Leurocephala chilensis Vargas & Moreira, 2017

Pereira, Cristiano M., Silva, Denis S., Gonçalves, Gislene L., Vargas, Héctor A. & Moreira, Gilson R. P., 2017, A new species of Leurocephala Davis & Mc Kay (Lepidoptera, Gracillariidae) from the Azapa Valley, northern Chilean Atacama Desert, with notes on life-history, Revista Brasileira de Entomologia 61, pp. 6-15 : 7-13

Leurocephala chilensis Vargas & Moreira sp. nov.

Type material. Male holotype: Azapa , Arica, Chile, August 2015, ex leaf miner larva on S. molle , July 2015, H.A. Vargas coll. (MNNC) .

Paratypes: Two males, two females, same data as holotype (MNNC) ; two males and three females, same data as holotype (IDEA) ; one male and one female Azapa , Arica, Chile, December 2010, ex leaf miner larva on S. molle , July 2010, H.A. Vargas coll. (IDEA) ; three males Azapa , Arica, Chile, November 2011, ex leaf miner larva on S. molle , October 2011, H.A. Vargas coll (IDEA) .

Genitalia dissected by H.A. Vargas (HAV) were deposited in IDEA, under accession numbers as follows, all from Azapa, Arica, Chile, ex leaf miner larva on S. molle: HAV 276, one male, December 2010 ; HAV358, 359 and 402, three males, November 2011; HAV108, one female, December 2010; HAV1020 and 1024, two males, August 2015; HAV1021 and 1023, two females, August 2015. H.A. Vargas coll. Immature specimens of L. chilensis were deposited in LMCI, dissected from leaf mines on S. molle from Azapa , Arica, Chile, August 2012, H.A. Vargas and G.R.P. Moreira coll. as follows: preserved in 100% alcohol below −10 ◦ C, used for DNA extraction (LMCI 191-3); preserved in 75% alcohol, used for microscopy studies, seven eggs (LMCI 191-37), six first instar larvae (LMCI 191-41), twelve last instar larvae (LMCI 43), seven pupae (LMCI 191-44).

Specimens of L. schinusae used for comparison were either dissected (immatures) or reared (adults) from leaf mines collected on Schinus terebinthifolius by C. M. Pereira (CMP) in Laranjeiras do Sul, PR, Brazil, as follows: LCMI 309-1, five larvae preserved in 100% alcohol below −10 ◦ C, used for DNA extraction, 25.VII.2015; LMCI 309-3, one female, preserved in 70% alcohol, with genitalia in slide preparation (CMP 001-16F), 07.XI.2015; LMCI 309-4, one male, pinned, with genitalia in slide (CMP 001-18M), 07.XI.2015; LMCI 309-5, one male, in 75% alcohol, with genitalia in slide (CMP-22M), 16.VI.2016; LMCI 309-6, one female, pinned, with genitalia in slide (CMP-31F), 16.VI.2016. Also , five last instar larvae, preserved in 100% alcohol below −10 ◦ C, used for DNA extraction, dissected from leaf mines on Schinus aff. polygamus, Coxilha das Lombas, Santo Antonio da Patrulha , Rio Grande do Sul, Brazil, April 2015, G.R.P. Moreira & S. L. Bordignon coll. (LMCI 295-19).

Diagnosis

Despite their morphological and life history resemblance, the two species of Leurocephala can be differentiated based on morphology of the adult and larval stages, and by the shape of the mine. The apex of the sacculus of male genitalia in L. chilensis is provided with a short, spine-like process that projects upwards, which is absent in L. schinusae ; the dorsal surface of the aedeagus of L. chilensis is sculptured with several small tooth-like projections on the concave area, which are absent in L. schinusae . In the female genitalia, two signa are found on the corpus bursae of L. chilensis , while only one signum is found in L. schinusae ; furthermore, the horn-like lateral extensions of the antrum are laterally projected in L. chilensis , while these structures are apically projected in L. schinusae At the larval stage, the ventral plate of the prothorax of the last instar of L. schinusae is uniformly sculptured by a great number of granular projections of similar size, about 12 of which are at the posterior margin of the plate, almost touching laterally, while in L. chilensis the greatest granular projections, almost 1.5 times the length of the smallest ones, are restricted to the posterior third of the ventral plate, with only four at the posterior margin, clearly separated from each another by a distance similar to the diameter of the respective projection. The serpentine mine constructed by the second instar of L. schinusae has a little blotch-like broadening a short distance from the empty chorion, while broadening is absent in the serpentine mine of the second instar of L. chilensis .

