A new Brazilian Passiflora leafminer: Spinivalva gaucha, gen. n., sp. n. (Lepidoptera, Gracillariidae, Gracillariinae), the first gracillariid without a sap-feeding instar

Abstract Male, female, pupa, larva and egg of a new genus and species of Gracillariidae (Gracillariinae), Spinivalva gaucha Moreira and Vargas from southern Brazil are described and illustrated with the aid of optical and scanning electron microscopy. A preliminary analysis of mitochondrial DNA sequences including members of related lineages is also provided. The immature stages are associated with Passiflora actinia, Passiflora misera and Passiflora suberosa (Passifloraceae), and build mines on the adaxial leaf surface. Initially the mines are serpentine in shape, but later in larval ontogeny become a blotch type. Although the larvae are hypermetamorphic as in other Gracillariidae, there is no sap-feeding instar in Spinivalva gaucha; the larva feeds on the palisade parenchyma, thus producing granular frass during all instars. Pupation occurs outside the mine; prior to pupating, the larva excretes numerous bubbles that are placed in rows on the lateral margins of the cocoon external surface. This is the second genus of gracillariid moth described for the Atlantic Rain Forest, and the second gracillariid species known to be associated with Passifloraceae.

listed 14,552 species of vascular plants for the entire Atlantic Rain Forest, of which 6,933 (49%) are endemic. Considering the wide range of host plants used and the high level of host specificity usually found for the leaf-mining gracillariids in general (Davis 1987), it seems reasonable to predict that hundreds of gracillariid species await description in this understudied, species-rich biome, to which probably most of them are also endemic.
In the course of an ongoing survey on the diversity of microlepidopterans in the Atlantic Rain Forest in southern Brazil, we recently found a leaf-mining gracillariid associated with Passifloraceae. A search of the literature indicated that this taxon is distinct from other described genera of Gracillariidae, and therefore a new genus is proposed herein. We describe and illustrate all the life stages of this new species, and provide a preliminary characterization of its life history, including histological aspects of the leaf mine. We also present a preliminary analysis of mitochondrial DNA sequences, including members of related genera.

Materials and methods
Specimens used in the study were reared in small plastic vials under controlled abiotic conditions (14 h light / 10 h dark; 25 ± 2 °C) in the Laboratório de Morfologia e Comportamento de Insetos, Departamento de Zoologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre city, Rio Grande do Sul State (RS), Brazil, from May 2011 through December 2012. They came from field-collected leaves with eggs, mines with feeding larvae inside, and pupae on plant shoots of Passiflora actinia Hook. (São Francisco de Paula municipality, RS), P. misera Kunth and P. suberosa L. (Porto Alegre municipality, RS).
Immature stages were fixed in Dietrich´s fluid and preserved in 75% ethanol. For descriptions of the gross morphology, the specimens were cleared in a 10% potassium hydroxide (KOH) solution and slide-mounted in either glycerin jelly or Canada balsam. Observations were performed with the aid of a Leica® M125 stereomicroscope. Measurements were performed with the aid of an ocular micrometer (precision = 0.01mm). Structures selected to be drawn were previously photographed with a Sony ® Cyber-shot DSC-H10 digital camera mounted on the stereomicroscope. Vectorized line drawings were then made with the software Corel Photo-Paint ® X3, using the corresponding digitalized images as a guide. At least five specimens were used for the descriptions of each life stage or instar.
For scanning electron microscope analyses, additional 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 examined and photographed in a JEOL® JSM5800 scanning electron microscope at the Centro de Microscopia Eletrônica (CME) of UFRGS.
Descriptions of plant anatomy were based on diaphanized, field-collected leafmines (n =5) from P. actinia shoots that were fixed in FAA (37% formaldehyde, glacial acetic acid, and 50% ethanol, 1:1:18, v/v), stained with rose bengal (aqueous solution: 200 mg/1) and mounted either whole or in freehand section in glycerin on slides, following a procedure described in detail by Brito et al. (2012).

