Systematics of Helioandesia tarregai gen. et sp. nov. (Lepidoptera: Yponomeutoidea: Heliodinidae) from the Andes of Northern Chile

The adult stage of Helioandesia tarregai gen. et sp. nov. (Lepidoptera: Yponomeutoidea: Heliodinidae) is described and illustrated from the arid western slopes of the Andes of northern Chile. The larvae of H. tarregai gen. et sp. nov. feed as leaf skeletonizers on Mirabilis acuta (Reiche) Heimerl (Nyctaginaceae). The mostly gray forewing of H. tarregai gen. et sp. nov., ornamented with strongly bulging metallic spots, resembles that of the representatives of the mainly Nearctic Lithariapteryx Chambers, 1876. However, the latter lacks CuP in the forewing, has a single bristle in the female frenulum, and lacks a well-developed cornutus. Helioandesia gen. nov. clustered as sister to Neoheliodines Hsu, 2004 in a cladistic analysis, although no synapomorphies were found for this cluster, while Lithariapteryx was sister to Helioandesia gen. nov. + Neoheliodines based on two synapomorphies. The genetic distance between a DNA barcode sequence of H. tarregai gen. et sp. nov. and representatives of other genera of Heliodinidae Heinemann, 1877 was 9.0–12.5% (K2P), and a maximum likelihood analysis based on this molecular marker confirmed the placement of H. tarregai gen. et sp. nov. as a member of this micromoth family. This contribution represents the first confirmed record of Heliodinidae for Chile.


Introduction
The Heliodinidae Heinemann, 1877 (Lepidoptera: Yponomeutoidea) is a family of metallic-colored mostly diurnal micromoths, whose monophyly has been supported by morphological (Hsu & Powell 2004) and molecular (Sohn et al. 2013) analyses. Four autapomorphies are recognized for the Heliodinidae: (1) forewing with M two-branched, (2) female genitalia with apophyses anteriores fused into a medial band, (3) male genitalia with tegumen enormously expanded posteriorly, and (4) dorsal and lateral bristles in the pupa (Hsu & Powell 2004). Sixty nine species of this family are currently described (van Nieukerken et al. 2011), most of which occur in the New World, while a few occur in the Palearctic and Australian regions (Hsu & Powell 2004). The larvae of Heliodinidae feed externally as leaf skeletonizers or flower feeders, or internally as leaf miners or stem borers (Harrison & Passoa 1995;Hsu 2002). Larvae of some species can feed on cecidomyiid galls (Walsingham 1897;Hsu & Powell 2004). The main host plant family of Heliodinidae is Nyctaginaceae, although some species are associated with Aizoaceae Martinov, Araliaceae Juss., Amaranthaceae Juss., Onagraceae L., Phytolaccaceae R.Br., Piperaceae Giseke and Portulacaceae Juss. (Hsu & Powell 2004).
Although the Neotropics harbor the largest number of described species of Heliodinidae, a great part of their diversity remains unknown (Heppner 1984(Heppner , 1987Heppner & Landry 1994;Becker 1999;Hsu & Powell 2004;Heppner 2008). There are no confirmed records of species of this family in Chile. Clarke (1967) indicated that Elachista rubella Blanchard, 1852 (Elachistidae), described from Valdivia, southern Chile, "has the fascies of a heliodinid", and also mentioned its resemblance to some species of Phyllocnistis Zeller, 1848 (Gracillariidae Stainton, 1854) in the shape of the two signa of the female genitalia, but did not find other characters in common with species of this genus. Heppner (1984) listed the species under Heliodines Stainton, 1854 (Heliodinidae). However, Hsu & Powell (2004) excluded E. rubella from the Heliodinidae based on their updated definition of the family. Currently, the species is again recognized as a member of the Elachistidae Bruand, 1851 (Kaila 2019).
Micromoths fulfilling the four autapomorphies of Heliodinidae listed by Hsu & Powell (2004) were recently reared from leaf skeletonizer larvae collected on a native Nyctaginaceae in the Andes of the northernmost part of Chile. Further examination revealed that these micromoths represent an undescribed species whose morphological peculiarities preclude its inclusion in any of the 13 genera currently recognized in this family of micromoths (Hsu & Powell 2004). Accordingly, the aim of this study is to provide the corresponding description of a new genus and species of Heliodinidae based on adult morphology, and to assess its phylogenetic relationships using morphological characters and DNA sequences.

