Molecular phylogenetic and morphological studies on the systematic position of Heracula discivitta reveal a new subfamily of Pseudobistonidae (Lepidoptera: Geometroidea)

Heracula discivitta Moore is an uncommon moth species currently recorded from India, Nepal and China. Although this species has traditionally been placed in Lymantriinae, its systematic position in Macroheterocera has been enigmatic due to its unique morphological features. Here we used molecular and morphological data to explore the systematic position of H. discivitta. Our molecular phylogenetic analyses indicate that this species is sister to Pseudobiston pinratanai Inoue, a member of a recently established monotypic family Pseudobistonidae. The examinations of morphological features further show that H. discivitta shares synapomorphies with Pseudobistonidae. Based on the analysis results, we propose a new subfamily of Pseudobistonidae (Heraculinae subfam.n.) to accommodate H. discivitta. The resemblance of the habitus to that of the brahmaeid genus Calliprogonos Mell & Hering is discussed.


Introduction
Lepidoptera, with over 157 000 recognized extant species, are one of the most speciose insect orders (Mitter et al., 2017;Triant et al., 2018). They play an important role in the natural ecosystem as major herbivores, pollinators and prey, and serve as model organisms for studying ecology, genetics, physiology and evolutionary biology (Wagner, 2001;Roe et al., 2010). Although the deep-level phylogeny of Lepidoptera remains largely unclear, recent advances in molecular phylogenetic studies are gradually contributing to our understanding of the phylogeny and evolution of such a huge insect order (Regier et al., 2009(Regier et al., , 2013Mutanen et al., 2010;Bazinet et al., 2013;Kawahara & Breinholt 2014, Timmermans et al., 2014Mitter et al., 2017), and have allowed us to address many previously unresolved problems, particularly for the systematic positions of mysterious taxa with unique morphological traits in some major Macroheterocera represent a mega-diverse Lepidopteran lineage of more than 72 000 known species (Nieukerken et al., 2011;Mitter et al., 2017). Within Macroheterocera, a rare and mysterious taxon, Heracula discivitta Moore, is currently known from India, Nepal and China, and has persisted as a monotypic genus as the type species in Lymantriinae of Noctuoidea since its original description in 1866 (Moore, 1866;Kishida, 1993;Chao, 2003). However, the systematic placement of this species has been problematic due to its distinctive morphological traits. For instance, H. discivitta has a pair of filiform antennae and a well-developed proboscis, whereas the antennae of Lymantriinae are strongly bipectinate and their proboscis is usually absent. Moreover, H. discivitta lacks distinctive metathoracic tympanal organs that are viewed as an apomorphic character of Noctuoidea. A molecular study of higher classification in Lymantriinae (Wang et al., 2015) that initially included Heracula showed that it was an outlier from the rest of the group and probably misplaced.    coxa; em, epimeron; f, furca; k, katepisternum; lc, laterocervicale; pa, patagium; pm, prospinasternum; pp, propleuron; pr, pronotal  Recently, the molecular phylogenetic study of an eight-gene dataset by Mutanen et al. (2010) addressed many long-standing questions of lepidopteran systematics. This base has been used to investigate the relationships of higher taxa such as families and superfamilies, especially in the so-called Macroheterocera clade (Nieukerken et al., 2011;Sihvonen et al., 2011;Zahiri et al., 2011Zahiri et al., , 2012Zahiri et al., , 2013aRajaei et al., 2015). In addition to confidently delineating families of Macroheterocera, the molecular data have led to the discovery of a new, currently monotypic family, Pseudobistonidae, consisting of the SE Asian species Pseudobiston pinratanai (Rajaei et al., 2015). In order to explore the systematic position of H. discivitta, we added the molecular data of this species to the dataset of Mutanen et al. (2010), augmented with sequences from Rajaei et al. (2015) and re-ran the molecular phylogenetic analyses, and also re-examined morphological characters of H. discivitta. Based on the results of our analyses, we propose here a new subfamily of Pseudobistonidae to accommodate H. discivitta.

