Revista Brasileira de Entomologia Revista Brasileira de Entomologia
Systematics, Morphology and Biogeography
Phyllocnistis hemerasp. nov. (Lepidoptera: Gracillariidae): a new species of leaf-miner associated with Daphnopsis fasciculata (Thymelaeaceae) in the Atlantic Forest
Júlia Fochezatoa, Rosângela Britoa, Rosy Mary dos Santos Isaiasb, Gislene Lopes Gonçalvesc,d, Gilson R.P. Moreiraa,,
a Universidade Federal do Rio Grande do Sul, Instituto de Biociências, Departamento de Zoologia, Porto Alegre, RS, Brazil
b Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Botânica, Belo Horizonte, MG, Brazil
c Universidade Federal do Rio Grande do Sul, Instituto de Biociências, Departamento de Genética, Porto Alegre, RS, Brazil
d Universidad de Tarapacá, Facultad de Ciencias Agronómicas, Departamento de Recursos Ambientales, Arica, Chile
Recebido 04 Outubro 2017, Aceitaram 01 Novembro 2017
Abstract

During recent studies performed in the Atlantic Forest, a new species of Phyllocnistinae (Gracillariidae), Phyllocnistis hemerasp. nov., leaf miner of Daphnopsis fasciculata (Thymelaeaceae) was discovered. The adults are described and illustrated as well as the immature stages, with notes on natural history including a description of the leaf mine. Additionally, DNA barcode sequences were compared to other representatives of Phyllocnistinae to test for the specific status of P. hemera and to infer phylogenetic relationships. This is the fifth species described for the genus Phyllocnistis in the Atlantic Forest and the first record of a gracillarid mining Thymelaeaceae leaves.

Keywords
Microlepidoptera, New species, Morphology, DNA barcoding, Neotropical region
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Introduction

The Gracillariidae (Lepidoptera) is one of the most diverse families of leaf mining microlepidoptera. Approximately 2000 species have been described in 106 genera, with cosmopolitan distribution but not found in the Antarctic region (Davis, 1987; De Prins and De Prins, 2017). Kawahara et al. (2017) carried out a phylogenetic analysis and proposed a new classification for the group, dividing Gracillariidae into eight subfamilies. The Phyllocnistinae, one of the subfamilies retained in the new classification, has been the recent focus of taxonomic studies of the Neotropical region (Kawahara et al., 2009; Davis and Wagner, 2011; Brito et al., 2012; De Prins et al., 2016; Brito et al., 2017a,b).

Phyllocnistinae is a monotypic subfamily represented by species of Phyllocnistis Zeller, 1848. This genus has currently 108 species described worldwide, but only 27 for the Neotropics (De Prins and De Prins, 2017; Brito et al., 2017a). Representatives of this genus can be distinguished from the other gracillariids by a set of typical fasciae and strigulae on the forewings, and by a set of tergal spines on the abdominal segments on the pupae. The larvae usually present three sap-feeding instars followed by a spinning instar (Davis, 1987). The sap-feeding instar feeds on cell fluid which is released by the laceration of leaf tissue; the spinning instar, which does not feed, is responsible for the construction of a silk cocoon within which pupation occurs. Only two species, P. citrella Stainton and P. tethys Moreira & Vargas, have information regarding the type of tissue used as food by the sap-feeding larvae. Those of P. citrella are known to feed on epidermal cells, while larvae of P. tethys feed upon the spongy tissue (Achor et al., 1997; Brito et al., 2012).

Hostplants are known only for a third of the species of Phyllocnistis. They belong to 34 families of angiosperms, 13 of which occur in the Neotropics (De Prins and De Prins, 2017). Recently, during collections performed in the Atlantic Forest, in southern Brazil, a gracillariid representative associated with Thymelaeaceae was found for the first time. The comparison at both morphological and molecular levels confirmed that it is a new Phyllocnistis species. Here we provide illustrations and description of the corresponding adult and immature stages, and highlight important characteristics regarding its life history and feeding habits in association with the characterization of the leaf mine. DNA barcode (COI) was obtained from some specimens in order to establish the specific status and its phylogenetic relationships with representatives of the Phyllocnistinae.

