The southwestern Andes of Peru harbors a hidden taxonomic diversity of Lepidoptera. Here a new leaf-mining species of Gracillariidae (Lepidoptera) is described, Phyllocnistis furcata Vargas & Cerdeña, sp. nov., from a dry Andean valley of southern Peru, at 2400 m above sea level. The morphological aspects of adults (male and female) and the immature stages associated with Baccharis alnifolia Meyen & Walp. (Asteraceae) are given, under optical microscopy and scanning electron microscopy. DNA barcodes show that its nearest neighbor is the Atlantic Forest species Phyllocnistis ourea Brito & Moreira, 2017 that feeds on Baccharis anomala DC. The importance of morphological characters from immature stages for diagnosis among congeneric species is also discussed. Phyllocnistis furcata represents the fourth species of Phyllocnistis Zeller for Peru, and first record from the south of Peru for the genus.

Andes, Arequipa, barcoding, immature stages, Phyllocnistinae

The Andes region of Peru contains hotspots of biodiversity for plants and animals (Myers et al. 2000). It includes global record highs of species richness and endemism rates for many taxa of Lepidoptera, particularly along the eastern slopes of the Andes (Lamas 2003; Pyrcz 2004; Hall 2005; Ignatov et al. 2011; Willmott et al. 2011; Pyrcz et al. 2014; Sublett et al. 2019). On the other hand, the level of knowledge of the lepidopteran fauna on the southwestern slopes of the Peruvian Andes is poor, based on a small number of studies of butterflies (Lamas 1977; Cerdeña et al. 2014; Farfán 2018; Farfán et al. 2020b), and with recent records of moths (Cerdeña et al. 2019; Farfán et al. 2020a), including the first record of a Gracillariidae species for this region (Davis et al. 2020).

Gracillariidae represents one of the most diverse families of micromoths with 1995 recognized species distributed in more than 100 genera (van Nieukerken et al. 2011; De Prins and De Prins 2020). Larvae are predominantly leaf-miners although some species mine stems or fruits (Guillén et al. 2001), and others bore into flowers (Vargas and Landry 2005), fruits (Hu et al. 2011) or stems (Davis et al. 1991), and may also be leaf-rollers or gall inducers (Hanson et al. 2014; Kawakita and Kato 2016; Vargas-Ortiz et al. 2019). In the Neotropics, more intense taxonomic work has been performed in the last decade, resulting in 28 newly described species (De Prins and De Prins 2020). Despite this effort of documenting the fauna, currently numbering 204 Neotropical species, there are gaps in information for gracillariids in several biodiversity hotspots, such as the Andes. Particularly in Peru, currently only 28 species of gracillariids are known (Kawakita et al. 2019; Davis et al. 2020; De Prins and De Prins 2020). From these, one is a non-native species introduced in the coastal area (Castillo and Cornejo 1996), two species were recently reported from southwestern Peru (Vargas 2010; Davis et al. 2020), five were described from northern Peru (Kawakita et al. 2019), and 19 species were described in the early last century by Edward Meyrick (Meyrick 1915, 1921) from material collected by Herbert Simpson Parish from the central Peruvian coast (Lima), central Andes (Matucana, Oroya, Huancayo, Jauja), and northeastern Peruvian Amazon (Iquitos, Yurimaguas) during two collecting trips to the tropics in 1914 and 1920 (Alexander 1916, 1921). These species in particular remain only known from the type specimens, some of which have deteriorated (Brito et al. 2017; De Prins et al. 2019).

Phyllocnistis Zeller, 1848 is a genus of Gracillariidae with 112 named species distributed in all biogeographic regions except Antarctica (Brito et al. 2016, 2017; Fochezato et al. 2018; Kirichenko et al. 2018; De Prins and De Prins 2020). A total of 28 species has been reported for the Neotropical region (De Prins et al. 2019), with 16 species recorded in the last ten years (Kawahara et al. 2009; Davis and Wagner 2011; Brito et al. 2012, 2017, 2019; Fochezato et al. 2018). However, only three species were registered from Peru: Phyllocnistis sciophanta Meyrick, 1915, P. sexangula Meyrick, 1915, and P. citrella Stainton, 1856; the first two species, collected from the center of Peru (Department of Lima) more than 100 years ago, remain with their host plant and immature stages unknown (Brito et al. 2017; De Prins et al. 2019). The third species, with a worldwide distribution, known to be a pest in citrus fruits, is a native from Asia (Castillo and Cornejo 1996; De Prins et al. 2019). This lack of data is mainly due to two conditions that prevailed for a long time, not only in Peru but also in other countries of the Neotropical region (Brito et al. 2016): low collection intensity and scarce taxonomy activity on Gracillariidae and Microlepidoptera in general.

