Overlooked gall-inducing moths revisited, with the description of Andescecidiumparrai gen. et sp. n. and Olierasaizi sp. n. from Chile (Lepidoptera, Cecidosidae)

Abstract There are still many gall systems associated with larvae of Lepidoptera in which the true gall-inducers have not been identified to species. Reports on misidentification of gall inducers have been recurrent for these galls, particularly in complex gall-systems that may include inquilines, kleptoparasites, and cecidophages, among other feeding guilds such as predators and parasitoid wasps. Here we describe and illustrate the adults, larvae, pupae and galls, based on light and scanning microscopy, of Andescecidiumparraigen. et sp. n. and Olierasaizisp. n., two sympatric cecidosid moths that are associated with Schinuspolygamus (Cav.) Cabrera (Anacardiaceae) in central Chile. Adults, immatures, and galls of the former did not conform to any known cecidosid genus. Galls of A.parrai are external, spherical, and conspicuous, being known for more than one century. However, their induction has been mistakenly associated with either unidentified Coleoptera (original description) or Olieraargentinana Brèthes (recently), a distinct cecidosid species with distribution restricted to the eastern Andes. Galls of O.saizi had been undetected, as they are inconspicuous. They occur under the bark within swollen stems, and may occur on the same plant, adjacent to those of A.parrai. We also propose a time-calibrated phylogeny using sequences from mitochondrial and nuclear loci, including specimens of the new proposed taxa. Thus in addition to clarifying the taxonomy of the Chilean cecidosid species we also tested their monophyly in comparison to congeneric species and putative specimens of all genera of Neotropical and African cecidosids.


Introduction
Cecidosidae are a small group of ancient gall-inducing and stem-mining micromoths restricted in distribution to the Southern Hemisphere. They include seven genera and 19 species that are distributed in southern South America, southern Africa, and New Zealand (for taxonomic reviews see Davis 1988, Hoare and Dugdale 2003, Moreira et al. 2017. In spite of their interesting biology, close association with their Anacardiaceae host-plants (Schinus L.) and broad geographical distribution, the diversity of Cecidosidae remains mostly unexplored in the Neotropical region. Their galls may consist of complex ecological systems that involve additional organisms, in addition to the host-plant and corresponding gall-inducer. For example, other insects can act in multi-trophic levels in these galls, as inquilines, kleptoparasites, or cecidophages, among other feeding guilds, including predators, parasitoids or successors that use them just for shelter (Moreira et al. 2017). In the case of kleptoparasites and inquilines, the original inducer might be killed and thereafter the gall is usurped by the invader (Ronquist 1994, van Noort et al. 2007). This type of phenomenon may bring important taxonomic consequences, preventing the correct identification of the real inducer when such systems are undersampled. This situation is found for many gall-inducing insects, including other Lepidoptera (e.g., Morris 2000, van Noort et al. 2007, Luz et al. 2015. This was recently demonstrated in detail for Cecidonius pampeanus Moreira and Gonçalves, a cecidosid moth whose identity was overlooked for more than one hundred years, as its galls had been erroneously described as induced by their hymenopteran inquilines (Moreira et al. 2017).
