Systematics and biology of the new genus Macrosaccus with descriptions of two new species (Lepidoptera, Gracillariidae)

Abstract The new genus Macrosaccus Davis & De Prins is proposed for three species formerly assigned to the genus Phyllonorycter: Macrosaccus robiniella (Clemens), Macrosaccus morrisella (Fitch), and Macrosaccus uhlerella (Fitch); two new, closely related species: Macrosaccus neomexicanus Davis and Macrosaccus gliricidius Davis, are also proposed. Descriptions of the adults, pupae, larvae, life histories, and distributions are supplemented with photographs, line drawings, and scanning electron micrographs. Larvae of all species are serpentine/blotch leaf miners on various genera of the plant family Fabaceae. The genus is endemic to the New World, with the invasive species Macrosaccus robiniella now widely established in Europe.


introduction
The gracillariid subfamily Lithocolletinae includes 503 species (De Prins and De Prins 2011) assigned to 7-9 genera according to different authors (Kumata 1993;Davis and Robinson 1998;Kuznetzov and Baryshnikova 2001). Lithocolletinae are very small moths (less than 10 mm in wing expanse) with often brilliantly coloured forewings of ochreous-orange or reddish-brown ground colour, marked by white or silvery white striae and fasciae (Chapman 1902;Vári 1961;Kumata 1961Kumata , 1963Kumata , 1993Watkinson 1985;Kuznetzov 1981;Stekol'nikov 1987, 2001;Davis and Robinson 1998;Kuznetzov and Baryshnikova 2001). The subfamily has a worldwide distribution, but is more species-rich in temperate zones: 273 species are known from the Palaearctic, 142 species from the Nearctic, 49 species from the Oriental, 26 species from the Afrotropical, 16 species from the Neotropical, and only 8 species from the Australasian region (De Prins andDe Prins 2005, 2011). The hostplant range within this subfamily is broad. Lithocolletinae feed on no less than 36 families of plants (De Prins andDe Prins 2005, 2011). Approximately 32 families of dicotyledonous plants serve as hosts for Phyllonorycter moths, compared to 11 families for Cameraria (De Prins andDe Prins 2005, 2011). Deschka (1993) lists 13 hostplant families for Cameraria, but we failed to find any published record related to Cameraria larvae feeding on either Oleaceae or Sapotaceae. Seven lithocolletine genera (Cameraria, Phyllonorycter, Chrysaster, Hyloconis, Neolithocolletis, Porphyrosela and Protolithocolletis) mine plants of the family Fabaceae, with the latter five genera feeding exclusively on Fabaceae (Robinson et al. 2007;De Prins and De Prins 2011). Probably many more taxa will be discovered in the southern hemisphere. However, even in the seemingly well-known European and North American Lithocolletinae fauna the generic assignment of some lithocolletine species is still questionable; for example, the species-rich genus Phyllonorycter, comprising about 400 species and having a world-wide distribution, has served for some time as a depository for several species of uncertain phylogenetic placement.
Characters defining genera within the Lithocolletinae are still being investigated. Most of these concern the life history and morphology of the preimaginal stages (Kumata 1993). With regards to adult morphology, Kumata (1993) diagnosed Lithocolletinae genera by the following characters: 1) wing venation, in particular the parallel condition of veins Rs and M near the base of the hindwing; 2) number of setae on apical part of tegumen in male genitalia; 3) development of the transtilla in male genitalia; 4) number and shape of signa in female genitalia; and 5) dark margins of whitish fasciae on forewings. Adult morphology does not always clearly separate genera: for example, 1) the wing venation in Phyllonorycter and Cameraria is identical (with the exception of C. fasciata); 2) a pair of setae on the apical part of the tegumen is present in Cameraria, Chrysaster and Porphyrosela (absent in Phyllonorycter); more than two pairs of tegumenal setae are found in Hyloconis and Cremastobombycia; 3) transtilla is incomplete in Hyloconis and Cameraria (with the exception of C. magnisignata, C. milletiae, C. palawanensis, C. pongamiae, and C. virgulata); 4) signa of the corpus bursae show a variety of shapes in Phyllonorycter; 5) most of the species in both Cameraria and Cremastobombycia show black margins distally on the whitish fasciae (Busck 1909;Ely 1918;Braun 1925;Kumata 1963Kumata , 1993Kumata , 1995Opler and Davis 1981;Powell and Opler 2009). Recently, a multidisciplinary approach was undertaken which incorporates adult morphology, chemical communication and DNA barcoding to resolve the generic assignment within Lithocolletinae (De Prins et al. 2009). Although this approach appears useful to assess the generic limits of Gracillariidae, it is applicable only to those few groups of species for which chemical communication has been studied (De Prins et al. 2009;Liblikas et al. 2009 and the references therein). Collecting efforts are beginning to reveal the existence of several previously unknown Lithocolletinae genera in the tropics. An evaluation of generic apomorphies will only be accomplished after a thorough phylogenetic study of Lithocolletinae genera, based on both molecular and morphological characters, is completed.