The hypermetamorphic development of the larvae of Phyllocnistis typically comprises two endophytic forms (e.g., Fochezato et al. 2018; Brito et al. 2019). The early, sap-feeding larva actively mines specific host tissues; later, the spinning larva does not feed and has most of the buccal apparatus atrophied, but has a functional spinneret that is used to expel silk to construct the pupal cocoon.

A variety of host plants are associated with Phyllocnistis in the Neotropical region, including 15 genera from 13 different plant families (Brito et al. 2017). Three species are known to be associated with the genus Baccharis (Asteraceae): P. baccharidis Hering, 1958, P. ourea Brito & Moreira, 2017, and an undescribed species associated with Baccharis trimera (Brito et al. 2017), the first from Argentina and other two from Brazil. This Neotropical plant genus is characterized by the tufted indumentum of leaves and stems, and by the unisexual florets generally in separate specimens (Müller 2006), currently comprising 440 species (Heiden et al. 2019).

Recently, as part of an ongoing study on the diversity of microlepidopterans in the Andes in southern Peru, we found a leaf-mining species of Phyllocnistis associated with Baccharis. Comparison at both morphological and molecular levels showed that it does not conform to any known Phyllocnistis species. The morphological description of adults (male and female) and immature stages of this new species is herein given. We also present a preliminary analysis of mitochondrial (COI) DNA sequences including congeneric Neotropical species.

Larvae and pupae found in mines on leaves of Baccharis alnifolia Meyen & Walp. (Asteraceae) in the locality of Characato (16°27'S, 71°28'W), 2400 m, Characato Municipality, Arequipa Department, Peru, were collected and reared in plastic cups, at constant abiotic conditions (20 ± 2 °C, 13:11 h photoperiod) in the laboratory of Area de Entomología, Museo de Historia Natural, Universidad Nacional de San Agustin, Arequipa city, Peru, during September 2018, April 2019, and November 2019.

In total, 58 specimens have been studied: 23 adults, 17 larvae, 20 pupae. Adults that emerged from the mines were pinned and dried, and immature stages were fixed with Dietrich´s fluid and preserved in 70% ethanol. Genitalia were cleared by heating in hot 10% KOH for ~ 15 minutes. They were subsequently stained with Chlorazol black and Eosin, and then slide-mounted with Euparal.

Morphological observations were performed with the aid of a Zeiss Stemi305, and structures selected to be illustrated were photographed with a Nikon SMZ25 stereomicroscope. Vectorized line drawings were then made with the software CorelDraw X4, using the corresponding digitalized images as a guide. The terminology used for descriptions of adult wing pattern, genitalia and immature stages follows Brito et al. (2017, 2019).

For scanning electron microscope analyses, specimens 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 then examined and photographed in a JEOL JSM6060 scanning electron microscope at the Centro de Microscopia Eletrônica (CME) of Federal University of Rio Grande do Sul (UFRGS).

For plant anatomical descriptions, field-collected leaf portions (approx. 0.3 cm²) of B. alnifolia containing mines of P. furcata were fixed in FAA (37% formaldehyde, glacial acetic acid, and 50% ethanol, 1:1:18, v/v) for 24 h. They were then dehydrated in a series of ethanol (40%, 70%, 90%, 96%); embedded in paraffin and sectioned transversely (7 µm) on a rotary microtome. The sections were adhered to a microscope slide glass, then observed and photographed without staining, by using a Nikon SMZ25 stereomicroscope.