Description

Adult ( Fig. 1 )

Male. Head. Front mostly whitish gray with brownish gray spots close to the compound eyes; vertex whitish gray; maxillary palpus whitish gray; labial palpus mostly whitish gray, second segment with brownish-gray spots distally; proboscis short, naked; antenna filiform, slightly shorter than forewing; scape elongated, brownish gray dorsally, whitish gray ventrally; with two narrow longitudinal stripes on the medial surface, one brownish gray in contact with the ventral area, the other whitish gray in contact with the dorsal area; pedicel and two first flagellomeres with coloration similar to the scape, remaining flagellomeres brownish gray.

Thorax. Mostly brownish gray dorsally with a few whitish gray scales; tegula dorsally brownish, ventrally whitish gray with a tuft of long scales at tip; lateral and ventral surfaces whitish gray. Foreleg mostly whitish gray, medial surface of femur and tibia brownish gray, tibial epiphysis whitish gray, a brownish gray ring at base of each tarsomere. Middle leg mostly whitish gray, tibia with two brownish gray rings, tibial spurs whitish gray, tarsomeres similar to those of the foreleg. Hindleg mostly whitish gray, a brownish gray ring at base of the femur, two brownish gray rings on the tibia, proximal tibial spurs brownish gray, distal tibial spurs mostly whitish gray with a brownish gray ring at middle, long whitish gray hair-like scales on the anterior and posterior surface of the tibia, tarsomeres whitish gray.

Forewing. Length: 4.0–4.5 mm (n = 10). Mostly brownish gray; a distinctive white transverse stripe arises in the middle of the coastal and hind margins, slightly projected apically at the longitudinal axis of the wing, sometimes interrupted by ground color scales; a short oblique, apically projected stripe arises from 3/4 of the hind margin reaching the longitudinal axis of the wing; a distinctive blackish gray dot subapically; fringe around apex short, concolor with the wing, a small apical tuft of plain scales; fringe on hind margin with long hair-like brownish gray scales. Venation as described by Davis et al. (2011) for L. schinusae .

Hindwing. Length: 3.2–3.4 mm (n = 10). Uniformly brownish gray with concolorous fringe of long hair-like scales. Venation as described by Davis et al. (2011) for L. schinusae .

Abdomen.Mostly brownish gray dorsally, with oblique segmental stripes of whitish gray ventrally until segment VI, completely whitish gray ventrally at apex. Segment VII with tergum and sternum reduced to fine transversal stripes. Segment VIII with sternum as a hood-like slightly sclerotized plate; sternum VIII as a slightly sclerotized fine transversal stripe. Membranous area between segment VII and VIII laterally with a pair of hair-like coremata at the apex of a finger-like mostly membranose lobe provided with a rodlike sclerite. Segment VIII with a second pair of hair-like coremata on the lateral apex of the sternum.

Male genitalia ( Figs. 8–11 ). Uncus absent. Tegumen as two fine stripes touching dorsally. Saccus U-shaped in posterior view, ventral area with the anterior margin slightly projected forward, posterior margin slightly convex. Gnathos ( Fig. 8 ) as two short, slightly sclerotized finger-like lobes. Valva ( Fig. 8 ) broadly joined basally to the posterior margin of the lateral arms of the saccus; costal margin straight; cucullus mostly membranous, ventral margin parallel to the costa; sacculus well sclerotized, basal part broad, triangle-like, delimited by a broad concavity on the ventral margin, with distal part straight, down-curved, slightly dilated subapically, bearing a short spine-like projection at apex ( Fig. 9 ). Transtilla as a slightly sclerotized transversal band joining the base of the costal margin of the right and left valvae. Juxta absent. Aedeagus ( Fig. 10 ) a bit shorter than valva, with insertion of the ductus ejaculatorius dorsally, close to the middle; basal half forward directed, tip blunt, diameter increasing toward the middle; distal half upcurved, with lateral sides slightly asymmetrical at apex, dorsal surface sculptured by several small tooth-like projections on the concave portion ( Fig. 11 ). A small sclerite joined dorsally on the middle of the aedeagus. Cornuti absent.

Female. Similar to male in size and color.