Material and methods

The specimens were reared in small plastic vials under controlled abiotic conditions (14h light/10h dark; 25±2°C) at the Laboratório de Morfologia e Comportamento de Insetos, Departamento de Zoologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul state (RS), Brazil, during May 2016, January, June and July 2017. They came from field-collected leaf mines associated with the host plant Daphnopsis fasciculata (Meisn.) Neveling (Thymelaeaceae), at the Centro de Pesquisa e Conservação da Natureza (CPCN Pró-Mata/PUCRS 29°28′36″S, 50°10′01″W), São Francisco de Paula, RS.

Morphological analysis

Adults were pinned and dried. Immature stages were fixed with Dietrich's fluid and preserved in 75% ethanol. At least 3 specimens of each stage were used to describe the immatures. For adult gross morphology the specimens were cleared in 10% potassium hydroxide (KOH) and slide-mounted in Canada balsam. Morphological analysis and descriptions were performed with the aid of a Leica® M125 stereomicroscope with an attached Sony® Cyber-shot DSC-H10 digital camera for photography. Genitalia structures were analyzed and photographed with a Nikon AZ 100M stereomicroscope. The software CorelDraw® X8 and Corel Photo Paint® X8 were used for vectorization and image processing. The terminology used for the description of forewing pattern and genitalia follows Brito et al. (2017b) and Kobayashi and Hirowatari (2011), respectively.

For scanning electron microscopy, specimens were dehydrated in a Bal-tec® CPD030 critical-point drier, mounted on metal stubs with double-sided tape and coated with gold in a Bal-tec® SCD050 coater. Photographs were taken under a JEOL® JSM5800 scanning electron microscope at the Centro de Microscopia Eletrônica (CME), UFRGS.

For histological sections, leaf fragments (0.5cm2) with mines (n=6) were fixed in FAA (37% formaldehyde, acetic acid, 50% ethanol, 1:1:18, v/v) for 48h, dehydrated in an n-butyl series, embedded in Paraplast and sectioned transversely (12μm) in a rotary microtome (Jung Biocut). The sections were stained in safranin-astrablue (2:8, v/v) (Bukatsch, 1972, modified to 0.5%), and mounted in colorless varnish (Paiva et al., 2006). Photographs were taken under a Leica DM 2500-LED light microscope with a Leica DFC 7000T camera.

Museum collections

The material examined was deposited in the following entomological collections:

LMCI  Laboratório de Morfologia e Comportamento de Insetos, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil 
MCTP  Museu de Ciências e Tecnologia da Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil 

Molecular analysis

Genomic DNA was extracted from larval tissue of three specimens of P. hemera (LMCI 292-25C, D and E) using the Purelink genomic DNA extraction kit (Invitrogen) following manufacturer's instructions. Polymerase chain reaction (PCR) was performed to amplify the DNA barcode region (sensu Hebert et al., 2003), i.e. a 660-base pair (bp) segment of the mitochondrial gene cytochrome c oxidase subunit I (CoI), with the universal primers and conditions proposed by Folmer et al. (1994). Variants obtained matched the region sequenced in other Phyllocnistis deposited in the GenBank and BOLD Systems databases. PCR products were treated with exonuclease I and FastAP thermosensitive alkaline phosphatase (Thermo Scientific), sequenced using BigDye chemistry and analyzed in an ABI3730XL (Applied Biosystems). Sequences were automatically aligned using the algorithm Clustal X in MEGA v5 (Tamura et al., 2011) running in full mode. Data generated in this study were deposited in GenBank and BOLD (Table 1).

Table 1.

Specimens used in this study to infer the specific status of Phyllocnistis hemera and phylogenetic relationship within the genus.

Group  Genus  Species  Acession number
      Genbank  BOLD systems 
Ingroup         
  Phyllocnistis       
    citrella  KF919121  GREC094-12 
    gracilistylella  AB614510  – 
    hemera sp.n.  MG264519  MISA019-17 
    hyperpersea  HQ971045  RDOPO395-10 
    magnoliella  KF492018  LTOL1038-11 
    ourea  KY006927  MISA013-16 
    saligna  AY521491  – 
    selene  KY006929  MISA015-16 
    phoebus  KY006928  MISA014-16 
    perseafolia  HM382097  RDOPO394-10 
    populiella  KR941466  CNRVE3067-15 
    tethys  JX272049  – 
    vitegenella  KR938941  – 
Outgroup         
  Angelabella       
    tecomae  KM983605  – 
  Marmara       
    arbutiella  FJ412783  LBCS922-07 

Phylogenetic trees were reconstructed to test the specific status of the new species and to infer its relationships within the genus. Representative taxa belonging to Phyllocnistis were incorporated, particularly Neotropical species already described (Table 1). The tree was rooted with species of Angelabella Vargas & Parra and Marmara Clemens and representatives of the subfamilies Oecophyllembiinae and Marmarinae, known to be closely related to Phyllocnistiane (Kawahara et al., 2017).