Morphological examination
The specimens examined in this study are deposited in the Insect Collection, Department of Entomology, South China Agricultural University (SCAU), Guangzhou, China, except for the holotype and the only female specimen of H. discivitta which are kept at the Natural History Museum, London, UK (NHMUK). Photos of adults were taken using a Nikon Coolpix S8000 digital Camera. Adults were entirely dissected in pure ethanol and were stained with Chlorazol Black E after the whole body (except wings) was macerated in hot 10% sodium hydroxide in an aqueous solution for 2-8 min. Wing venation was observed after removing the scales in absolute ethanol and staining the wings in an acetocarmine solution. The dissected structures of adults (including head, thoracic exoskeleton, abdomen, legs, genitalia and wing venation) were mounted on microscope slides in glycerin, and photographed using a KEYENCE VHX-5000 digital microscope or a Zeiss Discovery V12 stereoscope. All images of the female H. discivitta were obtained from NHMUK. All of the images were edited with Adobe Photoshop CS6. Label data for the illustrated specimens in this study are given in Table S1. Terminology largely follows Scoble (1992) and Kristensen (2003). Some terms for the cephalic and thoracic morphology follow Matsuda (1970), Brock (1971), Fänger (1999, Kawahara et al. (2012) and Minet (2003). The naming of the vein venation follows Wootton's (1979) system.

Molecular data analyses
The DNA sequences of Macroheterocera in the study of Mutanen et al. (2010) were downloaded from NCBI Genbank, including also four taxa of Pyraloidea to root the tree. In addition, the sequences of Pseudobiston from the study of Rajaei et al. (2015) were downloaded from NCBI Genbank. For H. discivitta, total genomic DNA was extracted from two legs of a dried adult specimen, using Qiagen DNeasy tissue extraction kit (Hilden, Germany) following the manufacturer's protocol. The eight gene regions (COI, EF-1 , RpS5, GAPDH, MDH, CAD, IDH and Wingless) used by Mutanen et al. (2010) were amplified using PCR. DNA amplification and sequencing protocols followed Wahlberg & Wheat (2008). All sequences and accession numbers used in this study are listed in Table S2.
The sequences obtained were edited and aligned using BioEdit v7.2.0 (Hall, 1999). Aligned DNA matrix used in the phylogenetic analyses of this study is provided in File S1. Phylogenetic analyses were conducted using two model-based methods: maximum-likelihood (ML) and Bayesian inference (BI). Eight partitions were made by gene regions. The GTR + G + I model was identified as the best-fit model of sequence evolution for each gene partition using the Akaike information criterion (AIC) implemented in MrModeltest2 (Nylander, 2004). BI analysis was performed using the GTR + G + I model in MrBayes v3.2.2 (Ronquist et al., 2012) on the CIPRES Science Gateway (Miller et al., 2010), with the default parameters and four independent runs for 50 million generations, each with one cold chain, sampled every 1000th generation, and three heated chains. For ML analysis, the GTR + G model was used, as the strong correlation between parameters G and I confounds their use together (Ren et al., 2005). ML analysis was executed with the web-server RAxML (Stamatakis et al., 2008) on CIPRES, using 1000 bootstrap replicates. Tree files and DNA matrices are deposited in TreeBASE with a study accession URL: http://purl.org/ phylo/treebase/phylows/study/TB2:S22416.
that Heracula Moore is sister to Pseudobiston Inoue which is the type genus of a recent established new family Pseudobistonidae  Head (Fig. 2). Antenna (in the male) filiform; scapus and pedicellus nearly cylindrical, entirely scaled; scapus thin basally, c.2× the length of the pedicellus; intercalary sclerite fairly small; flagellum dorsally scaled and ventrally covered with tiny sensory setae. Frontoclypeus, vertex and occiput densely covered with piliform scales. Compound eyes well-developed, naked. Ocelli absent. Chaetosemata small, remote from each other, sparsely covered with yellow hairs. Two discrete, minute cranial pits present on the vertex and each side of the postgenal-occipital area, respectively. Maxillary palpus rather short, three-segmented. Labrum scaled, without distinct projection medially; pilifers absent. Mandibles absent. Labial palpus porrect, covered with dense scales, three-segmented; basal segment almost as long as median one; distal segment short, <1/3 of median one in length. Proboscis present, unscaled; galea with a row of brush-like setae along the posterior-lateral margin.
