Antispila oinophylla new species (Lepidoptera, Heliozelidae), a new North American grapevine leafminer invading Italian vineyards: taxonomy, DNA barcodes and life cycle.

A grapevine leafminer Antispila oinophylla van Nieukerken & Wagner, sp. n., is described both from eastern North America (type locality: Georgia) and as a new important invader in North Italian vineyards (Trentino and Veneto Region) since 2006. The species is closely related to, and previously confused with Antispila ampelopsifoliella Chambers, 1874, a species feeding on Virginia creeper Parthenocissus quinquefolia (L.) Planchon., and both are placed in an informal Antispila ampelopsifoliella group. Wing pattern, genitalia, and DNA barcode data all confirm the conspecificity of native North American populations and Italian populations. COI barcodes differ by only 0-1.23%, indicating that the Italian populations are recently established from eastern North America. The new species feeds on various wild Vitis species in North America, on cultivated Vitis vinifera L. in Italy, and also on Parthenocissus quinquefolia in Italy. North American Antispila feeding on Parthenocissus include at least two other species, one of which is Antispila ampelopsifoliella. Morphology and biology of the new species are contrasted with those of North American Antispila Hübner, 1825 species and European Holocacista rivillei (Stainton, 1855). The source population of the introduction is unknown, but cases with larvae or pupae, attached to imported plants, are a likely possibility. DNA barcodes of the three European grapevine leafminers and those of all examined Heliozelidae are highly diagnostic. North American Vitaceae-feeding Antispila form two species complexes and include several as yet unnamed taxa. The identity of three out of the four previously described North American Vitaceae-feeding species cannot be unequivocally determined without further revision, but these are held to be different from Antispila oinophylla. In Italy the biology of Antispila oinophylla was studied in a vineyard in the Trento Province (Trentino-Alto Adige Region) in 2008 and 2009. Mature larvae overwinter inside their cases, fixed to vine trunks or training stakes. The first generation flies in June. An additional generation occurs from mid-August onwards. The impact of the pest in this vineyard was significant with more than 90% of leaves infested in mid-summer. Since the initial discovery in 2006, the pest spread to several additional Italian provinces, in 2010 the incidence of infestation was locally high in commercial vineyards. Preliminary phylogenetic analyses suggest that Antispila is paraphyletic, and that the Antispila ampelopsifoliella group is related to Coptodisca Walsingham, 1895, Holocacista Walsingham & Durrant, 1909 and Antispilina Hering, 1941, all of which possess reduced wing venation. Vitaceae may be the ancestral hostplant family for modern Heliozelidae.

Introduction

There are several cases known of leafmining Lepidoptera developing into important agricultural pests, such as Stainton, 1856 (Gracillariidae) on citrus, now a worldwide problem (Heppner and Dixon 1995) and Guérin-Méneville, 1842, Ghesquière, 1940and related species (Lyonetiidae) on coffee, that are amongst the more important coffee pests (Le Pelley 1973). Leafmining moths apparently often disperse easily, possibly due to their small size, and some have shown rapid invasions over large areas, e.g.: Deschka & Dimić, 1986, (Zeller, 1850), (Kumata, 1963)and (Clemens, 1859) (Šefrová 1999; Hellrigl 2001; Šefrová 2002a, b; Šefrová and Laštůvka 2002; Davis and De Prins 2011).

Lepidopteran leafminers of grapevine ( L.) have not yet developed into serious pests in Europe, although one North American species did recently invade European vineyards; Clemens, 1859 (Lepidoptera: Gracillariidae) became established in Italy and elsewhere in Europe around 1995 (Posenato et al. 1997)PageBreak. The only native European leafminer of grape is (Stainton, 1855) (Lepidoptera: Heliozelidae) (Hering 1957), a minor pest in vineyards in southern Europe and western Asia (see references below). was described from Malta and later reported from Italy. It develops two to three generations annually (Mariani 1942; Marchi 1956; Camporese and Marchesini 1991; Dal Rì and Delaiti 1992; De Tomaso et al. 2008; Baldessari et al. 2009). Infestations leading to damage are infrequent, probably because pest populations are controlled by a complex of eulophid parasitoids (Hymenoptera) (Camporese and Marchesini 1991; Alma 1995). European populations of occur in northern Italy (Marchesini et al. 2000; Villani 2002; Reggiani and Boselli 2005; Duso et al. 2011), Slovenia (Seljak 2005) and Switzerland (Cara and Jermini 2011). Itcan produce up to four generations annually and has given rise to local outbreaks in northeastern Italy (Posenato et al. 1997; Marchesini et al. 2000). The larvae of both moths produce characteristic mines in grapevine leaves; in a narrow initial gallery leads subsequently to an oval full-depth blotch, from which the larva cuts out an oval pupal case or shield, in which it pupates, leaving an oval hole in the leaf. makesa long tortuous gallery mine in the upper epidermis, with a distinct dark central frass line that ends in a pupal chamber. Both species can easily be detected in a vineyard based on the presence of their diagnostic leafmines.

In the summer of 2007, leafmines similar to those caused by were observed in a vineyard in northeastern Italy (Borgo Valsugana, Trento province). However, the initial gallery mine was immediately enlarged into a larger blotch, indicating a different species. Adults reared from these mines differed from in size and wing pattern. On their external characters they were identified as belonging to the genus Hübner, [1825], but not to one of the two species currently known in Europe, i.e., (Fischer von Röslerstamm, 1843) and ([Denis & Schiffermüller], 1775) (Karsholt et al. 1995; Ellis 2010; van Nieukerken 2011) which both feed on dogwood ( spp.). Thus, we determined that we had either an undescribed species or an alien species introduced from another continent. This pest has been reported previously as sp. (Baldessari et al. 2009; Duso et al. in press). Because taxonomic knowledge of the family Heliozelidae is poor, very few species being described up to modern standards, and because many of the known species are associated with species or related Vitaceae, it took some time to establish that this species was an undescribed, but common, North American species, hitherto confused with the North American Chambers, 1874, described from Virginia creeper . Unfortunately this confusion has already led to the incorrect introduction of the name into European literature (Laštůvka 2009; van Nieukerken 2011; van Nieukerken et al. 2011a). In this paper we describe the species as van Nieukerken & Wagner, sp. n., provide a diagnosis for its identification, and characterize its geographic distribution and life cycle. We also sequenced a part of the cytochrome C oxidase subunit I (COI) gene (DNA barcode) (Hebert et al. 2003b; Hebert et al. 2003a) PageBreakas well as those of a selection of other Heliozelidae and the other two European grape leaf-mining micro-moths ( and ). DNA barcode data played important roles in revealing the original source of the infestation and unravelling the taxonomy of the new grape pest.

Family Heliozelidae

The family Heliozelidae (superfamily Adeloidea) comprises 123 described species in 12 genera (van Nieukerken et al. 2011b), with the greatest diversity in North America and Australia. Larvae of the Heliozelidae produce leafmines (rarely galls) in various trees and vines, rarely herbs, and typically cut-out an oval case or shield from the leafmine, in which they moult once into a non-feeding final instar or prepupa, and finally pupate in the leaf litter or on plant parts. All are thought to overwinter in temperate regions as prepupae. Eight species of Heliozelidae occur in Europe (van Nieukerken 2011), belonging to four genera: , Hering, 1941, Herrich-Schäffer, 1853 and Walsingham & Durrant, 1909. Four species of Heliozelidae were previously known from Italy: (Zeller, 1850), (Haworth, 1828), and (Fischer von Röslerstamm, 1843) (Karsholt et al. 1995), but it is likely that the fauna is incompletely sampled, and that most European species occur in Italy as well. In addition to , we record here ([Denis & Schiffermüller], 1775) as new from Italy (see Appendix B).

The grapevine family Vitaceae comprises an important group of hosts for the genus worldwide; out of 32 species for which host plants are known, 13 feed on Vitaceae, of which at least ten are associated with the genus (Table 1). Several more unnamed species are also associated with Vitaceae. In North America, Chambers, 1877 makes galls in leaves and shoots on (McGiffen and Neunzig 1985). There are only a few previous records of Heliozelidae as minor pests on grape: in addition to , as mentioned above, has been recorded as a pest in Japan (Kuroko 1987; Ueno et al. 1987). Clemens, 1860 is listed by McGiffen and Neunzig (1985) as occurring on bunch grape leaves, but not as a pest.

