Phyllocnistis tethys Moreira & Vargas

Brito, Rosângela, Gonçalves, Gislene L., Vargas, Hector A. & Moreira, Gilson R. P., 2012, A new species of Phyllocnistis Zeller (Lepidoptera: Gracillariidae) from southern Brazil, with life-history description and genetic comparison to congeneric species, Zootaxa 3582, pp. 1-16 : 4-10

Phyllocnistis tethys Moreira & Vargas , sp. nov.

Figs. (1–8)

Type material. BRAZIL: Centro de Pesquisas e Conservação da Natureza Pró-Mata ( CPCN Pró-Mata; 29o28’36’S, 50º10’01’W; 900 m), São Francisco de Paula Municipality, Rio Grande do Sul State, Brazil. All adults were preserved dried and pinned, and reared by the senior author from larvae and pupae collected on 05-11.V.2011 by G.R.P. Moreira, R. Brito & K. Barão, on Passiflora organensis Gardner (Passifloraceae) . HOLOTYPE: 3 ( LMCI 155-58), deposited in DZUP (22.623). PARATYPES: 2 ƤƤ ( LMCI 155-41 and 155-43), deposited in DZUP (22.633 and 22.643); 1 3, 1 Ƥ ( LMCI 155-31 and 155-26), deposited in MCNZ (81901 and 81902); 1 3, 1 Ƥ ( LMCI 155-35 and 155-30), deposited in MCTP (28635 and 28636).

Other specimens examined. Adults, dried and pinned, 4 3, with the same collection data, deposited in LMCI (155-25, 27, 32, 33); 2 ƤƤ, fixed in Dietrich’s fluid and preserved in 70% ethanol, with the same collection data, deposited in LMCI (155-20). Genitalia preparations, mounted in Canada balsam on slides, with the same collection data, deposited in LMCI under the following accession numbers: 5 3 ( GRPM 50-10, 13, 14, 15 and 16); 4 ƤƤ ( GRPM 50-8, 17, 18, and 19). Immature stages, fixed in Dietrich’s fluid and preserved in 70% ethanol, with the same collection data, deposited in LMCI under the following accession numbers: 3 eggs ( LMCI 155-14), 2 first-instar (sap-feeding) larvae ( LMCI 155-3 and 4), 5 third-instar (sap-feeding) larvae ( LCMI 155-12 and 13), 4 fourth-instar (spinning) larvae ( LMCI 155-16), and 8 pupae ( LMCI 155-18 and 19). Mature leaf mines (n = 24) containing exuvia of all instars, mounted in glycerin on slides and stained with rose bengal, with the same collection data, 26.III.2012, deposited in LMCI, under accession numbers LMCI 174-1 to 24.

Diagnosis. Adults of P. t e th ys can be readily distinguished from all other known species of Neotropical Phyllocnistis in the forewing pattern, primarily by the absence of longitudinal and costal fasciae. Of the five species of Phyllocnistis known from neighboring Argentina and Chile ( Davis & Miller 1984, Davis 1994), only two ( P. abatiae Hering and P. puyehuensis Davis ) lack the basal longitudinal fascia. However, P. abatiae possesses a pair of small, isolated costal fasciae; and P. puyehuenis has a single, broad, isolated pale-gold costal fascia that crosses the wing. In addition, in these species the presence of yellowish-orange scales on the subapical part of the forewing is restricted to a small circular area adjacent to the black spot. Also, in contrast to P. tethys , in these species the tornal fringes are uniform in color.