A similar case of such a complex system was recently found for a cecidosid-induced gall in central Chile. In fact, such a spherical, external gall was briefly described for the first time by Kieffer and Herbst (1905), in association with the branches of the Anacardiaceae Duvaua dependens (Ortega) DC. [= S. polygamus (Cav.) Cabrera] as induced by an unidentified species of Bruchus L. (Coleoptera, Chrysomelidae). One year later, the same authors reported this type of gall again, providing in addition illustrations that included a cross-section view of it (Kieffer and Herbst 1906). In his monograph of galls from South and Central American plants later, Houard (1933) reproduced this description and illustrations, associated with the same country, gall-inducer, and host-plant. More than 50 years later, Núñez and Sáiz (1994) working with these galls in the Parque Nacional La Campana, Valparaiso Region, cited the corresponding findings that were repeated by Houard (1933); however, a note was added by the authors recognizing that the true inducer of the galls belonged to Lepidoptera. Later, Sáiz and Núñez (1997) stated that these were in fact induced by Cecidosidae, but they did not describe the moth. Based on scarce material sent by these authors to the Universidad de Concepción, Parra (1998) described for the first time the corresponding gall-inducer cecidosid moth as conspecific to Oliera argentinana Brèthes, a species at that time only known to occur on the eastern side of the Andes (Argentina) and with poorly known immatures. He also synonymized Oliera Brèthes with Cecidoses Curtis, and proposed Cecidoses argentinana (Brèthes), thereafter considered to be the valid species responsible for the induction of Schinus galls in Chile. However, Moreira et al. (2012) in a broad study including abundant material of galls from Argentina and Brazil, also including type material, confirmed the original description of O. argentinana given by Brèthes (1916), showing that Oliera galls are formed under the bark instead, and have different morphology from material described by Parra (1998) based on the larvae, pupae and adults, thus revalidating the genus Oliera. In addition to morphological characters, Moreira et al. (2012) based their study on a preliminary analysis of mitochondrial (COI) DNA sequences, including putative members of the four Neotropical cecidosid genera recognized at that time. They also concluded that material described by Parra (1998) should belong to a different cecidosid genus yet to be identified, which is described in detail in the present study.
While searching later in the field for additional material of this new cecidosid genus, three of the authors of this study (G. R. P. Moreira, G. San Blas and H. A. Vargas) found that there was a second undescribed, cecidosid species in Chile occurring in sympatry, forming galls on swollen stems of the same S. polygamus plants, as already suggested by Sáiz and Núñez (1997). These internal, inconspicuous galls, from which Parra (1998) had not received material to work at that time, turned out to be an additional, unknown species of Oliera (Brèthes), which is also described here. Thus the main goal of this report is to clarify the peculiar and somewhat confused taxonomic history of these two cecidosid moths in Chile. Under light and scanning electron microscopy, their galls, larvae, pupae and adults are described and illustrated, and preliminary information on their life history is provided. A phylogenetic analysis of concatenated mitochondrial (COI and 16S) and nuclear (Wingless) DNA sequences, including putative members of all lineages of extant Cecidosidae from South America and Africa, was also conducted to provide support for the proposition of these new taxa.

Morphology
Adults used in the study were reared by H.A. Vargas from galls either attached to Schinus polygamus branches or detached ones found in litter, under the canopy cover, which were maintained in small plastic vials at room temperature in the entomology laboratory of the Facultad de Ciencias Agronómicas, Universidad de Tarapacá, Arica, Chile. They were periodically inspected for emerged adults, which were then pinned and dried. Larvae and pupae were obtained by dissecting additional galls. They were fixed in Kahle-Dietrich´s fluid and preserved in 70% EtOH. For DNA analyses, additional specimens were preserved in 100% EtOH at -20 °C.
For gross morphology studies, the specimens were cleared in a 10% potassium hydroxide (KOH) solution and mounted on slides with either glycerin jelly or Canada balsam. Observations were made with the aid of a Leica M125 stereomicroscope. Structures selected to be drawn were previously photographed with an attached Sony Cyber-shot DSC-H10 digital camera. The vectorized line drawings were made with the software CorelPhotoPaint X4, using the corresponding digitalized images as a guide. At least four specimens were used for the descriptions of each life stage. Measurements were made with an attached ocular micrometer; values are presented as mean ± standard deviation unless noted otherwise.
Specimens used in scanning electron microscope (SEM) analyses were dehydrated in a Bal-tec CPD030 critical-point dryer, mounted with double-sided tape on metal stubs and coated with gold in a Bal-tec SCD050 sputter coater. They were examined and photographed in a JEOL JSM6060 scanning electron microscope at Centro de MicroscopiaEletrônica (CME) of Federal University of Rio Grande do Sul, (UFRGS), Porto Alegre, RS, Brazil.