Total genomic DNA was extracted from larval tissue (last sap-feeding instar) of five specimens (H86–H90), using the CTAB method (Doyle and Doyle 1987), to support the hypothesis that the morphologically distinct specimens studied confirm a new Phyllocnistis species, and explore the phylogenetic placement among Neotropical congeners. We amplified part of the mitochondrial gene cytochrome oxidase I (COI – 639 bp) using primers and conditions described by Folmer et al. (1994). PCR products were purified using Exonuclease I (GE Healthcare Inc.) and Shrimp Alkaline Phosphatase (SAP), sequenced with forward and reverse primers using a BigDye kit, and analyzed on an ABI3730XL (Applied Biosystems Inc.). Chromatograms obtained from the automatic sequencer were read and sequences were assembled using the software CodonCode Aligner (CodonCode Corporation). The new sequences obtained in this study are publicly available in GenBank and BOLD (DS-GRANEO) databases (Table 1). To explore the phylogenetic position of the new taxon and its specific classification we combined our COI data with a published dataset of ten species and 13 undescribed lineages of Neotropical Phyllocnistis (Davis and Wagner 2011; Brito et al. 2012, 2017, 2019; Lees et al. 2013) (Table 1). This includes P. ourea that feeds on Baccharis anomala, and Phyllocnistis sp. 12 (Brito et al. 2017) associated to Baccharis trimera (Less) DC. Angelabella tecomae Vargas & Parra, 2005 (Oecophyllembiinae) and Marmara arbutiella Busck, [1904] (Marmarinae) were used as outgroups as they represent subfamilies closely related to Phyllocnistinae (Kawahara et al. 2017). A distance tree based on Neighbor-joining (NJ) method was generated from 31 nucleotide sequences using Kimura 2-parameters (K2P) model in MEGA X (Kumar et al. 2018). The evolutionary history was inferred by using the Maximum Parsimony (MP) analysis using the Tree-Bisection-Regrafting (TBR) algorithm with search level 1, with initial trees obtained by random addition of sequences (10 replicates) also in MEGA X. A Maximum Likelihood (ML) analysis was also performed, with the substitution model GTR+G+I according to the Akaike Information Criterion (AIC) estimated by JMODELTEST (Posada 2008), using PHYML 3.0 (Guindon et al. 2010). Initial trees for the heuristic search were obtained automatically with BioNJ algorithm to a matrix of pairwise distances. Monophyly-confidence limits of all analysis were assessed with the bootstrap (BS) method after 1000 bootstrap iterations. Sequence divergences were quantified using K2P model for (i) the genus Phyllocnistis (using 35 named species deposited in BOLD; Table 1), (ii) the Neotropical Phyllocnistis (10 described + 13 undescribed species, Table 1), and (iii) the new species vs. Baccharis-feeding lineages.

Abbreviations for the museum collections and institutions from which specimens were examined are:

LMCI Laboratório de Morfologia e Comportamento de Insetos, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.

MUSA Museo de Historia Natural, Universidad Nacional de San Agustín de Arequipa, Arequipa, Perú.

MUSM Museo de Historia Natural, Universidad Nacional Mayor San Marcos, Lima, Peru.

Phyllocnistis is one of the most species-rich genera of gracillariids, in which a number of taxa were recently described in the Neotropical region, predominantly from tropical and subtropical forests (Davis and Wagner 2011; Brito et al. 2012, 2016, 2017; Lees et al. 2013). Herein we describe an Andean new species, P. furcata, based on morphological and molecular characters that clearly separate it from congeneric species. The COI tree showed a monophyletic status for the new species, and different methods of reconstruction support the inference of its sister relationship with P. ourea among Neotropical lineages.

Overall, adults of P. furcata resemble the majority of Neotropical Phyllocnistis in general aspects of forewing pattern (Brito et al. 2017, 2019; Fochezato et al. 2018); nevertheless, comparing P. furcata with P. ourea and P. baccharidis, all associated with Baccharis as host plants, there are no characters in the wing pattern that group them together; however, they share the presence of a small stout spine at the apex of the valva in the male genitalia. However, a high genetic distance (11.4%) is found between P. furcata and P. ourea, suggesting either an ancient divergence of these sister species or incomplete sampling of Neotropical species, taking into account that much more Phyllocnistis species could be expected associated with the large genus Baccharis (440 sp), and what could also be expected the close relationships among these micromoths.