Molecular analysis
High-quality DNA was purified from larval tissue using the organic method of Cetyl Trimethyl Ammonium Bromide (CTAB) to investigate (i) levels of genetic variation within Spinivalva specimens collected in different localities and from different host plants (Passiflora misera, P. suberosa and P. actinia) and (ii) reconstruct phylogenetic relationships of this new genus among and within the Parectopa group of gracillariids. A total of nine field-collected specimens from three populations: 1) Porto Alegre, RS, from P. suberosa and P. misera (Pop. 1); 2) São Francisco de Paula, RS, from P. actinia (Pop. 2) and 3) Curitiba, PR, also from P. actinia (Pop. 3). They were used to amplify 1.5 kb of mitochondrial genes cytochrome c oxidase subunit I (CO-I), transfer RNA (tRNA-Leu), and cytochrome c oxidase subunit II (CO-II). For the PCR amplification we used the primer pairs Jerry + Pat II for the first segment (700 bp), and Patrick + Eva for the second (800 bp), following the procedure described by Caterino and Sperling (1999). Additionally, we amplified genetic material from three specimens of Spinivalva, using the universal barcode primers LCO1490 (5'-ggtcaacaaatcataaagatattgg-3') and HCO2198 (5'-taaacttcagggtgaccaaaaaatca-3'), following the procedure of Folmer et al. (1994). We obtained variants that exactly matched the region previously sequenced in 6 representative taxa of the Parectopa group of gracillariids, downloaded from GenBank and incorporated into our analysis (Table 1). The remaining PCR products were treated with exonuclease I and shrimp alkaline phosphatase (ExoSAP) (Fermentas Inc.), sequenced using the BigDye sequencing kit and analyzed in an ABI 3730XL DNA Analyzer (Applied Biosystems Inc.). Sequences were aligned and visually inspected using the algorithm Clustal X in MEGA 5 (Tamura et al. 2011) running in full mode with no manual adjustment. The dataset of 1.5 kb generated for specimens of Spinivalva from three different localities was deposited in GenBank and BOLD, under the accession numbers KC512114 to 512123 and GRABR001-13 to 010-13, respectively. The phylogenetic tree was reconstructed based on Bayesian inference and implemented in BEAST 2.0 (Drummond et al. 2012) to recover (i) the evolutionary distance within Spinivalva taxa from different localities and host plants, and (ii) relationships of Spinivalva among the lineages of gracillariids surveyed in this study. In both trees, the HKY85 model of sequence evolution (Hasegawa et al. 1985) was used with empirical base frequencies and 4 gamma categories. A relaxed uncorrelated log-normal clock was used, with no fixed mean substitution rate and a Yule prior on branching rates. We used four independent runs of 10 million generations, with the first 500,000 of each run discarded as burn-in. Posterior probabilities were used as an estimate of branch support. The species-level tree was unrooted, while the genus-level was rooted with a species of Bucculatricidae (Bucculatrix ulmella Zeller, 1848).

Museum collections
Abbreviations of the institutions from which specimens were examined are: extension of valva abruptly narrowing distally, forming a single, medially bent process bearing a stout sensillum at the apex; 2) aedeagus tubular, slender, straight and long, ending as a sharply pointed spine; 3) saccus with anterior process long and tubular; 4) two pairs of coremata, each with two unit types that are formed by an external hair pencil and a tubular, membranous, corrugated pouch. In the female genitalia, the circular ostium bursae is located near the anterior margin of sternum VII, having a membranous corpus bursae associated with an accessory bursa, with no signum. The larvae construct mines on the adaxial surface of passion-vine leaves; initially the mines are serpentine in shape but later in ontogeny become a blotch type. Unlike all known stages of other leaf-miner gracillariids, S. gaucha has no larval sap-feeding instars; all instars of its larvae have a conspicuous spinneret and mandibles of the chewing type, and feed on the palisade parenchyma after hatching. Pupation occurs outside the mine; the larva excretes numerous bubbles