Phylogenetic reconstruction used distance (neighbor-joining [NJ]) and model-based methods (Maximum Likelihood [ML] and Bayesian Inference [BI]). The substitution model GTR+G was used for ML and BI according to the Akaike Information Criterion (AIC) performed in MEGA v5. The NJ and ML analyses were run in MEGA v5 using default parameters for tree inference. Monophyly confidence limits were assessed with the bootstrap method (Felsenstein, 1985) at 60% cutoff after 1000 bootstrap iterations. The BI analysis was implemented in BEAST v1.8.4 (Drummond et al., 2012), using an uncorrelated lognormal clock and a Yule prior on branching rates. Four independent runs of 10 million generations and a burn-in period of 10,000 (the first 1000 trees were discarded) were implemented; the remaining trees were summarized in TreeAnnotator v1.6.2 (Drummond and Rambaut, 2007) and used to infer a maximum a posteriori consensus tree. Bayesian posterior probabilities (BPP) were used as an estimate of branch support. Consensus trees were visualized and edited in FigTree 1.4.2 (http://tree.bio.ed.ac.uk/software/201/). The genetic distances between the same pairs of taxa used in the phylogenetic analysis (including outgroups) were analyzed using the Kimura 2-parameter (K2P) model, with 1000 bootstrap replications in MEGA v5.

ResultsPhyllocnistis hemera Brito & Fochezato sp. nov. (Figs. 1–7)

Type material. MALE HOLOTYPE: São Francisco de Paula municipality, Rio Grande do Sul (RS), Brazil; preserved pinned and dried, reared from a mine associated with D. fasciculata, 22–24.VI.2016, G.R.P. Moreira, R. Brito and J. Fochezato colls. (LMCI306-47). PARATYPES: Same locality, 28-30.VI.2017, G.R.P. Moreira and J. Fochezato coll., two males (LMCI 319-30 and 319-36) and two females (LMCI 319-35 and 319-45); 22–24.VI.2016, G.R.P. Moreira, R. Brito and J. Fochezato coll., one male (LMCI 306-46) and one female (LMCI 306-43) donated to MCTP (60251, 60252, respectively); 01–02.VII.2017, G.R.P. Moreira and J. Fochezato coll., one female (LMCI 320-43), donated to MCTP (60253).

Additional specimens examined from the same locality and host plant, all preserved pinned and dried: 22–24.VI.2016, G.R.P. Moreira, R. Brito and J. Fochezato colls., four males (LMCI 306-26, 32, 36 and 40) with genitalia on slides (GRPM 50-144 to 147, respectively); three females (LMCI 306-34, 35 and 49) with genitalia on slides (GRPM 50-148 to 150, respectively); 28–30.VI.2017, G.R.P. Moreira and J. Fochezato colls., one male (LMCI 319-69).

Additional immature specimens of P. hemera were deposited at LMCI, all dissected from leaf mines of D. fasciculata collected at the type locality: 10–13.II.2015, G.R.P. Moreira and R. Brito coll., preserved in 100% ethanol at −10°C and used for DNA extraction (LMCI 292-25); 28–30.VI.2017, G.R.P. Moreira and J. Fochezato coll. and preserved in 75% ethanol. Eight sap-feeding larvae (LMCI 319-9), three spinning larvae (LMCI 319-8) and three pupae (LMCI 319-7) were used for microscopic studies. Additionally, nine leaf mine fragments containing sections of P. hemera mines (LMCI 320-9 to 11) from the type locality were fixed and preserved in FAA as described above, and used in the histological sections, 01–02.VII.2017, G.R.P. Moreira and J. Fochezato coll.