Sampling, rearing and morphological observations
The sampling site (18°19′45″ S, 69°34′56″ W) is at 3400 m elevation on the western slopes of the Andes of northern Chile, about 1.5 km northeast of Zapahuira Village, Parinacota Province. The area has a tropical xeric climate, with seasonal rains between December and March (Luebert & Pliscoff 2006). The vegetation cover is seasonal, with higher levels shortly after the rains (Muñoz & Bonacic 2006). Leaf skeletonizer larvae were collected on Mirabilis acuta (Reiche) Heimerl (Nyctaginaceae) in April 2018 and March 2019. The collected larvae were placed in plastic vials with leaves of the plant and paper towel at the bottom and brought to the laboratory. The vials were cleaned periodically and fresh leaves of M. acuta were provided until the larvae finished feeding. Vials were observed regularly after pupation. One pupa was placed in ethanol 95% at −20°C until DNA extraction. The adults obtained were mounted, their abdomens were removed, cleared in hot KOH 10% for a few minutes, stained with Eosin Y and Chlorazol black and slide-mounted with Euparal. Images were captured with Sony CyberShot DSC-HX200V and Micropublisher ver. 3.3 RTV-QImaging digital cameras attached to a Leica M125 stereo microscope and an Olympus BX51 optical microscope, respectively. Morphological descriptions follow the terminology of Hsu & Powell (2004). The distribution map was generated using SimpleMappr (Shorthouse 2010). Specimens will be deposited in the Colección Entomológica de la Universidad de Tarapacá, Arica, Chile (IDEA).

Cladistic analysis
The phylogenetic relationships of the new taxon were assessed using morphological characters in a cladistic analysis. The morphological characters of the new taxon were coded and included in the data matrix provided by Hsu & Powell (2004). The data matrix and the resulting trees were edited in the software WINCLADA (Nixon 2002). The cladistic analysis was performed using the software NONA ver. 2.0 (Goloboff 1993). The heuristic search for trees was undertaken under equal weighting, hold*100 000, mult*2000 and hold ⁄100, with all characters treated as unordered and non-additive. Node support was assessed with 200 bootstrap replicates.

DNA extraction and analysis
DNA extraction from one pupa was performed by staff of the Laboratorio de Biología Molecular de Plantas (Universidad de Tarapacá, Arica, Chile) following the procedures described by Huanca-Mamani et al. (2015). Genomic DNA was sent to Macrogen Inc. (Seoul, South Korea) for purification, PCR amplification and sequencing of the DNA barcode fragment with the primers LCO-1490 and HCO-2198 (Folmer et al. 1994) and the PCR program described in Escobar-Suárez et al. (2017). DNA barcode sequences of 658 base pair (bp) length of other genera of Heliodinidae were downloaded from BOLD (Ratnasingham & Hebert 2007). Sequences were aligned with ClustalW, sequence divergence was assessed by the Kimura 2-parameter (K2P) method, and a maximum likelihood (ML) analysis was performed in the software MEGAX (Kumar et al. 2018). Sequences of the families Bedelliidae Meyrick, 1880 andScythropiidae Friese, 1966 were also downloaded and included as outgroups in the ML analysis following the phylogenetic study of Yponomeutoidea Stephens, 1829 of Sohn et al. (2013). The nucleotide substitution model (GTR+I) was chosen using the lowest value of Bayesian information criterion (BIC). The statistical support of the nodes was assessed by 1000 bootstrap replicates. The Xia test (Xia et al. 2003) was used to assess the presence of phylogenetic signal in the alignment with a substitution saturation analysis in the software DAMBE ver. 7.2.1 (Xia 2018).

DNA barcodes
Five DNA barcode sequences of Heliodinidae and two of outgroups (Bedelliidae and Scythropidae) were included in the alignment of 657 bp length with 178 variable sites ( Table 2). The genetic distance between the new genus and species (GenBank accession MT782162) and other genera of Heliodinidae was 9.0-12.5% (K2P). No evidence of stop codons and no substitution saturation (ISS < ISS.C; p < 0.001) were detected in the alignment, indicating that the data set was suitable for phylogenetic analysis. The ML analysis confirmed the monophyly of Heliodinidae and the placement of the new genus and species as a member of this family. Furthermore, although with low support, the ML clustered the new genus and species with Neoheliodines nyctaginella (Gibson, 1914), the only representative of Neoheliodines Hsu, 2004 in the alignment (Fig. 2).