Table 1.

Heliozelidae species associated with Vitaceae, type country and hostplant species. For the American species where the identity is not fully established we added [cf] between genus and species name.

Species	Type country	Hostplants	source
Antispila oinophylla	USA	Vitis aestivalis, Vitis labrusca, Vitis riparia, Vitis vinifera, Vitis vulpina, [Parthenocissus]	this paper
Antispila ampelopsifoliella Chambers, 1874	USA	Parthenocissus quinquefolia	Chambers 1874a, this paper
Antispila sp. “vitis1”	(USA)	Vitis aestivalis	this paper
Antispila [cf] isabella Clemens, 1860	USA	Vitis aestivalis, Vitis labrusca, Vitis riparia	Clemens 1860, this paper
Antispila sp. “vitis2”	(USA)	Vitis aestivalis, Vitis riparia	this paper
Antispila viticordifoliella Clemens, 1860	USA	Vitis vulpina	Clemens 1860, this paper
Antispila cf viticordifoliella Clemens, 1860	(USA)	Parthenocissus quinquefolia	this paper
Antispila voraginella Braun, 1927	USA	Vitis arizonica	Braun 1927, DLW
Antispila ampelopsia Kuroko, 1961	Japan	Ampelopsis brevipedunculata, Vitis flexuosa	Kuroko 1961
Antispila inouei Kuroko, 1987	Japan	Vitis coignetiae, Vitis labruscana	Kuroko 1987
Antispila iviella Kuroko, 1961	Japan	Parthenocissus tricuspidata	Kuroko 1961
Antispila orbiculella Kuroko, 1961	Japan	Ampelopsis brevipedunculata	Kuroko 1961
Antispila tateshinensis Kuroko, 1987	Japan	Vitis coignetiae	Kuroko 1987
Antispila uenoi Kuroko, 1987	Japan	Vitis coignetiae, Vitis labruscana	Kuroko 1987
Antispila argostoma Meyrick, 1916	India	Cayratia trifolia	Meyrick 1916, Fletcher 1920
Antispila aristarcha Meyrick, 1916	India	Vitis sp.	Meyrick 1916, Fletcher 1920, Fletcher 1933
Antispila isorrhythma Meyrick, 1926	India	Vitis sp.	Meyrick 1926
Antispila species	Indonesia, Borneo	Leea indica	EJvN
Antispila species	Australia	Cissus antarctica	Common 1990
Holocacista rivillei Stainton, 1855	Malta	Vitis vinifera	see text
Heliozela aesella Chambers, 1877	USA	Parthenocissus quinquefolia, Vitis vulpina, Vitis sp.	McGiffen and Neunzig 1985

Material and methods

Material

adults were collected from Borgo Valsugana for sequencing and larvae were collected and reared for morphological studies. and adults were also collected from northeastern Italy (Appendix B). To obtain material of the new speciesand related species from North America for comparisonPageBreak, various mines and larvae were collected by EJvN and CDo during a field trip September-October 2010 in the states of Georgia and Tennessee and by EJvN in September 2011 (partly with DLW) in Connecticut, Massachusetts, Vermont and New York state. Other material included in the taxonomic and DNA analyses was collected by DLW, who has been collecting and rearing and other leafminersPageBreak from across North America for three decades (Appendix B). Further material was studied or borrowed from the following collections.

Abbreviations for depositories:

ANSP

Academy of Natural Sciences in Philadelphia, Pennsylvania, USA

CNC

Canadian National Collection of Insects, Arachnids and Nematodes, Ottawa, Ontario, Canada

DLW

Research collection of David L. Wagner, Storrs, Connecticut, USA

MCZ

Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts, USA

RMNH

Netherlands Centre for Biodiversity Naturalis, former Leiden Zoology collections, Leiden, Netherlands

UMDC

University of Maryland, College park, USA

UPI

University of Padova, Department of Environmental Agronomy and Crop Science, Italy

ZMUO

Zoological Museum University of Oulu, Finland

Rearing

Collected leaves were kept in polystyrene jars or bags, with some moss and or tissue added, until the larvae had prepared the shields. It was often necessary to remove the cut/out shields from the leaves, which were then removed from the breeding jars and dried as vouchers. Breeding jars were kept during winter in an outbuilding, and brought indoors in March, where they were kept until emergence of adults. Specimens collected during fall 2011 were still in hibernation diapause when this manuscript was accepted.

Morphology

Methods for preparation of the genitalia follow Nielsen (1980a) and van Nieukerken (1985), with some minor changes. Nielsen’s unrolling technique does not work well for Heliozelidae, so we usually embedded the total genitalia in dorso-ventral position. For staining male genitalia we used (Mayers) haemaluin or phenosafranin. Wings were stained with phenosafranin and mounted in euparal. Photographs of moths, leafmines, genitalia slides and wing slides were taken with a Zeiss AxioCam digital camera attached, respectively, to a Zeiss Stemi SV11 stereo-microscope, a motorized Zeiss SteREO Discovery.V12 (only Figs 1, 40, 41) or a Zeiss Axioskop H, using Carl Zeiss AxioVision software.

Figures 1–5.

, adult habitus. 1 Male holotype, RMNH.INS.24204 2 Female paratype, RMNH.INS.24039, Italy, Borgo Valsusana. 3–5 Alive male,Georgia, paratype, emerged 29.iv.2011.

Figures 33–41.

and adult habitus in dorsal or lateral(40, 41)view. 33 , male, Italy 34 , male, USA: Arizona, genitalia slide EJvN3918 35 , female, USA, Vermont: Salisbury, genitalia slide JCK15220 36 , female, USA: Georgia, Chattahoochee NF 37 cf viticordifoliella, female, Canada: Ottawa 38, 39 cf isabella, male, upper and underside (39) with androconial scales, USA: Connecticut, Mansfield, DLW90J8 40 “vitis1”, female, USA: Florida, genitalia slide EJvN4205 41 cf viticordifoliella, female, USA: Florida, genitalia slide EJvN4207. Arrows indicate white tipped antennae in and cf viticordifoliella.

The Distribution Map for North America was prepared with DMap 7.0 (Morton 2000).

Molecular analysis

DNA was extracted destructively from larvae or adult specimens preserved in 96% or 100% ethanol or extracted in a non-destructive fashion from the abdomen of voucher specimens, which were then used to prepare genitalic dissections (protocol in Knölke et al. 2005). From some larvae used for DNA extractions, the cuticle was also cleared and saved. In Padova, total DNA was extracted applying a salting-out protocol (Patwary et al. 1994). In Leiden extractions were carried out with the Qiagen DNeasy Blood and Tissue kit (QIAGEN), using the protocol “purification of total DNA from animal tissues (spin‐column protocol).”

A 665 bp or a 658 bp fragment of the mitochondrial COI gene was amplified using the following primers: in Padua LCO1490 and HCO2198 (Folmer et al. 1994), in Leiden the Lep primers (Hebert et al. 2004), often tailed with T7 promotor and T3 tails in the shorter (amplifying 665 bp) and longer versions (amplifying 658 bp): T‐LepF1-short and T‐LepR1-short or T‐LepF1 and T‐LepR1, or when not tailed LepF1-short and LepR1-short. For some older museum specimens, the DNA was too degraded for amplifying sections over 400 bp long. For these we used internal primers (Hajibabaei et al. 2006). For details of primers see the BOLD site (http://www.barcodinglife.com/).

In Padova, amplification was carried out in 20ml volumes containing 2ml from the nucleic acid extract, 200mM dNTPs, 0.5mM of each primer, 4mM 10x PCR buffer, 2.5 mM MgCl2 and one unit of Taq polymerase (Promega). The reaction was performed in an INC PTC-100 thermal controller (MJ Research Inc.). Amplification conditions were as follows: the first period of denaturation was 94°C for 5 min, followed by 38 cycles of denaturation at 94°C for 1 min, annealing at 48°C for 1 min, and extension at 72°C for 1 min; the final extension cycle had a step at 72°C for 5 min. A negative control with no template was included for each series of amplifications, to detect instances of contamination. The amplified products were separated on a 1% agarose gel and visualized under UV following staining with Sybr Safe (Invitrogen). PCR products were purified with the ExoSAP-IT kit (Amersham Biosciences).