Diagnosis. P. hemera adults are easily distinguished from the other Neotropical Phyllocnistis by a longitudinal fascia on the forewing with superior border enlarged, reaching the costal margin. Its pupal stage is similar to that of P. drimiphaga Kawahara, Nishida & Davis in having the cocoon-cutter divided into three processes, the central longer than the lateral ones, and by the similar arrangement of tergal spines on abdominal segments. However, P. hemera has lateral processes shorter and wider than P. drimiphaga, and two pairs of setae on the clypeus, while P. drimiphaga has only one pair. The spinning larva of P. hemera is similar to that of P. ourea Brito & Moreira, as both share ventral ambulatory, single callus on central meso- and metathorax. However, these species differ in the location of the ventral calli on the abdominal segments (Ab); P. hemera has ventral calli on abdominal segments 3 to 7 (Ab 3–7), while P. ourea has calli on Ab 3–6.

Description. Adult (Fig. 1). Male and female similar in size and color. Forewing length 3.51–4.17mm (n=5). Head: antennae silver, ∼ the length of forewing. A pair of tufts formed by a set of scales emerging from the base of antenna are directed to the frons.

Fig. 1.
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Adult of Phyllocnistis hemera, dorsal view: (A) wings spread, pinned and dried (LMCI 306-47); (B) wings folded, on Daphnopsis fasciculata leaf surface. Scale bars: 1mm.

Labial palp slender, silver, ∼0.5mm in length. Proboscis without scales. Thorax: Forewing ground color white silver, with light yellow fasciae bearing brown borders. Longitudinal fascia with well-marked border, which is much wider and convex on the basal half, reaching the costal margin; longitudinal fascia emerges from the wing base toward the median region, being completely connected to the first transverse fascia. The latter emerges on the costal margin and is slightly connected to the second transverse fascia. The second transverse fascia crosses the wing from the costal margin toward the inner margin; it is disconnected from the third and fourth fascia. Last two fasciae fused, forming a blotch on the distal region. Costal strigulae emerge from second, third and fourth transverse fasciae. Apical strigulae emerge from apical spot. Inner marginal fringes mostly light brown. Abdomen: covered with silver scales.

Male genitalia (Fig. 2A–D). One pair of coremata located between the intersegmentary membrane of Ab 8 and 9; the coremata are formed by a set of long, fine scales, reaching ∼0.4× the size of valvae (Fig. 2B and C). Uncus absent. Tegumen narrow at base, widening toward the apex, forming a dorsal sclerotized arch; small setae occur next to the lateral borders of the tegumen; tuba analis narrow and membranous, surpassing the distal margin of tegumen. Valvae digitiform, slightly narrower and finer on base, widening toward the apex. Setae vary in size from small to medium on ventral distal region, forming a line; along the valva, setae varying in size are randomly arranged (Fig. 2A and C). Saccus U-shaped. Phallus elongated, weakly sclerotized, cylindrical and partially wrinkled, with fine apex. Cornuti absent (Fig. 2D).

Fig. 2.
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P. hemera genitalia under light microscopy: (A–D) male genitalia; (E–G) female genitalia. (A) apex of left valva, mesal view (LMCI 319-69); (B) left corema, ventral (LMCI 306-26); (C) male genitalia, ventral; (D) aedeagus, lateral (LMCI 306-36); (E) female genitalia, ventral; (F) female last abdominal segments, lateral (LMCI 306-49) with the ostium bursae indicated by arrow; (G) signum in detail, ventral (LMCI 306-49). Scale bars: 50 (A, B, D), 100 (C, F, G), 400μm (E).

Female genitalia (Fig. 2E–G). Anterior and posterior apophyses similar in shape; the posterior half the size of the anterior; the posterior apophyses reach Ab 8 and the anterior ones the posterior portion of Ab 7. Anal papillae with medium-sized setae, randomly arranged. Ostium bursae located on median region of the eighth abdominal sternum; ductus bursae long, membranous and slender; corpus bursae ellipsoid and membranous; signum wide, slightly rectangular with two spines on the proximal margin; one acute, well developed, the other of reduced size (Fig. 2E–G). Variation in these structures was found, such as: (1) one of the signa with an acute spine, the other without spines; (2) both signa bearing well developed spines; (3) one single signum containing a spine with bifurcated apex.