Adult ( Figs. 1 , 2 ). Male and female similar in size and color ( Fig. 1 ). Forewing length 2.41–2.72 mm (n = 5). Head: Vestiture moderately smooth, with a pair of latero-dorsal light-gray scale tufts that curve forward to the frons. Eyes medium in size (interocular index ranging from 0.51 to 0.72; n = 4). Antenna mostly dark gray, ~ equal to length of forewing, covered with lanceolate scales; a single row of scales encircling each flagellomere. Labial palpus slender, ~ 0.3 mm in length, covered with dark-gray scales. Proboscis without scales, slightly longer than labial palpus. Thorax: Forewing light gray; longitudinal and costal fasciae absent; transverse fascia C-shaped, with faint dark border filled in with sparse light-gray scales; apical to subapical area bright yellowish orange, medially interspersed on costal strigulae and transverse fascia, and with large black spot; three slender, dark costal strigulae, three slender dark apical strigulae, and one dark tornal strigula arising from the apical black spot; fringe along tornal margin light gray with a wide dark basal band of scales; ventral surface dark gray. Hindwing dark gray. Legs light gray; foretibia and tarsomeres mostly dark gray. Abdomen: Length ~ 1.7 mm, covered with dark-gray scales. Male genitalia: Tergum VIII small, semicircular; sternum VIII reduced to a narrow transverse band. A pair of coremata present meso-laterally on segment VIII, consisting of inflatable tubular extensions bearing a terminal cluster of long, wide and flat scales ( Fig. 2 D). Tegumen formed by a basal, narrow transverse band that continues caudally up to approximately the length of the valvae, as an elongate, mostly membranous, basally spinose cylinder that encloses the anal tube ( Fig. 2 A); saccus well developed, ~ 0.3 length of valve, U- shaped with rounded anterior end and sinuous posterior margin having pronounced concavity medially; valvae digitiform, slightly curved medially and long, ~2.0 length of saccus, with moderately broad base formed by two wide dorsal and ventral projections that converge, reaching each other medially; setae of medium size are scattered found on median surface of valve, and short setae distally. Aedeagus ( Fig. 2 B) subcylindrical, weakly sclerotized, ~ equal to length of valva, having basal 2/3 portion slightly dilated and with subapical, dorsally located concave aperture. Vesica with several short spiniform cornuti ( Figs. 2 B, E). Female genitalia: Sternum VII subrectangular, with concave anterior margin more heavily sclerotized, and posterior margin slightly concave ( Fig. 2 C); tergum VIII reduced to narrow transverse band, with large subtriangular, latero-ventral projections; anterior apophysis similar in length to subtriangular projections of sternum VIII; anal papillae connected dorsally, covered with long piliform setae and microtrichia ( Figs. 2 C, F); posterior apophyses similar in length to anterior ones; ostium bursae broad, located on posterior margin of sternum VII; ductus bursae membranous, broader at base and narrow distally; corpus bursae membranous, pear-shaped, ~ twice length of ductus bursae, with a conspicuous, proximal, diagonally oriented, and hook-shaped signum that is directed posteriorly into the lumen ( Figs. 2 C, G); ductus seminalis membranous, narrow, inserted in apex of corpus bursae.

Immature stages. Egg ( Fig. 4 A; 7C). Flat, slightly ellipsoid; chorion translucent, without external ornamentation, and white at deposition; larva can be seen by transparency before emergence; aeropyles and micropylar area were not observed.

Larva ( Figs. 3 A–C; 4B–I; 5; 7B, E, G). Leaf-miner, with hypermetamorphic development and four instars, all endophyllous. The first three instars are sap feeders, prognathous and apodous, with highly modified buccal apparatus and depressed body; maximum length of larvae examined 4.79 mm. The prothorax and mesothorax of first-instar larvae are somewhat longer than the metathorax, which is not the case in the following instars. However, we found no stable differences either in shape or coloration among the sap-feeding instars of P. te t hy s. Instars can be correctly identified through measurements of the head capsule, since there is no overlap between the head-capsule size of succeeding instars (Table 2). For the three sap-feeding instars, the following exponential growth equation was adjusted for the head-capsule width: y = 0.073e ^0.504x; n = 45; r = 0.98; p <0.0001. The fourth instar (= non-feeding, “spinning”) is also prognathous and apodous, but has the mouth parts either reduced or absent, except for the functional spinneret; maximum length of larvae examined 4.17 mm. Body color uniformly white in all instars.

