Biology and systematics of the New World Phyllocnistis Zeller leafminers of the avocado genus Persea (Lepidoptera, Gracillariidae)

Abstract Four New World species of Phyllocnistis Zeller are described from serpentine mines in Persea (Family Lauraceae). Phyllocnistis hyperpersea,new species, mines the upper leaf surfaces of avocado, Persea americana Mill., and red bay, Persea borbonia (L.) Spreng. and ranges over much of the southeastern United States into Central America. Phyllocnistis subpersea,new species, mines the underside and occasionally upper sides of new leaves of Persea borbonia in southeastern United States. Phyllocnistis longipalpa, new species, known only from southern Florida also mines the undersides of new leaves of Persea borbonia. Phyllocnistis perseafolia,new species, mines both leaf surfaces and possibly fruits of Persea americana in Colombia, South America. As in all known species of Phyllocnistis, the early instars are subepidermal sapfeeders in young (not fully hardened) foliage, and the final instar is an extremely specialized, nonfeeding larval form, whose primary function is to spin the silken cocoon, at the mine terminus, prior to pupation. Early stages are illustrated and described for three of the species. The unusual morphology of the pupae, particularly the frontal process of the head, is shown to be one of the most useful morphological sources of diagnostic characters for species identification of Phyllocnistis. COI barcode sequence distances are provided for the four proposed species and a fifth, undescribed species from Costa Rica.

. COI distance tree of five Persea-mining species of Phyllocnistis based upon neighbor-joining analysis with Kimura 2-parameter model.
CuA2 are lost in the forewing; the base of M is absent within the forewing discal cell, and the cell is open in the hindwing. The apex of the forewing is unusual in being greatly attenuated with Rs4 extending to the apex.
The morphology of the male genitalia is relatively simple and usually characterized by a relatively broad vinculum, narrow tegumen and slender valvae, and specieslevel differences are modest relative to other gracillariids or Lepidoptera in general.
Homology of what appears to be segment 10 is uncertain and consists of a mostly membranous cylinder extending caudally from the sclerotized tegumen that mostly encompasses the anal tube (Fig. 16A).
The female genitalia, although characteristic for the genus, likewise appears relatively uniform among species, consisting primarily of very short papillae anales, a comparatively large, oblong corpus bursae often containing a pair of similar, fusiform signa bearing a short median projection (Fig. 16D); a pair of similar, slender, elongate ducts that extend from usually opposite ends of the corpus bursae, one (ductus bursae) leading to the ostium and the other (ductus spermathecae) to the spermatheca.
The larvae of Phyllocnistis are among the most specialized Lepidoptera (Trägårdh 1913). Four instars appear to be the norm (Condrashoff 1962, Wagner and Davis unpublished data), with the first three instars possessing a sapfeeding morphology and behavior (Davis 1987). Sapfeeding instars create a long serpentine, subepidermal mine on either the upper or lower surfaces of the host leaf. A few species also form subepi-dermal mines on stems and various fruits, including avocado. A characteristic, median frass trail extends the length of the mine, usually as a dark, unbroken line. The fourth instar is a highly specialized, apodal, non-feeding instar whose primary function is to spin the cocoon, at the mine terminus, prior to pupation.
In contrast to the conservative morphology of the larval and adult stages, the pupae of Phyllocnistis are structurally diverse, particularly with regard to the development of the frontal process (cocoon-cutter) of the head (Figs 6A, B; 12A, B; 14A, B; see also Kawahara et al. 2009). In addition, the mid-dorsal areas of abdominal terga 3-7 possess a mostly symmetrical cluster of recurved spines that frequently differ in their arrangement and form among species. Given the uniformity in both male and female genital characters in Phyllocnistis, it is surprising to us to find reliable species-level differences in pupal morphology across what appear to be closely-related congeners feeding on Persea. We certainly encourage others to collect and illustrate pupae whenever new species are described in the genus.
Consistent differences in wing patterns were noted for each of the four species described here-although such are easily abraded if specimens are not collected and prepared with care. We note that our descriptions are based solely on reared material and thus might differ in appearance from flown specimens. Because of the general similarity of both male and female genitalia that exists among most members of Phyllocnistis, species identification relying upon standard genitalic characters may be impossible at times. We expect that the application of COI barcoding will be especially useful in this large genus of minute moths.