Phyllocnistis ourea clustered in all COI analysis as the closest related to P. furcata, sharing the same genus of host plant (Baccharis anomala), which could indicate a clade which is associated only with Baccharis. However, the undescribed Phyllocnistis sp. 12 (Brito et al. 2017), which feeds on Baccharis trimera, did not cluster in that group. Moreover, this lineage presents a high genetic divergence (ca. 15%) from P. furcata, near to the mean divergence found of all Phyllocnistis species, suggesting a convergent evolution of host plant use. Such pattern is distinct to described for a species group of Phyllocnistis that feed on Salicaceae, recovered as a single evolutionary clade in a COI tree (Kirichenko et al. 2018). Further inclusion of P. baccharidis, and eventually more species/lineages (currently unknown) associated with Baccharis, together with a multi-locus approach, will allow one to make more robust phylogenetic inferences and shed light on the diversification of Neotropical Phyllocnistis related to its host plants.

Interestingly, males of P. furcata possess two pairs of coremata on abdominal segment VIII. Only one pair, formed by long, slender, flattened scales is found generally in species of Phyllocnistis in which the coremata are described (e.g., Kawahara et al. 2009; Brito et al. 2012, 2019; Kirichenko et al. 2018). Phyllocnistis furcata presents one pair similar to those in other congeneric species, and a second pair formed by wide rounded flat scales, which may represent the first report of this type of structure for the genus. Furthermore, another interesting aspect is observed in females of P. furcata, where one of the three signa of the corpus bursae is prominent (~0.5 × length of corpus bursae) with a particular fork-shaped appearance not observed in any other species of Phyllocnistis. This partially resembles the only signum of P. tropaeolicola Kawahara, Nishida & Davis, 2009, which has the form of a narrow band with two spines projecting inwards (Kawahara et al. 2009), but the spines are more prominent in P. furcata. Phyllocnistis drimiphaga Kawahara, Nishida & Davis, 2009 is another species with one of its two signa of large size, but five short spines arise from this signum. The remaining species of this genus mostly contain a small pair of fusiform signa or a single signum that occupies less than 2/3 of corpus bursae (Kirichenko et al. 2018; De Prins et al. 2019). Also, there is no information about the variability of coloration pattern between females and males in other species of Phyllocnistis, notably that males are slightly darker than females in P. furcata. This has not been observed in other species of the genus, and should be reviewed in detail in the future.

The knowledge of immature stages of Phyllocnistis is also insufficient. In the majority of the species of the genus whose pupal morphology is described, pupae are characterized by a well-developed cocoon cutter and some abdominal terga (generally A2–A7) with a pair of prominent laterally curved spines and many small spines medially (Kobayashi and Hirowatari 2011; Brito et al. 2012, 2019; Fochezato et al. 2018; Kirichenko et al. 2018; Liu et al. 2018). However, a few species do not match this pattern perfectly. For instance, P. subpersea Davis & Wagner, 2011 has the cocoon cutter in the form of a pair of stout conical processes with a strongly recurved subapical spine (Davis and Wagner 2011). Curved spines of the abdominal terga are absent in two Neotropical species (Brito et al. 2012, 2019) and are present only on two segments of P. citrella (Kobayashi et al. 2013). The pupa of P. furcata matches well the general pattern of the genus. In addition, the prominent curved spines are present on A1–A7, a condition also found in P. ourea, whose larvae also feed on Baccharis (Brito et al. 2019). The apex of the lateral setae of A6 and A7 is clavate in the pupae of these two Baccharis-feeding Phyllocnistis. However, this condition is also found in other species whose larvae are associated with other plant families (Kawahara et al. 2009; Davis and Wagner 2011; Brito et al. 2019). In the spinning larva, a single ambulatory callus placed ventrally at the center of the meso- and metathorax is found in P. ourea (Brito et al. 2019), P. furcata, and also in the Thymelaeaceae-feeding P. hemera Brito & Fochezato, 2018, while lateral sensilla on abdominal segments are found in P. hemera (Fochezato et al. 2018) and P. furcata. In the sap-feeding larva, the presence of two stemmata, like in P. furcata, has been described for P. ourea and the Winteraceae-feeding P. selene Brito & Moreira, 2017 (Brito et al. 2019). Certainly, the external morphology of the immature stages should be explored in additional species of Phyllocnistis to have a more realistic perspective of the actual variation and its relationship with ecology and evolution.