TABLE. 2. Variation in size among head capsules of sap-feeding instars of Phyllocnistis tethys (n = 15 per instar). Instar Head capsule width (mm)

Mean ± standard error Range Growth rate I 0.121 ± 0.003 0.116–0.158 - II 0.197 ± 0.005 0.179–0.242 1.63 III 0.333 ± 0.004 0.305–0.368 1.69

Sap-feeding instars ( Figs 3 A, B; 4B–I; 7B, E). Head prognathous, greatly depressed ( Figs. 4 B–D, G); primary setae either lost or reduced; stemmata absent. Antenna 3-segmented ( Fig. 4 F); second segment more slender than first, with 2 moderately stout sensilla; third segment less than 1/3 the length of second, with 2 apical sensilla. Labrum ( Figs. 4 D, E) with well-developed lateral lobes; antero-lateral margins rounded; anterior submargin densely spinose; posterior margins slightly concave. Mandibles large, rounded, flattened plates; anterior surface smooth, lateral area with single tooth, and mesal area with minute serrations. Labium with well-developed lateral lobes, conspicuous rugose cuticular band extending across anterior margin, and cluster of short hypopharyngeal spines laterally. Spinneret rudimentary ( Fig. 4 H), without extension of cuticle covering aperture. Maxillary and labial palpi absent. Thorax and abdomen without setae. Legs and prolegs absent; one latero-dorsal pair of rounded lobes on each of terga A1–6 ( Fig. 3 B, 4I).

Spinning instar ( Figs. 3 C; 5; 7G). Body cylindrical, with all appendages and setae greatly reduced. Head capsule weakly sclerotized, with anteriorly pronounced trophic lobe ( Figs. 5 A–D); integument finely corrugated. Stemmata absent. Antenna short ( Fig. 5 F), one-segmented, nearly flush with head capsule, with 4 short sensilla. Maxilla rudimentary ( Fig. 5 E), flush with head capsule, represented by one moderately long and a pair of short sensilla chaetica. Spinneret short, with simple terminal opening ( Fig. 5 E). Legs and prolegs absent. Two pairs of weakly differentiated, ventral and dorsal callosities ( Fig. 5 G) on A1–8; pair of microsetae laterally between the ventral callosities; pair of ventral and dorsal lobes laterally on A4–8. Pleural region of body and last two abdominal segments partly covered by microtrichia ( Figs. 5 H, I).

Pupa ( Figs. 3 D–F; 6B–O; 7H–K). Maximum length of specimens examined ranging from 2.59 to 3.20 mm. Coloration changing from light yellowish during early stage of pupation to yellowish brown ( Fig. 7 J) later in development. Vertex with large, subtriangular acute process (= cocoon cutter; Figs. 6 B–E) with serrated anterior edge. Frons with 2 pairs of short frontal setae ( Fig. 6 F). Antenna long and straight, extending almost to abdominal segment A7; forewing extending almost to A6 ( Figs. 3 E, F). A pair of relatively long setae, latero-dorsally on meso-, metathorax and A1–8, those of A2–8 on chalaza ( Fig. 6 J); a second pair of micro-setae, meso-dorsally on anterior margin of A3–8; spiracles ( Figs. 6 K, L) on prothorax and from A1–8, anterior to latero-dorsal setae ( Fig. 6 J). Six mid-dorsal spine clusters, arranged in V-shaped pattern ( Figs. 6 G–I) on anterior margin of A2–7; each cluster with row of similar, low, posteriorly curved spines. Tenth abdominal segment with two pairs of relatively short, stout, digitate caudal projections located latero-dorsally and latero-ventrally ( Figs. 6 M–O). Pleural region of body and last two abdominal segments partly covered by microtrichia ( Figs. 6 J, M–O).

Pupal cocoon ( Figs. 6 A; 7I). Endophyllous, constructed at the end of the mine; spherical, covered by sparse silk threads ( Fig. 6 A), and without external ornamentation ( Fig. 7 I). Spun by the non-feeding (spinning) fourthinstar larva prior to molting.

Etymology. Phyllocnistis tethys is named after Tethys , a Titan goddess in the Greek mythology; the wife of Oceanus, and the mother of rivers, springs, streams, fountains and clouds. Thus, the name also alludes to the cloudy and humid nature of the area of the Brazilian Atlantic Rain Forest where the new species was first found. Proposed as a noun in apposition.

Host plant ( Fig. 7 A). The only host plant known for the immature stages of P. tethys is the passion vine Passiflora organensis Gardner (Passifloraceae) ( Fig. 7 A). This passion vine is found mainly on forest edges in the coastal mountains of southern Brazil, where it is endemic, ranging in distribution from the states of Minas Gerais to Rio Grande do Sul (details of the biology and distribution of P. organensis were given by Mondin et al. 2011 and Moreira et al. 2011, respectively).