Prothorax (Fig. 3). Patagia approximately kidney-shaped, anterior surface sclerotized, posterior surface membranous. No distinct parapatagia (they may be membranous or reduced). Pronotal anteromedian plate small, articulated with a short finger-like posteromedian process. Anterolateral pronotal plates c.3× as long as wide. Laterocervicale consists of well-developed anterodorsal and ventral arms, the former relatively straight and the latter concave laterally. Katepisternum small, subtriangular; trochantin absent. Propleuron with a weakly sclerotized epimeron caudad of the ventral half of the pleural sulcus. Dorsal processes of furca tapering toward apex, shorter than a digitate prospinasternum that is articulated with median longitudinal sulcus on the posterior margin of prosternum.
Mesothorax (Figs 4,5). Tegula falcate, broader at anterodorsal surface, covered with dark brown fine hairs. A pair of prescutal clefts convergent dorsally, their dorsal extremities almost as wide as the length of the antecostal sulcus. Anterolateral margins of prescutum prolonged along the prescutal clefts, forming long finger-like prealar arms. Phragma 1 membranous, inconspicuous, slightly bilobed. A complete median longitudinal sulcus present on the mesoscutum. Subtegula elongated, fused to a tergopleural apodeme. Suralare short, forming an anterior notal wing process, being separated from the mesoscutum by an anterolateral scutal sulcus that produces an internal ridge and ends posteriorly in a short transverse bar. Median notal wing process well-developed, anterobasally bordered by a small notal incision, and associated with a short posterior notal wing process through a plate-like sclerite. Mesoscutellum approximately rhombic in dorsal view, slightly round at the middle of posterior margin. Mesopostnotum concave anteriorly. Phragma 2 large, with a small notch on the ventral-medial edge. Laterophragmata 2 well-developed. Anepisternum relatively large, its ventral margin being clearly demarcated by an anapleural cleft that extends backwards to the pleural sulcus. Katepisternum small, triangular with posterior margin almost equal to that of the preepisternum in length. The marginopleural sulcus short, anastomosing anteriorly with an oblique precoxal sulcus. The parepisternal flexion zone broad, extending upward to the anterior edge of the anapleural cleft. Parepisternum moderately sclerotized, connected to the basisternum, ventral half of posterior margin bordered by a parepisternal sulcus. Mesoclidium shaped like an inverted-Y, with the divergent ventral arms reaching to the anterolateral area of the basisternum. Subalare spacious, irregularly sclerotized at the dorsal side. Epimeron deeply concave on the upper margin; its anterior arm narrow, tapering dorsally. A large subtriangular sclerite located between the posterior area of the epimeron and meron. The furcal arm large, strongly sclerotized, fused with the posterior arm of the epimeron. Meron dorsally broad, separated from eucoxa by a long mesocoxal sulcus.  Metathorax (Figs 5, 6). Metascutum concealed mesally under the metascutellum; its lateral part large and broad, with a pigmented sulcus arising near the anterolateral margin of the metascutellum. No distinct fenestra media. Fenestrae laterals minute. Supraphragmal sclerite elongate, delimited by an internal ridge ventrally. Lateropostnotal region with a well-defined subtriangular sclerite (termed 'laterophragma' by Brock, 1971) that is ventrally adjacent to the true lobe-like laterophragma. Euphragma (def. : Fänger, 1999) developed only in the lateral region along the tergal rim (sensu Brock, 1971), with a large, central oval foramen. Anepisternum small. Preepisternum gradually narrowed toward the ventral surface. Parepisternal sulcus long, reaching to the anapleural cleft. Marginopleural sulcus moderately long, linking the pleural sulcus posteriorly. Subalare well-developed. In lateral view, an unsclerotized flexion zone across the posteroventral arm of the metepimeron. Furco-furca band (sensu Minet, 2003) broad. The internal ridges of stem of furca relatively slender, extending dorsally to the secondary furcal arms (termed 'laminae of secondary arms' by Brock, 1971). A pair of struts (sensu Brock, 1971) connected ventrally with the stem of furca, their dorsal extremities reaching to the posterior ventral laminae that unite anteriorly with the furcal apophyses and merge posteriorly into an enlarged medial region between the secondary furcal arms. Eucoxa and meron bordered by a metacoxal sulcus.
