Spinivalva gaucha Moreira & Vargas

Brito, Rosangela, Goncalves, Gislene L., Vargas, Hector A. & Moreira, Gilson R. P., 2013, A new Brazilian Passiflora leafminer: Spinivalva gaucha, gen. n., sp. n. (Lepidoptera, Gracillariidae, Gracillariinae), the first gracillariid without a sap-feeding instar, ZooKeys 291, pp. 1-26 : 9-15

Spinivalva gaucha Moreira & Vargas sp. n. Figs 111

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 light-gray 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 dark-gray 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.

Male genitalia (Figs 3 A–D; 4A, B, D, E). As described for genus.

Female genitalia (Figs 3E; 4C, F). As described for genus.

Type material.

BRAZIL: Condomínio Alpes de São Francisco, 29°27'9.2"S, 50°37'6.6"W, São Francisco de Paula Municipality, Rio Grande do Sul State (RS), Brazil. All preserved dried and pinned, reared by the senior author from larvae and pupae collected on Passiflora actinia Hook. ( Passifloraceae ): LMCI 186, 26.V.2012, by G.R.P. Moreira, H.O. Vargas and S. Bordignon; LMCI 199, 19.XII.2012 by G.R.P. Moreira, R. Brito and F.A. Luz. HOLOTYPE: ♂ (LMCI 199-01), donated to DZUP (24.976). PARATYPES: 1♀ (LMCI 199-02), donated to DZUP (24.986); 1♂, 1♀ (LMCI 199-03 and 186-12), donated to MCNZ (81900 and 81903, respectively); 1♂, 1♀ (LMCI 199-04 and 186-15), donated to MCTP (31442 and 31443, respectively).

Other specimens examined.

LMCI 156: Floresta Nacional de São Francisco de Paula, 29°25'21.4"S, 50°23'26.6"W, São Francisco de Paula Municipality, RS, Brazil, collected by K.R. Barão, 13-15.V.2011, on Passiflora actinia . LMCI 157: Condomínio Alpes de São Francisco, 29°27'9.2"S, 50°37'6.6"W, São Francisco de Paula Municipality, RS, Brazil, collected by G.R.P. Moreira, R. Brito and G.L. Gonçalves, 28.V.2011, on Passiflora actinia . LMCI 164: Campus da Vale, Universidade Federal do Rio Grande do Sul (UFRGS), 30°04'12.9"S, 51°07'11.5"W, Porto Alegre Municipality, RS, Brazil, collected by R. Brito, on Passiflora misera Kunth and Passiflora suberosa L. ( Passifloraceae ).

LMCI 169: Centro Politécnico da Universidade Federal do Paraná, 25°26'44.1"S, 49°13'56.8"W, Curitiba Municipality, Paraná State, Brazil; 5 larvae dissected from mines collected by G.R.P. Moreira, on Passiflora actinia ; used for DNA extraction only. Adults, dried and pinned, with the same collection data, deposited in LMCI: 4♂♂ (LMCI 156-9, 164-6, 7, 10); 1♀, (LMCI 164-9). Genitalia preparations, mounted in Canada balsam on slides, with the same collection data, deposited in LMCI: 4♂♂ (GRPM 50-11, 13, 21, 22); 4♀♀ (GRPM 50-12, 23, 32, 34). Immature stages, fixed in Dietrich’s fluid and preserved in 70% ethanol, with the same collection data series, deposited in LMCI: 2 eggs (LMCI 157-2), 4 first-instar larvae (LMCI 157-8), 5 third-instar larvae (LCMI 157-4), 6 fifth-instar larvae (LMCI 157-10), and 9 pupae (LMCI 157-5, 6). Mature leaf mines (n = 5) containing larval exuvia, mounted in glycerin on slides and stained with rose bengal, with the same collection data, deposited in LMCI, under accession numbers LMCI 186-3, 7 and LMCI 199-5, 6, 7.

Etymology.

The specific name is derived from the Portuguese “Gaúcho”, a term commonly used for natives of Rio Grande do Sul, the southernmost state of Brazil, where this new species was first found.

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.

First instar (Figs 5A, 6 A–F). Body depressed, without setae, legs or pseudopodia. Antennae (Fig. 6C) reduced, one-segmented, nearly flush with head capsule, with four short sensilla. Stemmata absent. Labrum (Fig. 5A) moderately bilobed, with slight central notch. Mandibles (Fig. 6B) of chewing type, with three blunt teeth. Maxilla (Fig. 6E) rudimentary, uni-segmented, with two finger-like, terminal lobes. Labium relatively broad, with well-developed tubular spinneret having flared terminal opening (Fig. 6D). Hypopharynx bearing few papilliform projections basally (Fig. 6D). Labial palpi (Fig. 6F) vestigial, reduced to pair of closely appressed, slender setae.

Third instar (Figs 7 A–F). Similar to first instar, but with body partly depressed and setae greatly reduced. Prothoracic shield slightly differentiated (Fig. 7F), nearly colorless; legs and pseudopodia absent. Antennae (Fig. 7E) bi-segmented, first segment stouter than second segment, each bearing three sensilla. Stemmata absent. Labrum (Fig. 7B) bilobed, with pronounced median notch, and several ventral, posteriorly bent papilliform projections. Mandibles of chewing type, with three teeth. Maxilla well developed, as shown in Fig. 7C. Labium broad, with spinneret similar to that of first instar, but shorter and stouter (Fig. 7C). Labial palpi (Fig. 7C) short, bi-segmented, each bearing apical sensillum. Hypopharynx with several papilliform projections basally (Fig. 7C).