Immature stages

Sap-feeding larva (Figs. 3A, 4 and 7D). Leaf miner, flattened dorsoventrally (Fig. 4D), hypermetamorphic, presenting three instars. Body light yellow, 5.39–7.67mm (min–max length); average last instar head capsule width ∼0.64mm (n=5) (Fig. 7D). Head: prognathous, setae absent; labrum slightly bilobed with small hypopharyngeal spines next to lateral margin. Labium shaped like the labrum, however wider and with greater number of spines (Fig. 4A–C and E); a small aperture on labium indicating the rudimentary spinneret (Fig. 4F). Labial and maxillary palpi absent. Antenna 3-segmented; the second segment with two sensilla, the distal one with a single apical sensillum (Fig. 4G). On anterior section of lateral region of the head, one rounded stemma followed by a microseta (Fig. 4H). Thorax: without setae, legs absent; presence of one pair of latero-dorsal prothoracic spiracles, with peritreme not differentiated (Fig. 4I). Abdomen: setae, prolegs and calli absent; a pair of laterodorsal lobes from first to eighth abdominal segment (Fig. 4J and M–N). These lobes are dorsoventrally flattened; on Ab 8 there is a second pair of lateroventral lobes with fine apex (Fig. 4K and L); last abdominal segment slightly divided, with pairs of microsetae on ventral region (Fig. 4O).

Fig. 3.
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Larval and pupal morphology of P. hemera under light microscopy: (A) sap-feeding larva, dorsal and ventral views; (B) spinning larva, dorsal and ventral; (C) pupa, dorsal, ventral and lateral, respectively. Scale bars: 500μm.

Fig. 4.
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Scanning electron micrographs of P. hemera sap-feeding larva: (A–D) head under dorsal, ventral, anterior and lateral views (arrow indicates stemma); (E) labrum, dorsal; (F) labium, ventral (arrow indicates spinneret aperture); (G) antenna, ventral; (H) stemma in detail (indicated by arrow in D), lateral; (I) prothoracic spiracle, dorsal; (J) segment A7, ventral; (K) segments A8-10, ventral; (L) detail of latero-ventral lobe indicated by arrow in K, ventral; (M, N) latero-dorsal lobe, highlighted by the red rectangle in K, lateral and dorsal; (O) last abdominal segment, ventral. Scale bars: 200 (A, B, D, K), 70 (C), 100 (E, F, O), 30 (G), 20 (H), 10 (I), 250 (J), 25 (L), 50 (M), 40μm (N).

Spinning larva (Figs. 3B, 5 and 7E). Endophyllous, cylindrical, with coloration similar to the sap-feeding larva, 5.47–6.04mm (min–max length). Body covered with microtrichia. Head: setae absent or reduced, except for three pairs located on the clypeal region; buccal apparatus modified into an anteriorly located, trophic lobe presenting corrugated tegument (Fig. 5A, B, D and E). Labial palpi absent; maxilla represented by three pairs of small setae. The trophic lobe in ventral view is long, with functional apical aperture (Fig. 5C). Antennae short, 3-segmented; three sensilla emerge from the second segment and one, bristle-like seta from the apical segment (Fig. 5H). Thorax: setae reduced or absent. Legs absent; prothoracic shield slightly evident, represented by an irregular, corrugated, central area (Fig. 5F). Laterally on prothoracic tergum one pair of rounded spiracles, with peritreme slightly elevated (Fig. 5G). A single ambulatory callus centrally on ventral region of meso- and metathorax; slightly divided on metathorax (Figs. 3B and 5I and J). Abdomen: one pair of ambulatory calli ventrally on Ab 3–7 (Figs. 3B and 5M and N), smaller compared to those on meso- and metathorax. One pair of small lateral sensilla on Ab 4–7 (Fig. 5L), which decrease in size antero-posteriorly. Last abdominal segment partially divided into two lobes (Fig. 5K) with two pairs of microsetae on ventral region (Fig. 5O).

Fig. 5.
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Scanning electron micrographs of P. hemera spinning larva: (A, B) head, dorsal and ventral views; (C) spinneret, antero-lateral (arrow indicates functional aperture); (D) head, lateral; (E) detail of trophic lobe, dorsal; (F) prothoracic shield, dorsal; (G) prothoracic spiracle, lateral; (H) antenna, anterior; (I) meso- and metathoracic calli, ventral; (J) mesothoracic callus in detail (indicated by rectangle in I), ventral; (K) abdominal segments Ab 7-10, dorsal; (L) latero-sensillum indicated by arrow in K, dorsal; (M) abdominal segment Ab 7, ventral (arrow indicates one of the calli); (N) callus in detail, ventral (indicated by arrow in M); (O) last abdominal segment, ventral. Scale bars: 200 (A, B, D, E, K), 150 (C,F), 10 (G, N), 20 (H, L), 250 (I), 80 (J, O), 100μm (M).