Legs (Figs 7, 8). Femora covered with long dense piliform scales at the inner side. Midfemora slightly longer than the other femora. Tibiae and tarsi smoothly scaled. Foretibiae with a stubby epiphysis. Tarsi composed of five tarsomeres that bear irregularly arranged spines on their inner surface. The tibial spur formula: 0-2-4 (hind leg with a minute pair of subapical spurs that are c. 0.5× length of the apical pair). Pretarsi with a pair of simple, robust claws; pulvilli finger-like, with numerous minute setae; pseudempodial setae fairly long; arolium heavily pigmented, membranous apically.
Wings (Figs 9-11). Forewing apex round, outer margin somewhat convex. Upperside of male forewing with a red-brown spot at the base, and a large brown reniform patch at the distal one-third area; medial transverse band broad, deep brown, interior margin surrounding the basal spot, exterior margin slightly concave, both interior and exterior margin with a narrow pale white border. Upperside of male hindwing dark brown, with a red-brown marginal band. Underside of both wings brown, without wing patterns; frenulum present at the humeral angle of the hindwing, interlocking with a retinaculum at the base of the forewing. Female larger than male in size; forewing with yellow brown spot and patch, and with a much wider pale white border, hindwing with a yellow marginal band. Forewing venation with a rather long Sc that extends beyond the middle of the costa; R free, arising just before the upper angle of the discal cell; Rs 1 to Rs 4 stalked, and Rs 4 branching before Rs 1 + 2 + 3 ; areole absent; M 1 free, arising near the upper angle of the cell; M 2 arising closer to M 3 than M 1 ; M 3 and CuA 1 separated; CuA 2 branching from the inner margin of the cell; 1 + 2A arising from the base of the forewing. Sc + R in the hindwing arising from the base, connecting with the costa of the cell at the basal area and forming a small basal cell; Rs and M 1 separated, branching from the upper angle of the cell; M 2 close to M 3 that arises from the lower angle of the cell; CuA 1 and CuA 2 arising from the inner margin of the cell, respectively; 1 + 2A originating from the base of the hindwing.
Pregenital abdomen (Fig. 12). Abdominal tympanal organs absent. Dorsal A1 densely covered with long piliform scales; neotergite long, band-shaped, narrower than its anterior membranous area, laterally fused with the marginotergites. Tergum A2 without distinct lateral rods; antecostal phragma small, tapering apically; its anterolateral angles somewhat produced and its lateral edges near to the spiracles. Terga A2-A8 relatively well pigmented. Sternum A2 sclerotized except the posterior area; its anterolateral processes short, without connection to the marginotergites. Sterna A3-A8 almost unpigmented, with the similar width as their corresponding terga. The abdominal segments from A5 to A8 being gradually narrower.
Male genitalia (Fig. 13a). Uncus broad basally, tapering toward the apex, slightly acute at the apex. Gnathos laterally sclerotized along the posterior margin of tegument, with a large inverted triangular plate mesally. Valvae simple, elongated, broad at basal half, being narrow from the distal half of sacculus; costa somewhat straight; cucullatus round; a short weakly sclerotized process located near the middle area of sacculus. Cristae of juxta with a short arm dorsally and a long finger-like one ventrally. Saccus small. Aedeagus short, vesica simple.
Subfamily diagnosis. The subfamily Heraculinae can be clearly separated from its sister group Pseudobistoninae by filiform antennae in the males and the hind tibia with four spurs. Pseudobistoninae have a pair of bipectinate antennae in the males and their hind tibia only possess two spurs. Another distinct difference between the two subfamilies lies in the position of vein M 2 : this vein arises closer to M 3 than to M 1 in the hindwings of Heraculinae, whereas it arises closer to M 1 than to M 3 in the hindwings of Pseudobistoninae.
Etymology: The subfamily name is derived from that of the type genus, Heracula Moore.

