A10). Twelve pairs of thoracic and abdomi-
nal 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 an-
other pair between spiracle and coxa; two pairs on meso and metathorax, one posterior
to supraspiracular scolus and another dorsal to coxa; one pair on A1–2 and A7–8, which
is ventral to lateral scolus; and one pair on A9, on the latero-ventral face of the segment.
216 Jackie Farfán et al. / ZooKeys 1113: 199–227 (2022)

Instar identification. The successive instars can be accurately distinguished by the

width of the head capsule, because they do not overlap (Table 2). The corresponding
exponential growth equation was adjusted for the five instars: y = 0.328eˆ0.403x;
n = 62; r2 = 0.980; p < 0.05. Thus, the growth pattern of the head capsule follows the
Brooks-Dyar´s rule (Daly 1985). The mean growth ratio among instars was 1.495,
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; da Silva et al.
2006, 2008; Barão and Moreira 2010; Vargas et al. 2014; Barão et al. 2015).
Pupa (Figs 5F–H, 8, 9D–F) General shape elongated, ground color non-uni-
form, consisting of a mixture of shades of gray, light brown, and ocher (Fig. 5F–H),
with variation between individuals in their intensity. Length (mean ± standard er-
ror; n = 5) = 19.98 ± 0.28 mm. Head with pair of short, angled cephalic projections
(Figs 8, 9D); epicranial suture absent; eyes with sculptured region near antenna, bearing
few short hair-like setae, 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 sur-
passing posterior margin of A4; antennae arising laterally on head, projected ventrally to
apex of maxilla, with many smooth, round tubercles on surface. Thorax with the three
segments exposed. Prothorax as a small hexagonal plate in dorsal view, with anterior
and posterior margins broadly excavated, with two pair of lateral tubercles. Mesothorax
broadly expanded laterally along anterior half with a meso-dorsal crest that is well devel-
oped, broadly rounded, bearing a pair of round lateral tubercles; two pairs of marginal
and submarginal tubercles close to base of wings; one pair of submarginal tubercles each
near apex of wing; mesothoracic spiracle opening laterally at anterior margin of seg-
ment. Metathorax as a narrow plate with anterior margin broadly excavated, with pair
of lateral tubercles, hindwings as straight stripes between forewings and abdominal seg-
ments. Abdomen with segments A1–A4 partially hidden by wings; with pair of lateral
tubercles on A1–A7 which are little developed on A1–A2, most developed on A3, and
decreasing in size posteriorly; one meso-dorsal tubercle on A5–A7; one supraspiracular
tubercle on A2–A4; one pair of subspiracular tubercle on A4, pair of ventral tubercles on
A5–A6; spiracles of A1 and A2 hidden and partially hidden, respectively, by forewings,
and spiracles of A3–A7 elliptical; anal segment with two prominent tubercles, ventrally.
Cremaster quadrate, with truncate apex, and a large number of short, curved hooks.

Table 2. 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
 New species of Dione from xeric western slopes of the Andes 217

Etymology. 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.
Distribution. Adults of Dione (Agraulis) dodona sp. nov. are known from dis-
tinct 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), Moque-
gua (1,800–2,100 m elevation), and Tacna (1,800 m elevation). In Chile, two speci-
mens were collected in 1951 and 1968 from two localities in the Tarapacá Region be-
tween 2,300 and 3,000 m elevation, and recently other two specimens were collected
from Arica Region (1,580 m elevation).
Host plant. 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
(Bull-Hereñu 2020), and was reported recently from southern Peru (Weigend et al.
2015; Beltrán et al. 2018), restricted to the western slopes of the Andes above 1,500
m. M. tenuifolia is a shrub with reddish or yellowish tubular flowers that grows to ca.
1 m in height (Fig. 10), associated with the dry beds and immediate surroundings of
seasonal rivers in Chile (Bull-Hereñu 2020), and steep scree slopes along the road cuts
in the type locality of Dione (Agraulis) dodona sp. nov. (Fig. 10A).
Life history. 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 disap-
pearing 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 vegeta-
tion, compared to dodona, which was found only in xerophytic areas associated with
the host plant (Fig. 10A, B). Females were observed laying eggs particularly on flow-
ers (Fig. 10D). Oviposition occurred with the female sitting on top of the flower and
curling the abdomen around the flower edge so that eggs were deposited underneath
(Fig. 10C). During visits made at different months of the year in the type locality,
freshly laid eggs were always collected, both on flowers and leaves. Between one and
eight eggs were found in the same plant, in different flowers or leaves, but sometimes
several eggs were obtained from the same flower (Fig. 10D). The eggs are laid isolated
from each other. Newly hatched larvae first consumed the chorion, and afterwards
began to feed inside of flower, leaving a hole through which they enter the flower
(Fig. 10E). Subsequent instars feed externally on leaves. Larvae were consistently soli-
tary in all instars, regarding all activities, such as feeding or resting. Pupae were found
218 Jackie Farfán et al. / ZooKeys 1113: 199–227 (2022)