The plant genus Persea includes approximately 200 species worldwide, with a majority of the species concentrated in Central America and southeast Asia (Mabberley 2008); Kopp (1966) recognized 81 species for the New World. The most important species economically is the avocado, Persea americana Mill., which is grown throughout the tropics for its fruit.
Half of the Phyllocnistis found in eastern North America are hostplant specialists on archaic families of woody plants: 3 species feed on Persea in the Lauraceae, 2 on Magnolia and Liriodendron in the Magnoliaceae, and 1 on Liquidambar in the Hamamelidaceae (or Altingaceae), all plant families that date to the Cretaceous. Ninety seven-million-year-old (more recently estimated at 102 mya, Brenner et al. 2000) phyllocnistine leafmines provide the oldest fossil evidence of Ditrysian Lepidoptera (Labandeira et al. 1994). Phylogenetic studies will be needed to ascertain if the association of the genus with archaic plant families is a testament to the ancient and often conservative nature of insect-plant associations (Farrell et al. 1992, Labandeira et al. 1994, Wilf et al. 2000 or the result of more recent host colonizations. The damage inflicted by Phyllocnistis larvae feeding on avocado may vary according to region and the species of miner involved. Wysoski et al. (2002) reported major damage to avocado caused by an unknown Phyllocnistis in Peru that could reduce tree vigor, but only minor damage on avocado by Phyllocnistis in Florida. Larval feeding by P. perseafolia on avocado leaves in Colombia is known to cause serious damage (Francisco Posada,in litt.,Fig. 4). Possibly the damage to avocado in Peru reported by Wysoski et al. (2002) was also produced by P. perseafolia.

Material
The material examined is deposited in the collections listed below: BMNH The Natural History Museum (formerly the British Museum (Natural History)), London, United Kingdom.

Collecting and rearing.
Nearly all of the adults in this study were reared from their plant hosts. Leaves containing mines with larvae were placed in plastic bags or large plastic tubes and examined daily thereafter. Newly-eclosed adults were killed with ammonium hydroxide fumes or frozen, pinned, and spread. Some larvae representing different instars and pupae were fixed in Pampel fluid and preserved in 75% ethanol. Samples of alcohol-preserved larvae and pupae were gently washed in 409® detergent, then dried in a critical point drier, sputter coated with 20-25 gold palladium 60:40 alloy, and photographed with an Amray 1810 scanning electron microscope. Genitalic dissections were cleared by heating in hot 10% KOH for ~ 30 minutes, and subsequently cleaned and stained with either 2% chlorazol black E or mercurochrome solutions. All genitalic illustrations were drawn from dissections temporarily stored in glycerine, which were later permanently embedded in Euparal or Canada balsam. Genitalic terminology follows Klots (1970). Molecular analysis. DNA sequences were produced at the Biodiversity Institute of Ontario, University of Guelph, Canada. DNA was extracted from legs or entire bodies of adult moths using a QIAGEN DNeasy Tissue Kit. Primers LepF1 and LepR1 (Herbert et al. 2004) were used to obtain a 658 base pair fragment of COI with a standard thermocycling regime (Hajibabaei et al. 2006). Sequences are available at the National Center for Biotechnology Information GenBank database and at the Barcode of Life Database (BOLD). Neighbor-joining (NJ) trees were generated from 14 nucleotide sequences as implemented in BOLD (Ratnasingham and Hebert 2007).

Results
Exemplars of all five species clustered as each other's closest neighbors. A compressed subtree ( Fig. 1) of the COI barcode sequences from the 14 specimen samples was computed using Molecular Evolutionary Genetics Analysis (MEGA) version 4 (Tamura et al. 2007). Only partial sequence data were obtained for P. subpersea. Uncorrected pairwise distances exceeded 10% between species. P. hyperpersea, the only species in our study that preferentially mines upper leaf surfaces, clustered outside of the group that mine lower leaf surfaces.