The material examined is deposited in the following collections:

Molecular phylogeny
We assembled data from two mitochondrial loci and one nuclear protein-coding locus from 12 species representing all Neotropical lineages of Cecidosidae (Cecidonius Moreira & Gonçalves, Cecidoses, Dicranoses Kieffer & Jörgensen, Eucecidoses Brèthes and Oliera) to infer the relationship of Andescecidium gen. n. and the phylogenetic status of A. parrai sp. n. and its congeneric undescribed taxon Andescecidium sp. (Table 1).
To infer the relationship of O. saizi sp. n., we added an undescribed taxon (Oliera sp.) in addition to the type species (O. argentinana). We also included distinct populations of A. parrai and O. saizi to cover putative geographic divergence. The South African genus Scyrotis Meyrick was also taken into account in the analysis. As outgroup, we in-cluded representative species of Adelidae, Incurvariidae, Heliozelidae, and Prodoxidae, which together with Cecidosidae comprise the superfamily Adeloidea (van Nieukerken et al. 2011, Regier et al. 2015. Voucher specimens used are listed in Table 1. Total genomic DNA was purified from fresh larval tissue of Andescecidium parrai, Andescecidium sp. and two additional species of Oliera (O. saizi and Oliera sp.), using the PureLink genomic DNA kit (Life, Invitrogen, USA) following the manufacturer's instructions. Sequences of Cecidonius pampeanus, Cecidonius sp., Cecidoses eremita Curtis, Dicranoses congregatella (Brèthes), Eucecidoses minutanus Brèthes, Oliera argentinana, Scyrotis granosa (Meyrick) were previously generated by Moreira et al. (2017). Adelidae, Incurvariidae and Prodoxidae sequences were incorporated from the GenBank/NCBI databases, as well as the African cecidosid S. pulleni Mey.
Approximately 2.5 Kb of gene segments were amplified by polymerase chain reaction (PCR): 1421 bp of the cytochrome oxidase subunit I (COI), 474 bp of the 16S ribosomal RNA (16S) and 395 bp of the Wingless (Wg) gene loci, with primers and conditions described in Moreira et al. (2017). PCR products were purified using the enzymatic method (exonuclease and alkaline phosphatase), sequenced with BigDye chemistry, and analyzed in an ABI3730XL (Applied Biosystems Inc.). Chromatograms obtained from the automatic sequencer were read and sequences were assembled using the software CodonCode Aligner (CodonCode Corporation). Sequences generated in this study will be deposited in GenBank and BOLD (Table 1).
We used BEAST v2.02 (Bouckaert et al. 2014) to perform relaxed clock Bayesian analyses of the concatenated data and of each gene. Input files contained partitions for each codon position of each gene, which were selected by PartitionFinder v.1.1.1 (Lanfear et al. 2012). We linked site models in accordance with partitions identified by Partition-Finder, unlinked clock models across all partitions, and linked topology across all partitions. We tested the null hypothesis of a strict molecular clock for each gene (data not partitioned) using likelihood-ratio tests in MEGA v7 (Tamura et al. 2013) and found that all genes rejected the strict molecular clock hypothesis. Thus we used uncorrelated lognormal relaxed molecular clock models for all partitions, with mean rates estimated from a gamma distribution relative to a partition with an arbitrary fixed rate of 1. For a tree prior, we used a Calibrated Yule (pure-birth) model with random starting trees. Initial runs with BEAST showed that cecidosids did not remain monophyletic in relation to the outgroup taxa (Prodoxidae, Adelidae, Heliozelidae, and Incurvariidae). Because the monophyly of Cecidosidae is not in question in this study, we constrained it to be monophyletic in the analyses. To adjust the molecular clock we used the fossil calibration point of Adeloidea, about 120±10mya, with a log-normal distribution (Walhberg et al. 2013).We used 1×10 5 generations (sampled every 5,000) for each individual gene tree, with the number of generations increased to 2.5×10 5 for the concatenated analysis. We assessed convergence of BEAST analyses using Tracer v1.6 (Drummond and Rambaut 2007) to examine whether effective sample sizes exceed 200. We used TreeAnnotator v1.7.5 (Drummond et al. 2006) to summarize the posterior distribution of trees from individual MCMC analyses into maximum clade credibility trees using a burn-in fraction of 25%. Trees were observed and edited in FigTree v1.4.3 (Rambaut 2009). Clades with Bayesian Posterior Probability (BPP) ≥ 95% were considered strongly supported. Pairwise genetic distances (p-distances) among lineages were calculated in MEGA v7 with 1,000 bootstrap replications.

