Reinstated status of the butterfly genus Agraulis (Lepidoptera, Nymphalidae, Heliconiinae) Author Penz, Carla M. text Zootaxa 2022 2022-11-17 5209 3 394 398 journal article 189257 10.11646/zootaxa.5209.3.8 07d27c12-d9e3-41f9-8222-638355d37abd 1175-5326 7329767 FA768332-156C-47D9-A115-84443D948177 Agraulis Boisduval & Le Conte , reinstated status * Agraulis incarnata (Riley) ** Agraulis incarnata incarnata (Riley) ** Agraulis incarnata nigrior Michener * Agraulis vanillae (Linnaeus) * Agraulis forbesi Michener * Agraulis insularis Maynard * Agraulis maculosa (Stichel) * Agraulis lucina C. Felder & R. Felder * Agraulis galapagensis Holland * Agraulis dodona (Lamas & Farfán) TABLE 1. Morphological characters that support the relationships between Agraulis and Dione plus those that distinguish each genus as listed by Penz (1999) . Note that a single species of Agraulis was recognized at the time of publication of such study. Character numbers correspond to the published analysis, which should be consulted for explanations and illustrations. Homoplasious characters are in parentheses. Abbreviations prior to character numbers: L, larva; P, pupa; A,adult. All taxa listed below are members of Nymphalidae , Heliconiinae . ( Agraulis , Dione )

(L6).	Setae D1 on thoracic segments 1–3 equal to, or longer than, the head height in the first instar larvae. This character
was also observed in Dryadula phaetusa (Linnaeus) , Dryas iulia (Fabricius) , and Philaethria dido (Linnaeus) within
Heliconiiti, and also Speyeria mormonia (Boisduval) (Penz pers. obs.).
(L22).	Aboral region of mandibles enlarged in the fifth instar larvae. This character is also found in Euptoieta hegesia
(Cramer) and Heliconius doris (Linnaeus) .
L42.	Secondary setae distributed around the center of anal plate in the fifth instar larvae.
(P44).	Gold spots absent. This implies a loss of gold spots in the ancestor of ( Agraulis , Dione ) and a gain of this pattern in
the ancestor of Dione glycera and D. moneta . This hypothesis is equally parsimonious to two independent losses in
Agraulis vanillae and D. juno .
P52.	Distal end of antennae acute. In this clade the distal ends of the antennae do not reach the edge of the wing case, thus
producing the acute pattern.
(P53).	Apex of the head adorned with ridges or tubercles. This character was also found in Boloria selene (Denis &
Schiffermüller) (Penz pers. obs.).
(P58).	Dark pigment concentrated on the distal end of the tracheae. This character was also observed in Dryadula
phaetusa .
(P61).	Leg 3 not visible. Reversal in Dione juno .
P63.	Region along the dorsal suture of the mesothorax with a single keel: highest point along the suture in the mid-portion
of the mesothorax.
(P74).	Crown hooks bifid. This character was also observed in Actinote pellenea Hübner and Cethosia biblis (Drury) (Penz
pers. obs.).
(A81).	Distal segment of the female leg 1 tarsi with a groove formed by the swelling of the ventro-lateral portions. This
character is also observed in Euptoieta claudia (Cramer) (Penz pers. obs.).
A96.	Forewing cross-vein m2–m3 out-curved.
A118.	Juxta curved longitudinally upwards.Although in Agraulis vanillae both the juxta and the phallus are curved upwards,
the phallus of Dione did not show a correlated curvature with that of the juxta.
(A122).	Anterior edge of costae expanded beyond the pedunculi.
A134.	Filiform scales on stink clubs predominantly bifid.

...Continued on the next page TABLE 1. (Continued) ( Agraulis )

(A79).	Distal segments of female leg 1 tarsi cylindrical. This homoplasious character is also present in Dione moneta , and
the outgroups Boloria selene and Euptoieta claudia .
(A87).	Dorsal process of pulvillus absent (legs 2 and 3). Michener (1942) and Emsley (1963) grouped Agraulis and Dione
on the basis of their reduced pulvillus (their ‘tarsal setae’ and ‘paronychial processes’, respectively). A distinction
was made between the complete absence of pulvillar processes (as in Agraulis ) and a reduced condition (as in
Dione ). This character was also observed in the outgroup species Actinote pellenea and Cethosia biblis .

( Dione )

(L26).	Presence of large spines in a cluster in the antero-lateral portion of the maxillulae in the fifth instar larvae. This
character is also observed in Podotricha telesiphe (Hewitson) and Actinote pellenea .
(L28).	Arms of the mentum bent markedly inward in the fifth instar larvae. This character is also observed in Podotricha
telesiphe and Heliconius sara (Fabricius) .
(L37).	Scoli on thoracic segment 1 present in the fifth instar larvae. Agraulis vanillae has tubercles in the homologous position
of the prothoracic scoli of Dione , although intraspecific variation was observed in the size of these tubercles.
(P56).	Wing outline scalloped. This character is also observed in Podotricha telesiphe .
(A82).	Vestigial leg 1 pre-tarsal claw in females. The pre-tarsal claw is absent in Dione moneta and Eueides aliphera
(Godart) .
A88.	Dorsal process of pulvillus reduced, not reaching half the length of the pre-tarsal claw (leg 2).
(A124)	Dorso-posterior extremity of harpe adorned. This character is also observed in Boloria selene , Euptoieta claudia ,
Dryadula phaetusa , and species of Eueides Hübner.
(A125).	Crista present. This structure is present in various taxa within the Heliconiinae .
A132.	Stink club tip squared.