Figure 11. Phylogenetic status of Dione (Agraulis) dodona sp. nov. based on 650 bp-sequences of the Cy-
tochrome 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.

Table 3. 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 1.

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 -

predominantly off the host plant, clinging to rocks near the host plant, and sometimes
on branches of the host plant close to the ground.
Molecular data. Dione (Agraulis) dodona sp. nov. was recovered as an independent
lineage within the Agraulis clade of the COI-tree (Fig. 11), diverging in ca. 5% to the
group formed by its other species, and 9.5% to the Dione clade (Table 3).

Discussion
 New species of Dione from xeric western slopes of the Andes 219

The identification key to adults of Agraulis provided by Núñez et al. (2022) based on
external morphology, indicated only one character of the wing pattern color to separate
Dione (Agraulis) dodona sp. nov. (listed as Agraulis sp.), an extra black spot on the up-
per hindwing cell Cu1-Cu2 (Fig. 1E). As already mentioned, we propose in addition
the divided silver discal spot on the hindwing underside (Fig. 1F); this last trait is not
found in other species of the Agraulis clade, but is shared with other species of Dione.
Núñez et al. (2022) found that Dione (Agraulis) galapagensis W. Holland, 1890 and Di-
one (Agraulis) dodona sp. nov. have similar size (forewing length 27 mm or less) but are
smaller in relation to other species of the Agraulis clade. These findings are supported
by the specimens studied by us, where the longest wings of Dione (Agraulis) dodona
sp. nov. were from a male and a female, reaching up to 28 and 27 mm, respectively;
however, this should not be a morphological attribute to be used to separate species.
Regarding the male and female genitalia, Dione (Agraulis) dodona sp. nov. are very
distinct from those of other members of the Agraulis clade, the most notable differ-
ences being in the valval termen without denticles, the narrow crista projection and
the shape of yuxta in males (see Suppl. material 1), as well as the shape of the signum
in females. According to Emsley (1963), these characters have an important value to
differentiate among species within Heliconiinae. Thus, morphological characters of
adults described for Dione (Agraulis) dodona sp. nov. allow its clear differentiation from
other species. Similarly, phylogenetic relationships inferred from DNA sequence data
support Dione (Agraulis) dodona sp. nov. as a distinct taxon, placed as external to the
group formed by its congeners, considered the earliest diverging (ca. 8 Mya) lineage
within the Agraulis clade (Núñez et al. 2022). Although sharing morphological traits
with the Dione clade, the genetic distance of nearly 10% is quite high, which together
with previous inferences using several molecular markers (Kozak et al. 2015; Mas-
sardo et al. 2015), supports the recognition of two evolutionary lineages that shared a
common ancestor around 14 million years ago (Núñez et al. 2022). Following Zhang
et al. (2019), we consider that the phylogenetically most informative hypothesis is to
treat Dione and Agraulis as equivalent subgenera. In summary, as previously accepted
by Núñez et al. (2022), Dione (Agraulis) dodona sp. nov. is evidenced as a valid species
based on morphological and molecular evidence.
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 helico-
niines at the larval stage (Fleming 1960; Tavares et al. 2002; Kaminski et al. 2008).
Also, Dione (Agraulis) dodona sp. nov. exhibits some unique characters in the fifth
instar that were not observed in other species in the genus Dione s. l. Among them, it
is worth highlighting the spiracles with pronounced peritrema, the predominance of
short chalaza-like setae covering abdominal segments including scoli, and the linear
220 Jackie Farfán et al. / ZooKeys 1113: 199–227 (2022)