Fifth instar (Figs 5B, 8 A–L, 10F, H). Body subcylindrical, covered by microtrichia and with setae well developed; thoracic legs reduced; pseudopodia present on A3-5, A10. Head and prothoracic shield brown (Fig. 10F); remaining parts of body yellowish (Fig. 10F), changing to red before pupation (Fig. 10H). Maximum length of larvae examined 5.52 mm. Antennae (Fig. 8B) three-segmented, second segment longer than third, each bearing four sensilla. Stemmata six in number, five of them arranged close to lateral margin of head, and one inconspicuous stemma located ventrally (Fig. 8C). Labrum (Fig. 8A) bilobed, with deep median notch, similar to previous instars, but with ventral papilliform projections curved anteriorly. Mandibles similar to those of previous instars. Maxilla well developed, as shown in Fig. 8F. Labium broad, with stout, tubular spinneret having subapical opening (Figs 8G). Labial palpi (Fig. 8E) bi-segmented, basal segment longer than distal one, each bearing apical sensillum, that of distal segment longer. Hypopharynx with two sets of dense papilliform projections (Fig. 8E). Chaetotaxy: A group trisetose; L group unisetose; S group trisetose; SS group bisetose.

Thorax with prothoracic dark-brown dorsal shield well developed; one pair of legs on each thoracic segment; each leg with one pair of tarsal setae and one curved hook-like tarsal claw; one circular spiracle on each side of prothorax, near posterior margin and slightly displaced dorsally (Figs 8 H–J). Protothorax chaetotaxy: D group bisetose, both located on dorsal shield; XD group bisetose, XD1 on dorsal shield and similar in length to D1 and D2; XD2 lateral to dorsal shield, about three times XD1 in length; L group bisetose, L1 dorsal to L2, slightly longer than XD2 and about three times L2 in length; SV group bisetose, posteroventral to L2, both similar to L1 in length; V group absent. Meso- and metathorax chaetotaxy: D group bisetose, length of both setae similar to prothoracic D2; D2 posterolateral to D1; L group unisetose, L1 similar to that on prothorax in length; SV group bisetose, similar to prothoracic SV group in size and position; V group absent.

Abdomen with paired, circular spiracle laterally on A1-8; prolegs on A3-5 and A10, bearing uniordinal crochets in lateral penellipse (Figs 8 K–L). Chaetotaxy of A1-2, 6-7: D group bisetose, both setae very small, D1 anterolateral to D2 and posterior to spiracle; SD group unisetose, SD2 anteroventral to spiracle, length of SD2 about half of D1; L group unisetose, L1 length similar to anterior segment; SV group unisetose, length of SV1 about half of L1; V group unisetose, length of V1 similar to L1. A3-5: Similar to anterior segment, but V1 located on proleg and extremely reduced. A8: Similar to anterior segment, but SV group absent. A9: All setae lost except L group, which is similar to anterior segment. A10: D group unisetose, D2 on posterior margin; SD group unisetose, SD1 lateral to D2; L group unisetose, L1 about 1/3 length of corresponding seta on A9; SV group on proleg, bisetose; V group unisetose.

Pupa (Figs 5C-E, 9 D–L, 10J). Maximum length of specimens examined ranged from 3.69 to 5.10 mm. General coloration yellowish, with head, thorax, and corresponding appendices darkening later in development (Fig. 10J). Vertex bearing subtriangular acute process (= cocoon cutter; Figs 9 D–F) with serrated anterior edge, formed by several pointed projections that are fewer and larger at apex. Frons with 2 pairs of short frontal setae (Fig. 9D). Antennae long and slender, extending longer than pupal length; forewing reaching anterior margin of A6; proboscis extending to A2; prothoracic, mesothoracic and metathoracic legs reaching A3, A5 and A9, respectively (Figs 5 C–E). Abdominal integument dorsally covered with michotrichia (Figs 9 G–I). Intermediate abdominal segments with lateral margin of terga corrugated (Figs 9 G–H). From A1 to A7, two micro-setae, located medially on anterior margin of terga; additional micro-setae are found laterally, located posteriorly to spiracles (Figs 9 H–I). Last abdominal segment with two pairs of spines dorsally and one pair laterally, on posterior margin of tergum (Figs 9 J–L).

Host plants. Passifloraceae : Passiflora actinia Hook, Passiflora misera Kunth and Passiflora suberosa L. The former, where Spinivalva 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 Passiflora 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 Passiflora 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 Grande do Sul (RS), Brazil. In the Porto Alegre population, Passiflora misera and Passiflora 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 Passiflora 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 9 A–C, 10 A–E, G–I, K, 11). Eggs of Spinivalva 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 Passiflora 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 Passiflora suberosa or Passiflora misera , together with those from São Francisco de Paula / Condomínio Alpes de São Francisco (Pop. 2) hosted on Passiflora actinia . The second group included individuals from Curitiba (Pop. 3) sampled on Passiflora 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.