Integrative taxonomy clarifies species limits in the hitherto monotypic passion‐vine butterfly genera Agraulis and Dryas (Lepidoptera, Nymphalidae, Heliconiinae)

Abstract

The Heliconiini genera Agraulis and Dryas are widely distributed throughout the Neotropics and into adjacent temperate regions, and although they are currently treated as monotypic, both show significant geographic phenotypic variation. In this work, we employ six genetic markers (4199 bp), two mitochondrial and four nuclear, to perform coalescent species delimitation analyses in Bayesian Phylogenetics and Phylogeography (BPP) and in integrated BPP (iBPP), the latter also includes linear measurements and wings landmarks. We also analyze cytochrome c oxidase I (COI) barcode sequences for each genus using genetic distances, haplotype networks and a character-based approach. Based on the model testing results, complemented with data from previous studies, we performed morphometric analyses to compare fore and hindwing size, aspect ratio and shape among the new species. In addition, we compared the forewing spot pattern of hypothesizes species using the r package patternize and, for Dryas, compared the colour patterns of mature larvae. Model testing of the molecular species delimitation outputs favoured a seven species hypothesis for Agraulis and a four species hypothesis for Dryas. Average distances among COI barcode sequences of these groups were from 1.09 to 5.81% in Agraulis and from 1.09 to 3.44% in Dryas. Within-group distances ranged from 0 to 1.11% and between 0 and 2.43%, respectively. NeighborNet haplotype networks showed that all but one of the species are monophyletic, and the character-based approach found exclusive diagnostic positions for most species, while the rest can be recognized by unique combinations of the 44 informative nucleotide positions analysed. Morphometric analysis supported all species of Agraulis and Dryas based on wing shape, and also in several cases on wing sizes and aspect ratio (hindwing length-forewing length), including A. v. galapagensis, which was absent from the molecular study. The analysis of the forewing spot pattern also revealed differences among most species hypothesis.

Figure 11 from: 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

“…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.…”

“…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;Massardo et al 2015), supports the recognition of two evolutionary lineages that shared a common ancestor around 14 million years ago (Núñez et al 2022).…”

“…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;Massardo 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.…”

“…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-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.…”

“…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.…”