Discussion
Our molecular phylogenetic analyses show that Heracula and Pseudobiston (the type genus of the monotypic family Pseudobistonidae established by Rajaei et al., 2015) form a strongly supported clade, which itself is the sister clade to the family Epicopeiidae with moderate support (Fig. 1). Although the systematic position of the Pseudobistonidae + Epicopeiidae clade has not been robustly resolved based on our eight-gene molecular analyses or the analyses of Rajaei et al. (2015), more extensive molecular evidence indicates that at least Epicopeiidae belongs to the superfamily Geometroidea (Bazinet et al., 2013;Regier et al., 2013;Heikkilä et al., 2015).
Molecular phylogenetic analyses from this study and Rajaei et al. (2015) indicate that Geometroidea are comprised of two main clades (namely, Pseudobistonidae, Epicopeiidae and Sematuridae form a clade, which is sister to Geometridae + Uraniidae), with weak support for the monophyly of this superfamily. Rajaei et al. (2015) proposed five morphological autapomorphies supporting the monophyly of Geometroidea, and provided a number of characters as derived traits among geometroid families, although several of these characters were considered to be continuous in nature (Heikkilä et al., 2015). Rajaei et al. (2015) also provided two synapomorphies for Pseudobistonidae + Epicopeiidae + Sematuridae as well as four synapomorphies for Geometridae + Uraniidae, but they did not compare the variation of male abdominal tympanal organs for the two clades. In fact, the male abdominal tympanal organs are present in Geometridae and Uraniidae, but absent in the remaining three families of Geometroidea (Minet & Scoble, 1998;Rajaei et al., 2015), this could be regarded as a diagnostic character if the sister relationship between the two clades is accepted.
In addition, the chaetosemata are present and remote from each other in both Heracula (Fig. 2i) and Pseudobiston, but they are not regarded as a synapomorphy of the two taxa, as similar structures also occur in many other Macroheterocera including some epicopeiid taxa (Minet, 2003;Rajaei et al., 2015). The chaetosemata are usually a small bundle of long setae in Epicopeiidae (Minet & Scoble, 1998), but absent in Mimaporia (Wei & Yen, 2017), and well-developed in Deuveia with a transverse shape (Minet, 2003). The long setae of chaetosemata often overhang the eyes in Sematuridae (Minet & Scoble, 1998). In Geometridae and Uraniidae, the well-developed, transverse chaetosemata have been treated as a synapomorphy of the two families, even though their sizes and shapes are variable in Geometridae (Scoble & Krüger, 2002;Rajaei et al., 2015).
At least nine morphological characters can be used to diagnose Heracula in the present paper. (i) The vertex, on each side of the postgenal-occipital area, has two discrete, minute cranial pits (Fig. 2b, f). The cranial pits are proposed in Libytheinae, a subfamily of nymphalid butterflies, and are suspected to be an attachment site for a sensillum (Kawahara et al., 2012: figs 3, 4). These structures also are found on the vertex between the antennae in many geometrid moths (see the 'extensions' of Sihvonen, 2005: fig. 100), but are not recorded in Pseudobiston. (ii) Maxillary palpus is rather small, consisting of three segments (Fig. 2d,  black arrow). There is only one segment in Pseudobiston, and one or two segments in Uraniidae and Geometridae (Minet & Scoble, 1998;Rajaei et al., 2015). (iii) Antennae are filiform in the males. The filiform antennae are seen widely in the males of Geometroidea (Minet & Scoble, 1998), whereas the male antennae are bipectinate in Pseudobiston (Rajaei et al., 2015: Fig. 5). (iv). Ocelli are absent. The absence of ocelli is common in the family-group taxa of Geometroidea, but they are often present in Sematuridae, reduced in Pseudobiston, (Minet & Scoble, 1998;Rajaei et al., 2015). (v) Euphragma in the metathorax is only developed in the lateral region along the tergal rim, with a large, oval central foramen (Fig. 6a). The foramen of the euphragma is usually central in the advanced lineages of the Ditrysia, whereas it is dorsal in Pseudobiston, Epicopeiidae, certain Uraniidae, at least two geometrid subfamilies: Geometrinae and Ennominae, and is absent in the sematurid genus Sematura (Fänger, 1999;Rajaei et al., 2015). (vi) Eucoxa and meron of the metathorax are bordered only by a single metacoxal sulcus (Fig. 6c, d).