Female genitalia ( Figs. 12–14 ). Anterior and posterior apophyses well sclerotized, with length similar to the sternum VII. Ostium bursae broad, covering completely the posterior margin of the sternum VII. Antrum broad, sclerotized, trapezoid-like in ventral view, length about half of the sternum VII, cephalic side about half of the posterior side, with two laterally directed horn-like extensions on the middle of the lateral sides. Ductus bursae membranous, with similar length to the antrum. Corpus bursae elliptical, elongated, mostly membranous, with two signa ( Fig. 12 ); the larger ( Fig. 13 ) one on the ventral surface, triangle-like, with a narrow sclerotized longitudinal stripe with the cephalic tip projected forward as a short spine into the corpus bursae; the smaller ( Fig. 14 ) one on the dorsal surface, elliptical, elongated, length about 3/4 of the larger, with a longitudinal sclerotized stripe. Ductus seminalis basally inserted on the ventral surface of the corpus bursae ( Fig.12 ).

Egg ( Fig. 2 ).

Round and flat, with a translucent chorion, allowing by transparence visualization of the embryo under development within.

Larva ( Figs. 15, 16 , 19–26 ).

There are three morphotypes and five intars, which are similar in morphology to those described by Davis et al. (2011) for the type species, except for the last instar described below. The first instar is of a “sap-feeding” type, having mandibles modified for slicing the epidermis cells, differing from the remaining four instars that are tissue-feeders, which have mandibles used for chewing the leaf parenchyma. These can be identified by measuring their head capsule width, which do not overlap in size ( Table 2). Corresponding exponential growth curve for the four tissue-feeding instars of L. chilenis reared on L. molle was: y = 0.0494e 0.4787 x; n = 50; r = 0.98; p <0.0001.

Last instar ( Figs.15, 16 , 19–26 ). Maximum body length = 5.5 mm (n = 10). Head ( Figs. 15, 16 , 19, 20 ). Brown, semicircular in dorsal view, vertex partially covered by the prothorax, slightly depressed dorsoventrally, mostly smooth with a rhomboid-like area covered by short spine-like microtrichia between the frontoclypeous and the stemmata; epicranial notch U-shaped, broad and deep; frontoclypeous rectangle-like, about two times longer than wide; six circular stemmata, with stemmata 1 and 2 close to seta A3, stemmata 3–5 in a diagonal line ventral to seta A1, stemma 5 slightly displaced to the ventral surface of the head, stemma 6 isolated on the ventral surface of the head, almost equidistant to setae S2 and SS2. Antenna three-segmented; first segment annular, second segment cylindrical, about two times longer than the first segment, with sensillae distally, and third segment cylindrical, similar in length to second segment, about a half the diameter the second segment, with sensillae at apex. Mouthparts of the chewing type; labrum bilobed, four short hair-like setae on the external surface; epipharyngeal spines close to the distal margin of the labrum, one pair of plain epipharyngeal sclerites close to the each group of epipharingeal spines; mandible well-developed, with five distal cusps; maxilla with well-differentiated galea and palpus; labium with a well-developed cylindrical spinneret at apex and a pair of bisegmented palpi laterally to the spinneret; hypopharynx provided with long hair-like projections. Chaetotaxy. AF group bisetose, AF1 and AF2 as microsetae close to the dorso-median apex of the patch of microtrichiae. A group bisetose, A1 close to antenna, A3 dorsal to stemma, about two times the length of A1. CD group of microsetae trisetose. C group unisetose, C1 as a microseta. F group of setae absent, Fa pore present. L group unisetose, L1 as a short hair-like seta posteroventral to A3. MG group of microsetae bisetose. P group bisetose, P1 at middle of the patch of microtrichiae, size similar to A1, P2 greatly reduced, slightly greatest CD setae. S group bisetose, S1 about halfway between stemmata 1 and 3, S2 about halfway between stemmata 1 and 6. SS group trisetose, SS1 ventromedial to stemma 5, SS2 about halfway between stemmata 4 and 6, SS3 posteromedial to stemma 6. Thorax and abdomen sculptured by short spine-like microtrichiae.

Thorax ( Figs. 15, 16 , 22–24 ). Prothorax. Dorsal shield grayish brown, smooth, in the form of two subtriangular plates separated medially by a narrow membranous stripe; each plate with the anterior margin slightly convex, medial margin straight, lateral margin widely concave close to the anterior margin and almost parallel to the medial margin on the distal 2/3, posterior vertex widely rounded. An ellipsoid shield postero-ventral to SV group. Ventral shield square-like,lateral sides slightly concave close to the anterior margin, posterior margin slightly convex; sculptured by granular projections variable in size, the largest restricted to the posterior third of the plate, clearly separated from each other by a distance similar to the diameter of the respective projection. A circular spiracle with slightly elevated peritrema laterally close to the posterior margin of the segment. A longitudinally oriented callus-like structure between the lateral margin of the dorsal shield and the SV group; another callus-like structure postero-lateral to the coxa. Chaetotaxy: D group bisetose, D1 greatly reduced, on the posterior half of the dorsal shield close to the lateral margin, D2 about three times the length of D1 between the dorsal shield and the callus-like structure. XD group bisetose, XD1 anterior to D2, XD2 ventral to XD1. SD group bisetose, lateroventral to the callus-like structure, SD1 similar in size to D2, SD2 about 4–5 times longer SD1. L group bisetose, similar to SD 2 in size, clearly anterior to the spiracle. SV group bisetose, antero-dorsal to the ellipsoid shield. V group unisetose, V1 between the coxa and the ventral shield.