distance between stemmata I and II larger than distance between stemmata II and
III in head (see Suppl. material 2). Since the phylogenetic relationships between the
Agraulis and Dione clades and their taxonomic consequences have been controversial,
we suggest to explore the evolutionary history of these genera taking into account
morphological characters herein found for Dione (Agraulis) dodona sp. nov. within a
cladistic approach.
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 spe-
cies of Heliconiinae that oviposits preferentially on flowers or larvae feeding on them
(de Castro et al. 2018). The host plant of Dione (Agraulis) dodona sp. nov., Malesherbia
tenuifolia, represents the first record of the genus as a host plant in Heliconiinae (Bec-
caloni et al. 2008). This genus represents one of the oldest lineages of the Passifloraceae
s.l. (Tokuoka 2012), considered until a few years ago as a separate family (Malesher-
biaceae) (Gengler-Nowak 2003), but recent phylogenetic studies proposed to include
it in Passilforaceae s. l. (Soltis et al. 2007; Bremer et al. 2009). Malesherbia are a little-
known group of xerophytic plants endemic to a variety of arid habitats in the Pacific
coastal desert and adjacent Andes of Peru, Chile, and neighboring Argentina (Gengler-
Nowak 2002; Bull-Hereñu 2020). The subgeneric classification of Malesherbia com-
prises five sections (Gengler-Nowak 2003), M. tenuifolia belongs to the Malesherbia
section Gengler-Nowak (2003: 343) along with other species that are distributed at
the northern limit of the genus, from central Peru to northern Chile (Gengler-Nowak
2003; Beltrán et al. 2018; Bull-Hereñu 2020). Coincidentally, the known populations
of Dione (Agraulis) dodona sp. nov. follow the geographical distribution of this section
of Malesherbia, and this could imply that Dione (Agraulis) dodona sp. nov. uses other
Malesherbia species as hostplants, considering that in the Department of Lima (central
Peru) where Dione (Agraulis) dodona sp. nov. was collected, four Malesherbia species
have been reported, but not M. tenuifolia (Beltrán et al. 2018).
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.
Peña (1951) reported it for the first time in Chile based on a male specimen collected
in the locality of Parca, and afterwards Pérez-D’Angello (1970) reported a second spec-
imen from Guatacondo, in the collection of Pedro Millas. Subsequently, all Chilean
butterfly checklists published cited “A. vanillae” as a species distributed in northern
Chile (Ureta 1963; Etcheverry 1970; Herrera 1972; Peña and Ugarte 2006; Benyamini
et al. 2014). By examining both specimens that are currently at MUSM (the second
one on loan from MHNS), we confirm their identity as Dione (Agraulis) dodona sp.
nov. Probably, the host plant used by this population from northern Chile is M. tenui-
folia, reported in these localities of Tarapacá region (Bull-Hereñu 2020).

Acknowledgements
 New species of Dione from xeric western slopes of the Andes 221

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 [MUSA]) for access to the Laboratorio de Sis-
temática Animal of Universidad Nacional San Agustin de Arequipa (UNSA). The first
version of the manuscript benefitted from valuable suggestions provided by André
Freitas (UNICAMP). JF was supported by a CAPES scholarship. GLG and GRPM
were assisted by FAPERGS and CNPq grants, respectively. This study was supported
in part by a project of the Universidad Nacional de San Agustin de Arequipa (UNSA)
with Contract IBAB-001-2019-UNSA.

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Supplementary material 1
Figure S1
Authors: Jackie Farfán
Data type: Image.
Explanation note: Male genitalia of Dione (Agraulis) species congeners to Dione (Agrau-
lis) 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.
Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/zookeys.1113.85769.suppl1
 New species of Dione from xeric western slopes of the Andes 227

Supplementary material 2
Figure S2
Authors: Jackie Farfán
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 ab-
dominal segment, lateral; (I) proleg with exposed crochets; (J) last abdominal seg-
ment, posterior; (K) stemmatal region, dorso-frontal; (L) short chalaza-like setae;
(M) proleg of six abdominal segments; (N) seventh abdominal segment, latero-dor-
sal; (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).
Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/zookeys.1113.85769.suppl2