Molecular phylogeny
The concatenated tree corroborated our hypothesis of monophyletic status for the new genus and species and their relationships within Cecidosidae with strong node support (BPP = 1) ( Figure 1). Individual trees based on COI and 16S showed nearly identical topologies, with a slight difference for Wg (likely due to evolving at a slower rate than the other genes), resulting in weaker phylogenetic resolution than the concatenated tree; however, all retrieved the monophyletic status of the new taxa (data not shown). The concatenated time-tree indicates that Andescecidium is sister of Cecidonius, a Pampean lineage found in the grasslands of Southern Brazil, and emerged around 35 Mya (95% HDP 20.6-49.1). Genetic distances of the new genus to other cecidosids ranged from 12 to 27%; a lower divergence was observed in relation to Oliera saizi and highest to Dicranoses (both D. congregatella and D. capsulifex) and Scyrotis (both S. pulleni and S. granosa (Table 2). Diagnosis. Andescecidium gen. n. resembles Cecidonius in having pupae bearing strong spines on the anterior margins of the abdominal terga and larvae with two stemmata, which make them unique among cecidosids. However, Andescecidium shows several adult, larval, and gall features which in conjunction differentiate it from the latter. Adults are much larger, bear mandibular rudiments and have reduced threesegmented maxillary palpi, which are respectively absent and with two in the small Cecidonius. The ovipositor in Andescecidium is reduced in size, and associated with an inconspicuous dorsal crest in the oviscapt cone that is used to lay eggs on superficial external buds. The ovipositor of Cecidonius is very long, having the oviscapt cone dorsally  anchored by a conspicuous crest, thus allowing oviposition deep into the stem. Contrary to the larvae of Cecidonius, which are unique among cecidosids in having setae of much longer size on the thorax, those of Andescecidium show these structures uniform on thorax and abdomen. The spherical galls of Andescecidium are associated with stem  (Figs 2A, B, 3). The adult male morphology was accurately described and illustrated in part by Parra (1998). We complement this description for the male here, adding illustrations that were not provided by Parra, and we also describe the female for the first time.

Taxonomy
Male. Forewing length ca. 12.1 mm (n = 1). Head ( Figure 3A): vertex and frons covered by narrow, elongated dark brown scales with a few scattered whitish gray scales. Compound eyes black. Antennae filiform (~ 0.7× length of forewing), with dark brown scales on scape, pedicel and dorsal surface of basal half of flagellum; filiform sensilla on remaining dorsal and ventral surface of flagellum. Labrum semicircular, short. Mandibles poorly developed, as small stubs. Pilifers absent. Maxillae with galeae reduced to small lobes (~2/3 labial palpus length); maxillary palpi tri-segmented (ratios of segments from base ~1.0:0.8:0.8). Labial palpi tri-segmented (ratios of segments from base ~1.0:0.9:1.2). Maxillary and labial palpi with brownish gray scales. Thorax. Mostly with brownish gray scales. Anterior arms of latero cervical sclerites not observed. Metafurca ( Figure 3B) with slender, elongate postero-dorsal apophyses, free from secondary arms. Forewings (Figure 2A, B) lanceolate, mostly covered with brownish gray scales; a broad dark brown spot close to the middle of the posterior margin of discal cell; a small dark brown spot at apex of discal cell. Hindwings lanceolate, brownish gray. Wing venation as described by Parra (1998: fig. 2). Prothoracic legs ( Figure 3C) dark brown with a few scattered whitish gray scales, bearing an epiphysis on distal portion of tibia. Mesothoracic legs similar in coloration to prothoracic legs, with two whitish gray tibial spurs. Metathoracic legs whitish gray with two pairs of tibial spurs; tibia with longitudinal stripe of narrow, elongated hair-like whitish gray scales. Tibial length proportion (anterior / medium / posterior legs) ~ 0.6:0.7:1.0. Abdomen brownish gray with a few dark brown and scattered whitish gray scales. Male genitalia as described by Parra (1998: figs 2, 3, 4A).