Diagnosis
Helioandesia gen. nov. can be recognized by the following combination of morphological characters: (1) presence of CuP on forewing of female and male; (2) two bristles of unequal length in the female frenulum; (3) forewing mostly gray, ornamented with strongly bulging metallic spots, (4) two triangular processes on anterior margin of tergum VIII in the male abdomen; (5) sclerotization of the widened proximal part of the ductus bursae not reaching the ostium; (6) signum with proximal portion coarser than distal portion; (7) presence of an appendix bursae; (8) slightly swollen distal end of the saccus; and (9) a well-developed cornutus on the vesica. The mostly gray forewing pattern of Helioandesia gen. nov., ornamented with strongly bulging metallic spots, resembles that of the mainly Nearctic Lithariapteryx. However, the latter lacks CuP in the forewing of female and male, has a single bristle in the female frenulum, and lacks a well-developed cornutus on the vesica. The female genitalia of H. tarregai gen. et sp. nov. are very similar to those of the Bolivian Lithariapteryx loriculata (Meyrick, 1932) in the shape of the transverse bridge, signum and antrum. However, H. tarregai gen. et sp. nov. lacks a V-shaped mark from costa on the forewing, has a sclerotization on the distal part of the ductus bursae and has the signum on the left margin of the corpus bursae, while L. loriculata has a V-shaped mark from the costa on the forewing, lacks sclerotization on the distal part of the ductus bursae and has the signum on the middle of the ventral part of the corpus bursae. The male of L. loriculata remains unknown, impeding comparison with H. tarregai gen. et sp. nov. Two bristles of unequal length in the female frenulum of Helioandesia gen. nov. resemble those of Epicroesa Meyrick, 1907, Heliodines, Lamprolophus Busck, 1900and Philocoristis Meyrick, 1927. However, all these genera lack CuP in the forewing.

Etymology
The genus name is derived from the Greek ʻheliosʼ (sun), the root of the family name Heliodinidae, and Andes, in reference to the Andes Range, among whose amazing landscapes the type species of Helioandesia gen. nov. was discovered. The name is considered feminine in gender.
Head. Vertex and frons silvery metallic. Pale orange yellow scales behind compound eyes. Antenna with alternate rings of creamy white and brownish gray on basal two third, distal third brownish gray. Labial palpus creamy white on two basal segments; brownish gray scales with creamy white margin on third segment.  (Meyrick, 1917), the geographically nearest heliodinid species to H. tarregai gen. et sp. nov. on the western slopes of the Andes, in Peru. Square = type locality of Lithariapteryx loriculata (Meyrick, 1932) on the eastern slopes of the Andes of Bolivia, the morphologically nearest species to H. tarregai gen. et sp. nov.
THorax. Variable proportion of silvery metallic and brownish gray with creamy white margin scales dorsally, brownish gray scales with wider creamy white margin latero-ventrally. Legs mainly concolorous with latero-ventral side of thorax. Forewing mostly covered by brownish gray with creamy white margin scales; five metallic silvery bulging spots with black proximal margin, three near costal margin, size progressively decreasing from base to apex, two near anal margin; small metallic silvery spot near tegula, slightly differentiated metallic silvery diagonal stripe from about middle of distal margin of discal cell to near costal margin; a few brownish orange scattered scales mainly on distal half; fringe brownish gray. Hindwing brownish gray; fringe concolorous.
abdomen. Mainly brownish gray dorsally, lighter near posterior margin of each segment; scales brownish gray with creamy white margin ventrally. geniTalia (Fig. 5). As described for genus.

Female
Similar to male. geniTalia (Fig. 6). As described for genus.

Host plant
The only known host plant is the native Mirabilis acuta (Nyctaginaceae) (Fig. 7), a Chilean endemic (Rodríguez et al. 2018). In the study zone M. acuta sprout and flower after the summer rains. The larvae of H. tarregai gen. et sp. nov. feed externally as leaf skeletonizers, remaining partially covered by a fine silk layer (Fig. 7C).

Distribution
Known only from the type locality (Fig. 8), near Zapahuira, Parinacota Province, at about 3400 m elevation on the western slopes of the Andes of northern Chile.