that are aligned on the lateral margins of the cocoon surface prior to pupation. Description. Adult (Figs 1-4). Male and female similar in size and color. Small moth, forewing length 2.78-3.61 mm (n = 5). Head ( Fig. 2A): Vestiture moderately smooth, with a large, light-gray dorsal scale tuft that curves forward to the frons; scales slender, with apices slightly rounded. Eye relatively large, rounded, with dorsal margin slightly concave; vertical diameter ~ double minimum interocular distance across frons (n = 6). Antenna filiform, long, exceeding length of forewing; scape slightly elongate, ~ 2.4× length of pedicel; flagellomeres completely encircled by single, dense row of slender scales. Labrum trilobed, pilifers well developed, triangular. Mandible absent. Haustellum naked, elongate, ~ 2.0× length of labial palpus. Maxillary palpus short, smoothly scaled, 4-segmented; ratios of segments from base: ~1.0 : 2.2 : 3.6 : 3.5. Labial palpus smoothly scaled, moderately long, bent anteriorly and upward; ratio of segments from base: ~1.0 : 4.6 : 0.3. Thorax: Forewing (Fig. 2B) lanceolate, with 12 veins, all arising separately from the cell and reaching the margin; L/W index ~ 7.3; retinaculum consisting of few subcostal, narrow, flat, longer, loosely coiled scales (Fig. 2C); discal cell ~ 0.8× length of forewing (n = 4) ending near distal fifth of wing margin; R5-branched; R1 ending near proximal third of wing margin; M3-branched, CuA not branched, and faded basally; CuP weak proximally and not stalked, with 1A+2A that is well developed, extending past midlength of posterior margin. Hindwing (Fig. 2B) extremely lanceolate, L/W index ~ 9.6, ~ 1/8 forewing in length; male frenulum ( Fig. 2D) a single stout bristle; female with frenulum divided at base, then fused for nearly its entire length and appearing as a single stout bristle; pseudofrenulum consisting of ~8 modified scales arising in two to three irregular rows near Sc+R 1 ending at circa 1/5 anterior margin; Rs faded proximally, ending at circa 1/3 anterior margin; M and CuA unbranched, both faded proximally and weak distally, ending at circa 1/3 and 2/3 of posterior margin. Legs with tibial spur pattern 0-2-4; epiphysis present. Tibial length ratios (anterior / middle / posterior legs) ~ 0.55/0.85/1.0. Abdomen: Male with segments VII-VIII complex and reduced, except for enlarged tergum VIII; segment VII reduced to narrow, almost completely sclerotized ring; tergum VIII elongate, hoodlike, partly covering tegumen; sternum VII bearing two pairs of coremata, arising from distal apex of rodlike sclerites that protrude from intersegmentary membrane VII-VIII; each coremata ( Fig. 3D) bearing two types of units -an external hair pencil ( ~ valva in length) and a tubular, membranous, corrugated pouch; pouches of anterior pair ~ ½ hair pencil in length; those Male genitalia (Figs 3A-C, 4A, B, D, E). Uncus absent. Tegumen broad, hoodshaped, mostly membranous, with shallow apical notch. Pair of long, distally narrower, membranous lobes arising ventrally beneath tegumen. Vinculum long, broadly Vshaped, extending laterally along base of valva. Saccus well developed, U-shaped; anterior process long and tubiform, ~1/2 length of valva, apex slightly capitate. Transtilla an arched, sclerotized plate joining bases of valvae. Juxta small, a dorsally concave, membranous plate, attached to middle of aedeagus. Aedeagus (Figs 3B, 4E) tubiform, slender, straight and long (~2× valve length), slightly dilated caudally, with subapical, dorsally located concave aperture and ending as sharply pointed spine; entry of ductus ejaculatorius located at anterior end; vesica without cornuti. Valva (Figs 3C, 4A, B, D) broad at base, and deeply divided; costal margin relatively straight and distally rounded; cucullus densely covered by long piliform setae; sacculus with broad lobe abruptly narrowing distally, ending as a medially bent process with apex bearing a stout, blunt sensillum.
Etymology. The genus name is derived from the Latin spina (spine) and valva (valve), in reference to the conspicuous spine-like process present on the male valvae. Gender feminine. Diagnosis. As discussed for the genus.