Distribution. Phyllocnistis tethys is known only from the type locality, the Dense Umbrophilous Forest (= Brazilian Atlantic Rain Forest sensu stricto) portions of the CPCN Pró-Mata, São Francisco de Paula Municipality, Rio Grande do Sul, Brazil.

Life history. Phyllocnistis tethys eggs ( Figs. 4 A, 7C) are deposited mostly on the abaxial leaf surface, adhered by a cement substance, usually on the secondary veins. Eclosion occurs through the surface of the egg adhered to the leaf; the first-instar larva enters progressively into the leaf, loading frass to the outside, empty space covered by the chorion ( Fig. 7 D), since initially the posterior part of the body remains within the chorion. Larvae are sapfeeding leaf miners during the first three instars. By feeding in circles, they form a blotch mine that widens as the larvae develop ( Figs. 7 B, D). The feeding paths of a larva can be traced by following the dark-green, non-granular frass lines left and head capsule exuvia shed in the mine ( Figs. 7 F, 8A, B). The three sap-feeding instars are specialized in the abaxial spongy parenchyma, leaving the two epidermis layers and generally the palisade parenchyma intact ( Figs. 8 C–E). In conditions of low larval density, the adaxial palisade parenchyma may be partly used by later instars ( Fig. 7 F), and in this case the feeding damage appears as white scars visible through the transparent upper leaf surface ( Fig. 7 A). However, if a leaf is intensively attacked, at the end of development the palisade parenchyma can be almost completely consumed; leaves then appear mostly deprived of green color ( Fig. 7 H, I). We could not find a distinct weaving pattern for the flimsy endophyllous cocoon constructed at the end of the mines by the last larval (spinning) instar ( Fig. 7 I). During adult emergence, the pupal cocoon is ruptured 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. 7 K).

At the type locality, P. tethys mines are common in P. organensis plants. One to several mines may be present per leaf (up to 13 young mines have been found in a single leaf) and may cover almost the entire lamina later in development ( Figs. 7 A, H). Our field collection data indicate that the species may have more than one generation per year, with adults emerging primarily in summer and autumn.

Molecular phylogeny. A total of 639 nucleotide sites were analyzed, in which 231 were variable and 173 parsimony-informative. ML and MP analyses showed identical topology and similar bootstrap supports, and we therefore show only the former ( Fig. 9 ). According to our phylogenetic hypothesis, P. tethys was strongly supported as a monophyletic clade, showing high branch length in relation to the other 11 species surveyed. Additionally, it was placed as the most basal lineage within Phyllocnistis .

Figures

FIGURE 1. Phyllocnistis tethys adult: wings spread, pinned, dorsal view (A); wings folded, on Passiflora organensis leaf, in dorsal (B) and lateral (C) views. Scale bars = 0.5, 1.0 mm, respectively.

FIGURE 2. Genital morphology of Phyllocnistis tethys under light and scanning electron microscopy: (A) male genitalia, ventral view (aedeagus omitted); (B) aedeagus, lateral view; (C) female genitalia, ventral view; (D) male coremata, lateral view; (E) male cornuti in detail, lateral view; (F) female papilla annalis in detail, latero-dorsal view; (G) female signum in detail, lateral view. Scale bars = 100, 200, 25, 50, 25, 25 µm, respectively.

FIGURE 3. Larval and pupal morphology of Phyllocnistis tethys under light microscopy: (A) first larval (“ sap-feeding ”) instar, dorsal and ventral views; (B) third larval (“ sap-feeding ”) instar, dorsal and ventral views; (C) fourth larval (“ cocoon-spinning ”) instar, dorsal and ventral views; (D – F) pupa, dorsal, ventral and lateral views. Scale bars = 100, 400, 400, 300 µm, respectively.

FIGURE 4. Scanning electron micrographs of Phyllocnistis tethys egg and third larval “ sap-feeding ” instar: (A) egg, on abaxial surface of a Passiflora organensis leaf; (B – D) head, lateral, anterior and dorsal views; (E) labrum and mandibles, dorsal view; (F) antenna, anterior view; (G) head, ventral view; (H) labium, ventral view (arrow indicates the spinneret); (I) abdominal lobe, dorsal view. Scale bars = 100, 100, 50, 100, 50, 10, 100, 50, 20 µm, respectively.