In Leiden, amplification was performed in volumes of 25 µl. The PCR cycle consisted of 3 min initial denaturation at 94°C, 15 sec cycle denaturation at 94°C, 30 sec cycle at 50°C, 40 sec cycle extension at 72°C for 40 cycles. After all cycles had finished, a final extension was performed at 72°C for 5 min. The amplified products were separated on a 1% agarose gel and visualized under UV following staining with ethidium bromide.

The sequencing at Padova was performed at the BMR Genomics Service (Padova, Italy) in an ABI PRISM automatic DNA sequencer (Applied Biosystems), in both forward and reverse direction, but for some samples only in forward direction. In Leiden PCR clean-up and sequencing was outsourced to MACROGEN on an ABI 3730XL, all samples were sequenced in both forward and reverse direction. The chromatograms were checked with Sequencher (Gene Codes Corporation) and the resulting sequences were aligned by eye in BIOEDIT 7.0.9.0 (Hall 2004).

Tree analysis

Neighbor-joining (NJ) trees based on DNA barcode sequences of all available specimens were reconstructed with Paup* 4.0b10 (Swofford 2003). Genetic distance calculations were performed both using the Kimura two-parameter (K2P) model and uncorrected P distance (Srivathsan and Meier 2011). After initial analyses with barcodes of Italian , we excluded this gracillariid from subsequent analyses (because it was so divergent from focal Heliozelidae: minimum K2P distance being greater than 18%). A Genbank sequence of (Denis & Schiffermüller, 1775) (Incurvariidae), another member of the superfamily Adeloidea, was used as the outgroup. Bootstrap values were calculated with 10,000 replicates.

Phylogenetic trees based on maximum parsimony were generated with PAUP using a heuristic search, 1,000 replicates, with tree-bisection-reconnection (TBR) as the branch-swapping algorithm. A bootstrap analysis was run with TNT (Goloboff et al. 2008), a program made available with sponsorship of the Willi Hennig Society, for 10,000 replicates. From the dataset we selected one sequence for all barcode clusters with less than 2% intraspecific distance, but we included four specimens of our target species , two from Italy and two from the USA.

A Bayesian Analysis was carried out with the same dataset. Model selection was performed using jModeltest 0.1.1 (Posada 2008). The best-fit model was chosen based on AIC value (Posada and Buckley 2004). Bayesian analyses were run in MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). Each analysis was run twice, starting from random starting trees, for 20 million generations and sampling every 1000 generations. Two partitioning schemes were explored: first, each codon position was given a separate partition and rate multipliers, while the second scheme combined first and second codon positions into a single partition with respect the third codon positions (Shapiro et al. 2006). Convergence of the Markov Monte Carlo chains was assessed by plotting the likelihood scores in Tracer v1.5 (Rambaut and Drummond 2007). A conservative burn-in of 5 million generations was chosen.

The sequence data generated and used in this study have been deposited in the public BOLD database (project “ Vine introduction” [ANTVI] and GenBank (Appendix B).

Field observations

Surveys were carried out from 2007 to 2011 to investigate the distribution in northeastern Italy. We sampled commercial vineyards but also isolated vine rows and plants of Virginia creeper, .

Observations on phenology and behaviour were carried out in 2008 and 2009 in Borgo Valsugana (Trentino Regione). The vineyard was planted with a Chardonnay cultivar and was trained with the local “pergola” system. The vineyardPageBreak received a number of fungicide treatments but insecticides were not applied. In 2008, a total of 180 leaves (30 plants, six leaves per vine) were sampled six times during the season, from May to September. In 2009, a total of 100 leaves (five replicates of ten plants, two leaves per plant) taken from the mid part of the shoots were sampled across ten dates, from May to September. In both years the number of mines produced by larvae was assessed on each leaf. In 2009, active mines containing living larvae were distinguished from those vacated by the larvae (mines with larval cut-outs).

Results

Identification

To identify the new Italian , we checked all descriptions of the Vitaceae miners, as well as all other known species. Unfortunately, outside Europe, genitalia have been illustrated and described only for Japanese species of , including all five Vitaceae miners (Kuroko 1961; Kuroko 1987). For the North American fauna, only a revision for three Cornaceae-feeding (with genitalia illustrations), has been published (Lafontaine 1973). The genitalia of the Italian populations (Fig. 9) did not match any published illustrations. An important external character of the moths is the silver apical spot on the forewing (Figs 1–2), a feature found in just a few members of the genus, whereas the other pattern elements that we examined are more general across the genus. Similarly-sized subapical spots were only noted in descriptions of some from the New World, although larger subapical patches occur in Japanese species, such as Kuroko, 1961. After excluding a poorly known species from Brazil as a less likely candidate, two North American Vitaceae miners with this spot were studied in more detail: Braun, 1927, occurring in Arizona and southern reaches of the Rocky Mountain area, and , which occurs widely acrosseastern North America. The genitalia of the male holotype of did not match, but several specimens identified as and reared from , had almost identical genitalia as the Italian populations. However, all specimens of reared from , were consistently different ( was described by Chambers from leafmines that he collected on in Kentucky). Leafmines that we collected in 2011 in northeastern United States on further showed that at least two species with different mines occur on that host. DNA barcoding results discussed below demonstrated that the Italian and North American examples from belong to the same species, and that American feeders belong to two different barcode clusters, supporting our morphological and biological findings that two species, co-occurred on in eastern North America. Material from the Chambers collection (see below) was insufficient to confirm the identity PageBreakof . Here we restrict the name to one of the two species feeding on . The miner from North America, previously misidentified in collections as being , is unnamed, morphologically identical to the Italian population, and described below.

Figures 9–16.

, male genitalia.Paratype, Italy,RMNH.INS.23920 (9, 15, 16), Paratype, Italy, RMNH.INS.15247 (12),Holotype, RMNH.INS.24204 (10, 11, 13–14). 9 Complete genitalia with separate phallus in ventral view 10–12 Phallus and juxta in ventro-lateral view 15–16 Complex of tegumen, uncus, valvae and transtilla 15 Detail of valval tips and pectinifers 16 Detail of spines near phallotrema.

Taxonomy

Antispila Hübner

Hübner, [1825]: 419. Type species Hübner, [1825]: 419 (a junior synonym of ([Denis & Schiffermüller], 1775), subsequent designation by ICZN (1988).

Van Nieukerken & Wagner sp. n.

urn:lsid:zoobank.org:act:F58A029E-A856-4414-B4EA-D7CAA6151948

http://species-id.net/wiki/Antispila_oinophylla

Figs 1 –6 9 –29 62 63

Figures 6–7.

, venation. 6 , male, Italy, RMNH.INS.24257 7 , male, Netherlands, Leiden, RMNH.INS.24258.

Figures 17–20.

, female genitalia. 17 Terminal segmentsandapophyses, ventral view, paratype, EJvN4211,USA, Kentucky (pseudotype ampelopsifoliella) 18 Internal genitalia, lateral view, showing sclerotisation in vestibulum,paratype, EJvN4206,USA, Connecticut 19 Ovipositor tip, dorsal view, EJvN4206 20 Detail of S8, ventral view,paratype, Italy,RMNH.INS.15244.

Figures 21–28.

, life history: leafmines on several species of and different localities. 21, 23, 24 Italy, Borgo Valsusana, , 25.vi.2009 22 USA: Vermont, Button Bay SP, 16.ix.2011 25 USA:Tennessee, NP Great Smoky Mts,, 2.x.2010, mine in shade leaf 26, 28 USA: Georgia, type locality, var. aestivalis, 14.x.2010 27 USA: Vermont, Button Bay SP, , 16.ix.2011, DNA barcode,RMNH.INS.18589.

Figure 29.

, distribution in North America.

Figure 62.

Map showing the distribution of in Italy up to 2010 (filled circles = sites of occurrence; arrow = site of collection specimens for sequencing).

Figure 63.

Incidence of the infestation at Borgo Valsugana (Trento province, Italy) in 2009 expressed as A the number of mines per leaf and B the percentage of infested leaves (mean ± SE).

Antispila sp.;

Antispila ampelopsifoliella ;

Antispila ampelopsiella ;

Type material.