Female. Forewing length ca. 11.5 mm (n = 2). Similar to male in general, except that sensilla are less abundant and smaller on the antennae, and abdominal sternum VII with caudal margin more sclerotized, bearing a dense ring of stout, elongate setae ( Figure 3E). Female genitalia formed by an oviscapt cone (sensu Kristensen 2003, San Blas andDavis 2013), with weak internal dorsal crest, reaching the anterior margin of tergum seven. Anterior apophyses extending to anterior margin of sixth abdominal segment. Posterior apophyses ~1.3× length of anterior apophyses. Posterior apophyses are caudally fused to form the short, compressed, and sagittate apex of the ovipositor. Ductus and corpus bursae membranous, the latter saculiform, without signum.
Etymology. The genus name is derived from a composition between the Portuguese Andes and Cecidia (a gall; from the Greek kekídion). Thus, the epithet refers to the Andes Mountains where the galls of Andescecidium were first found.  (Brèthes, 1916) (misidentification).

Diagnosis.
As discussed for the monotypic genus.
Etymology. Named in honor of Prof. Dr. Luis E. Parra, from the Universidad of Concepción, who for the first time characterized such a gall-inducing moth, for his great contribution to the development of Lepidopterology in Chile.
Life history. The large, short pedunculated galls of A. parrai develop externally from the beginning, on stem buds of S. polygamus terminal and sub-terminal branches ( Figure 8B, C). A few galls of different sizes may be found on the same branch. They are elliptical and usually reddish when young, becoming to spherical and green when mature. The larval chamber is almost cylindrical in shape (maximum width and length  varying from 9-10 and from 10-14 mm, respectively; n = 10), and transverse to the stem axis. Their external wall becomes dark brown and almost cylindrical during late development. At least some are apparently deciduous, falling to the ground. This morphotype has a rough external wall ( Figure 8D), and was found among the litter under the S. polygamus plants, where they are easily confused with fecal pellets of the European rabbit (Oryctolagus cuniculus L.: Leporidae), that is abundant in the region. Like C. pampeanus galls, those of A. parrai also lack an operculum. Using the frontal process and abdominal spines present on the pupae to make pressure, together with body contortions, are likely the means the pupa uses to open an orifice on the distal, weaker wall ( Figure 8E), in which it pushes itself partially out of the gall.