Description. Adult (Fig. 1). Head. Frons light gray; vertex covered mostly by white scaled tuft that curves forward to the frons. Antennae mostly dark gray; scape white ventrally with pecten of light-gray hairlike scales; pedicel and flagellum ventrally whitish gray. Maxillary and labial palpi mostly white, with scattered dark-gray scales laterally. Thorax. Forewing mostly covered by dark-gray scales. Narrow stripe of white scales along posterior margin; a zigzag edge, formed by short, oblique white fascia, separates this stripe from the remaining, mostly dark-gray area; distal portion of apical fascia bearing brownish scales. Apical portion with transverse bar of lightgray scales that separates distally two well-defined dots, one dark gray (toward anterior margin) and one white (toward posterior margin). Fringe with scales of two sizes, mostly white at base and dark gray apically. Hindwing completely covered by darkgray scales and with concolorous fringe. Forelegs mostly dark gray, with some white scales basally and apically on each podite, particularly on coxa. Midlegs mostly white with scattered light-brown scales, and transverse dark-gray stripes on femur, tibia, tibial spurs and tarsomeres. Hindlegs similar to midlegs, but with hair-like scales on tibia. Abdomen. Mostly dark gray, with transverse, V-shaped white stripes on ventral surface of segments III-VI.
Immature stages. Egg (Fig. 10C). Flat, ellipsoid, laid on the abaxial surface, usually close to the leaf veins; chorion translucent, larva visible through transparent area of leaf before eclosion; chorionic ultrastructure, aeropyles and micropylar area not observed. Larva (Figs 5A, B, 6, 7, 8, 10F, H). Head brown, thorax and abdomen yellowish. Leaf-miner, with hypermetamorphic development and five instars, all endophyllous, prognathous and tissue feeders; that is, there is no sap-feeder instar, and all larvae have a typical spinneret and functional mandibles of the chewing type. Instars change gradually in external morphology during ontogeny, and can be identified through measurements of the head capsule, since there is no overlap between the head-capsule size of succeeding instars ( Table 2). The following exponential growth equation was adjusted for the head-capsule width from larvae reared on Passiflora actinia: y = 0.078e^0.420x; n = 25; r = 0.99; p < 0.0001. Preliminary observations suggested that the number of instars may vary from four to five as a function of the host plant, which should be further explored.
Host plants. Passifloraceae: Passiflora actinia Hook, P. misera Kunth and P. suberosa L. The former, where S. gaucha was most frequently collected, is found primarily in forest edges in the coastal mountains of southern Brazil, where it is endemic, distributed from the Brazilian states of Espírito Santo to Rio Grande do Sul. Passiflora suberosa and P. misera have broader distributions, extending to Central America, and also occur in relatively open areas occupied by shrubs and herbaceous vegetation. Details about the biology and distribution of these passion-vine species in southern Brazil were provided by Mondin et al. (2011) and Moreira et al. (2011), respectively.
Distribution. Spinivalva gaucha is known from the type locality (Condomínio Alpes de São Francisco) and the Floresta Nacional de São Francisco de Paula, both located in São Francisco de Paula Municipality, where P. actinia plants are used as larval host plants. A few scattered specimens were also collected from an additional population located in Porto Alegre Municipality. Both municipalities are located in Rio showing frass and damage to leaf parenchyma left by last-instar larva within the mine F fourth-instar larva in the mine G exit hole (arrow) used by a last-instar larva to leave the mine h last-instar larva after changing color, building the cocoon outside the mine on the leaf surface i cocoon, with pupa visible through transparency J pupa in detail, after removing the cocoon K pupal exuvium protruding from the cocoon exit hole onto plastic substrate, just after the adult emergence. Scale bars = 20, 10, 0.2, 1.5, 1, 0.5, 0.5, 2, 2, 1, 2 mm, respectively. Grande do Sul (RS), Brazil. In the Porto Alegre population, P. misera and P. suberosa are used as hosts. We could not find conspicuous morphological differences among the specimens collected in RS, as also corroborated by the DNA analyses. Additional Spinivalva specimens were collected farther north in Curitiba Municipality, Paraná, also mining P. actinia leaves. However, as discussed below, analyses of the molecular data suggested that this population may correspond to a new species, a possibility that should be further explored. All these populations are located within the Atlantic Rain Forest domain sensu lato (Morellato and Haddad 2000).
Life history (Figs 9A-C, 10A-E, G-I, K, 11). Eggs of S. gaucha are deposited on the abaxial leaf surface, adhered by a cement substance, close to the midrib or secondary veins (Fig. 10C). Hatching occurs through the surface of the egg adhered to the leaf; the first-instar larva moves directly into the leaf lamina, easily reaching the adaxial side of the leaf (Fig. 10D). Initially, the mine is narrow, slightly serpentine in shape, increasing in width progressively during ontogeny and becoming a blotch during the last larval instar. The larva feeds on the palisade parenchyma from the beginning to the end of the mine (Figs 10B, 11). Dark-green granular frass pellets of increasing size are found in the larva's feeding path (Figs 10D, E), as are the head-capsule exuviae.