Holotype ♂, USA: Georgia, Murray Co., Chattahoochee Nat. Forest, E of Chatsworth, GA rd 52, 523 m, 34.74066N, 84.71852W, hardwood forest along highway, leafmines on var. aestivalis, 14.x.2010, EvN2010266, emerged 14.iv–4.v.2011, E.J. van Nieukerken & C. Doorenweerd, Genitalia slide EJvN 4204, RMNH.INS.24204 (RMNH).

Paratypes.

32♂, 31♀. Italy: 1♂, 3♀ (all dissected), Trento, Borgo Valsusana, leafmines 2007, on , emerged 1.iii–26.iv.2008, M. Baldessari; 3♀, same locality, 13.viii.2008; 10♂, 1♀ (1♂ RMNH.INS.23920 dissected & DNA barcode), same locality, 18.viii.2008; 17♂, 18♀ (1♂ RMNH.INS.24038, 1♀ RMNH.INS.24039 dissected & DNA barcode), same locality, 29.vi.2009, leafmines on , EvN no 2009903, emerged in Leiden, 14.vii–6.viii.2009, M. Baldessari (all RMNH). Canada: 1♂, Ontario, Ottawa, mines on , rearing 57–112, emerged 31.iii.1958, Freeman & Lewis (CNC); 1♀, Quebec, Hull, mines on , rearing 55–228, emerged 26.vi.1956, T.N. Freeman (CNC). USA: 1♂, ConnecticutPageBreakPageBreak, Tolland Co., Mansfield, 22.viii.1989, leafmines on Vitis, DLW89H37 breeding, emerged 4.v.1990, D.L. Wagner (DLW); 1♀ (dissected), Connecticut, Windham Co., Hampton, 916 Pudding Hill Rd., leafmines on 1–5.ix.1988, DLW 88J7, emerged 20.vii.1989, D.L. Wagner (DLW); 1♂ 1 ♀, Georgia, same data as holotype; 1♀, Georgia, Murray Co., Chattahoochee Nat. Forest, Cohutta Overlook, 730 m, 34.785356N, 84.627323W, shrub in forest clearing, leafmines on var. bicolor, 14.x.2010, EvN2010270, emerged 19.iv.2011, E.J. van Nieukerken & C. Doorenweerd (RMNH); 1♀ (dissected, EvN 4211), Kentucky, [Covington], bred, [19th century], Chambers, “pseudotype,” MCZ Type 1367 (MCZ); 1♂, 1♀ (♂ dissected), Vermont, Chittenden Co., South Burlington, leafmines on 11.viii.1988, DLW 88H23, emerged 30.iii–15.v.1989, D.L. Wagner (DLW).

Non-type material

(all in RMNH). Italy: leafmines & larvae, Borgo Valsusana, 29.vi.2009, on , EvN no 2009903, M. Baldessari. USA: 1 larva, Connecticut, Tolland Co., Storrs campus, on , 185 m, 8.ix.2011, EvN2011168, B. Gagliardi; leafmines and larvae (being reared), Connecticut, New London Co., Connecticut College Arboretum, 34 m, 41.37929N, 72.11121W, on , 10.ix.2011, EvN2011193, E.J. van Nieukerken; leafmines and larvae (being reared), Connecticut, New Haven Co., West Rock Ridge SP, 125 m, 41.33353N, 72.96423W, on var aestivalis, 10.ix.2011, EvN2011198, E.J. van Nieukerken; leafmines & larvae (DNA barcode RMNH.INS.18394), Georgia, same data as holotype; leafmines & larvae (DNA barcode RMNH.INS.18392), Georgia, Murray Co., Chattahoochee Nat. Forest, Cohutta Overlook, 730 m, 34.78535N, 84.62732W, shrub in forest clearing, leafmines on var. bicolor, 14.x.2010, EvN2010270, E.J. van Nieukerken & C. Doorenweerd (RMNH); leafmines & 2 larvae (DNA barcode RMNH.INS.18533), Massachusetts, Berkshire Co., Beartown State forest, SW margin, 480 m, 42.19814N, 73.28928W, on , 12.ix.2011, EvN2011208, E.J. van Nieukerken; leafmines & larvae (DNA barcode RMNH.INS.18558), New York, Essex Co., Hwy 9N, 3.5 km WSW Keeseville, 142 m, 44.49233N, 73.52042W, on , 14.ix.2011, EvN2011237, E.J. van Nieukerken; leafmines & larvae (DNA barcode RMNH.INS.18555), New York, Essex Co., Wilsboro, Noblewood Park, 62 m, 44.35216N, 73.36435W, on , 14.ix.2011, EvN2011244, E.J. van Nieukerken; leafmines & larvae (DNA barcodes RMNH.INS.18298, 18300), Tennessee, Blount Co., NP Great Smoky Mts, Rich Mountain Gap, 619 m, 35.64557N, 83.80537W, rich forest on limestone ridge, leafmines on , 2.x.2010, EvN2010119, E.J. van Nieukerken & C. Doorenweerd (RMNH); mine and larva, (DNA barcode LGSME035–06), Tennessee, Cocke Co., Cosby, ATBI house, 35.77771N, 83.21359W, on sp. 12.viii.2006, DLW 2006H55, D.L. Wagner (DLW); leafmines & larvae (being reared and DNA barcode RMNH.INS.18669), VermontPageBreakPageBreak, Addison Co., Button Bay SP, Lake Champlain borders, 44 m, 44.18154N, 73.36892W, on , 16.ix.2011, EvN2011253, E.J. van Nieukerken.

Differential diagnosis.

In North America, at least four other species have an apical silver spot (together forming the ampelopsifoliella group): , , which has a darker head, an unnamed species from (here “vitis2”) and Chambers, 1874. The latter, which is closely similar in appearance, can be separated by the greater number of white flagellomeres at the antennal tip (six segments) and feeds on L. (Hydrangeaceae). Dissection of genitalia is needed to distinguish from other members of the ampelopsifoliella group. Male genitalia are characterised by the long carinal spine at the phallotrema and several other details; female genitalia differ by the number of cusps on the ovipositor from at least .

In Europe, differs from all other Heliozelidae with a similar forewing colour pattern (species of , and ) by the presence of a small silvery spot in the apical part of forewing and the distinctly white head. Some Elachistidae are superficially similar, but differ in long-pointed and upcurved palpi, longer antennae and more elongate habitus.

The leafmine of differs from that of by its short initial gallery, which is later usually completely incorporated into the blotch, whereas the initial gallery of mines is usually as long as or longer than the blotch, and remains intact. In Eastern North America other -feeding do not show the concentric arrangement of frass that is typical for – particularly in thinner leaves –and the mines are often larger. Mines of cf isabella and related species are much larger, and also have much larger cut-outs, 5 mm or longer. Since not all miners have been comprehensively studied, mine identification cannot yet be relied on.

Description.

Adult (Figs 1–5). Head face and vertex covered with appressed, strongly metallic, silvery-white scales, more prominently raised in male. Palpi porrect, white; base of proboscis covered with white scales. Antenna fuscous, apical 1 or 2 flagellomeres white. Labial palp silvery white, slightly upturned. Thorax lead-coloured, shiny, contrasting with forewings. Legs grey, tarsi mostly yellowish white, especially on undersides. Forewing dark fuscous with silver-golden patterning; an outwardly oblique fascia from 1/8 of posterior margin to 1/4 of costa, narrowing towards costa; triangular (dorsal) spot at middle of posterior margin, reaching to middle of wing, smaller triangular costal spot just beyond middle, sometimes touching dorsal spot; small, silvery subapical spot in middle of wing at 3/4; fringe line distinct. Terminal fringe paler. Hindwing pale grey. Abdomen lead-coloured, including vestiture on external genitalia.

Measurements: male: forewing length 2.5–2.8 mm (2.6 ± 0.10, n=11), wingspan 5.5–6.2 mm, 25–31 antennal segments (29.1 ± 1.9, n=11); female: forewing length 2.3–2.8 mm (2.5 ± 0.16, n=10), wingspan 4.8–5.6 mm, 25–29 antennal segments (27.2 ± 1.4, n=8).

Venation (Fig. 6). Forewing with Sc barely visible. R1 a separate vein, connected by persistent trachea to Rs+M stem. Rs+M terminating in five branches, interpretedPageBreak as Rs2 (possibly with 1) to costa, Rs3+4 to costa just before apex, M1 to dorsum just beyond apex, M2+3 to dorsum and a weakly developed CuA. A1+2 a strong separate vein. Hindwing with Sc barely or not visible, Rs+M a strong vein, bifurcate from ca. 1/4th, upper vein ending in two branches: Rs and M1, lower vein single (M3); Cu and A1+2 separate veins.