Diagnosis:
Phyllocnistis hyperpersea is the smallest of the species treated here with FW lengths < 2.3 mm. The short labial palpi (less than height of eye) and prominent black apical spot, taken together, distinguish hyperpersea from the other Persea-feeding Phyllocnistis. The second costal fascia is weakly developed and does not fuse with the transverse fascia as in other Phyllocnistis treated here. Hind tarsomere 3 is more likely to be black than that of the other species. The black fringe scales about the tornus are less conspicuous: fewer in number, narrower, and less blackened relative to those of Phyllocnistis subpersea with which it commonly co-occurs. The frontal process of the pupa extends forward as a relatively large, broadly triangular, acute spine (Figs 6A-B). Adult ( Fig. 2A): Length of forewing: 1.9-2.2 mm. Head: Frons shiny white, smooth glabrous, with subtle faint orange tints over vertex. Flagellomeres with orange-fuscous luster above. Labial palpus white, reduced, length less than height of eye.
Thorax: Patagia and tegulae with silvery stramineous to orange tints. Forewing with longitudinal fascia usually ending before joining transverse fascia, thinly edged with black scales above and below except distad. Transverse fascia usually complete, leaving costal margin at 45° angle; usually more thickly edged with black scales along proximal side; distal side somewhat rounded with black, edge-scaling weakened medially. Second costal fascia poorly differentiated, not fusing with transverse fascia. Apical spot of black scales well developed. Costal and apical strigulae modestly differentiated, often only two of latter evident. Black fringe scales about tornus only modestly differentiated: few in number, not strongly raised, and not appreciably broadened. Dorsal and outer surfaces of foretibiae and foretarsi, and to lesser extent those of mesothoracic legs, with fuscous metallic orange; third tarsomere of hindleg often darkened; otherwise legs mostly silvery white and unmarked.
Abdomen: Silvery white and unmarked.
Male Genitalia (Figs 16A-C): Uncus absent. Tegumen complex, consisting of narrow, sclerotized dorsal arch, continuing caudally as far as apex of valva as an elongate, mostly membranous, basally spinose cylinder which encloses anal tube. Vinculum well developed, ~ 0.5× length of valva, U-to V-shaped with relatively narrow anterior end. Valva simple, relatively long, ~ 2.0× length of vinculum, very slender and straight; broad at extreme base, then narrowing along middle, becoming slightly broader over apical third; apex of valva evenly rounded; basal apodeme of valva directed mesad at nearly right angle to valva. Transtilla arising from mesal base of valva as an elongate, acute process, and continuing mesally to articulate at midline with process from opposite valva. Aedeagus slender, weakly sclerotized, externally finely wrinkled cylinder ~ half length of valva; cornuti absent; phallobase greatly extended as membranous tube ~ 6× length of aedeagus; terminal hood of phallobase abruptly inflated and curved at ~ right angle to phallobase.
Last instar larva not examined, but probably similar to that of P. subpersea. Larval Mine (Fig. 3A): A long, slender, serpentine gallery, with a relatively broad, dark brownish, median frass trail, almost always located on the upper (adaxial) side of the leaf (only 1 under (abaxial) side mine found). The egg is deposited on the upper leaf surface away from the midrib. Mine begins on one side of the blade, but after much of one side is consumed, crosses over near the leaf apex to the other side. The median frass line is unusually broad for a species of Phyllocnistis, resembling more that of the Chilean genus Prophyllocnistis (Davis 1994). As previously noted (Davis 1994),   the mines of hyperpersea are also similar in general morphology to early Cenomanian phyllocnistine leafmines (Labandeira et al. 1994). Pupation occurs in the lamina, away from the leaf edge (~ 5-7 mm in diameter) in a circular nidus, similar to that fashioned by Prophyllocnistis. The serpentine portion of the mine begins as a narrow tract ~ 0.3 mm wide and gradually enlarges before the pupation chamber to a width of ~ 2-2.5 mm. The median frass line is ¼ of the mine width in the early instars and gradually broadens to more than half the mine width.