After the fifth-instar larva leaves the mine through a slit made in the blotch section (Fig. 10G) and prior to pupal moulting, it spins the pupal cocoon, usually on the adaxial leaf surface of adjacent leaves. The pupal cocoon is exophyllous, elliptical in general outline, transparent, from 7.76 to 8.74 mm long in the specimens examined. Silk filaments are woven in a tight pattern, forming a compact, flat wall that covers the pupa (Figs 9A, 10I). The cocoon periphery is adorned with several irregularly spaced, minute, light-yellow bubbles (Fig. 10I). These are not compartmentalized, showing a finely granular structured surface (Fig. 9C). They are discharged from the anus by the mature larvae to the outside through a slit made with the mandibles in the cocoon wall, which is closed soon after deposition (Fig. 9B). Throughout this process, the bubbles are manipulated by the spinneret. During adult emergence, one end of the pupal cocoon is split by the frontal process of the pupa (cocoon cutter). Generally after the adult emerges, the anterior half of the pupal exuvium (head and thorax) protrudes outside, while the posterior half remains in the pupal cocoon (Fig. 10K).
At the type locality, S. gaucha mines occur at low numbers on P. actinia plants (Fig. 10A). In most cases, only one mine was found per leaf, but up to three were collected in a single leaf, and several leaves may be used per plant. There was no indication that this behavior differed from that of the other passion-vine species and populations studied. We could not find a clear pattern for the number of generations per year and the flight period.
Molecular phylogeny (Fig. 12). A total of 1583 nucleotide sites were analyzed for Spinivalva from different localities and host plants; 110 (7%) were variable. An unrooted Bayesian tree recovered two major groups (Fig. 12A). The first included specimens from Porto Alegre (Pop. 1), hosted on either P. suberosa or P. misera, together with those from São Francisco de Paula / Condomínio Alpes de São Francisco (Pop. 2) hosted on P. actinia. The second group included individuals from Curitiba (Pop. 3) sampled on   Kawahara et al. 2011), based on 610 bp of the barcoding region (cytochrome oxidase c subunit I gene). Numbers above branch indicate node support (posterior probability); those located below represent the raw branch length. A species of Bucculatricidae (Bucculatrix ulmella) was used to root the tree. P. actinia. The genetic divergence between these major groups was 7% (Fig. 12A). The intraspecific difference between localities in the first group was 1%. In addition, the barcode fragment analyzed recovered 610 nucleotides, including 236 (38%) variable sites. According to our phylogenetic hypothesis, Spinivalva was strongly supported as a monophyletic lineage within the Parectopa group of gracillariids (Fig. 12B). Despite the strong statistical support within this lineage of gracillariids, the internal relationships for the genera included in the Parectopa group were poorly resolved. Leurocephala schinusae and Liocrobyla desmodiella were placed as closest to Spinivalva (showing lower genetic divergence), but with weak posterior probability of node support.