Compared to the complicate venation of many other species, including the type species , (example in Fig. 7, ) venation reduced with loss of forewing cell, separate M stem and connection between R1 and Rs, loss of Rs1 and in hindwing loss of M2. The venation more closely resembles that of (Fig. 8), which is even more reduced and also lacks Cu in the forewing.

Figure 8.

, venation.Female, Italy, RMNH.INS.24259.

Male genitalia (Figs 9–16). Uncus bar-shaped, with two large setae dorsally. Vinculum very long, anteriorly rounded, posteriorly shallowly bilobed. Valva more or less triangular, pecten on pedicel, with 10–13 comb teeth (Fig. 15); inner margin of valva with setose lobe anterior to pecten pedicel; basally with a triangular protuberance, almost touching that of other valva; transtilla with trapezoid medial plate, sublateral processes relatively short. Juxta anteriorly spade-shaped, about half as long as phallus. Phallus long, anteriorly much widened, at phallotrema with a comb of about 10–12 strong teeth and at left side a very long curved process (Figs 10–12, 16).

Female genitalia (Figs 17–20). Ovipositor with 4–5 cusps at either side (Fig. 19). S8 medially indented, with many papillate setal sockets. Vestibulum with broad, indistinct sclerotization and no spines (Fig. 18).

Biology.

Host plants.In North America reared from or found as larva on summer grape Michx., both var. aestivalis and var. bicolor Deam, fox grape L., riverbank grape Michx. and frost grape L. Literature PageBreakPageBreakPageBreakrecords of “ampelopsifoliella” from or grape likely refer to this species (Chambers 1874a, b; Forbes 1923; Needham et al. 1928). We did not find any reports of this species occurring in vineyards in North America. In Italy mines produced by were detected on various cultivars, hybrids (e.g. x rupestris) and French-American grapes (e.g. Clinton). Infestation levels on the latter were comparable with those observed on commercial vineyards. A preference for some grape cultivars (e.g. Cabernet Sauvignon, Chardonnay, Muscat) is suggested from observations carried out in mixed cultivar vineyards. It is interesting that we also found active mines on Virginia creeper in Italy (Levico and Caldonazzo, Trento province) (identification of larvae confirmed by DNA barcodes, no rearing attempted), whereas we have as yet no records of from this host in North America.

Leafmines (Figs 21–28). The egg is inserted on the underside of a leaf, usually within 1–2 mm from a vein. The mine starts as a rather straight or slightly contorted gallery towards the vein, usually forms a right angle and often follows the vein for a short distance, then again turns away from the vein where it expands into a blotch. The gallery portion, of variable length, is usually later incorporated into the blotch mine. The frass is linear, usually occupies the complete mine width, but occasionally is deposited in a thin line (Fig. 27). In the blotch much of the blackish-brown frass is deposited close to the origin in semicircular concentric frass lines. This characteristic pattern is best seen in thin shade leaves (e.g., Figs 25, 26); in sun-exposed leaves the frass pattern is often obscured. The whole mine occupies as a rule an area of less than 10 × 10 mm; only in thin leaves are mines appreciably larger. The larva cuts out an elliptic case of about 3.2–4.0 mm long.

Distribution

(Fig. 29, 62). In North America, is known with certainty (material cited) from Canada: Ontario, Quebec; USA: Connecticut, Georgia, Kentucky, New York, Tennessee, and Vermont. Records under from Maine, Missouri, and Ohio (Brower 1984, Forbes 1923) may partly refer to this species. In Europe introduced into northern Italy, see below. In our experience in the southern Appalachians and New England, at least in the fall, is often the most abundant species occurring on .

Etymology.

The epithet oinophylla, a noun in apposition, is from the Greek οινος (oinos = wine) and φυλλον, plural φυλλα (phyllon, phylla = leaf), “wine leaves,” because the larva lives in the leaves of the grapevine from which wine is made.

Justification for status as new species.

Four species feeding on Vitaceae have been named previously from North America. No name-bearing types are available for three species, only for is a holotype extant. The latter is clearly different from , and restricted to western North America. For the eastern species , and , we have only the original descriptions and subsequent interpretations to establish identities. The fact that our preliminary sampling of DNA barcodes for grape-feeding show great diversity, complicates matters further. Below, we will discuss these three species in the chronological order of their descriptions.

was described from mines on “Isabella grape” (a cultivar of ) and adults (Clemens 1860). The description unequivocally describes a relatively large species without a silvery apical spot. Clemens characterizes the case (shield) as large and almost roundish – both features exclude our species. We have tentatively named one larger barcode cluster as cf. isabella, because mines and adults conform to this description.

was also described by Clemens in 1860, from mines on “wild grapes” only, differing by a smaller case (shield) and a larva “without dots.” Although the foodplant was not explicitly mentioned by Clemens, from the species name it is evident that the host must have been Michx. (a synonym of ). In fact his very brief description could fit the mines of , but subsequently the name has always (e.g. Forbes 1923) been used in the sense of Chambers (1874a), who first described the moth (as “viticordifoliella N. sp.?”), without an apical spot and with several, white, distal flagellomeres. He reared that moth from the same hostplant ()as Clemens did, and was not able to find the mine on any other (Chambers 1874a: 169). One of the species that we studied from has similar externals, and is named here cf viticordifoliella (Fig. 37). Because we haven’t been able to find or rear any similar adults from we are at the moment unable to establish if the miner is indeed the same as , but clearly it is not our species (because it lacks an apical spot). In a future revision a neotype will need to be selected to firmly anchor the identity of this species, material from the Chambers’ collection (two extant “syntypes”, see Miller and Hodges 1990) probably is most suitable for that goal. In collections and websites (e.g., http://mothphotographersgroup.msstate.edu/) the name is often misinterpreted as the species that we call cf isabella or a closely related one.

:Chambers(1874a: 168) only briefly described the mine and larva from “” [= ] (and stated that he “never succeeded in breeding it.”). Only a month later he described the moth under the name “” [sic, considered as a subsequent incorrect spelling], writing: “Since that paper was placed in the hands of the Editor, many months ago, I have succeeded in rearing it from the mine [from ]” (Chambers 1874b). Theconfusionof the new specieswith dates from Chambers’ original description, because he also described a moth that he reared from and shows the external characters of both species:

“Last summer I found its leaves [referring to a species] mined by a larva closely resembling that of , supra, and which I suspect to be the same. ….. From it I bred the species described below, which I do not now name, as it may prove to be identical with .” (Chambers 1874a). One month later he wrote: “but I believe it to be the same” (Chambers 1874b). Ever since these two publications, the species has been considered to feed both on and (e.g., Forbes 1923; Brower 1984). However, our rearing and barcode data show that two or three species of are feeding on , which show large barcode distances to or other miners (Fig. 30), and thus are not identical.

Figure 30.

Neighbor-joining tree for heliozelid COI barcodes, based on uncorrected pairwise distances. Numbers on branches are bootstrap values, 10,000 replicates. Vitaceae-feeding clusters are coloured differently, others in black. Labels include species name or informal name, codes for country and state (in North America) and sample numbers (Genbank numbers for sequences taken from Genbank).

In Chambers’ collection at MCZ there are three specimens under the name that probably served as the basis for the adult description. These specimens were termed pseudotypes (Miller and Hodges 1990), since they were not available at the time of the original description, because then Chambers only had mines and larvae available. Of the three specimens, one is completely missing from the pin. The one labelled as from unfortunately is heavily damaged, only a forewing and hindwing being present. A third specimen, a female, is complete and was dissected (Fig. 17). This specimen, however, appears to be . This is no surprise, since Chambers (1874a, 1874b) considered the miner to be the same as the miner, and thus he would have placed specimens reared from both hosts under the same name. There is no indication of the hostplant or the collecting year on this particular specimen, so it is useless for confirmation of the identity of .

We restrict here the usage of the name to the species feeding on , with an apical spot (The generic name for was at the time Chambers described the species.) Although we have not obtained a DNA barcode form such an adult, the fact that an adult from the other cluster on this host (see below) does not have such a spot and is tentatively identified as cf viticordifoliella, we can associate adults with one of the larval types.PageBreak When adults are available for all barcode clusters, we suggest that a neotype be selected from material reared from from the vicinity of Covington, Kentucky, to fix the identity of Chambers’ name.