Meso and metathorax without dorsal, lateral or ventral shields. A transversally oriented callus-like structure anterior to D2; another callus-like structure postero-lateral to the coxa. Chaetotaxy: D group bisetose, D1 anterior and D2 posterior to the callus-like structure. SD group bisetose, ventro-lateral to the callus-like structure. L group bisetose. SV and V groups unisetose. Legs moderately well developed, bearing large tarsal claws.

Abdomen ( Figs. 15, 16 , 25, 26 ). All segments bearing dorsal and ventral smooth shields varying in shape. Dorsal shield of A 1–6 in the form of a small irregular plate, those of A7 and A8 little developed, in the form of a small dot; dorsal shield of A9 ellipsoidal, well-developed, transversally arranged; dorsal shield of A 10 in the form of two widely separated plates close to the posterior margin of the segment. Ventral shield of A1–2 and A8 circle-like, little evident on A3–5, between the respective prolegs; those of A6–7 similar in size and shape to those of A3–5; ellipsoidal on A9, transversally arranged, smaller than the dorsal shield of the same segment; absent on A10. Chaetotaxy: A1–2, 6–7: D group bisetose, D1 anterior and D2 posterior to the callus-like structure. SD group bisetose, SD1 latero-ventral to the callus-like structure, SD2 greatly reduced, dorsal to the spiracle. L group unisetose, L1 postero-ventral to the spiracle. SV bisetose, dorso-lateral to the callus like structure. V group unisetose, V1 between the callus-like structure and the ventral shield. A3–5: similar to the preceding segments; SV dorso-lateral to the proleg. A8 similar to preceeding segment, except that SV group unisetose. A9 similar to preceeding segment, except that SD group unisetose. A10 with D and SD groups bisetose; D1 anterior and D2 posterior to the dorsal shield, SD1, SD2 on the margin of the lateral part of the dorsal shield; L, SV and V groups unisetose. Spiracles round, with moderately elevated peritreme. Prolegs present on A3–5 and A10; crochets arranged in a staggered caudal varying from 10 to 16 hooks on A3–5 ( Fig. 25 ); A10 with crochets reduced to 4 hooks.

Pupa ( Figs. 17, 18 , 27–32 ).

Maximum body length: 4 mm (n = 10). Similar in color and general appearance to L. schinusae , as described by Davis et al. (2011) for L. schinusae . Minutely and densely spinose, particularly the dorsal abdomen. Cocoon cutter subtriangular, with outer ridge having numerous minute teeth, the central three teeth the largest. Antennae long, surpassing the abdomen in length. Labial palpi ca. 1/3 the length of proleg. Proboscis as long as the proleg. Forewings narrow, well separated, extending to abdominal segment A7. Hindlegs extending to abdominal segment 9 + 10. Setae D1, SD1, and L1 present on A1–7; only SD1 present on A8. Abdominal spiracles round, with elevated peritreme ( Fig. 31 ). Cremaster formed by three pairs of slightly curved spines, two lateral and one dorsal ( Fig. 32 ).

Etymology. The specific epithet is derived from the country of the type locality.

Distribution. L. chilensis is known only from Azapa (type locality) Valley in the Atacama Desert of northern Chile.