Pupa (Figs. 3C, 6 and 7G). Dark brown (Fig. 7G), 3.92–4.08mm (min–max length). Cocoon-cutter with three projections; the central one lanceolate, longer and wider, with serrated border; the lateral ones shorter, hook-shaped (Fig. 6A–B and D). Clypeus slightly bilobed, with two pairs of small setae (Fig. 6B). Antenna long and filiform, reaching the last abdominal segments (Fig. 3C); proboscis extending to anterior margin of Ab 2; anterior, median and posterior legs reaching Ab 3, Ab 4 and Ab 7, respectively; forewings extending to the posterior portion of Ab 5 (Figs. 3C and 6E). A set of small, posteriorly directed spines, dorsally at the center from second to seventh abdominal segments (Fig. 6I); on tergum of the same abdominal segments, also one pair of stout spines and one pair of posteriorly directed small setae (Fig. 6E, F and I). A pair of medium-sized setae laterally on meso- and metathorax. One pair of setae with fine apex on pleura from Ab 2-5 (Fig. 6G); from Ab 6–7 the setae have clavate apex (Fig. 6H). Open spiracles present on Ab 2–7 (Fig. 6G). The eighth abdominal segment presents one pair of microsetae dorsolaterally on tergum, one pair of spiracles partially closed and one pair of medium-sized setae posteriorly directed (Fig. 6J and K). One pair of posteriorly directed, digitiform caudal processes on last abdominal segment (Fig. 6J–L).

Fig. 6.
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Scanning electron micrographs of P. hemera pupa: (A) head, lateral view; (B) setae over clypeus, ventral; (C, D) cocoon-cutter, ventral and dorsal; (E) terga of abdominal segments Ab 3-4, dorsal; (F) detail of segment Ab 3, dorsal; (G) lateral seta with fine apex, adjacent to spiracle on abdominal segment Ab 4, dorsal; (H) lateral seta of Ab 7 with clavate apex, dorsal; (I) detail of tergum of Ab 3, lateral; (J–L) last abdominal segments, lateral, dorsal and ventral. Scale bars: 200 (A), 80 (B), 100 (C, D, G, K, L), 400 (E), 150μm (F, H, I, J).

Fig. 7.
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Natural history of P. hemera: (A) Host plant, Daphnopsis fasciculata at the type locality; (B) seedling of D. fasciculata with several mines; (C) leaf with a single P. hemera mine on adaxial surface (closed and open arrows indicate the beginning and final portions of the mine); (D) sap-feeding larva, latero-dorsal view; (E) spinning larva, latero-dorsal; (F) pupal cocoon, dorsal; (G) pupa, dorsal; (H) pupal exuvium protruded from cocoon after adult emergence. Scale bars: 40 (B), 3 (C), 1 (D, E, G, H), 0.3mm (F).

Etymology. Hemera, in Greek mythology is the daughter of the night and represents the divinity that personifies the daylight, here making an allusion to the light and bright color of the forewings of this species.

Distribution. P. hemera is known only from its type locality, the Dense Umbrophilous Forest (=Atlantic Forest sensu stricto), CPCN Pró-Mata, São Francisco de Paula municipality, Rio Grande do Sul, Brazil.

Host plant (Fig. 7A). D. fasciculata (Meisn) Neveling (Thymelaeaceae). The host plant of P. hemera occurs as either a shrub or a small tree, endemic to Brazil and occurring in the following regions: Midwest (Distrito Federal), Southeast (Espírito Santo, Minas Gerais, Rio de Janeiro and São Paulo) and South (Paraná, Santa Catarina and Rio Grande do Sul) (Rossi, 2017).