Discussion
The following characteristics suggest that Spinivalva gen. nov. belongs to the subfamily Gracillariinae (sensu : 1) flat, scaled head; 2) maxillary palpi with four segments; 3) male abdomen bearing two coremata; 4) pupation occurring outside the mine; 5) adults resting with the anterior portion of the body inclined circa 45°. Our molecular analysis placed it within the Parectopa group (sensu Kawahara et al. 2011) in the Gracillariinae, near the genera Leurocephala Davis and McKay and Liocrobyla Meyr. From a morphological perspective, the forewing of adults of Spinivalva resembles those in the Parectopa group in general coloration, fascia arrangement, presence of apical dot, and venation pattern (Vári 1961). When compared to Leurocephala, a recently described genus also found in the Atlantic Rain Forest of Brazil , additional similarities are found in the males, in particular regarding the reduced segment VII that bears two pairs of coremata, the elongated tergum VIII, and the presence of paired gnathal lobes. However, as noted above, males of Spinivalva differ markedly from those of Leurocephala and the remaining genera of the Parectopa group in relation to the valva. Unlike them, it has a saccular extension with a conspicuous process bearing a stout sensillum, in association with an aedeagus that is tubular, long and slender, and a saccus with the anterior process long and tubular. These differences extend to additional lineages related to the Parectopa group that were not included in our molecular analysis, for example Micrurapteryx Spuler, 1910, Neurobathra Ely, 1918(Kawahara et al. 2011), and Chileoptilia Vargas and Landry, 2005. data), and other genera described by Vári (1961). As far as we are aware, the existence of a saccular tubiform portion associated with the hair pencils in the coremata of Spinivalva has not been reported within Gracillariidae. However, detailed descriptions for coremata structures are rarely provided in the gracillariid literature, and thus one should use caution regarding the validity of this apomorphy. Bubbles adorning the pupal cocoon similar to those described here for S. gaucha have been found not only in other phylogenetically related genera such as Conopomorpha Meyrick, 1885, Epicephala Meyrick, 1880and Leurocephala Davis & Mc Kay, 2011, but also in other lineages that are not closely related to the Parectopa group (e.g., Wagner et al. 2000, Hu et al. 2011). Wagner et al. (2000 speculated they provide a physical barrier, thus protecting the pupa against the attack of parasitoids and predators. The existence of at least one "sap feeding" instar early in larval ontogeny has been considered a synapomorphy for all Gracillariidae (Kumata 1978, Davis 1987). However, our data showed clearly that this is not the case for S. gaucha, where all instars are of the "tissue feeding" type. That is, although the larvae are hypermetamorphic (early instars apodal and without stemmata, later instars with legs) as in other Gracillariidae, there is no sap-feeding in S. gaucha. Early-instar larvae also have mandibles of a chewing type combined with a well-developed spinneret, and with the remaining mouth parts differentiated; and after they hatch, these larvae feed on the palisade parenchyma. Palisade cells typically have well-developed, compact walls compared to those in the spongy parenchyma. The morphological characteristics in S. gaucha are associated with feeding on tough tissues after hatching, contrary to other gracillariid species that have sap-feeder early instars that feed by dilacerating either the leaf epidermis layers or the spongy parenchyma (e.g., Kumata 1978, Wagner et al. 2000, Brito et al. 2012. The absence of a sap-feeding instar was suggested for the life cycle of Chileoptilia yaroella Vargas & Landry, 2005, although the first instar was not described by the authors at that time. Additional studies using scanning electron microscopy recently conducted by two of us (Vargas and Moreira, unpublished data) confirmed this prediction; in this case, however, the first instar is not a leaf miner, but feeds on the tiny gynoecia within the calyx of flowers of Acacia macracantha Willd. (Mimosaceae) in northern Chile. These discoveries will certainly have important implications for future studies concerning the evolution of the wide diversity in feeding habits known to exist within Gracillariidae.
We found no conspicuous morphological differences at any life stage among populations of Spinivalva occurring in Rio Grande do Sul, independently of the host plant. These observations were corroborated by the molecular data, which showed a low divergence rate among the different populations. Consequently, we consider all these specimens to be conspecific; that is, a set of variations exists within S. gaucha species boundaries and among the host plants used. However, comparison of these specimens from Rio Grande do Sul with those collected from Curitiba revealed a greater divergence in mitochondrial DNA sequences. We did not study genitalia morphologies of the latter, or their immature stages, and so a decision about their taxonomic status awaits further investigation. Thus, specific diversity within the genus Spinivalva might be higher than described here. As discussed for the flora in general, many passion-vine species occur in the Atlantic Rain Forest, and several of them are endemic to this biome (Stehmann et al. 2009). In the future, they should be searched for the presence of this and other lineages of gracillariids. Another gracillariid species, Phyllocnistis tethys Moreira & Vargas, 2012, has been associated with a different passion-vine species in southern Brazil (Brito et al. 2012). However, Phyllocnistis larvae use a wide range of plant families as hosts (Kawahara et al. 2009). Therefore, Spinivalva is the first genus that is known to be particularly associated with the Passifloraceae. Passion vines are toxic to most lepidopterans, and the biological implications, if any, for such a peculiar association in herbivory also remain unknown (Brito et al. 2012).