FIGURE 5. Scanning electron micrographs of Phyllocnistis tethys fourth larval (cocoon-spinning) instar: (A) head, general, dorsal view; (B – C) detail of head, dorsal and lateral views; (D) head, general, anterior view; (E) spinneret (indicated by arrow), anterior view; (F) antenna, lateral view; (G) invaginations of integument on abdominal sterna, ventral view; (H) caudal end of abdominal, ventral view; (I) dorsal view of fig. H (last segment retracted). Scale bars = 100, 50, 50, 50, 10, 10, 50, 50, 50 µm, respectively.

FIGURE 6. Scanning electron micrographs of Phyllocnistis tethys pupal cocoon and pupa: (A) weaving pattern of lower surface of pupal cocoon; (B) head, lateral view; (C) cocoon-cutter, lateral view; (D) head, ventral view; (E) cocoon-cutter, ventral view; (F) frons, ventral view; (G) abdominal segments 4 and 5, dorsal view; (H) spines on tergum 5, dorsal view; (I) spines on tergum 4, lateral view; (J) spiracle (arrow) and lateral seta on A 5, dorsal view; (K) spiracle A 3, lateral view; (L) spiracle A 8, lateral view; (M – O) last abdominal segments in ventral, dorsal and posterior views, respectively Scale bars = 100, 100, 50, 150, 50, 50, 200, 25, 25, 25, 10, 5, 100, 100, 100 µm, respectively.

FIGURE 7. Life history of Phyllocnistis tethys: (A) Passiflora organensis shoot twining around on a fern at the type locality, showing several leaves with leaf mines at different development stages; (B) leaf mine on abaxial leaf surface (open and closed arrows, respectively, indicate empty chorion on leaf surface, and sap-feeding larva seen through transparent mine); (C) egg containing developing embryo; (D) freshly hatched larva (indicated by closed arrow; open arrow indicates green frass lines left within the egg chorion; (E) third-instar (sap-feeding) larva; (F) detail of frass lines and damage on leaf parenchyma, left by the larva within the mine; (G) fourth-instar (spinning) larva; (H) Passiflora organensis containing several pupae, seen by transparency (indicated by arrows); (I) a pupal chamber in detail, showing a pupa by transparency; (J) pupa, lateral view; (K) pupal exuvium protruded (arrow) from mine exit hole, just after the adult emergence. Scale bars = 100, 1, 0.2, 0.3, 1, 1, 1, 20, 5, 0.5, 2 mm, respectively.

FIGURE 8. Diaphanized portion and histological sections of a Passiflora organensis leaf, showing by transparency the organization levels of a Phyllocnistis tethys mine in relation to larval ontogeny: (A) general aspect of the mine, containing a third-instar “ sap-feeding ” larva; asterisk indicates feeding area of the first instar; Roman numerals indicate larval instar numbers and corresponding positions of head capsules in the mine; open and closed arrows indicate the limit areas of nongranular frass lines left by second- and third-instar larvae, respectively; (B) detail of head capsule shed by the second-instar larva (bar indicates position for measurement of head-capsule width); C) transverse section of a mine; (D) transverse section of intact portion of leaf lamina (indicated by left arrow in C); (E) transverse section of mined portion of leaf lamina (indicated by right arrow in C). Ab, abaxial surface of epidermis; Ad, adaxial surface of epidermis; Lm, leaf mine; Pp, palisade parenchyma; Sp, spongy parenchyma. Scale bars = 1 mm, 50, 100, 25, 50 µm, respectively.

FIGURE 9. Maximum-likelihood tree of Pyllocnistis species based on 639 bp of the mitochondrial gene cytochrome oxidase subunit I (COI). Numbers above branches indicate bootstrap support higher than 60 %; branch lengths are indicated below. Asterisks indicate bootstrap values <60 % and branch lengths <0.001. Species of Bucculatricidae (Bucculatrix canadensisella) and Gracillariidae (Acrocercops and Caloptilia) were used to root the tree, according to the phylogeny proposed by Kawahara et al. (2011); see Table 1 and text for further description.