Life history (Fig. 7B–H). Eggs of P. hemera are deposited on the adaxial leaf surface. After eclosion, the sap-feeding larva (Fig. 7D) penetrates the leaf blade starting the mine construction. In the beginning the mine is narrow and serpentine-shaped, increasing in width during development (Fig. 7B). Centrally along the mine a path of black feces left by the larva can be seen by transparency (Fig. 7B). Mines are initially constructed within the adaxial epidermal cells (Fig. 8A) by cutting the anticlinal cell walls (Fig. 8B). Later the larva goes deeper into leaf tissues and install within palisade parenchyma cells, also by cutting the anticlinal cell walls (Fig. 8C). The epidermal cells remain intact over the intermediary mine (Fig. 8C–E), while fragments of the anticlinal cell walls of the palisade parenchyma cells remain in the lateral portions of the mine (Fig. 8D) but are totally consumed in the central portion (Fig. 8E).

Fig. 8.
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Transverse histological sections of P. hemera mine on Daphnopsis fasciculata leaf. (A) initial portion (location indicated by dashed line in Fig. 7C); (B) detail of initial portion (enlarged area marked with a rectangle in A); (C) intermediate portion (location indicated by unbroken line in Fig. 7C); (D, E) details of intermediate portion (enlarged areas marked with rectangles in C). Asterisks indicate intact cells of epidermis. Closed and open arrows indicate cellular fragments left on epidermis and palisade parenchyma after feeding. Ab, abaxial surface of epidermis; Ad, adaxial surface of epidermis; Lm, leaf mine; Pp, palisade parenchyma; Sp, spongy parenchyma. Scale bars=100 (A, C), 50μm (B, D, E).

The spinning larva (Fig. 7E) does not feed and is responsible for the construction of the cocoon. This is endophylous and constructed at the final portion of the mine, and completely covered by whitish silk that provokes a slight leaf wrinkling (Fig. 7F). The pupal cocoon is ruptured by the pupa's cocoon-cutter (Fig. 7G) during adult emergence. Later, the pupal exuvia is seen partially protruding from the cocoon (Fig. 7H). More than one mine were found in most of the mined leaves. A few mines were found on full grown D. fasciculata trees. The greatest density of leaves mined by P. hemera was found on young plants, especially those located in humid sections of trail borders existing in the type locality. The larvae were found active in the field from February to August, suggesting the species is multivoltine.

Phylogenetic inference

A total of 660 nucleotide sites were analyzed, of which 268 (40%) were variable. In accordance with our phylogenetic hypothesis, the monophyly of the new species was recovered in both methods of inference (distance and model-based), with high node support values (Fig. 9). Since the topologies were slightly different, all are presented. The sister relationship of P. hemera was not well resolved: node supports were quite low in all trees (NJ, Bayesian and ML). In the NJ and ML inferences, P. hemera clustered with P. saligna (Fig. 9). In the Bayesian analysis, the closest related lineage was P. phoebus; however, the node support (BPP) was very low. The genetic distance estimated between P. hemera and other taxa ranged from 14% to 20% (±2%) (see Supplementary Material; Fig. S1). The distance of the new species to the outgroups was 24% (±3%).

Fig. 9.
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Phylogenetic reconstruction for P. hemera based on 660bp of the mitochondrial cytochrome oxidase c subunit I gene (‘DNA barcode’ region) using three methods: (A) distance (Neighbor-joining), (B) Maximum likelihood, and (C) Bayesian inference. Numbers above branches indicate node support (bootstrap for A and B and posterior probability for C). Samples in blue highlight the new taxon.

Discussion

P. hemera is described here based on morphological and molecular characters, showing enough stable characters in both types of analysis to separate it clearly from other congeneric species. Phylogeny showed a monophyletic status for the new species, but did not resolve close relationships. Different methods of reconstruction retrieved different results for sister taxa of P. hemera, although with low support. In the NJ and ML trees it was close to P. salignela, whereas in Bayesian inference it clustered with P. phoebus, a sympatric species from the same region of Atlantic Forest (Brito et al., 2017a). A comparative assessment of genetic distance to other Neotropical Phyllocnistis indicates a minimum of 12% for P. hemera to P. citrella and P. vitegenella, which indicates a great amount of divergence sampled in Phyllocnistis. Such high diversity is likely reflected in the evolutionary history reconstructed for the genera, e.g. by the absence of unknown lineages in the phylogeny, suggested by the putative long-branch attraction apparently found in P. hemera and P. salignela relationship (NJ and ML trees).