Discussion
The taxonomic diversity of Yponomeutoidea has been little explored in the Neotropics in recent years (Cepeda 2016;Vargas 2018;Moreira et al. 2019). Hsu & Powell (2004) suggested that a great part of the diversity of the Heliodinidae remains unknown in Central and South America. The discovery of H. tarregai gen. et sp. nov. supports their suggestion, revealing a previously unknown Neotropical lineage of this micromoth family.
Among the morphological characters of H. tarregai gen. et sp. nov., the presence of CuP in the forewing is remarkable, as its absence or reduction to a vestigial vein was mentioned as a probable autapomorphy for Heliodinidae (Kyrki 1984). However, the placement of H. tarregai gen. et sp. nov. in this family, suggested by the presence of the autapomorphies listed by Hsu & Powell (2004), was confirmed in the cladistic analysis. Since CuP is either absent or present in the outgroups used in the analysis, an additional heuristic search was undertaken with the character ʻCuP in forewing: (0) absent, (1) presentʼ added to the matrix, but H. tarregai gen. et sp. nov. remained as a member of Heliodinidae (not shown).
The presence of two bristles of unequal length in the female frenulum of H. tarregai gen. et sp. nov. resembles the basal lineages of Heliodinidae: Epicroesa, Heliodines, Lamprolophus and Philocoristis. However, the cladistic analysis indicated that Helioandesia gen. nov. is only distantly related to these four genera. In contrast, Helioandesia gen. nov. was clustered as sister to Neoheliodines, although this clustering was based on two plesiomorphies: (1) base of ductus bursae heavily sclerotized into a cylinder immediately subtending the ostium bursae (character 21, state 1), and (2) one SV seta on A9 of late instar larva (character 29, state 1). The two genera can easily be separated, as the latter lacks CuP and the strongly bulging metallic spots on the forewing of female and male, has a single bristle in the female frenulum and the ostium bursae surrounded by a sclerotized ring, and lacks a welldeveloped cornutus on the vesica. Two synapomorphies were found for the clustering of (Lithariapteryx (Helioandesia gen. nov. + Neoheliodines)): (1) anterior margin of tergum VIII of the male with a pair of triangular processes extending anteriorly beneath tergum VII (character 11, state 1), and (2) signum with the proximal portion coarser than the distal portion (character 17, state 1). However, the low bootstrap support of this cluster suggests that further studies are needed to understand the phylogenetic relationships of Helioandesia gen. nov. better.
The sclerotization of the widened proximal part of the ductus bursae not touching the ostium appears to be a distinctive character of Helioandesia gen. nov. and could represent an autapomorphy. As shown by Hsu & Powell (1997), the same state is found in L. loriculata, the only Neotropical representative of the otherwise Nearctic Lithariapteryx, suggesting that this Bolivian species could represent another member of the Helioandesia gen. nov. The remarkable similarity in their genitalia suggests that they could be a pair of cryptic allopatric species separated by the Andes range, with one inhabiting the arid high elevation environments on the western slopes and the other the moist low elevation (type locality 750 m) environments on the eastern slopes (Fig. 7). Similar patterns have been described in other families of micromoths (Pereira et al. 2017;Silva et al. 2018). However, as the current knowledge of L. loriculata is based only on the female holotype (Hsu & Powell 1997), an assessment of the phylogenetic relationships of this species should wait until additional specimens are available for examination and analysis of morphological and molecular characters. In the meantime, Helioandesia gen. nov. is a monotypic genus.
Although the relationships of the genera of Heliodinidae were not well resolved in the ML analysis based on DNA barcode sequences, this analysis was useful to confirm the placement of Helioandesia gen. nov. as a member of this family despite the limited taxon sampling. Additional markers and species of additional genera would be included in further analyses to understand better the phylogenetic relationships of the genera of Heliodinidae using molecular approaches.
The discovery of H. tarregai gen. et sp. nov. represents the first confirmed record of Heliodinidae for the Chilean fauna. The nearest records of the family west of the Andes are those of Aetole demarcha (Meyrick, 1917), a little-known species occurring from near sea level to about 2400 m elevation in the Lima Department, central Peru (Meyrick 1917), about 1000 km northeast of the type locality of H. tarregai gen. et sp. nov. (Fig. 7). The discovery of H. tarregai gen. et sp. nov. highlights the currently overlooked diversity of micromoths harbored by the arid environments of the Andes that deserves further attention (e.g., Farfán et al. 2020;Vargas et al. 2020). The host plant records of the different genera of Heliodinidae, summarized in detail by Hsu & Powell (2004), can be used as an extremely valuable guide to explore the Neotropical diversity of this family of micromoths. This approach would be valuable to find immature stages, providing a great opportunity to gain insights on their biology and external morphology, two aspects still little-known in the Heliodinidae of South America.