Pupa (Figs 6A-7D): Length of largest pupa 2.8 mm, maximum diameter 0.7 mm. Vertex with relatively large, broadly triangular, acute frontal process (cocoon-cutter) similar to that of Metriochroa psychotriella Busck, but with base constricted slightly on each side (Fig. 6A); lower frons with 2 pairs of short frontal setae. Antenna long and straight, extending almost to 7th abdominal segment (A7); forewing extending almost to A6. Abdominal setae generally short except for greatly lengthened SD1 on A2-7; apex of SD1 on A2-7 slightly enlarged, but not spatulate; abdomen with 6 mid-dorsal pairs of spine clusters (Figs 6C-D) beginning near anterior margins of terga 2-7; each cluster with series of similar, low, strongly recurved spines arranged in 4 irregular columns of about 4-6 ranks; pair of much larger, strongly recurved spines immediately lateral to central cluster and adjacent to seta D1 on A4-7; sternum A6 with spinules evenly scattered over surface (  Distribution: This species has been found from Nansemond and Virginia Beach Counties, Virginia, USA, south along the lowland Atlantic coastal region to the Florida Everglades. Adults in the collections of the USNM, from avocado, some with associated mines and collected at various localities in Honduras, may also represent this species. No pupae were available for study and attempts to barcode two specimens were unsuccessful. Mines with associated pupae of what appear to be hyperpersea have also been intercepted on shipments of avocado within the United States from unspecified localities in Mexico. Some fluctuation in the northern limits of this leafminer may have occurred in recent years. As late as June 8-11, 1974, DRD and Mignon Davis found mines of P. hypersersea common on leaves of Persea borbonia within First Landing State Park and Dismal Swamp, Virginia. On March 14, 1992 and during August 1993 no mines could be found at First Landing State Park (Dismal Swamp was not visited in 1993). These localities have not been surveyed for leafminers since 1993.
Etymology: The specific name is derived from the Greek, hyper (above, over) and the generic plant name of its host, Persea, in reference to the characteristic leafmining habit of the larva on the upperside of the leaf. The specific epithet is a noun in the nominative singular. Phyllocnistis subpersea Davis and Wagner, sp. n. urn:lsid:zoobank.org:act:20FB0553-E021-401D-9475-9F0B06E115DA http://species-id.net/wiki/Phyllocnistis_subpersea Figs 2B, 3B, 5B, 8A-13F, 17A-E Diagnosis: P. subpersea is the phenotypic outlier among the four Persea-feeding species that we treat here: the row of raised, broadened, black-tipped fringe scales along the tornal margin of the forewing is unique. The apical dot tends to be poorly developed. It is the only Persea feeder that consistently has a subbasal fuscous spot along the inner margin of the forewing (P. hyperpersea sometimes has fuscous scales in the subbasal area of the forewing, but these do not form a spot but rather extend as a diffuse patch to the wing base). The hind tarsomeres (especially segments 2-3) often bear orange to fuscous scaling that is somewhat more pronounced than that of the species that follow. The ductus bursae is broadly joined to corpus bursae. The frontal process of the pupa consists of a pair of stout conical spines arising near the apex, and a single, more subapical, strongly curved spine from the upper frons (Figs 12A-C).
Thorax: Patagia and tegulae with stramineous to orange tints. Longitudinal fascia joining transverse fascia but weakened distad, edged with black scales above and below, with those below more consistently present distad. Transverse fascia leaves costal margin at 45° angle; lower arm where it leaves inner margin poorly defined, often fusing with diffuse subbasal patch of fuscous scales. Second costal fascia usually fusing with transverse fascia distally. Apical spot weakly developed, small, fuscous but not black in our material, composed of apices of a few to several scales. Apical strigulae vague and poorly differentiated. Black fringe scales about tornus broadened, conspicuously blackened apically, raised appreciably above plane of wing. Legs silvery white with exception of faint orange luster to dorsal and outer surfaces; foretibiae, foretarsi, and distal tarsomeres sometimes modestly darkened; hind tarsi with tarsomeres 2-4 with faint orange to fuscous tint.

Spinning (last) instar
Larval Mine (Figs 3B): Similar to that described for P. longipalpa. A long, slender, serpentine gallery, containing a dark, median frass trail, on the underside or occasionally the upperside of the leaf, with pupation occurring in a slightly enlarged, elliptical chamber at the mine terminus along a leaf edge. The egg is deposited away from the midrib, usually on the lower side of the leaf. Mine width increases from ~ 0.3 mm broad to a maximum width of ~ 2-2.5 mm; width of the frass trail is usually about half the mine width.
Pupa (Figs 12A-13F): Similar to P. hyperpersea except: length of largest pupa 3.2 mm. Vertex with pair of stout conical, spines arising near apex, and single, slender, more subapical, strongly recurved spine from upper frons (Figs 12A-C). Abdomen with 6 pairs of small, sclerotized, oval, median pits near anterior margins of terga 2-7; each sclerotized pit giving rise to 2 columns of low, strongly recurved spines, relatively larger than in hyperpersea and fewer in number, arranged in about 1-3 ranks (Figs 12D-E); pair of slightly larger, strongly recurved spines immediately lateral to caudal end of median cluster and nearly contiguous to seta D1; A2-7 with SD1 setae greatly lengthened, apices spatulate (Fig. 13A); sternum A6 with spinules evenly scattered over surface as in hyperpersea (Fig. 13D); A10 with pair of relatively large, stout, caudal processes directed mostly laterally (Figs 13E-F). Etymology: The specific name is derived from the Greek, sub (under) and the generic plant name of its host, Persea, in reference to the characteristic leafmining habit of the larva usually on the underside of the leaf. The specific epithet is a noun in the nominative singular. Diagnosis: Phyllocnistis longipalpa can be distinguished from other Persea-feeding Phyllocnistis in the southeastern United States, by its long, slightly upcurved labial palpi (> height of head). The apical spot is poorly developed, which distinguishes it from hyperpersae. It lacks the run of raised black scales from the forewing tornus characteristic of P. subpersea.
Thorax: Patagia and tegulae with subtle, silvery, stramineous to orange tints. Longitudinal fascia joining transverse fascia, edged with black scales above and below, with those below more consistently present distad. Transverse fascia leaves costal margin at 45° angle; proximal edge of transverse fascia where it leaves the inner margin vague, composed of 2-3 rows of dark scales. Second costal fascia fusing with transverse fas- cia distally. Apical spot poorly differentiated; likewise apical strigulae vague and poorly developed. Black fringe scales about tornus little broadened and not conspicuously elevated above plane of wing. Legs essentially silvery white and unmarked with exception of faint orange luster to dorsal and outer surfaces of foretibiae and foretarsi; distal tarsomeres sometimes modestly darkened.
Abdomen: Silvery white and unmarked. Male Genitalia (Figs 18 A-C): Similar to P. hyperpersea and subpersea except apex of valva not evenly rounded, instead more oblique and extended dorsad; total length of valva ~ 2.0× length of vinculum; basal apodemes of valva less divergent than in other species, with ventral apodeme strongly curved (Fig. 18B). Aedeagus ~ 0.65× length of valva.
Larva and pupa: Not examined. Larval Mine: Similar to that described for P. subpersea. A long, slender, serpentine gallery, containing a dark, narrow, median frass trail, present on the underside of the leaf, with pupation occurring in a slightly enlarged, elliptical chamber at the mine terminus along the leaf edge. Etymology: The specific name is derived from the Latin longus (long) and palpus (feeler), in reference to the elongate labial palpi, which are diagnostic for this species. The specific epithet is a noun in the nominative singular.
Remarks: We initially "discovered" Phyllocnistis longipalpa intermixed among our series of P. subpersea in 2009. As noted in the diagnosis, adults are reliably distinguished from that species by their longer labial palpi, the absence of the numerous, broad, raised tornal scales, and absence of the fuscous subbasal spot along the inner margin on the forewing which occurs in most subpersea. The larvae form serpentine mines on the undersides of new leaves, similar to those of P. subpersea. Diagnosis: Phyllocnistis perseafolia is the largest of the Persea-feeding species: forewing lengths typically exceed 2.6 mm, with that of intact specimens often reaching lengths of 2.9 or more mm. The well-developed black apical dot distinguishes P. perseafolia from all but P. hyperpersea. The forewing is the palest of the four Phyllocnistis described here: the black scaling--in the subbasal and tornal areas, as well as that which edges the longitudinal and transverse fascia--is reduced relative to the other species described here. The transverse fascia is often interrupted through the center of the forewing because the arms are so strongly angled outward that they may not meet; likewise the longitudinal fascia frequently does not join the transverse fascia in persaefolia for the same reason.