In Pseudobiston, merocosta is divided by a short gap into two sections that delimit laterodorsally a subtriangular region of the meron (Rajaei et al., 2015: Fig. 9). (vii) hind tibia possess four spurs (Fig. 7c). The same situation also occurs in Epicopeiidae, Sematuridae, as well as most taxa of Geometridae and Uraniidae (Minet & Scoble, 1998), however, only two hind tibial spurs appear in Pseudobiston (Rajaei et al., 2015: fig. 26). (viii) Vein M 2 arises closer to M 3 than to M 1 in both fore-and hindwings (Fig. 11). In Pseudobiston, M 2 arises closer to the upper angle of the cell than to the base of M 3 in the forewings, and closer to M 1 than to M 3 in the hindwings (Rajaei et al., 2015: fig. 31). (ix) Socii are absent in the male genitalia (Fig. 13a). These structures are very distinctive in Pseudobiston (Rajaei et al., 2015), and are considered as a part of the ground plan in several superfamilies including the Geometroidea (Schmidt, 2017).
As mentioned above, although Heracula and Pseudobiston form a well-supported monophyletic clade from our molecular analysis, morphological evidence shows that there exist great differences between two taxa. Heracula can be easily distinguished from Pseudobiston by the nine morphological characters enumerated in the above paragraph. Many of these characters are conservative in Lepidoptera, and their variations are often regarded as derived characters of higher-level taxa (Scoble, 1992;Kristensen, 2003). In addition, the shared synapomorphies of Heracula and Pseudobiston, except for the absence of anterolateral brush organs, are three thoracic features, which are usually not clear-cut and difficult to observe without a dissection of thoracic skeleton. For these reasons, here we formally propose two independent monotypic subfamilies (Heraculinae and Pseudobistoninae) of Pseudobistonidae to accommodate Heracula and Pseudobiston, respectively.
With regard to the clade Pseudobistonidae + Epicopeiidae, Rajaei et al. (2015) interpreted its sister relationship using seven imaginal synapomorphies, among which a dorsal foramen of the euphragma cannot now be used as synapomorphy, because Heraculinae, with an oval central foramen (Fig. 6A), are placed as a subfamily of Pseudobistonidae. Epicopeiidae currently include 26 species in 10 genera, and exhibit high morphological diversity and various complex mimicry features (Wei & Yen, 2017). The monophyly of Epicopeiidae is well studied based on morphological traits (Minet, 2003;Rajaei et al., 2015), but it has not been verified by molecular data. Recently, Wei & Yen (2017) did a preliminary molecular phylogenetic analysis of Epicopeiidae with incomplete sampling, but their results show significant differences from those of the morphological analysis in Minet (2003). Further molecular phylogenetic studies are needed for clarifying the monophyly of Epicopeiidae and the relationships among the genera of this family.

Notes on the resemblance of the habitus in Heracula and Calliprogonos
In the course of this work we noted the resemblance of the unusual habitus of Heracula to that of the similarly monobasic bombycoid brahmaeid genus Calliprogonos Mell & Hering, reviewed by Naumann (2009), particularly in the large pale ring at the base of the forewing (Fig. 9). However, morphological and molecular evidence precludes any close phylogenetic relationship between the two genera. For instance, the lower edge of hindwing discal cell is longer than vein M 3 in Heracula (Fig. 11), but is markedly shorter than vein M 3 in Calliprogonos (Lemaire & Minet, 1998: fig. 18.5H). Moreover, male of Heracula has a retinaculo-frenate wing coupling (Fig. 10), whereas male wing coupling is amplexiform in Calliprogonos ( (Lemaire & Minet, 1998). Furthermore, DNA barcoding indicates that Calliprogonos is very close to the brahmaeid genus Dactyloceras Mell (Naumann, 2009). Therefore, the resemblance between the two genera can only be through convergence. The resemblance may be purely fortuitous were not the two taxa sympatric over the more localized range of the brahmaeid, occurring at similar altitudes of 1500 m a.s.l. upwards, leading to the possibility of some interaction between them, with the ranges suggesting that Heracula would be the model in the case of mimicry. Field observations of the species at rest would be valuable to assess whether the rings might appear as eye spots to a potential predator.