Little is known about biology of A. parrai galls. Our field observations suggest that the species is univoltine, galls starting to develop during late spring and summer, when most leaf buds are found on S. polygamus plants (Sáiz and Núñez 1997). Deciduous galls were field-collected from the ground near the end of May, from which a few adults used here for description successfully emerged in the laboratory after ca. 25 days. Description of adults. Male. Forewing length varying from 5.5 to 6.3 mm (n = 3). Head: Vertex and frons covered with smooth, narrow, brownish gray scales. Compound eyes black. Antennae filiform, about half the length of forewing, with brownish gray scales dorsally, ventral half with short sensilla. Labrum semicircular, short. Mandibles poorly developed, as small stubs. Pilifers absent. Maxillae with galeae reduced to small lobes; maxillary palpi tri-segmented (ratios of segments from base ~1.0:0.9:0.6). Labial palpi tri-segmented (ratios of segments from base ~1.0:0.8:0.7). Maxillary and labial palpi with brownish gray scales. Thorax: Mostly with brownish gray scales. Wing venation ( Figure 9A) as in O. argentinana (Moreira et al. 2012). Prothoracic legs with coxa, trochanter and femur dark gray (occasionally brownish gray); tibia and tarsus brownish gray; apex of tibial epiphysis reaching the apex of tibia. Mesothoracic leg brown- ish gray; one pair of tibial spurs. Metathoracic legs brownish gray; two pairs of tibial spurs; tibia with longitudinal band of narrow, elongated hair-like scales. Tibial length proportion (anterior / medium / posterior legs) ~ 0.5:0.6:1.0. Fore-and hindwings lanceolate, brownish gray. Abdomen: Brownish gray. Genitalia ( Figure 9D) with narrow tegumen; uncus bilobed; socii as two small setigerous lobes. Saccus with dorsal arms narrow, U-shaped, with anterior projection prominent, slightly widened close to apex. Valva narrow, about 3/4 the length of the anterior projection of the saccus; dorsal and ventral margins sub-parallel, and distal portion dorsally dilated. Pectinifer ( Figure 9E) as a straight band on the ventral part of the medial surface of the valva. Transtilla as an inverted "V", with ventral arms straight, slightly widened close the vertex. Juxta ( Figure 9G) narrow, elongated, slightly spatulate distally. Phallus ( Figure 9F) narrow, somewhat sinuous, with similar length to valva, with anterior apex laterally widened forming a semicircle; vesica without cornuti.
Additional pinned-dried adults examined. Puente El Yeso, Cajón del Maipo, Metropolitan Region, R. Charling C. leg., 4 males, with one genitalia (DRD 16536) and one wing (DRD 29925) preparations; 2 females, with one wing and genitalia preparation (DRD 16537), USNM. Etymology. Named in honor of Prof. Dr. Francisco Sáiz, from the Universidad Católica de Valparaiso, who first noticed the existence of these galls, for his great contribution to the development of Cecidology in Chile.
Thorax (T) and abdomen (A) creamy white, mostly covered with rounded microtrichia; prothoracic shield weakly defined by a pair of slightly differentiate areas, with reduced microtrichia. A1-7 with well-developed callus ( Figure 11H), mesally, close to posterior margin of terga. Thoracic legs ( Figure 11G) reduced to circular, unsegmented tubercles; prolegs absent. Spiracles circular, without elevated peritreme, laterally on T1 and A1-8. Abdominal segment 10 composed of three lobes, one dorsal, wider and two ventral ( Figure 11I). Chaetotaxy of thorax and abdomen composed of setae reduced in size. T1 with eight pairs of setae; XD group bisetose, SD group unisetose outside prothoracic shield, L group trisetose, SV group unisetose, V group unisetose. T2-3 with four pairs of setae; D group bisetose, L and V groups unisetose. A1-5 with two  pairs of setae; D group bisetose. A6-7 with three pairs of setae, D group bisetose and V group unisetose. A8 with four pairs of setae; D group bisetose, L group unisetose and SV group unisetose. A9 with five pairs of setae; SD group unisetose and L group tetrasetose. A10 with four pairs of setae; SD unisetose, SV unisetose and V bisetose.
Life history. Oliera saizi develops spindle-shaped galls enclosed within swollen stems of S. polygamus subterminal branches ( Figure 14A, B). The larval chamber is elliptical in shape and transversally located in relation to the stem axis, and as in O. argentinana, the gall lacks an operculum. A progressive necrosis extends up the gall wall with the advent of pupation; meanwhile the pupa opens an orifice on the wall with the aid of its frontal process and through body contortions. The abdominal spines help the pupa during these movements to anchor itself on the wall; it pushes part of its body out of the gall, when the exuvia splits and the adult emerges ( Figure 14B, D). The outer wall eventually collapses, leaving the empty galls appearing as small holes on the host plant branches ( Figure 14E, F). Little is also known about the biology of O. saizi. Similar to what has been found for O. argentinana (Moreira et al. 2012), their galls have been collected occasionally, either on isolated plants or in groups. They may occur at high densities per branch, sometimes adjacent to those of A. parrai. Our field collection data indicate that the species is univoltine, with adult emergence occurring from late spring to early summer (November / December). Schinus polygamus branches bearing galls with full-grown pupae were field-collected during November, from which a few adults used for description emerged under laboratory conditions ca. 25 days later.