DNA barcoding and species relationships

Barcode analysis

Neighbor-joining trees of all sequenced barcodes, both based on Kimura 2P distances and uncorrected distances give highly similar results in topology and branch lengths, we illustrate here the last one (Fig. 30). All species clusters have a bootstrap value of 100, and within-species variation is usually low or absent. We caution, however, that for several species, such as or most sequences are from just one or two populations. Two species clusters show large intraspecific distances: the two specimens of have 5.22% K2P distance and 4.99% uncorrected pairwise distance, and the species tentatively named cf viticordifoliella forms two clusters with around 4% distance in both methods. Although the mines of these clusters look superficially the same we have not studied the adults of one cluster, so it is possible that these clusters represent separate species.

We have 20 sequences representing , seven of which are 100% identical, five from Italy (including one from ) and two from North America (RMNH.INS.18392 from Georgia and RMNH.INS.18558 from New York). The others are very similar, with at most five nucleotides differing from those of the core group (RMNH.INS.18394 from Georgia). The genetic distance varies from 0 to 1.23% K2P distance (1.22% uncorrected). The differences occur in 16 different positions, of which six cases are found in more than one specimen (e.g., a G instead of A in position 82 combined with a T in 316; the seven specimens forming a “clade” in Fig. 30 with RMNH.INS.18533 and BVS04; position 550: C instead of T; four specimens forming the “clade” in Fig. 30 with RMNH.INS.18533, position 634 a T instead of A in RMNH.INS.18298 and RMNH.INS.18300, both from Tennessee). Several haplotypes are found both in Italy and North America. The largest distance is between two North American specimens, one from Georgia and one from Tennessee (RMNH.INS.18394 and LGSM035–06). The genetic distance to the closest congeneric species is large: more than 10%.

Phylogenetic analyses

The maximum parsimony analysis of the barcode sequences resulted in three shortest trees, of which the 50% majority rule tree is illustrated (Fig. 31). The semi-strict tree differs only in the position of , which forms a polytomy with the three main heliozelid clades in Figure 31. Of the 658 characters, 243 characters are parsimony PageBreakPageBreakinformative. Bootstrap values are taken from the TNT analysis. The two Bayesian analyses of the same dataset showed few differences, we here illustrate the consensus tree based on three partitions (Fig. 32).

Figure 31.

Cladogram, 50% majority rule consensus of three shortest trees from maximum parsimony analysis of COI sequences. CI = 0.361, RI = 0.456, RC = 0.168. Figures are bootstrap values from a TNT analysis (10,000 bootstrap replicates). Purple-coloured taxa are feeding on Vitaceae. The semi-strict tree differs only in the position of (see text).

Figure 32.

Cladogram from Bayesian analysis on three partition dataset. Figures are posterior probabilities. Purple-coloured taxa are feeding on Vitaceae.

Both cladograms are rather similar. forms a highly supported clade. Clades for , a core grouping and a clade with several smaller genera and the group were recovered, with strong support in the Bayesian analysis for thelatter clade (0.97) and for (1) and less support for core (0.74). Within the core clade, the two Vitaceae species form a clade, well supported in the Bayesian tree, nested in or sister to the Cornaceae-feeding species.

The Bayesian analysis recovered a monophyletic group. In both analyses this group clusters with the small genera , and . These all share the reduced venation as described here for . Relative positions of these small genera and the two clades of vary amongst various analyses. In the Bayesian tree there is low support for a clade of and . In none of the analyses was Heliozelidae recovered as a monophyletic group.

Vitaceae-feeding taxa are indicated in the cladograms by a purple colour. If these cladograms correctly represent the phylogenetic history of the Heliozelidae, it appears that Vitaceae were the ancestral hosts for the family.

Comparative notes to other species

Below we will briefly treat the other Vitaceae miners amongst North American and European Heliozelidae and one other closely related species, in order to distinguish them from . As there are several more species in North America than currently described, this is a preliminary treatment until a thorough revision can be completed. Because we have not yet been able to link some larval barcode clusters to their associated adults, the number of leafmine types described below is higher than the number of adult “species”. Material examined for each of these “taxa” is listed in the Appendix A.

Chambers

http://species-id.net/wiki/Antispila_ampelopsifoliella

Figs 35 42, 43 53 56

Figures 42–47.

species, male genitalia. 42–43 , USA, New York state, genitalia slide EJvN4200 44–45 , USA, holotype, genitalia slide EJvN3916 46 cf isabella, USA: Kentucky, Morehead, genitalia slide CNC MIC1859 47 , USA: North Carolina, NP Great Smoky Mts., genitalia slide EJvN4198.

Figures 48–53.

, male and female genitalia, , female genitalia(53). 48–50 Male genitalia, Italy, slides RMNH.INS.15248, 15250, 15251 51–52Female genitalia, slide RMNH.INS.15252 53 Ovipositor and tergum 8, genitalia slide JCK15220.

Figures 54–61.

and species, life history. 54–55 on , Italy: Rovereto 56 on , USA: Button Bay SP, 16.ix.2011 57  cf viticordifoliella on , same locality 58, 60 cf isabella on , USA: Button Bay SP, 16.ix.2011 59, 61 “vitis2” on , samelocality. In the last four photos also parts are visible of gallery mines of .

Antispila ampelopsifoliella Chambers, 1874a: 168. Syntypes: leafmines [USA: Kentucky, Covington] on

Antispila ampelopsisella Chambers, 1874a: 197. Subsequent incorrect spelling.

Antispila ampelopsiella Chambers, 1874a: 198. Subsequent incorrect spelling.

Antispila ampelopsifoliella ;

Antispila ampelopsiella ;

Differential diagnosis.

We cannotseparate (Fig. 35) from based on external characters: it may average a bit smaller, but our samples are too few in number to make statistical comparisons. In the male genitalia (Figs 42–43), uncus not bilobed; valva with pecten with ca. 11–13 comb spines, base of valva with rounded lobe, not triangular; juxta rather wide, with lateral groups of spines; phallus with much shorter terminal spines and a comb of rather short triangular spines near phallotrema. Female genitalia (Fig. 53): ovipositor only with 3 cusps at either side. Vestibulum with some spines.

Biology.

Hostplant: .

Leafmines

(Fig. 56). Egg usually inserted in leaf under- or upperside close to a vein, mine starting with a relatively long contorted gallery with thin broken frass, or when it runs along margin in a straighter course, later abruptly enlarged into elongate blotch or wide gallery; frass dispersed in middle. The early narrow gallery may be as long as the elongate blotch. The mine can be found in any part of the leaf. Larva yellowish white, black head, cut-out ca 3.5–4 mm long. The mine resembles that of . It was most frequently found in the larger and thinner ground leaves of Virginia creeper.

Distribution.

Eastern North America, confirmed from USA: Connecticut, Kentucky, New York, Vermont and Canada: Ontario.

Braun

http://species-id.net/wiki/Antispila_voraginella

Figs 34 44, 45

Antispila voraginella Braun, 1927: 191. Holotype male:

Adult (Fig. 34) very similar to and about same size as , but head and thorax covered with brassy shining scales rather than silver. In male genitalia (Figs 44–45) uncus clearly bilobed, valva with fewer pecten spines: 8–10, triangular lobe absent; transtilla with narrower central plate and phallus with rather different set of spines: the long one of oinophylla absent, and row along phallotrema less comb-like, whereas there is a row of many spines along both sides. Female genitalia not examined.

Hostplant: . Seems to be univoltine, larvae found in June–July northward; through September in monsoonal areas to south; adults emerging the following spring April to June.

Leafmines.

Mine illustrated by Powell and Opler (2009: plate 59:7). Mines rather different from those of : larvae usually gregarious with mines forming large pale blotches.

Evidently allopatric to and only recorded from the Rocky Mountains: Utah, Arizona and West Texas.

Antispila “vitis1”

Fig. 40

From this barcode cluster we have just two females from Florida (Fig. 40, one barcoded) and one larva from Connecticut, of which it is unclear to what type mine it belongs. The female is indistinguishable externally from . Almost certainly this represents another new species.