The few studies in which the damage pattern caused by leaf miner larvae of Gracillariidae has been characterized using histological sections, suggest that its feeding activity can either be restricted to specific tissues throughout the leaf miner stage or the consumed tissues can change with larval ontogeny (Brito et al. 2012; Body et al. 2015; Moreira et al. 2018; Pereira et al. 2019; Vargas-Ortiz et al. 2019). In the case of Phyllocnistis, the larvae of P. citrella feed only in the epidermis of Citrus (Rutaceae) (Achor et al. 1997), and those of P. tethys Moreira & Vargas, 2012 on the spongy parenchyma of Passiflora organensis Gardn. (Passifloraceae) (Brito et al. 2012). Furthermore, larvae of P. hemera feed initially on the epidermis and later on the palisade parenchyma of Daphnopsis fasciculata (Meisn) Neveling (Thymelaeaceae) (Fochezato et al. 2018). The feeding behavior of larvae of P. furcata was found to be restricted to the palisade parenchyma of B. alnifolia.

The mines of P. furcata are found on adaxial and abaxial surfaces of the leaf. In contrast, those of P. ourea, another Baccharis-feeding Phyllocnistis, are restricted to the adaxial surface of the leaf of its host B. anomala (Brito et al. 2019). Despite histological descriptions of leaves of B. anomala mined by P. ourea not being available, it appears that the remarkable difference between the distribution pattern of the mines of the two species in the leaves of their hosts could be due, at least in part, to differences in leaf anatomy of the two hosts and the ability of the larvae to feed on palisade parenchyma. The organization of the mesophyll in B. alnifolia is isobilateral, with two or three layers of palisade parenchyma in each side and one or two layers of spongy parenchyma in the middle, a pattern reported for several species of Baccharis (Budel et al. 2018; Ornellas et al. 2019), while the organization of the mesophyll of B. anomala is dorsoventral, with two or three layers of palisade parenchyma and approximately three layers of spongy parenchyma (Budel and Duarte 2008). Given this diversity in leaf tissue structure, additional studies are needed to better understand the ecology and evolution of herbivory in Phyllocnistis. Certainly, histological descriptions of leaves mined by additional species of this genus will be helpful to propose hypotheses.

The known distribution of endemic species of Phyllocnistis in the western slopes of Peruvian Andes was previously restricted to the type localities of the two species described at the beginning of the last century by Edward Meyrick (1915, 1921) around the Department of Lima, in central Peru. Thus, the discovery of P. furcata in the Arequipa Department, as a result of recent evaluations of the Microlepidoptera fauna, provides the first record of this genus from southwestern Peru, at 2400 m, where the vegetation is dominated by xeric shrublands with abundant cacti (Montesinos et al. 2012, Heim 2014). However, as already mentioned, the taxonomic diversity of gracillariid species remain poorly studied in Peru, due to the absence of local specialists and collections of micromoths in general. As discussed in Brito et al. (2016), there exists a “taxonomic impediment” for the progress of studies on Neotropical gracillariids in general. Therefore, regional revisions of micromoth faunas would represent an important advance to the knowledge of this diverse group in Peru (e.g., Davis et al. 2020), particularly in areas that are subject to the highest rates of anthropic environmental degradation, like the environments of the southern Andes of Peru.

Thanks are due to staff members of Centro de Microscopia Eletrônica (UFRGS) for assistance and use of scanning electron microscopy facilities. RB was supported by fellowships from CNPq. GRPM was supported by a CNPq grant. JC and JF were supported, respectively by CNPq and CAPES scholarships. AL was supported by a project of the Universidad Nacional de San Agustin de Arequipa (UNSA) with Contract reference IBAIB-01-2019-UNSA. We acknowledge the Subject Editor, Erik J. van Nieukerken, for his reading and corrections. Our thanks also go to the reviewers of the document for their valuable comments.