TABLE 2. Morphological characters that distinguish Agraulis from Dione listed by Nuñez et al. (2022 , Appendix I). Numbers were maintained from the original publication, which should be consulted for illustrations and further information. Comments following characters are mostly quoted verbatim from the original publication, but references were generally omitted. Character comparisons to Dione appear in parentheses after the characters listed for Agraulis . Adult

1-	antennal club: 2 times as long as broad (3 times as long as broad in Dione ). Figured by Michener (1942) , Agraulis
antennal clubs are the broadest among Heliconiini .
2-	distribution of androconia: FW (FW & HW in Dione ). Condition shared only with Dryadula among the Heliconiini .
3-	FW outer margin: smooth, slightly concavous at mid portion (variously emarginated, deeply concavous at its mid portion in
Dione ). Emarginated in Cu1 and 1A and barely at M 3 in D. juno , emarginated in M2 and M 3 in D. glycera and D. moneta .
4-	Separation of HW anal veins ends: ≈ 2X distance 1A to Cu2 (≈ 2.5 X distance 1A to Cu 2 in Dione ).
5-	dorsal process of pulvillus: absent (present in Dione ). See A 87 in Table 1 .
6-	sexual dimorphism in origin of Cu veins at FW: present (absent in Dione ).
7-	crista on male genitalia valva: absent (present in Dione ). Nuñez et al. (2022) examined male genitalia of Agraulis
specimens at the ZSM representing incarnata (Rh1766) , insularis (Rh1754) , lucina (Rh1770, Rh1771), maculosa
(Rh1761) , nigrior (Rh1753, Rh1769) and vanillae (Rh1755, Rh1762). See A 125 in Table 1 .
8-	process on valva tip of male genitalia: short, with spines, posteriad pointed (elongated, without spines, anteriordorsal
pointed in Dione ). Modified from A 124 in Table 1 .
9-	dorsal process of valva: short, blunt and posteriad pointed (elongated, posterodorsal pointed, surpassing projection of tip
in Dione ).
10-	stink-club tip of female 8 th abdominal segment: rounded (squared in Dione ). Nuñez et al. (2022) examined female
genitalia of Agraulis specimens at the ZSM representing incarnata (Rh1768) , insularis (Rh1772) , maculosa (Rh1767) ,
and vanillae (Rh1760) . See A 132 in Table 1 .
11-	scales on stink club: longly bifid (hairlike, tip simple or shortly bifid in Dione ).
12-	signum of female genitalia: constricted transversely near anterior end, spines large (without constriction, spines tiny in
Dione ).

... Continued on the next page TABLE 2. (Continued) Egg

13-

ratio vertical/horizontal ridges & egg cells shape:>1 (≤ 1 in Dione ). The number of vertical/horizontal and egg ridges

is a variable condition but average ratio values are useful. Agraulis counts: 14/ 10–11 in incarnata , 15–19/ 9–12 in maculosa , and 16–17/ 10–11 in vanilla . Dione counts: 13–15/ 12–16 in juno , 15–16/ 16–18 in moneta , and 14–16/ 18– 23 in glycera . These differences result in Agraulis eggs having ca. squared cells while Dione ’s eggs possess thin horizontal rectangles. See Nuñez et al. (2022) for the sources of published counts.

First instar larva

14-	meso & metathorax SV setae: SV1 (SV1 & SV 2 in Dione ). Among Heliconiini , besides Dione species , the SV2 seta
is only present on mesothorax of Heliconius doris .
15-	length of D2 seta on A1–A8: ≈ 1/10 length of D1 (≈ 1/2–2/3 length of D 1 in Dione ). The extremely short D2 setae
on A1–A 8 in Agraulis is unique among all Heliconiini with immature stages described to date.

Fifth instar larva

16-	cluster spines on the anterolateral portion of the maxillula: absent (present in Dione ). See L 26 in Table 1 .
17-	arms of mentum bent inward: slightly (markedly in Dione ). See L 28 in Table 1 .

Pupa

18-	size of cephalic projections: large, more than half length of head (small, slightly surpassing anterior margin of head
in Dione ).
19-	texture of thoracic dorsal keel: smooth (rugose in Dione ).
20-	ornamentation on A4 dorsum: small size tubercles (enlarged flanges in Dione ).

This study proposes, and provides a justification for, the reinstatement of Agraulis to generic status. Sister-taxon relationships between Agraulis and Dione have been supported independently by analyses using morphology ( Penz 1999 ) and multiple DNA markers demonstrated to be informative from species to family levels ( Wahlberg et al. 2009 , Kozak et al. 2015 , Zhang et al. 2019, Chazot et al. 2021 ). In support for the genus rank of Agraulis , morphological characters that distinguish Agraulis and Dione are listed in Tables 1 and 2 . Finally, Nuñez et al. (2022) provided intra and intergeneric mean genetic distances for 12 molecular markers that range from 0.2–2.1% among species of Agraulis , 0.87–5.12% among those of Dione , and 2.85–10.47% between these genera. Given their findings, a comprehensive taxonomic revision of Dione is warranted.