The photograph of Pseudobiston at rest in Rajaei et al., 2015, Fig. 4) shows a resting posture that, if transposed to the other two taxa, would place the pale rings near the front of the moth and, if in the head-up position, could resemble the eyes of a bird, with the pale wing-margins forming a band below the eyes. The upper part of the Pseudobiston at rest does suggest, to a fertile imagination, the head of a bird, with the black-centred pale lunules at the bases of the forewings being the eyes.

Biogeographical distribution of Pseudobistonidae and their relatives
The Pseudobistonidae, Epicopeiidae and Sematuridae are placed in a clade sister to the rest of the Geometroidea: the Uraniidae and the Geometridae. They are all tropical or subtropical in distribution. Three of the family-group taxa are monobasic: the southern African subfamily of the Sematuridae, Apoprogoninae, and the two subfamilies of the Oriental but allopatric Pseudobistonidae discussed here. Both the Sematurinae and the Epicopeiidae contain several genera, the former mainly Neotropical and the latter mainland Oriental apart from a species of Epicopeia in Sumatra.
The Uraniidae (Lees & Smith, 1991;Holloway, 1998) are pantropical, a range spanned by the Uraniinae and the most species-rich subfamily, the Epipleminae. The two other subfamilies, the Auzeinae and the Microniinae, are found only in the Palaeotropics.
The family Geometridae has a cosmopolitan distribution, and is currently divided into eight subfamilies including four major ones: Larentiinae, Sterrhinae, Geometrinae and Ennominae (Sihvonen et al., 2011;Mitter et al., 2017). The Ennominae are the most species-rich, but the Larentiinae are more diverse at high latitudes and, in the tropics, high altitudes.
In the present study we have resolved the systematic position of the mysterious taxon Heracula, transferring it from Noctuoidea to Geometroidea as a new subfamily of Pseudobistonidae, using multilocus molecular phylogenetic data together with the re-examination of its morphological characters. In fact, Heracula is not the only mysterious taxon to have been misclassified in the Lymantriinae. Another example within Macroheterocera is the Oriental genus Allotoma Roepke which was originally placed in the Notodontidae but transferred to Lymantriidae by Kiriakoff (1968, Genera Insectorum 217c: 2) without comment, a placement that still stood in Watson et al. (1980). The genus was associated with Bombycoidea by Holloway (1999: 153-154), where it cannot be clearly assigned to any particular family based on the existing morphological traits. A more extreme transfer in phylogenetic placement was that of Shisa excellens Strand from the Lymantriinae to the cossoid family Ratardidae (Owada, 1993).
Mysterious taxa, with dubious placements from morphological information, also have been found in some other Lepidopteran lineages, and molecular phylogenetic data have been increasingly applied in addition to morphology to ascertain their systematic status with some particularly successful cases. For example, the systematic position of the rare Asian butterfly genus Calinaga had long been a topic for debate until it was recently verified as a monobasic subfamily Calinaginae in Nymphalidae based mainly on molecular phylogenetic data (Wahlberg, et al., 2003;Todisco, et al., 2017). The moth tribe Diptychini Janse, with a controversial placement, was demonstrated to be a group of Ennominae in Geometridae using an eight-gene molecular dataset . The mysterious African moth Prodidactis mystica (Meyrick) was finally confirmed as a monotypic family Prodidactidae belonging to Hyblaeoidea using molecular and morphological data (Kaila et al., 2013). The enigmatic Palaearctic moth genus Lypusa Zeller was historically assigned to Tineoidea, but later clarified as a family-level taxon in Gelechioidea based on the phylogenetic analyses of a seven-gene dataset (Heikkilä & Kaila, 2010;Kaila et al., 2011). The small Aenigmatinea glatzella Kristensen & Edwards was elucidated as the type of a new extant family Aenigmatineidae sister to Neopseustidae with evidence from 25 genetic loci (Kristensen et al., 2015). Thus, molecular phylogenetic data, as an additional powerful tool, may shed light on the systematic placements of such mysterious taxa in Lepidoptera where morphological studies have failed.

Supporting Information
Additional supporting information may be found online in the Supporting Information section at the end of the article. File S1. Aligned DNA matrix used in the phylogenetic analyses.nex. Table S1. Label data for the illustrated specimens in this study. Table S2. List of taxa with voucher codes and Genbank accession numbers.