Life history ( Figs. 2–7 ). Eggs are laid individually ( Fig. 2 ) on the adaxial surface of the leaflet, mostly close to the main leaflet vein. After hatching, the first, sap-feeder instar introduces itself into the leaflet, constructing a small, superficial, blotch-like mine a short distance from the empty chorion ( Fig. 3 ). The feces are deposited into the lumen of the chorion during the time that the anal apex of the first instar remains there. The second instar constructs a narrow serpentine mine ( Fig. 4 ) on the adaxial surface of the leaflet. Subsequent instars construct a conspicuous blotched mine on the adaxial surface whose diameter increases with the sequence of the instars, generally covering more than 50% of the leaflet when fully developed ( Fig. 5 ). A large number of feces are glued on the internal side of the “epidermal” surface of the blotch mine. The fully developed fifth instar makes a short slit on the margin of the mine to exit from it to search for a site for pupation. Pupation mostly occurs on the abaxial surface of a leaflet of the same plant; previously the fifth instar constructs an ellipsoidal smooth cocoon, generally with one of the lateral margins touching the lateral margin of the leaflet, and externally deposits silk bubbles ( Fig. 6 ) secreted by the anus. When metamorphosis is completed, the pupa makes a slit on the cocoon using the cephalic cocoon-cutter to enable the adult to emerge, after which the pupal exuvium typically appears protruded with the posterior body portion remaining in the cocoon ( Fig. 7 ). The first, sap-feeding instar feeds only on the epidermis cells ( Fig. 33 ), while the other four tissue-feeding instars feed on the palisade parenchyma, leaving the spongy parenchyma intact ( Figs. 34, 35 ).

Molecular analyses. Two reciprocally monophyletic lineages were found within the genus Leurocephala : the currently recognized L. schinusae and the new species L. chilensis ( Fig. 36 ). Genetic divergence estimated between these lineages was 12% (±1%) and of both species together versus the outgroups ( Spinivalva , Parectopa and Epicephala ), varied from 16 to 18% (±1%, for any comparison).

Figures

Figs. 1–7. Adult and life-history of Leurocephala chilensis on the abaxial surface of Schinus molle leaves: (1) pinned-dried male, dorsal view; (2) egg; (3) freshly hatched first instar larva, seen by transparence within the mine (empty chorium is indicated by arrow aside); (4) middle age mine (arrow indicates the egg-chorium at the beginning of the mine; letters and associated dashed-lines correspond to the locations of tissue sections, presented in transversal view in Figs.33–35); (5) old, empty mine (seta indicates exit of last instar larva); (6) pupal cocoon, ornamented with bubbles (arrow); (7) pupal exuvium, partially protruding from the cocoon. Scale bars= 1, 0.5, 0.5, 5, 5, 3 and 1 mm, respectively.

Figs. 8–14. Genitalia morphology of Leurocephala chilensis under light microscopy: (8) male, posterior view; (9) distal end of valva, in detail (enlarged, rectangular area marked in Fig. 8); (10) aedeagus, lateral view; (11) dorsal spines of aedeagus, in detail (enlarged area indicated by seta in Fig. 10); (12) female, ventral view; (13, 14) signa, in detail (indicated by open and closed arrows in Fig.12). Scale bars= 200, 50, 100, 20, 200, 50 and 50 µm, respectively.

Figs.15–18. Last instar (15,16) and pupal (17–18) morphologies of Leurocephala chilensis under light microscopy,in dorsal and ventral views,respectively.Scalebars= 0.5mm.

Figs. 19–26. Last larval instar of Leurocephala chilensis under scanning electron microscopy: (19–20) head, under dorsal and lateral views,respectively; (21) antenna, dorsal; (22) callum of first abdominal segment,ventral; (23) sternal prothoracic plate, ventral;(24) mesothoracic leg, lateral;(25) pseudopodium of fifth abdominal segment,ventral; (26) spiracle of eighth abdominal segment, lateral. Scale bars= 100, 50, 20, 50, 25, 50, 50 and 10 µm, respectively.

Figs. 27–32. Pupa of Leurocephala chilensis under scanning electron microscopy:(27) head,ventral view; (28) “cocoon-cutter” in detail,ventral; (29) labrum in detail,ventral; (30) sixth and seventh abdominal segments, dorsal, (31) spiracle of eighth abdominal segment, lateral; (32) last abdominal segments, dorso-posterior. Scale bars =100, 25, 100, 100, 5 and 100µm, respectively.

Figs. 33–35. Variation in transversal histological sections of Leurocephala chilensis on Schinus molle leaf, according to larval ontogeny; (33) first, sapfeeding instar (position indicated by letter “a” in Fig. 4); (34) early tissue-feeding instar (letter “b” in Fig. 4); (35) last tissue-feeding instar (letter “c” in Fig. 4). Ab, abaxial surface of epidermis; Ad, adaxial surface of epidermis; Lm, leaf mine; Ep, epidermis; Pp, palisade parenchyma; Sp, spongy parenchyma.Scale bars =0.2 mm.

Fig. 36. Maximum likelihood phylogenetic tree of Leurocephala inferred based on 658bp of DNA barcode sequences (cytochrome oxidase subunit I gene). Asterisk above branch indicate bootstrap support lower than 50%. Species of Spinivalva, Parectopa and Epicephala were used as outgroup; see Table 1 and text for further description.