Chambers

http://species-id.net/wiki/Antispila_hydrangaeella

Figs 36 47

Antispila hydrangaeella Chambers, 1874a: 170. Syntypes leafmines and larvae: [USA: Kentucky, Covington] on

DNA barcodes suggest that two species might be involved, and leafmines from a population in North Carolina (Smoky Mts NP) and northern Georgia do show some differences. Described adults and larvae are from the Georgia population.Externally, adult (Fig. 36) is extremely similar to the other species of the group, but it differs by the last six antennal segments being white and by genitalia and hostplant data. In male genitalia (Fig. 47) uncus only shallowly bilobed; valva with long pecten with more comb spines: ca. 20, triangular lobe absent, at base of valva beardlike setation; juxta rather wide, with groups of spines laterally; phallus with two very long terminal spines and many small spines near phallotrema, not forming a comb. Female genitalia not examined.

Hostplant: .

Leafmines.

One type (North Carolina) with long gallery mines, often following a vein, ending in a blotch with greenish to brown frass. The mines from Georgia with early gallery mine much contorted in a small area, with black frass, ending in elongate mine with blackish dispersed frass.

Distribution.

USA: Georgia, Illinois, Kentucky, North Carolina, presumably widespread in eastern United States.

Clemens

http://species-id.net/wiki/Antispila_viticordifoliella

Antispila viticordifoliella Clemens, 1860: 209. Syntype mines, larva [USA: Pennsylvania, Easton], larvae on “wild grapes” [

Antispila viticordifoliella ;

In the interpretation of this species by Chambers (1874a), as discussed above, differs from the group in missing the apical spot on the forewing and its long white antennal tip, the latter character is shared with . We have as yet not seen such specimens originating from .

Hostplant: . Leafmines not described in detail.

USA: Kentucky, Pennsylvania. Many records are unreliable and often refer to the isabella complex.

Clemens

http://species-id.net/wiki/Antispila_cf_viticordifoliella

Figs 37, 41 57

Remarks.

Two females (Figs 37, 41), reared from mines, match Chambers’ (1874a) description of adults. Because the possibility exists that two species with similar externals, feeding respectively on and , are involved here, we cannot decide whether the miner is conspecific with viticordifoliella or not, before we have studied genitalia and/or DNA barcodes from specimens originating from both hostplants (to date we have only barcodes from miners and no males from either form). Moreover, there is a deep split in the barcodes from miners, here tentatively identified as cf viticordifoliella, one cluster from New York and Vermont, the other from Connecticut and Florida. We did not see differences in mine or larva between these clusters, and thus tentatively regard them as one species.

Hostplant: .

Leafmines

(Fig. 57). Egg often inserted on leaf margin, position often hard to find, rarely near midrib, mine without a gallery at the start, an elliptic elongate blotch mine, often running along or near leaf margin; frass sometimes grouped in a clump, more typically spread in an irregular broad line. Larva yellow with almost black head, cut-out ca 3.5–4 mm long. This mine was most frequently seen in thicker leaves borne from climbing shoots.

Canada: Ontario. USA: Connecticut, Florida, New York, Vermont.

Clemens

http://species-id.net/wiki/Antispila_cf_isabella

Figs 38, 39 46 58, 60 (59, 61

Antispila isabella Clemens, 1860: 209. Syntypes: [USA: Pennsylvania, Easton], larvae on “Isabella grape”, September, adults emerged May, Brackenridge Clemens (ANSP if extant).

Antispila isabella ;

Under this name there is probably a complex of species, often with conspicuous androconial scales in males. Among the barcodes we distinguish two clusters, here tentatively named as cf isabella and “vitis2”. The adults described here do not necessarily belong to one of the described mine types.

Moths (Figs 38–39) of this species complex are easily distinguished from the group by the missing apical spot on forewing and larger average size. Moreover males have conspicuous yellow or brown androconial scales on forewing underside (Fig. 39). The venation is also more complete (as in Fig. 7).

Male genitalia were examined of one of the species (Fig. 46) the valva is more elongate, and the pecten includes 10–13 teeth. Phallus lacks larger spines at phallotrema, but has many scale-like, small spines, and posteriorly possesses an asymmetric broad lobe; anteriorly not widened. Other individuals have not been examined; as noted above, the group is in need of revision.

Hostplant: , [incl. “Isabella” grapes], .

Mines of cf isabella (Figs 58, 60) are relatively large mines, with the egg deposited near a vein. No gallery visible, mine a large blotch, with a roundish patch of reddish frass near beginning, probably attached to upper epidermis, and dispersed black frass throughout mine. Cut-out large, around 5 mm long.

Mines of “vitis2” (Figs 59, 61) also start on a vein, without gallery, and are relatively compact blotches, with frass concentrated in a mushroom shape or reversed triangular near beginning of mine. Cut-out large, around 4.8 mm.

Canada: Ontario. USA: Connecticut, Georgia, Kentucky, New York, Pennsylvania, Vermont.

Both COI sequences and external sexual secondary characters show that more species are involved. We have tentatively named the most common form as cf isabellaPageBreakPageBreak, and research of types or material from the collections of Clemens and Chambers is needed for establishing the identities of these names.

(Stainton)

http://species-id.net/wiki/Holocacista_rivillei

Figs 8 33 48–52 54, 55

[Unnamed]

Alucita vitella Vallot, 1822: 253. [Preoccupied by

Elachista rivillei Stainton, 1855: 87. [Malta, Godeheu de Riville, 18

Antispila rivillei ;

Antispila rivillella Rondani, 1877: 288 [Redescription, parasitoids].

Holocacista rivillei ; Walsingham and Durrant 1909: xxix [new genus, first recorded from France].

Moth (Fig. 33) much smaller than species, with 3.5–4 mm wingspan. Forewing pattern without apical spot, costal spots further away from wingbase than dorsal spots. Male genitalia (Figs 48–50) with slightly bilobed uncus, valva more elongate, pecten with 8–10 teeth; juxta with pair of lateral teeth; phallus extremely slender and long, ending posteriorly in long curved spine and row of small spines below that. Juxta bilobed apically. Venation reduced, rather similar to that of (see Fig. 8).

Hostplant: .

(Figs. 54–55). Mine beginning with relatively long, slender gallery, later a small blotch with small cut-outs. Cocoons often attached to stems or leaves.

Southern Europe, western and Central Asia: Spain, France, Italy, Malta, Slovenia, Croatia, Bulgaria, Greece, Ukraine, Turkey, SE Russia, Georgia, Kazakhstan, Uzbekistan, Turkmenistan (Voigt 1931; Berro 1934; Marchi 1956; Dovnar-Zapol’skij 1969; Bournier 1977; Puplesiene 1996; Maček 1999; van Nieukerken 2011).

Distribution of A. oinophylla in Italy (Fig. 62)

In Italy, was detected for the first time in the summer of 2007 in a vineyard located in Valsugana (Borgo Valsugana, Trento province, Trentino-Alto Adige Region). Additional surveys conducted in the late summer of 2007 revealed its occurrence also in the neighbouring Vicenza and Belluno provinces (Veneto Region), particularly in neglected vineyards. In 2008, the distribution of the species did not differ greatly in the Trento province; elsewhere the insect was recorded in commercial vineyards of three provinces of the Veneto Region (Vicenza, Belluno and Treviso), sometimes at significantPageBreak densities. In a number of vineyards occurred together with , rarely with . In 2009 and 2010, a dense infestation was detected in commercial vineyards located in the Vicenza province (Breganze), about 80 km south of Borgo Valsugana. In this area severe symptoms had been detected as early as 2006 but they were misidentified as being caused by . Since viticulture of this area is much more extensive than that around Borgo Valsugana, it is likely that was introduced first in the Vicenza province and dispersed from there to the other areas. Also in 2010 the species was recorded in the Verona province, 90 km west of Breganze (E. Marchesini, pers. comm.). The distribution of in Italy in 2010 is presented in Fig. 62.

Field observations in Italy

Observations carried out in winter 2008 showed that fully fed, final instar larvae of overwintered inside their cases, fixed to the vine trunks or training stakes. Most larvae pupated in May and the first adults were seen in early June. Mines were detected first in the second half of June. Larvae of the penultimate instar cover the internal surface of the mine with a thin layer of silk, cut away an oval leaf section from both the upper and lower leaf surfaces, and then formed a case by joining the excised leaf sections with silk. Case-bearing larvae move slowly on the leaf surface and then descend with a silken thread until they contact a trunk, training stake, or other solid object to which they affix their case. In the experimental vineyard, the first cases were observed in the first half of July. An additional generation occurred from the second half of August onwards. In 2008, 86.9% of the leaves were infested with a density of 3.26 ± 0.25 (mean ± standard error) mines per leaf by the end of the first generation. In PageBreaklate summer, by the end of the second generation, 95.6% of leaves were infested with an average of 5.44 ± 0.37 mines per leaf.