The purpose of this paper is to propose and diagnose a new lithocolletine genus, Macrosaccus, and to document the five species we recognize within this New World group. This study is long overdue because one species, Macrosaccus robiniella (Clemens), has become a serious invasive pest on the introduced Robinia pseudoacacia L. (Fabaceae) over much of Europe. With this contribution we also attempt to broaden the understanding of the generic definitions within Lithocolletinae. We transfer three previously known Phyllonorycter species to Macrosaccus, clarify the synonymy, and designate lectotypes whenever possible for the species-group taxa. Additionally, we provide DNA barcodes as identification aids and descriptions of two new congeneric species which also were reared from Fabaceae.

Methods
Collecting and rearing. Field investigations were carried out in Europe (Belgium), Canary Islands (La Palma), and in several states within the United States (Arizona, Illinois, Maryland, New Mexico). All specimens examined in this study were reared from species of Fabaceae which are summarized in Table 1.
Leaves containing mines with larvae were placed in plastic bags or rearing containers periodically moistening the lids protecting the specimens from drying out. Specimens were pinned, spread and mounted in the usual way for morphological examination. Some voucher samples of reared specimens were fixed in 100% ethanol for DNA analysis. Larvae and pupae collected on Robinia pseudoacacia L. were preserved in 75% ethanol.

Morphology
Adults were examined externally using either MZ12.5 or Nikon SMZ 1500 stereomicroscopes (maximum magnification 200×). Genitalia were prepared following table 1. Macrosaccus and four Phyllonorycter species that feed on Fabaceae.

Moth species
Host plant species Country Reference Macrosaccus gliricidius Davis, sp. n. Gliricidia sepium (Jacq.) Robinson (1976) with some modifications. After maceration of the abdomen in 10% KOH for 24 hours or by heating in hot 10% KOH for ~ 30 minutes, and subsequent cleaning and deionization, the male genitalia were stained with 2% eosine B, a mixture of 2% azophloxine and 2% acid fuchsin; the female genitalia were stained with a 1% chlorazol black E solution and embedded in Euparal or Canada balsam. Genital morphology was examined using a Leica DMLB microscope under magnifications of 150×, 200×, and 400×. The terminology follows Vári (1961), Klots (1970), Kumata (1993Kumata ( , 1995, and Kristensen (2003). Microslides for studies of wing venation were prepared following the technique suggested by Vári (1961) and applying modifications used by Hoare (2000). Some wing slides were cleaned, stained with saffranin, and mounted dry beneath a glass coverslip. For scanning electron microscopy, the immatures were immobilized by moment freezing at -27°C. Pupae were sputtered-coated with gold using a Bal-TEC/SCD 005 Sputter Coater. The images were taken with a Jeol MP 35060 camera combined with a Jeol JSM-5400 LV Electron Scanning Microscope and processed using the Orion 4 High Resolution Image Grabbing System software. Larval terminology follows Davis (1987).
The spellings of all species names were retained as originally proposed.

Molecular analysis
Sequences of the 658bp Cytochrome Oxidase I were generated 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 Extraction Kit (Qiagen, Inc., Valencia, CA). Primers LepF1 and LepR1 (Herbert et al. 2004) were used to obtain the barcoding fragment of COI following methods previously described (Hajibabaei et al. 2006 Figs 10,11). Vertex covered with long dense tuft of piliform scales; frons with smooth appressed scales; eyes of midsize; interocular index (= vertical eye diameter/interocular distance) ~ 0.75-0.96. Antenna about 0.7× the length of forewing (n=9), smooth scaled, with a single row of scales per segment; scape with dense pecten. Proboscis well developed, naked, ca. 1.8-2.5× length of labial palpus. Maxillary palpus very short, rudimentary, ~ 0.5× length of labial palpomere II, and directed laterally; consisting of 2 articulated segments; basal 2 segments fused; segment 3 free, spherical. Labial palpus slender, drooping, with ratio of segments from base 1.5: 1: 2.
Thorax (Fig. 12). Forewing slender, maximum width/length ratio ca. 0.2, narrow at apex. Venation with 8 veins, apical part with 5 veins; Sc strong, extending nearly to costa, basal half of R indistinct, Rs2 present, Rs3 arises from apex of the cell, position of Rs4 variable, arising either from base of Rs3 or stalked with Rs3, M and CuA1 separate, CuP indistinct (fold) for entire length, 1A strong, separate, discal cell either open (with absence of crossvein between Rs2 and Rs3) or closed, extending ~ 0.78 of wing length. Hindwing lanceolate, maximum width 0.12 that of length, venation reduced, similar to Phyllonorycter; Sc very short, Rs very long, extending almost to apex; basal 2/3 of M indistinct, parallel to Rs, distal part of M ends near distal 3/5: basal half of Cu strong, distal half indistinct, ending slightly before midway along dorsum; frenulum a single stout bristle in male, 2 tightly appressed bristles in female, retinaculum in male consisting of a broadly triangular curved fold from the ventral base of Sc and a few stiff, forward directed scales situated on the posterior part of Cu.
Abdomen. The margins of the abdominal opening strongly sclerotized and broad laterally, the sclerotized margination of abdomen opening unconnected on T2, S2 apodemes long, ~ half the length of S2, generally slender but more stout at basal 1/3 and very slender at distal 2/3; two pairs of tiny spinules on S2 sublaterally, and a pair of tiny spinules on S3-S6 sublaterally. Sternum 8 in male undeveloped.
Male genitalia. Tegumen relatively short, broad, moderately sclerotized laterally. Caudal portion covered with numerous tiny setae. A pair of long, slender setae present at apex of tegumen. Vinculum broad, U-shaped with very slender, elongate saccus which ranges from 1.1-1.7× the length of valva. Valvae symmetrical, moderately broad, costal margin nearly straight to slightly curved; ventral margin variable between species from slightly convex to slightly concave over distal half with apex varying from fully round to abruptly narrowing; median surface of valva with sparse setae of medium length; apex of valva densely covered with longer, more stout setae. Transtilla complete and well developed, laterally expanded into rounded lobes. Aedeagus very long, nearly as long as entire genital capsule (from apex of tegumen to anterior end of saccus), straight and slender, of uniform diameter along its length; caudal end of vesica usually with long, slender cornuti; phallobase ~ ¼ total length of aedeagus.
Female genitalia. Papillae anales flattened, strongly interconnected, covered with short setae mostly along apical margin; basal bar broad but weakly sclerotized. Posterior apophyses slightly longer than width of papillae anales, straight and slender. Segment 8 short, weakly sclerotized. Anterior apophyses as long or slightly shorter than posterior apophyses, with moderately broad bases, then slender extending to caudal 1/3 of segment 7. Ostium bursae opens medially, near caudal margin of segment 7; sterigma simple, without cuticle folds, antrum funnel-shaped, narrowing anteriorly. Subcaudal area of segment 7 mottled with numerous tubercles. Ductus bursae ~ 2× times longer than segment 7; a membranous accessory bursae ~ 2/3 the length of corpus bursae, arising from middle to anterior 1/3 of ductus bursae, with a smaller lateral pouch arising ~ midway along side of accessory bursae. Corpus bursae 1.0-2.0× the length of segment 7, subcaudal region of corpus bursae usually with scattered spicules or with spicules arranged in linear rows in M. robiniella.
Pupa. Head with vertex terminating in a relatively short, broadly triangular, acute frontal process (cocoon cutter). Abdomen mostly covered dorsally and ventrally with dense, minute spines; dorsum of A2-7 with a single anterior row of short, stout spines; caudal half of sternum 7 with a transverse ridge (accessory cremaster) bearing ~ 18-21 mostly longitudinal rows of short, blunt spines; cremaster of A10 greatly reduced, nearly absent, consisting of 1-2 pairs of minute tergal spines.
Etymology. The generic name is derived from the ancient Greek μακρο-(long) and σάκκος (bag) in reference to the elongate saccus in the male genitalia. Gender masculine.
Generic relationships and species diversity. Several morphological specializations closely associate Macrosaccus with the genera Chrysaster, Cremastobombycia, and Phyllonorycter. Some of these involve the moderately produced proboscis (~ 2× the length of the labial palpi) and the very reduced, two-segmented (with basal segment relatively enlarged), broad maxillary palpi (Figs 10, 11). The wing venation of all three genera is nearly identical and is among the most reduced within Gracillariidae. Only three branches of Rs are present in the forewing, accompanied by single branches of M and Cu (Fig. 12). Venation in the lanceolate hindwings is similarly reduced with only three major veins usually preserved (Rs, M, and Cu) in addition to the extremely basal Sc+R1. The position of Rs4 in Macrosaccus differs somewhat from that in the aforementioned three genera in arising either from the base of Rs3 or stalked with Rs3. Perhaps more significantly is that the discal cell is usually open in Macrosaccus due to the total or near absence of the Rs2-Rs3 crossvein. This crossvein is usually present in the other genera.
The most distinguishing feature in the male genitalia of Macrosaccus is the extremely long, rodlike saccus, whence the generic name is derived. The male saccus in Chrysaster, Cremastobombycia, and Phyllonorycter is either undeveloped or much shorter and stouter (except in two Afrotropical species P. farensis and P. obandai). Likewise sternum 8 in all males of these three genera is extended caudally as a variably lengthened plate beneath the genitalia, compared to being unmodified in Macrosaccus. The female genitalia of Macrosaccus typically possess a relatively large, variably shaped accessory bursa arising approximately midway along the long, slender ductus bursae. The corpus bursae contains dense patches or faint rows of minute spines. The accessory bursae in Phyllonorycter originates more caudally near the ostium, and usually two, circular and variably sclerotized signa are present (Davis and Deschka 2001).
The pupa of Macrosaccus is characterized by an accessory cremaster on abdominal sternum 7 that is unlike that of any other known gracillarid genus. This consists of a raised transverse ridge bearing ~ 18-21 mostly longitudinally oblique rows of short, blunt spines (Figs 84,85). The accessory cremaster when present in Phyllonorycter dif-fers greatly in consisting of a raised triangular area located midventrally on sternum 7 with 1-2 pairs of stout spines projecting laterally (Davis and Deschka 2001).
In addition to the foregoing morphological characters, a preliminary molecular phylogeny based on ten genes also strongly places Macrosaccus apart from Phyllonorycter (Kawahara 2010). Morphological characters distinguishing Macrosaccus from Phyllonorycter are summarized in Table 3.
Five species, all indigenous to the New World, are currently recognized in the new genus Macrosaccus. The high sequence divergence of the barcoding region of COI (> 7%) between species (Fig. 1, Table 2) further confirms the species concepts previously determined by morphological and larval host information. Sequence divergences within species for the 12 samples with multiple specimens were low and varied between 0-0.62% (M. gliricidius), 0% (M. morrisella), 0-0.46% (M. neomexicanus), and 0-0.71% (M. robiniella). The latter included specimens from Belgium and the United States.

Key to species of Macrosaccus (based primarily on male genitalia and larval host)
1 Valva of male of uniform width to broadly rounded apex (Fig. 32) Forewing with a short, oblique white streak from base of costa; median white fascia complete, slightly curved outward (Fig. 5). Valva constricted before apex; arms of transtilla reduced (Fig. 18) Head: Frons smooth, shiny white. Vertex extremely rough; vestiture consisting of a tuft of elongate, piliform, mostly dark brown, intermixed with white, scales. Labial palpus white. Antenna mostly dark fuscous dorsally for most its length, with dark area narrowing to a more slender dark streak toward basal 1/4-1/3 its length; antenna mostly white ventrally; apical segment entirely white. Thorax: Dark brown dorsally, white ventrally; tegula dark brown, with pale grey to white suffusion anteriorly. Forewing pattern complex, costal half mostly light orange brown crossed by 4 equally spaced, white costal strigulae, each bordered basally, sometimes faintly, by black to dark grey and distally by light grey scales; basal 2 strigulae strongly oblique; a fifth, minute, white strigula sometimes arising from black apical spot before forewing apex. Basal third and dorsal half of forewing usually darker, mostly black to sometimes pale golden grey between strigulae; slender white streak from base of wing usually indistinct or absent; a greyish, oblique strigula often evident near base of wing which connects with a larger, more distinct greyish strigula from dorsal margin; dorsal margin also with 3, usually less distinct white strigulae approximately opposite to distal 3 white strigulae from costa; basal dorsal strigula usually contiguous with second costal strigula. Apex of forewing with a large black apical spot, which is rarely reduced; fringe mostly light grey. Hindwing, including fringe, uniformly grey. Foreleg mostly dark fuscous dorsally, white ventrally, with 2 white annuli around basal tarsomeres; midleg with 2 oblique bands of white dorsally over tibia; tarsomeres more broadly banded with white dorsally; hindleg mostly white with much of tibia pale fuscous dorsally, and with 3 broad, pale fuscous annuli dorsally over tarsomeres.
Abdomen: dark fuscous dorsally and white ventrally with greyish suffusion on anterior portion of segments 2-7 laterally and sometimes ventrally on A8.
Female genitalia (Figs 15-17): Ductus bursae long and slender, nearly half the length of elongate corpus bursae. Accessory bursae ~ 2/3 the length of corpus bursae, arising from anterior 1/3 of ductus bursae; with a smaller lateral pouch arising ~ midway along side of accessory bursae. Corpus bursae gradually broadening anteriorly, with faint longitudinal striae in wall which bear longitudinal rows of low, dentate ridges around anterior third of corpus bursae; walls of anterior end (distal 1/5) of corpus bursae entirely membranous.
Larval mine . The mine begins as an elongate serpentine track ( Fig. 37) which enlarges to an elongate-oval, whitish blotch Fig. 36, 38) located on one side of the midrib and usually on the under (abaxial) side of the leaflet. Eventually the mine becomes slightly tentiform due to the silk laid down by the later instar larvae.
Hosts. (Table 1). Fabaceae: Robinia pseudoacacia L. (Clemens 1859: 320), Robinia viscosa Vent. (Chambers 1878: 111), Robinia hispida L. (Chambers 1878: 111;Needham et al. 1928: 288). The primary host, Robinia pseudoacacia, is believed once to have occurred primarily in two regions within the United States -one centered in the Appalachian Mountains from central Pennsylvania to northern Georgia and Alabama, and the other in the Ozark Plateau of southern Missouri, eastern Arkansas to eastern Oklahoma. This tree has since spread over much of the continental United States, portions of northeastern Canada, and parts of South America, Europe, Asia, Africa, and Australia (Stone 2009).
Life history . The egg of M. robiniella is deposited externally usually some distance from the leaf edge or midrib. Five larval instars have been observed by counting head capsules within mines in North America and Belgium. Kasch and Nicolai (2002) reported up to six instars based on head capsule measurements in Germany.
The larvae typically form elongate-oval, whitish blotch mines on usually the under (abaxial) side of the leaflets. Upon eclosion, the apodal, prognathous sap-feeding larva enters the leaf and begins a slender, subepidermal, serpentine mine (Fig. 37). Eventually the mine is expanded into an oval blotch (Fig. 38) which usually encompasses and obliterates the previous serpentine mine. As is true for the larvae of Phyllonorycter (Davis and Deschka 2001), the last sap-feeding instar probably begins expanding the mine laterally. Initiation of the tissue-feeding instar is indicated by deeper feeding into the spongy and palisade tissue layers of the leaflet as the larva begins to ingest solid tissue. The resulting injury becomes visible from the opposite leaf surface, particularly in the underside mines, as dense, whitish punctures. As the tissue-feeding larva matures, it begins to lay down silken strands across the inner surface of the mine causing the leaflet to roll inwards and the mine to become tentiform (Fig. 39). Pupation occurs inside a silken cocoon (Fig. 40) within the mine without any precut exit opening. Especially during heavy infestations, the mines of adjacent larvae may coalesce resulting in multiple pupal cocoons. The phenology of this species has not been accurately determined over its range within North America. Normally two to three generations per year have been reported in Europe, which can occasionally reach as many as four (Nicolai 2005). Braun (1908) noted that the mines could occur on both leaf surfaces. Weaver and Dorsey (1967) described the larval mining behaviour of M. robiniella in great detail and observed several differences between the upper side mines, which reportedly were more common at higher elevations (~ 760 m), and the under side mines. The latter were found most frequently at elevations of ~ 270 m at their West Virginia study sites. Some of the distinctions they observed were that upper side mines occurred usually more basal on the leaflet and often extended across the midrib, with the larval frass concentrated more basally within the mine. Under side mines are situated less basally and usually restricted to one side of the midrib, with frass scattered more uniformly throughout the mine. Only a single, somewhat loosely woven cocoon was observed in the upper side mines, compared to as many as three, densely woven cocoons in the lower mines. DRD compared males reared from the upper and lower side mines and found no significant morphological differences (Weaver and Dorsey 1967). A search for the Weaver specimens in the collections of the University of West Virginia at Morgantown yielded no material associated with the upper side mines from the higher elevation sites (~ 760 m). Hopefully specimens from the higher elevation, upper side mines can be collected in order to examine their genetic distances.
In addition to Hymenoptera parasitoids, other Lepidoptera larvae have been noted within the mines of M. robiniella (Weaver and Dorsey 1967). These were observed to alter the appearance of the mine by removing all mesophyll and largely destroying the frass pattern created by M. robiniella. Packard (1890) identifies a species of Gelechiidae, Filatima pseudacaciella (Chambers), which sometimes feeds within the mine in addition to feeding externally.
Natural enemies. (Table 4). Fifty seven species (including two unidentified) of Hymenoptera, the great majority of which are members of Eulophidae (Noyes 2010), have been reported as parasitoids of Macrosaccus robiniella in Europe and North America. Weaver and Dorsey (1967) also list two species of predators in the families Reduviidae and Vespidae that preyed on M. robiniella.  Pupa (Figs 82-89, 97, 98). Maximum length 3.6 mm; width 0.9 mm. Vertex with frontal process (cocoon cutter) relatively short, broadly triangular, acute (Figs 81, 82). Forewing extending to anterior margin of A6; antenna slightly longer to middle of A6; hindleg extending to A7. Abdomen mostly covered dorsally and ventrally with dense, minute spines; dorsum of A2-7 with a single anterior row of short, stout spines (Figs 83, 98); caudal half of sternum 7 with a transverse ridge (accessory cremaster) bearing ~ 18-21 mostly longitudinal rows of short, blunt spines (Figs 84, 85). Cremaster of A10 greatly reduced, nearly absent, consisting of 1-2 pairs of minute tergal spines.
Types  Distribution. Macrosaccus robiniella occurs naturally over much of eastern North America from Ontario, Canada south to South Carolina and west to Missouri and Texas. Macrosaccus robiniella was first reported in Europe in 1983, near Basel, Switzerland (Whitebread 1990: 344) and has since spread through 23 European countries (Table 5).
Remarks. The synonymous names Lithocolletis robiniella Clemens and Argyromiges pseudacaciella Fitch were both published in 1859. The month of publication  (1891) first treated pseudacaciella as a junior synonym of robiniella and no subsequent author has considered it otherwise, and (2) that the name robiniella has been recognized as the valid name for this taxon in more than 25 publications (141 publications using robiniella as the valid name are actually known) by more than 10 authors, this name must be preserved as the valid name for this taxon in accordance with the provisions of article 23.9.1 of the International Code of Zoological Nomenclature (ICZN 1999).

Macrosaccus morrisella
Head: Vestiture of head and antenna similar to M. robiniella. Thorax: Dark brown to fuscous dorsally, sometimes with a coppery to purplish luster; shiny white ventrally; tegula dark brown, with white suffusion anteriorly. Forewing pattern similar to M. robiniella except basal two costal strigulae less oblique; a slender white, slightly oblique streak usually well developed extending distad from tegula at base of wing to sometimes as far as first dorsal strigula; 3 white dorsal strigulae usually present, but these oriented less obliquely than in M. robiniella; basal strigula white, but sometimes obscure; median strigula connected to second costal strigula to form a narrow white fascia; black apical spot present as in M. robiniella; cilia light grey to white. Hindwing, including fringe, uniformly grey. Legs similar to M. robiniella in colour pattern.
Abdomen: Similar to M. robiniella, dark fuscous dorsally and white ventrally with greyish suffusion on anterior portion of segments 2-7 laterally and sometimes ventrally on A8.
Larva and pupa. Similar to that of Macrosaccus robiniella.
Note: In the lower right corner of drawer Mi 4423, a label with the following handwritten text is present: "Nb. Chambers' syntypes. The specimens bearing the serial numbers 1-68, and the specimens of G. hermannella var. lingulacella immediately following, were received by Stainton from Chambers. The numbers refer to a list made by Chambers; many of the specimens are likely to be syntypes. See Stainton foreign correspondence, letters nos. 94, 97 and 111 (by Chambers) and Stainton's "translations"  Distribution. Macrosaccus morrisella occurs widespread across eastern North America from Manitoba and Ontario, Canada, south and west to Texas and Colorado (Chambers 1877).
Remarks. Chambers in his 1877 description of Lithocolletis amphicarpeaeella expressed doubt if this was a new species or new variety (as he did on the same page and line for "Lithocolletis amorphaeella n.sp.? or var.?"). Their descriptions included long, detailed comparisons of the forewing patterns of these two new moths as well of robiniella and "texana" (misspelled). No locality for either name was mentioned except for Colorado in the title of the publication (Chambers 1877). Each of the three "type" specimens of amphicarpeaeella in the collections of the MCZ have the name "Kentucky" crossed out on the specimen label "Kentucky, Chambers", and no mention of Colorado appears. A male specimen in best condition has been selected as lectotype.
The holotype specimen of Lithocolletis texanella Zeller upon examination was found to be a male and not a female as stated originally by Zeller (1875). Diagnosis. As discussed in the diagnosis of M. robiniella, this species most resembles the former in general appearance. They differ in distribution, host preference, in genital morphology (see diagnoses of M. robiniella), and possibly overall size, with the wingspan of neomexicanus being slightly larger.
Head: Vestiture of head and antenna similar to M. robiniella; apical segment of antenna white to grey.
Thorax: Dark brown dorsally, with whitish suffusion anteriorly and laterally; white ventrally; tegula dark brown, with pale grey to white suffusion anteriorly. Forewing and hindwing patterns very similar to M. robiniella. Vestiture of legs similar to robiniella.
Abdomen: Similar to M. robiniella, dark fuscous dorsally and white ventrally with greyish suffusion on anterior portion of segments 2-7 laterally and sometimes ventrally on A8.
Male genitalia (Figs 23, 24): Valva relatively simple, gradually narrowing before apex without abrupt constriction; apex narrowly rounded, densely setose, particularly along costal margin. Saccus a slender, elongate rod ~ 1.3× length of valva. Aedeagus very long and uniformly slender, ~ 2.5× length of valva.  Female genitalia : Ductus bursae moderately long and slender, ~ 1/3 the length of elongate corpus bursae. Accessory bursae nearly as long as corpus bursae, arising from junction of ductus bursae and corpus bursae; with a smaller lateral pouch arising ~ midway along side of accessory bursae. Corpus bursae relatively slender, anterior end only slightly broader; a dense scattering of minute spicules encircling middle; remaining walls of corpus bursae entirely membranous.
Larva and pupa. Similar to that of Macrosaccus robiniella. Larval mine (Figs 46-50). The mine begins as a relatively short, serpentine track which enlarges to an elongate-oval, whitish blotch located on the under (abaxial) side of the leaflet. As the larva develops and begins laying down silk, the mine becomes strongly tentiform, causing the upper (adaxial) surface to roll over (Figs 48,49).
Host. (Table 1). Fabaceae: Robinia neomexicana Gray. The host is a moderately small, spiny shrub growing to as high as 5 meters and usually forming dense thickets (Fig. 45). It occurs from California to Texas and north to Wyoming.
Life history . Some collections of this species from southern Arizona have been from dense infestations. In such populations, oviposition tends to be concentrated on fewer available leaflets with as many as 45 short, initial serpentine mines observed on a single leaflet. These soon coalesce resulting in a single large blotch covering nearly the entire lower side of the leaflet (Fig. 47). Larval mortality is probably high under these conditions. One large composite mine opened contained 14 live and 11 dead, late instar larvae and no pupae. A maximum of 11 cocoons with pupae (5 on one side of the midrib and 6 on the other side) were found in one leaflet (DRD rearing lots 541, 541.1, Fig. 50).
Distribution. Known only from the southwestern United States from Arizona and New Mexico.
Etymology. The specific name is derived from the specific name of its plant host. The specific epithet is an adjective in the nominative singular.

Diagnosis
Abdomen: Similar to M. robiniella, dark fuscous dorsally and white ventrally with greyish suffusion laterally on anterior portion of segments 2-7 and sometimes ventrally on A8.
Larva and pupa. Not examined. Larval mine (Figs 51-53). The mature mine is an elongate-oval, whitish blotch located on the under (abaxial) side of the leaf usually near the edge of the leaflet. Eventually, as the mine becomes tentiform, the leaf edge is slightly curled (Fig. 51).
Hosts. (Table 1). Amorpha fruticosa L., (Chambers 1878: 58;Frey and Boll 1878: 276;Walsingham 1889: 119;Braun 1908: 292). Amorpha sp., Robinia sp. (Robinson et al. 2002: 357). The primary host, Amorpha fruticosa, is a shrub up to 4 meters high which occurs naturally from Louisiana to Florida and north to Wisconsin and Pennsylvania. Because no adults reared from Robinia are known or have been examined, the host record reported in Robinson et al. (2002)  Remarks. For over 120 years Argyromiges uhlerella Fitch has been regarded as the senior synonym of amorphaeella Chambers. The inadequacy of the original description of Argyromiges uhlerella (quoted below), together with the disappearance of any type material, has caused some uncertainty regarding this insect's identity. In his review of the insects feeding on Robinia pseudoacacia, Fitch (1859) proposed two names now assigned to Macrosaccus which he believed to be related to the Robinia leafminer: Argyromiges pseudacaciella (now considered a synonymn of M. robiniella) and Argyromiges uhlerella. Fitch stated that he did not know the hosts for these two moths (i.e., neither had not been reared). Walsingham (1889) concluded that Fitch's description of uhlerella agreed with that of M. amorphaeella, proposed 18 years later by Chambers (1877), and he consequently synonymized the latter name. All later workers treating this complex accepted Walsingham's decision. Closer examination of wing pattern variation within large series of reared M. robiniella, however, suggests that Fitch's description more approximates the greyish forewing colour of some specimens of M. robiniella than it does the non-greyish, more brownish forewing colour prevalent in the smaller sample of adult Macrosaccus reared from Amorpha fruticosa examined. Fitch's description also contains such abnormalities as the "five white spots along their outer sides" and "the tip of the wings is here replaced by a short black stripe thrice as long as it is wide", both of which may possibly reflect the poor condition of his specimen(s). Worn specimens of M. robiniella have been observed with one or two strigulae indistinct or missing, as well as some with reduced apical spots. Similar variation might also be found to occur within moths reared from Amorpha, once more specimens become available for examination.
No type material of "Argyromiges" uhlerella is believed to exist. In 1977, during a search for Fitch's Lepidoptera types deposited in the USNM, Tim McCabe found a pin bearing Fitch's label 8158 (the type number for uhlerella) in the main collection. The moth was missing and was presumed destroyed. The pin with that number has since disappeared. Because the name uhlerella has been used consistently as the valid name for this taxon since before 1899, we believe that this usage should continue even though some doubt now exists regarding the correct application of the name.
Original description of Argyromiges uhlerella Fitch: "337. Uhler's leaf-miner, Argyromiges Uhlerella, new species." "This resembles Pseudacaciella, but is throughout of paler color, the fore wings being golden gray, with five white spots along their outer sides, of which the hindmost ones are small, the others quite large and bordered with blackish upon their anterior sides; and the black dot on the tip of the wings is here replaced by a short black stripe thrice as long as it is wide; whilst the hind wings and their fringes are pale silvery gray. These marks will suffice to distinguish this from the two preceding species." Diagnosis. The forewing pattern of this species is similar to M. robiniella and M. neomexicanus in possessing 4 white costal and 3 dorsal, mostly sharply oblique strigulae, with a median fascia often formed by the junction of the 2 nd costal and median dorsal strigulae. The pale golden brown ground colour of M. gliricidius is distinctly paler than that of the other species. The forewing of M. gliricidius also differs from other Macrosaccus in possessing a small, elongate white subapical spot and a more reduced dark fuscous apical spot. The male valva of M. gliricidius is distinct in having the distal half more broadly rounded than that of other Macrosaccus.    Adult (Fig. 9). Forewing length 2.2-2.6 mm. Head: Vestiture of head and antenna similar to M. robiniella except vertex generally paler and with more white scales concentrated toward occiput.

Macrosaccus gliricidius
Thorax: Dorsum with a narrow, median, longitudinal band of light golden brown bounded laterally with white; tegula light golden brown; venter white. Forewing pale golden brown with 4 equally spaced, oblique, white costal strigulae and 3 white, dorsal strigulae, each bordered by dark brown scales; 2 nd costal strigula connected to median dorsal strigula; subapical dorsal strigula directed inward toward small, white subapical spot; dark fuscous apical spot poorly developed, with a more elongate subapical spot immediately basad to rudimentary apical spot; fringe mostly pale greyish white, with narrow, dark brown median band and broad, grey inner band. Hindwing, including fringe, uniformly grey. Foreleg mostly dark fuscous dorsally, white ventrally, with 2 white annuli around basal tarsomeres; midleg mostly white with oblique bands of white extending dorsally over tibia; tarsomeres more broadly banded with white dorsally; hindleg mostly white with much of tibia fuscous dorsally, and with 3 broad fuscous annuli dorsally over tarsomeres.
Female genitalia (Figs 34,35): Ductus bursae long and slender, ~ 1.25× the length of corpus bursae. Accessory bursae ~ 0.8× the length of corpus bursae, arising from near middle of ductus bursae at a point where the ductus is slightly constricted; a smaller lateral pouch arising near caudal end of accessory bursae. Corpus bursae elliptical, with numerous acute spicules somewhat evenly scattered over much of inner surface but less dense near anterior end.
Larva and pupa. (Figs 56,58). Similar to that of Macrosaccus robiniella. Larval mine (Figs 54-58). The mine begins as an elongate serpentine track which abruptly enlarges to an elongate-oval, whitish blotch located on either the upper (adaxial) or lower (abaxial) side of the leaflet. When present on the under side, the blotch mines usually develop along the midrib. Only the upper side blotch mines occurred directly on top of the midrib (Fig. 55, Cave, in litt.).
Host. (Table 1). Fabaceae: Gliricidia sepium (Jacq.). Gliricidia sepium is a small to medium-sized, thornless tree growing to a height of 10-12 meters. It is believed to have originated in Central America and has been introduced into many tropical countries around the world. It can be grown as dense hedges and is frequently used as "living fences".
Etymology. The species name is derived from the generic name of its host, Gliricidia. The specific epithet is a noun in the nominative singular.
Philadelphia, USA, and Ronald Wielgus, Kneeland, California, USA for the gift or loan of essential material and pertinent information. We kindly thank Zdenek Laštůvka (Figures 36, 37) of Macrosaccus robiniella. We cordially thank John Noyes, Natural History Museum, London, UK for providing some of the references on the parasitoids of M. robiniella. The DNA barcode sequences were generated at the Biodiversity Institute of Ontario with funding to Paul D.N. Hebert from Genome Canada through the Ontario Genomics Institute, NSERC, and the Ontario Ministry of Research and Innovation. Stephanie Kirk, Jeremy deWaard, and Megan Milton of the Biodiversity Institute of Ontario, and Jean-François Landry also were helpful in assisting with specimens for barcoding. Apostolis Pekas (Westerlo, Belgium) is kindly acknowledged for explaining the finesses of old Greek spelling, which were implemented into the formation of the genus-group name Macrosaccus. Belgian Science Policy Office is gratefully thanked for supporting financially the taxonomic study on Gracillariidae. DRD wishes to acknowledge the former Smithsonian Research Foundation, Smithsonian Research Initiatives, and the Research Opportunity Fund for supporting his research on the biology of leaf-mining Lepidoptera.