Host plant and distribution of both species. Galls of both A. parrai and O. saizi
have been found only on branches of Schinus polygamus (Cav.) Cabrera (Anacardiaceae), a small tree with single and glabrous leaves and slender spiny branches (Barkley 1957, Fleig 1989, commonly known in Chile as Huingan. The taxonomy of Schinus L. in southern South America is rather complex, as its wide geographic distribution and phenotypic plasticity have lead historically to a confused taxonomy. In fact, the genus is currently under review (CLS Luz, USP, pers. comm.), and S. polygamus (sensu lato; Cabrera 1938, Fleig 1987, 1989) may be split into several species, as already proposed by Barkley (1957). Nevertheless, according to this author in this case the true S. polygamus would be restricted in distribution to Chile. Andescecidium parrai and O. saizi galls were found on populations of S. polygamus on the central area, also known as the Mediterranean portion of the country (Quintanilla et al. 2012), which extends from 32°45'N to 37°30'S, abridging the V, VI, VII, VIII and Metropolitan (RM) regions ( Figure 15). The main characteristics of this area are the presence of two mountainous ranges, the Andes on the east and the Coastal Range on the west, as well as the central valley located in between them. Plants bearing galls of both cecidosid species were found mainly in the Coastal Range (Figs 8A, 14A). The climate in these valleys is typically austral Mediterranean; the vegetation is autochthonous, composed of sclerophyllous forests, palm communities and coniferous forests and steppe at higher altitudes (Quintanilla et al. 2012, Valencia et al. 2015.

Discussion
This study illustrates further how important are intensive studies including the immature stages to achieve correct identification of true gall-inducing in Lepidoptera. It confirms preliminary findings of Sáiz and Núñez (1997), which were further supported by Parra (1998), showing that the cylindrical external galls associated with S. polygamus in Chile are induced by a cecidosid moth, and not by a beetle as originally suggested by Herbst (1905, 1906) and Houard (1933). In fact, during dissection of these galls we occasionally found an unidentified species of Coleoptera associated with them but only later in ontogeny; small, early developing galls always contained a larva of Andececidium parrai. We believe that this beetle acts as a kleptoparasite (sensu Luz et al. 2015), since apparently it does not change substantially the shape of the galls after usurping them. In addition, we found that galls of A. parrai may host other unidentified parasitoids, predators, and successors, thus being complex systems, similar to what has been previously demonstrated for Cecidonius pampeanus (Moreira et al. 2017). Thus we attribute in part the historical confusion involving the taxonomy of cecidosids in Chile to the scarce sampling activity from a taxonomic perspective in the past, and to the fact that galls of A. parrai and O. saizi are sympatric, occurring sometimes on the same host-plant and branch, as already mentioned. Furthermore, galls of the former are deciduous and those of the latter restricted to under bark, which makes them difficult to detect on plants in the field, and as a consequence also hard to be reared to the adult stage by non-specialists.
Andescecidium parrai and O. saizi resulted as unique lineages in the present study from both morphological and molecular analyses. The former appeared as one of the most derived lineages to be evolved within the extant cecidosids. The genetic distance of A. parrai is ca. 10% from the nearest related species, an additional undescribed cecidosid existing in Mendoza, Argentina, which was included in the present study for comparison. Oliera saizi diverged ca. 6%, from an undescribed Oliera species collected at the same latitude in Argentina, and ca. 5% from the type species O. argentinana, sampled in southern Brazil. Thus molecular data suggest that although described as monotypic, there is at least one more species belonging to Andescecidium, and another to Oliera. In fact, these species share morphological characteristics with congenerics regarding the gall and larval stage, but unfortunately they have not been reared yet to the pupal and adult stages, upon which their descriptions are pending.