The forewing pattern of P. hemera resembles those described for congeneric species in the Neotropical region (Brito et al., 2017a), regarding number of fasciae and strigulae, presenting one well-marked longitudinal fascia, two visible transversal fasciae, three costal and four apical strigulae, the last emerging from the apical spot. Comparing P. hemera to the other congeneric Neotropical species the greatest similarity is found with P. bourquini Pastrana, a species described for Argentina; both species share forewing light yellow fasciae, but they can be contrasted by the morphology of the third and fourth fasciae, which are separated in P. bourquini and united in P. hemera. Another character that differentiates P. hemera from P. drimiphaga is the male valva; in P. drimiphaga the valva is divided into two lobes (Kawahara et al., 2009), the dorsal being more prominent than the ventral, while P. hemera has the distal portion of the valva undivided.

As already mentioned, the presence of ambulatory calli on the ventral region of meso- and metathorax in the spinning larva has already been described for P. ourea, also a species from the Atlantic forest. Ambulatory calli are not exclusive characters of Phyllocnistinae, and they also occur in representatives of the Oecophyllembiinae – Angelabella Vargas & Parra, Eumetriochroa Kumata, Metriochroa Busck and Prophyllocnistis Davis, a gracillariid subfamily closely related to the Phyllocnistinae (Vargas and Parra, 2005; Kumata, 1998; Busck, 1900; Davis, 1994; Kawahara et al., 2017). Stemmata have already been described for the sap-feeding larvae of some Phyllocnistis species of the Neotropical region, such as P. ourea Brito & Moreira and P. selene Brito & Moreira, which have two stemmata on the lateral region of the head. Thus P. hemera is the only species known so far with a single stemma followed by a microseta (Brito et al., 2017b).

The species described here is the first gracillariid associated with a Thymelaeaceae plant. As already described, D. fasciculata has a broad distribution in southeast Brazil (Rossi, 2017), suggesting that P. hemera might be distributed in other regions not evaluated in this study, which should be further explored. Interestingly, results presented here regarding the histology of the mine give further support for the existence of a broad feeding habit in relation to use of leaf tissues within Phyllocnistis. In other words, our data suggest that although highly species-specific to a given type of leaf tissue, species within this genus may use any kind of tissue, including the epidermis (e.g. P. citrella, Archor et al., 1997), spongy parenchyma (e.g. P. tethys, Brito et al., 2012), and palisade parenchyma (e.g. latter instars of P. hemera, as demonstrated here). The ultimate factors that lead to this variation in tissue usage remain to be determined. P. hemera uses the epidermis initially, and moves to the palisade parenchyma in the intermediary phase of the mine, which may indicate a search for better nutritional resources. Epidermal cells may function as lenses for capturing sunlight and as a consequence have large water-filled vacuoles and scarce cytoplasm. Palisade parenchyma cells, on the other hand, are commonly cytoplasm-rich, and may accumulate energetic molecules (Evert, 2006; Bowes and Mauseth, 2008).

The Atlantic forest is known for its extreme diversity with approximately 50% of the species considered endemics (Stemann et al., 2009). However, only five species of Phyllocnistis are known for the region (Brito et al., 2012, 2017a). As suggested by Brito et al. (2016), the vast majority of Neotropical gracillariid species remain to be discovered. Data presented in this study regarding a new species of Phyllocnistis for the Atlantic forest support further the hypothesis proposed by such authors in the sense that the scarcity of species described for the region in largely due to a lack of sampling, associated with a taxonomic impediment.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

We thank the Instituto de Meio Ambiente (IMA/PUCRS) for allowing access to the study area and for providing assistance with fieldwork at the CPCN Pró-Mata. We are also grateful to Thales O. Freitas (UFRGS) for use of laboratory facilities for molecular analyses, and to CME-UFRGS for the use of equipment and assistance in scanning electron microscopy. We thank Lafayette Eaton for editing the text. We are also grateful to Shigeki Kobayashi (Osaka Prefecture University) and two anonymous referees, whose comments improved substantially the first version of the manuscript. J. Fochezato was supported by a CAPES Master's Program Fellowship. GL Gonçalves received a research fellowship from FAPERGS (Process 16/2551-0000485-4). GRP Moreira and RS Isaias were supported by CNPq fellowships.

Appendix A
Supplementary data

The following are the supplementary data to this article:

mmc1.docx

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Corresponding author. (Gilson R.P. Moreira gilson.moreira@ufrgs.br)
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