Observations carried out during 2009 in the same vineyard, confirmed the existence of two generations. Adults were detected from early June to early July. The first mines were observed in mid-June and the first cases in late June (Fig. 63). Larval densities of the first generation peaked in early July, and by late July most mines had been abandoned by the larvae. Mines of the second generation were visible beginning in the second half of August. In the first generation, 96% of leaves were infested with an average of 4.6 ± 0.53 mines per leaf. In the second generation 97% of leaves were mined with an average density of 6.67 ± 0.72 mines per leaf. Active larvae were found until mid-October.

Discussion

Taxonomy and identification

Identification of the unknown leafminer proved to be difficult. Many groups of Microlepidoptera remain poorly studied taxonomically. Even in North America, Powell and Opler (2009) estimated that at least one third of the microlepidopteran fauna is still undescribed. There seems to be little chance of overcoming this situation, and even groups feeding on economically important plants such as species, remain unstudied. Although we had assembled substantial material of , the morphological similarity across the genus was confusing, and only after checking several genitalia slides and COI barcodes did it become clear that what was previously called “” was composed of at least two cryptic species on different hostplants. Finding COI barcode matches, in order to rule out the possibility of a non-American sibling species, took more time, because of lack of fresh material and because the miners in North America are more diverse than previously thought. An initial matching of the Italian pest’s barcode with that of an record in the BOLD identification system collected in the Great Smoky Mountains, helped focus our research efforts, and underscored the importance of a public DNA barcode reference database. In 2010 and 2011, with increased geographic and taxonomic sampling, we were able to confirm initial results and match additional sequences to those of the introduced Italian populations. The facts that several North American specimens show a 100% identical barcode to the majority of Italian specimens, the overall small genetic distances across all Italian specimens, and that the largest COI distance found was between two North American specimens, corroborate our position that the Italian populations represent a recent introduction from North America. All Heliozelidae species in this study differed sufficiently in their barcodes to allow reliable identification. The barcode data of North American material in addition showed us that the groups of and miners are more diverse than currently recognized and that we cannot identify all taxa with certainty based strictly on morphological grounds. We also note that the North American Vitaceae-feeding exhibit important differences in male secondary characters and genitalia. A revision of the genus is much needed, but was not possible in the context of this study, where a name was urgently needed for a pest of grapevines. Elsewhere, for example in mainland Asia, the group of miners is completely unworked, and in need of taxonomic study (before new outbreaks occur).

We emphasize here the importance of combining traditional morphological descriptions with the additional dataset of DNA sequences for taxonomic groups whose identification is particularly difficult and mainly based on the description of genitalia.

An interesting observation is that we did not find any occurrence of on in its natural habitat in North America, although it utilizes that host in Italy. We found mines on growing intertwined with vines, that harboured two different species of , all occurring within a few centimetres of each other. Despite this sympatry, we did not find any indication of host shifts.

Phylogeny

While it is generally inadvisable to rely solely on DNA barcodes for phylogenetic inferences, several recent studies suggest that some phylogenetic information could be taken from both the sequences themselves or translated amino acids (Wilson et al. 2011). Our phylogenetic results show that on the basis of the COI barcode, is a paraphyletic genus in relation to the genera , and . A generic revision of Heliozelidae has not been published, but the late Ebbe Nielsen made a primer to such a revision in his unpublished thesis (Nielsen 1980b) that has been examined by the senior author. Nielsen recognised three clades, one with and some related genera, one with and and a final one with Bradley, 1961 (from the Solomon Islands), , and a new South American genus. The only difference with our findings is the position of . Interestingly the clade of the group with , and , as we find it, is characterised by the very similar reduced venation. A reduced venation has been reported before from some exotic (Kuroko 1961), but for instance all Japanese species seem to share the complex venation of the core as illustrated here (Kuroko 1961; Kuroko 1987). Another character noted by Nielsen to group and is the habit of larvae to attach their cases to stems rather than the soil. This behavioural character is shared with and other members of the group. Despite the poor support for this clade on the basis of barcodes, the mitochondrial and behavioural data collectively suggest that Nielsen’s groups could be good and, should such prove to be the case, the genus will need to be subdivided into at least two genera. Alternatively, many of the smaller genera would need to be synonymised into one large , or the group and the smaller genera should be combined in one genus. In the latter case, the generic name would become , which is unfortunate, since the genus in its current circumscription is well recognisable both in morphology and biology. In any case, such decisions are outside the scope of the present paper and should be made after a careful phylogenetic generic revision.

Another interesting result from our provisional phylogenetic analyses is the hypothesis that Vitaceae may form the ancestral hostplants for modern Heliozelidae. For the basal genus Karsholt & Kristensen, 2003 no host plant information is available (Karsholt and Kristensen 2003). Vitaceae occupy a rather isolated position in the angiosperm phylogeny, as sister to all core rosids (Wang et al. 2009). Other Heliozelidae feed on a wide variety of angiosperm families, but most on “eudicots”. species feed mostly on rosids (Fagaceae, Betulaceae, Myrtaceae), species usually on asterids (Cornaceae, Rubiaceae), species on both rosid and asterid trees or shrubs (Davis 1998, van Nieukerken unpublished). Still, these results should be regarded as provisional hypotheses, and should be vigorously tested by analysing additional taxa and genes, as well as using morphological characters.

Introduction in Italy

is the first alien species of Heliozelidae introduced into Europe (Lopez-Vaamonde et al. 2010). Since our manuscript was finished a second species of Heliozelidae from North America was reported as introduction to Italy: a species on (Bernardo et al. 2011).

Factors leading to the introduction of in Italy are unknown. is the most recent Nearctic insect species reported to be damaging grapevines in Italy (first in Europe). Its invasion follows those of (Posenato et al. 1997) and Fitch, 1851 (Hemiptera: Cicadellidae) (Duso et al. 2005). Trade of vines from North America to Italy is limited while that of the alternative host seems to be more intense. However, the absence of records of on in North America makes introduction with Virginia creeper a less likely pathway. Anyway, because the caterpillars routinely attach their cocoons to debris, stems or stakes, transport of cases is probably common, and thus not unlikely to have happened. With the frequency of modern air traffic even the transport of adults, and in particular gravid females is not impossible. The fact that is an abundant and widespread species in eastern North America, together with its life history, makes such a possibility even more likely. However, it is also a warning that other species with similar life styles could be the next introduction, with an unpredictable outcome. Introduction from North America apparently occurs rather commonly; 16.5% of alien Lepidoptera species in Europe originate from North America (Lopez-Vaamonde et al. 2010).

The presence of several North American haplotypes of the DNA barcode in Italian material of may indicate that the introduction could have involved more than a single introduction event.

Infestation

Early observations, carried out during 2007 and 2008 in the Trento province, showed that the incidence of infestation by was significant in vineyards not treated with insecticides. By 2009 significant infestation levels were observed in several commercial vineyards in the Trentino and Veneto Regions despite the application of insecticides. is also increasingly important in commercial vineyards in northeastern Italy. Native parasitoids showed some effects in keeping below economic thresholds (Marchesini et al. 2000). Local outbreaks could be associated with the use of broad-spectrum insecticides, probably because they knock out many egg and larval parasitoids, thereby disrupting the interactions between the pest and its natural enemies. Similar mechanisms could affect the relationships between and its parasitoids. Knowledge of such relationships will be required to understand fully what pest status might reach in the future. In Trento PageBreakprovince, presently, the role of predators and parasitoids in controlling appears to be negligible. However, in the Veneto the situation is different, with 32 to 48% of the larvae and pupae in late summer being parasitized (C. Duso and A. Pozzebon, unpublished data). The identification of parasitoids from Italian vineyards is in progress. and share a number of parasitoid species (Mariani 1942; Camporese and Marchesini 1991; Alma 1995; Marchesini et al. 2000). It is therefore likely that some of these will also be found to attack populations.