Thorax: Scaling of patagia and tegulae damaged. Longitudinal fascia ending before transverse fascia; anterior side ill-defined with orange scales often reaching to costa, especially towards base of wing; lower side straight and clearly delineated; fuscous scales, if present, only along lower edge. Transverse fascia edged inwardly and outwardly with black scales; upper arm leaving costal margin at 30-35° angle, with distal reach curving toward apical dot; often interrupted through cell; arm of transverse fasciae from inner margin more strongly edged with black along outer edge; proximal edge of fascia where it leaves the inner margin vague, with faint dark scaling. Second costal fascia ill-defined, with little black scaling, sometimes conjoined with transverse fascia. Three costal and three apical strigulae modestly differentiated. Apical spot well developed. Black fringe scales about tornus reduced in extent, many replaced with smoky orange fringe scales; none raised appreciably above plane of wing. Legs essentially silvery white and unmarked with exception of faint orange luster to dorsal and outer surfaces foretibiae and foretarsi and distal tarsomeres sometimes with smoky overscaling.
Larva: Not examined. Larval Mine (Figs 3C, 4A-B): Similar to that described for P. subpersea. A long, slender, serpentine gallery, containing a dark, narrow, median frass trail, present on either the underside or upperside of the leaf, with pupation occurring in a slightly enlarged, elliptical chamber at the mine terminus along the leaf edge. Serpentine mines of possibly this species have also been observed by Francisco Posada on avocado fruit at the type locality.
Pupa (Figs 14A-15D): Similar to P. hyperpersea except: Length of largest pupa 3.4 mm. Vertex similar to that of P. vitegenella Clemens in possessing single, large apical spine (tip of spine broken in all 3 pupae examined) with minutely serrated, low ridge descending laterally from spine (Figs 14A-B). Abdomen with apices of greatly lengthened SD1 seta on A2-7 spatulate; mid-dorsal cluster of spines on abdominal terga 2-7  (Figs 14C-D) with median series of low, strongly recurved spines relatively larger than in hyperpersea and fewer in number, arranged instead in 2 short columns as in subpersea in about 2 ranks; 3-4 smaller, scattered spines immediately caudad to larger, median spine rows; pair of slightly larger, strongly recurved spines immediately lateral to caudal end of median cluster and nearly contiguous to D1 seta; sternum A6 with spinules arranged in ~ 20 longitudinal ridge-like rows (Fig. 15D); A10 with pair of relatively large, stout, caudal processes directed mostly laterally (Figs 15A-C)  Distribution: Currently reported only from the type locality in the Department of Caldas, west-central Colombia, but probably widespread over northern South America wherever avocado is cultivated.
Etymology: The specific name is derived from the generic plant name of its host, Persea and the Latin, folium (leaf ), in reference to its leafmining habit. The specific epithet is a noun in the nominative singular.
Remarks: All adults examined were received unpinned, unspread, and slightly damaged to the extent that we are uncertain of some scaling characters. The apex of the large frontal process was broken in all 3 pupae available for study. A fragment of one spine remaining in a vial with a pupa of perseafolia was observed to be slightly recurved, but not to the extent observed in pupae of the North American Phyllocnistis vitegenella. The spatulate apex of abdominal SD1 setae of perseafolia (Fig. 14F) is notable in being the broadest of the three species examined.
One other species of Phyllocnistis, P. aurilinea (auriinea [sic]) Zeller, has been described from Colombia (Bogotá). However, that species mines the leaves of a distinctly different host in the family Ericaceae, "Uva camarona" (Zeller 1877), (probably Macleania rupestris A. C. Smith, according to W. and J. De Prins 2011), Because larvae of Phyllocnistis and related gracillariids are known to be stenophagous, P. aurilinea is believed to represent a different species from P. perseafolia, whose larvae are leafminers in Lauraceae.