The present study also provides support to previous statements in the literature in the sense that inclusion of immature stages and galls is essential for elucidation of the taxonomy of Cecidosidae (Moreira et al. 2012(Moreira et al. , 2017. Our findings indicate that these moths have adults with relatively uniform morphologies, especially in relation to the genitalia, which apparently vary less compared to the immature stages at the generic level (see also Mey 2007). The adult mouth parts, frontal and caudal processes, tergal spines and arrangement of setae on the pupa, number and size of secondary setae on larva, and the plant tissue from which the galls are differentiated in association with their shape and size are apparently the main sources of morphological variation within the group. The wide genetic divergence among congenerics (6% in Oliera, 7% in Cecidonius and 10% in Andescecidium) found up to now at the molecular level (mitochondrial, COI sequences) is also notable, which demonstrates the importance of this kind of analysis in taxonomic studies.
A complete scenario of relationships within Cecidosidae, inferred in the Bayesian calibrated-time tree, retrieved high posterior probabilities for all nodes. It was somewhat unexpected to obtain a fully resolved genus-level tree, based on only two to three markers (see Kawahara et al. 2011). Therefore, results at this level in our study should be interpreted cautiously, as the support provided by the Markov Chain Monte Carlo-based Bayesian method may be overestimated, since it is more dependent on the model suitability than other methods, such as bootstrap (Simmons et al. 2004). The use of more loci usually increases the robustness of phylogenies, particularly the support of nodes (Regier et al. 2009. Although COI alone may not accurately reflect the evolutionary relationships among species (Wahlberg and Wheat 2008), it is suitable as a simple estimate of genetic distance among moth lineages, as a first approach to evaluating the amount of difference. Regier et al. (2015) sequenced 15 nuclear genes (ca. 7,500 bp) in distinct Adeloidea families and found 25% of variation within Cecidosidae (Cecidoses vs. Dicranoses), quite similar to the 23% we found in COI. A comparative analysis using multilocus data generated by Regier et al. (2015) on variation within Adeloidea supports that Cecidosidae present greater variation in relation to the other families included (Adelidae, 13%; Incurvariidae, 13%; Prodoxidae, 17%). The high variation found by Regier et al. (2015) and in our study (also shown by long branches in the reconstructed phylogeny of Cecidosidae) might be related to the old age of this group (i.e. with more time to accumulate differences between lineages), incomplete sampling, and/ or early extinctions. Additionally, differences in patterns and/or rates of molecular evolu-tion of cecidosids (also observed in the genus Scyrotis, retrieved as polyphyletic) specifically related to COI might be due to metabolism-related factors (Pentinsaari et al. 2016).
In this study we recorded for the first time two new species of cecidosids for Chile that belong to two relatively recent genera (Andescecidium and Oliera) that apparently have representatives distributed on both the west and east sides of the Andes. Based on a broader survey exploring several southern South America biomes, Silva et al. (2018) suggested that the uplift of the Andes had a major role on speciation of Eucecidoses (Brèthes), a related genus with distribution restricted to the eastern Andes. Similar studies should be conducted for the west Andean cecidosid species described here. As already mentioned, galls induced by O. saizi had not been detected on S. polygamus before this study. But gall morphotypes similar to them, and also to those described in this study for A. parrai, were reported for the stems of S. latifolius (Gillies ex Lindl.) Engl. by Sáiz and Núñez (1997). The geographic distribution of S. polygamus is broader than that covered in this study, and there are a few other native species of Schinus in Chile (see Rodríguez et al. 2018), which should be also explored for galls. In other words, the diversity of cecidosids in Chile is believed to be greater, and thus should be better studied from a taxonomic perspective, involving other Anacardiaceae species, before a phylogeographic approach can be undertaken.