Corresponding author: Erik J. van Nieukerken (
Academic editor: D. Lafontaine
A grapevine leafminer
There are several cases known of leafmining
Lepidopteran leafminers of grapevine (
In the summer of 2007, leafmines similar to those caused by
The family
The grapevine family
|
|
|
|
---|---|---|---|
|
USA | this paper | |
USA |
|
||
(USA) |
|
this paper | |
USA | |||
(USA) | this paper | ||
USA |
|
||
(USA) |
|
this paper | |
USA |
|
||
Japan |
|
||
Japan |
|
||
Japan |
|
|
|
Japan |
|
|
|
Japan |
|
|
|
Japan |
|
||
India |
|
||
India | |||
India |
|
||
Indonesia, Borneo |
|
EJvN | |
Australia |
|
|
|
Malta |
|
see text | |
USA |
|
Abbreviations for depositories:
Academy of Natural Sciences in Philadelphia, Pennsylvania, USA
Canadian National Collection of Insects, Arachnids and Nematodes, Ottawa, Ontario, Canada
Research collection of David L. Wagner, Storrs, Connecticut, USA
Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts, USA
Netherlands Centre for Biodiversity Naturalis, former Leiden Zoology collections, Leiden, Netherlands
University of Maryland, College park, USA
University of Padova, Department of Environmental Agronomy and Crop Science, Italy
Zoological Museum University of Oulu, Finland
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.
Methods for preparation of the genitalia follow
The Distribution Map for North America was prepared with DMap 7.0 (
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
A 665 bp or a 658 bp fragment of the mitochondrial COI gene was amplified using the following primers: in Padua LCO1490 and HCO2198 (
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 (
Neighbor-joining (NJ) trees based on DNA barcode sequences of all available specimens were reconstructed with Paup* 4.0b10 (
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 (
A Bayesian Analysis was carried out with the same dataset. Model selection was performed using jModeltest 0.1.1 (
The sequence data generated and used in this study have been deposited in the public BOLD database (project “
Surveys were carried out from 2007 to 2011 to investigate the
Observations on
To identify the new Italian
32♂, 31♀.
(all in RMNH)
In North America, at least four other species have an apical silver spot (together forming the
In Europe,
The leafmine of
Adult (
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 (
Compared to the complicate venation of many other
Male genitalia (
Female genitalia (
Host plants.In North America reared from or found as larva on summer grape
Leafmines (
(
The epithet
Four species feeding on
“Last summer I found its leaves [referring to a
Neighbor-joining tree for heliozelid COI barcodes, based on uncorrected pairwise distances. Numbers on branches are bootstrap values, 10,000 replicates.
In Chambers’ collection at MCZ there are three specimens under the name
We restrict here the usage of the name
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 (
We have 20 sequences representing
The maximum parsimony analysis of the barcode sequences resulted in three shortest trees, of which the 50% majority rule tree is illustrated (
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
Cladogram from Bayesian analysis on three partition dataset. Figures are posterior probabilities. Purple-coloured taxa are feeding on
Both cladograms are rather similar.
The Bayesian analysis recovered a monophyletic
Below we will briefly treat the other
We cannotseparate
Hostplant:
(
Eastern North America, confirmed from USA: Connecticut, Kentucky, New York, Vermont and Canada: Ontario.
Adult (
Hostplant:
Mine illustrated by Powell and Opler (2009: plate 59:7). Mines rather different from those of
Evidently allopatric to
From this barcode cluster we have just two females from Florida (
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
Hostplant:
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.
USA: Georgia, Illinois, Kentucky, North Carolina, presumably widespread in eastern United States.
In the interpretation of this species by
Hostplant:
USA: Kentucky, Pennsylvania. Many records are unreliable and often refer to the
Two females (
Hostplant:
(
Canada: Ontario. USA: Connecticut, Florida, New York, Vermont.
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
Moths (
Male genitalia were examined of one of the species (
Hostplant:
Mines of
Mines of
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
Moth (
Hostplant:
(
Southern Europe, western and Central Asia: Spain, France, Italy, Malta, Slovenia, Croatia, Bulgaria, Greece, Ukraine, Turkey, SE Russia, Georgia, Kazakhstan, Uzbekistan, Turkmenistan (
In Italy,
Map showing the distribution of
Observations carried out in winter 2008 showed that fully fed, final instar larvae of
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 (
Incidence of the
Identification of the unknown leafminer proved to be difficult. Many groups of
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
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 (
Another interesting result from our provisional phylogenetic analyses is the hypothesis that
Factors leading to the introduction of
The presence of several North American haplotypes of the DNA barcode in Italian material of
Early observations, carried out during 2007 and 2008 in the Trento province, showed that the incidence of infestation by
We thank Claudia Savio, Isabel Marinez-Saňudo, Alberto Pozzebon (Department of Environmental Agronomy and Crop Science, Padova, Italy), René Glas, Frank Stokvis and Dick Groenenberg (NCB Naturalis, Leiden, Netherlands) for their kind assistance in field trials and molecular laboratory work. Jean-François Landry (Agriculture Canada, Ottawa, Canada), Jason Weintraub (Academy of Natural Sciences in Philadelphia, Pennsylvania, USA) and Philip D. Perkins (Museum of Comparative Zoology, Harvard University, Cambridge, USA) loaned us material of American
Leafmines and larvae (barcodes RMNH.INS.18536, 38),
Canada: 1♀, Ontario, Ottawa, 14.iv.1971, “Vir. creeper” [
Italy: 28 adults (4♂, 1♀ dissected), Trento, Borghetto,
List of samples used for the DNA barcoding. (doi: