Research Article |
Corresponding author: Natalia Kirichenko ( nkirichenko@yahoo.com ) Academic editor: Erik J. van Nieukerken
© 2015 Natalia Kirichenko, Peter Huemer, Helmut Deutsch, Paolo Triberti, Rodolphe Rougerie, Carlos Lopez-Vaamonde.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Kirichenko N, Huemer P, Deutsch H, Triberti P, Rougerie R, Lopez-Vaamonde C (2015) Integrative taxonomy reveals a new species of Callisto (Lepidoptera, Gracillariidae) in the Alps. ZooKeys 473: 157-176. https://doi.org/10.3897/zookeys.473.8543
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Europe has one of the best-known Lepidopteran faunas in the world, yet many species are still being discovered, especially in groups of small moths. Here we describe a new gracillariid species from the south-eastern Alps, Callisto basistrigella Huemer, Deutsch & Triberti, sp. n. It shows differences from its sister species C. coffeella in morphology, the barcode region of the cytochrome c oxidase I gene and the nuclear gene histone H3. Both C. basistrigella and C. coffeella can co-occur in sympatry without evidence of admixture. Two C. basistrigella specimens show evidence of introgression. We highlight the importance of an integrative approach to delimit species, combining morphological and ecological data with mitochondrial and nuclear sequence data. Furthermore, in connection with this study, Ornix blandella Müller-Rutz, 1920, syn. n. is synonymized with C. coffeella (Zetterstedt, 1839).
COI, DNA barcoding, histone H3, mitochondrial-nuclear discordance, leaf-mining moths, contact zone, new species
Lepidoptera – butterflies and moths – are one of the most well-documented insect orders, but it is estimated that thousands of species, especially small-sized ones inhabiting the tropics, are still awaiting formal description. The integration of genetic data into taxonomic studies, especially with the advance of DNA barcoding campaigns (the construction of libraries of DNA barcodes for identification), has revealed many cases of cryptic or overlooked species in the tropics (
Leaf-mining micro-moths in the family Gracillariidae are no exception. A study based on the analysis of DNA barcodes recently revealed a considerable number of undescribed species in the Neotropical region (
Here we focus on the gracillariid Callisto coffeella (Zetterstedt, 1839), an arctic-alpine species, which has been recorded from northern Europe, the Alps and a few other mountain areas of Europe. Its larvae initially mine leaves of several species of Salix and later feed in a folded leaf (
Callisto adults in dorsal view. 1 C. coffeella, male, Austria, Leitnertal, Oberer Stuckensee, 2150 m, 07.IX.2013, leg. Deutsch (PCHD) | voucher specimen № 3 | sample ID – NK318 | process ID CALCO003-142 C. coffeella, male, Austria, Nordtirol, Bodenalpe, 2000 m, 9.–10.VII.1984, leg. Burmann (TLMF); 3 C. coffeella, male, Austria, Vorarlberg, Brandnertal, Böser Tritt, 1700-1800 m, 04.VII.1983, leg. Huemer (TLMF) 4 C. coffeella, female, Austria, Nordtirol, Obergurgl, 2000 m, e.l. M.III.1970, leg. Burmann (TLMF) 5 C. basistrigella sp. n., male, East Tyrol, Lienzer Dolomiten, Laserz, Dolomitenhütte, 1600 m, 12.VII.2013, leg. Deutsch (TLMF) | voucher specimen № 10 | sample ID – NK325 | process ID CALCO010-146 C. basistrigella sp. n., male, Italy, Prov. Udine, Mte. Sernio, Forcella Nuviernulis, 1700 m, 16.VII.1988, leg. Huemer (TLMF) 7 C. basistrigella sp. n., male, Italy, Prov. Udine, Mt. Canin N, Rif. Gilberti, 1850–1950 m, 29.VII.2001, leg. Huemer (TLMF) 8 C. basistrigella sp. n., female, Italy, Prov. Udine, Montasio, 16.IX.1951, leg. Pinker (TLMF).
In a recent DNA barcoding study,
Here we present new genetic, distribution and morphological data that support the hypothesis that individuals of C. coffeella from the south-eastern Alps represent a distinct lineage that we formally describe as a new species – Callisto basistrigella Huemer, Deutsch & Triberti, sp. n.
Specimens examined in this study were obtained by rearing adults from leaf mines and by collecting adults flying by day around Salix bushes, mainly S. glabra Scop., 1772 and S. waldsteiniana Willd., 1806, but also a few S. appendiculata Villars, 1789 and S. hastata L., 1753. Some adults were collected at light trap or flew in the early morning hours. Data for all specimens studied morphologically and genetically can be found in the Suppl. material
We examined the morphology of 135 dried, pinned and mostly set specimens belonging to C. coffeella s.l., the majority originating from the Alps, and half a dozen from Scandinavia. Pinned specimens were photographed with an Olympus E 3 digital camera and an Olympus SZX 10 binocular microscope, and processed with Helicon Focus 4.3 software, resulting in multiple images. Images were later edited by using Adobe Photoshop Lightroom 2.3 software. Genitalia were photographed with an Olympus E1 digital camera through an Olympus BH2 microscope.
Genitalia dissections and slide mounts followed
All measurements were done on a Leica M 165C stereomicroscope by P. Triberti and expressed in mm. The dataset resulting from these measurements was analyzed using a multivariate approach – one-way ANOVA (
DNA extracts were prepared from a single hind leg removed from each of 21 specimens of C. coffeella s.l. DNA extraction, PCR amplification and sequencing of the barcode region were carried out at the Canadian Centre for DNA Barcoding (CCDB, Biodiversity Institute of Ontario, University of Guelph) following standard protocols (
Furthermore, 21 samples with DNA barcodes were also sequenced for the nuclear gene histone H3, a ~350 bp fragment, at INRA, Orléans. PCR for this gene was performed using primers Hex AF (5' -ATG GCT CGT ACC AAG CAG ACG GC -3') and Hex AR (5' -ATA TCC TTG GGC ATG ATG GTG AC-3') (
Sequence divergences were quantified using the Kimura 2-parameter model implemented within the analytical tools on BOLD (www.boldsystems.org) (
Details on the collecting data for each specimen, as well as a photograph of vouchers, sequence records, trace files, and primer sequences used for PCR amplification, together with GenBank accession numbers are available through the following dataset (https://doi.org/10.5883/DS-CALLISTO) in BOLD (www.boldsystems.org).
LMK Landesmuseum Kärnten; Klagenfurt, Austria.
MCSN Museo Civico di Storia Naturale, Verona, Italy.
MCSNB Museo Civico di Scienze Naturali “E. Caffi”, Bergamo, Italy.
SMNK Staatliches Museum für Naturkunde, Karlsruhe, Germany.
TLMF Tiroler Landesmuseum Ferdinandeum, Innsbruck, Austria.
UO University of Oulu, Finland.
VND inatura Erlebnis Naturschau Dornbirn, Austria.
ZSM Zoologische Staatssammlung, Munich, Germany.
PCHD Helmut Deutsch, Bannberg, Assling, Tyrol, Austria.
PCJR Jurij Rekelj, Kranj, Slovenia.
PCJS Jürg Schmid, Illanz, Switzerland.
PCJW Josef Wimmer, Steyr, Austria.
PCJWdP Jurate and Willy De Prins, London, UK.
PCSG Stanislav Gomboc, Slovenia.
Morphological analysis of the 135 specimens confirms the differences observed in wing pattern in the south-eastern alpine population. Eighty-two of these individuals were diagnosed as Callisto coffeella and 53 as the new species C. basistrigella. In addition, we detected two moths which morphologically corresponded to C. basistrigella but with a COI barcode they fell into the cluster of C. coffeella (see below Molecular divergences).
Oecophora coffeella
Oecophora interruptella
Ornix caelatella
Ornix blandella
Annickia alpicola
Oecophora coffeella was described from an unspecified number of male specimens collected on the 14th of July near Bjerkvik [according to original description ´Bjoerkvik” in Norwegian Lappland] (
Annickia alpicola was described from a single male specimen collected in the French Alps (
Ornix caelatella was described from a single male collected in Montenero (Tuscany, Italy) in May by Josef Mann (
Ornix blandella was described by
Adult (Figs
Genitalia and eighth segment male (Figs
Callisto, male genitalia. 9 C. coffeella, Vorarlberg Zürs, 1800 m, 29.VI.1939, leg. Burmann, gen. slide TIN 1 (TLMF) 10 C. coffeella Teriol sept., Vent 2000 m, e.l. 01.III.1956, leg. Burmann, gen. slide TIN 4 (TLMF) 11 C. basistrigella sp. n., Italia sept. Prov. Udine, Mte. Sernio, Forcella Nuviernulis 1700 m, 16.VII.1988 leg. Huemer gen. slide TIN 2 (TLMF) 12 C. basistrigella sp. n. Italia sept. Prov. Udine, Mte. Sernio, Forcella Nuviernulis 1700 m, 16.VII.1988 leg. Huemer gen. slide TIN 3(TLMF).
Callisto, male, segment 8. 13 C. coffeella, Vorarlberg Zürs, 1800 m, 29.VI.1939, leg. Burmann, gen. slide TIN 1 (TLMF) 14 C. coffeella, Teriol sept., Vent 2000 m, e.l. 01.III.1956, leg. Burmann, gen. slide TIN 4 (TLMF) 15 C. basistrigella sp. n., Italia sept. Prov. Udine, Mte. Sernio, Forcella Nuviernulis 1700 m, 16.VII.1988, leg. Huemer, gen. slide TIN 2 (TLMF) 16 C. basistrigella sp. n., Italia sept. Prov. Udine, Mte. Sernio, Forcella Nuviernulis 1700 m, 16.VII.1988 leg. Huemer gen. slide TIN 3 (TLMF).
Genitalia female (Fig.
The species is restricted to higher mountain areas and shows an arctic-alpine distribution pattern. According to various publications (i.e.
The larval stage feeds on various species of mountainous Salix such as Salix arbuscula L., 1753 (which may refer to S. arbuscula in northern Europe or S. waldsteiniana in Central Europe), S. phylicifolia L., 1753 (
Holotype (Fig.
33 males and 11 females.
Austria: 3 males, East Tyrol, Lienzer Dolomiten, Lavanter Almtal, 1200-1400 m, 07.VI.1998, leg. Deutsch (TLMF); 1 male, East Tyrol, Lienzer Dolomiten, Laserzgebiet, 1800-2000 m, 21.VI.1999, leg. Deutsch (TLMF); 1 male, East Tyrol, Carnic Alps, Leitnertal, Oberer Stuckensee, 2150 m, 14.VII.2013, leg. Deutsch (PCHD) | voucher specimen № 8 | sample ID – NK323 | process ID CALCO008-14; 2 males, East Tyrol, Carnic Alps, Leitnertal, Oberer Stuckensee, 2150 m, 07.IX.2013, leg. Deutsch (PCHD) | voucher specimens № 1 and № 2 | sample IDs – NK316 and NK317 | process Ids CALCO001-14 and CALCO002-14; 1 female, East Tyrol, Lienzer Dolomiten, Hochstadel, 2000 m, VII.1952, leg. Pinker (TLMF); 2 females, East Tyrol, Carnic Alps, Leitnertal, Oberer Stuckensee, 2150 m, 07.IX.2013, leg. Deutsch (PCHD) | voucher specimens № 4 and № 6 sample | sample IDs – NK319 and NK321 | process Ids CALCO004-14 and CALCO006-14.
Italy: 4 males, Prov. Belluno, Passo di Valparola E, 2200-2300 m, 20.VII.2009, leg. Huemer (TLMF); 1 female, same data but gen. slide TRB3893 and BC TLMF Lep 01801 (TLMF); 1 male, A. Carniche, Sappada, Casera Sesis, 1800 m, 12.VI. unknown year, leg. Rocca, gen. slide TRB 1778 (MCSN); 1 male, A. Carniche, Sappada, Passo Siera, 1600 m, 04.VII.1933, leg. Rocca, gen. slide TRB 1785 (MCSN); 1 male, A. Carniche, Sappada, Hosthaus, 1800 m, 14.VII.1936, leg. Rocca (MCSN); 2 males, 2 females, A. Carniche, Sappada, L. d’Olbe, 2000 m, 02.VII.1933, leg. Rocca, gen. slide TRB284 male, TRB3894 male (MCSN); 1 male, Prov. Udine, Mte. Sernio-Massiv Forcella Nuviernulis 1700 m, 16.VII.1988, leg. Huemer, GU TIN2 male P. Huemer ’Callisto coffeella Zett. det. Triberti’ (TLMF); 1 male, Prov. Udine, Mte. Sernio-Massiv Forcella Nuviernulis 1700 m, 16.VII.1988, leg. Huemer, GU TIN3 male (TLMF); 1 male, 1 female, Prov. Udine, Montasio, 16.IX.1951, leg. Pinker, gen. slide TIN8 female (TLMF); 11 males, 1 female, Prov. Udine, Monte Canin N, Rif. Gilberti Umg., 1850-1950 m, 29.VII.2001, leg. Huemer (TLMF); 1 male, 1 female, Prov. Udine, Monte Canin, Biv. Marussich, 2040 m, 06.VII.2002, leg. Wieser (LMK); 3 males, Prov. Udine, Monte Canin, Sella di Grubia, 1700 m, 20.VI.2003, leg. Wieser (LMK).
Slovenia: 1 female, Crna Prst, 1400 m, 18.VII.1899, leg. Penther (TLMF).
In external appearance C. basistrigella is distinguishable from C. coffeella by its forewing pattern. In C. basistrigella, the sub-basal whitish silvery line of the forewing is almost parallel and lies in the fold, whereas in C. coffeella this line is transverse to the wing axis or reduced to a spot. On average, the forewings are slightly narrower than in C. coffeella (visible in series). Sexual dimorphism, as observed in C. coffeella, is absent in C. basistrigella. Genitalia do not provide obvious diagnostic differences but the length of the phallus is significantly longer in C. basistrigella than in C. coffeella although more specimens would be needed to confirm this difference (see Genital morphometrics).
Adult (Figs
Genitalia and subgenital segments male (Figs
Genitalia female (Fig.
Only known from a small area in the south-eastern Alps, ranging from the Dolomites (Italy) in the west to the Julian Alps (Slovenia) in the east and the Carnic Alps and Lienzer Dolomiten (Austria) in the north (Fig.
A sampling area of Callisto coffeella and C. basistrigella in Europe. B close up of the distribution of C. coffeella (green circles) and C. basistrigella (white squares) in the Alps; two C. basistrigella specimens (red triangles) show evidence of introgression. On Figs 19A, 19B, the 35 barcoded specimens are shown with numbers (1-5). The red circle on Fig. 19B shows the contact zone where both species occur together (Leitnertal, Eastern Tyrol, Austria and Sappada, Italy). When several samples were investigated per locality, the samples with the same coordinates have been slightly shifted in order to visualize overlapping data points on Fig. 19B.
The name refers to the characteristic wing markings.
Early stages are undescribed. Both C. basistrigella and C. coffeella adults have been collected during the day, flying around low bushes of alpine Salix glabra and S. waldsteiniana. The flight period is largely dependent on exposure and snow coverage and usually extends between early June and late July. Under extreme conditions such as harsh winters adults have been collected as late as mid-September. The habitats are related to the dwarf-shrub zone and include subalpine meadows, rock formations and scree with Salix-bushes and shrubs. C. basistrigella is restricted to limestone with an altitudinal range from about 1200 to 2300 m.
Multivariate ANOVA analysis based on morphometric of seven genital characteristics of the male moths failed to find any significant difference between C. coffeella and C. basistrigella (Wilks’ λ = 0.36, F = 2.07, p = 0.16). Six out of seven parameters, i.e valva, saccus, anellus and anellus process lengths, valva width and valva constriction were not found to differ in the two species. Non-parametric Mann-Whitney test however indicated that the phallus is significantly longer in Callisto basistrigella than in C. coffeella (MWT: Z = 2.36, N = 16, p = 0.02), although sample sizes remain relatively small (C. basistrigella N = 5, C. coffeella N = 11) (Fig.
DNA barcodes. We obtained DNA barcodes for 21 specimens of C. coffeella and 14 specimens of C. basistrigella. Their analysis revealed that the samples of these species form two distinct clusters in the NJ tree (Fig.
A neighbor joining tree based on the COI barcode fragment and B based on the histone H3 gene. The two specimens (ISSIK141-14, ISSIK274-14) with the C. basistrigella phenotype, but branching within the C. coffeella DNA barcode and within the C. basistrigella histone H3 cluster are marked with red triangles (as in Fig.
Excluding these two records, pairwise interspecific distances range between 1.39% and 2.37%, with a mean value of 1.75% (sd = 0.2). Within C. basistrigella and C. coffeella, respectively, genetic distances range from 0 to 0.31% (mean-value 0.17%, sd = 0.11) and from 0 to 1.23% (mean-value 0,56%, sd = 0.31). Sequence comparison revealed eight diagnostic substitutions (Table
Diagnostic substitutions in COI-DNA barcode sequences of Callisto coffeella and C. basistrigella.
Position | 70 | 88 | 145 | 206 | 271 | 295 | 547 | 631 |
---|---|---|---|---|---|---|---|---|
C. basistrigella | T | T | T | C | T | A | A | A |
C. coffeella | A | A | C | T | C | C | C | G |
Histone H3. We obtained sequences of the nuclear gene histone H3 (328 bp) for the same 21 moths that were barcoded. H3 showed a high conservatism, with a single diagnostic nucleotide substitution at position 151, dividing the studied specimens into two clusters matching exactly the morphology-based separation of C. coffeella and C. basistrigella (Fig.
The Slovenian (ISSIK141-14) and Italian (ISSIK274-14) specimens, morphologically assigned to C. basistrigella and whose DNA barcodes clustered within C. coffeella (Fig.
Contact zone. Both Callisto basistrigella and C. coffeella were found to occur in the same localities in the Carnic Alps (Leitnertal, Eastern Tyrol, Austria) at the altitude up to 2150 m (Fig.
Our study used newly generated mitochondrial and nuclear data in combination with morphological and morphometric data to characterize the variability of Callisto coffeella across its range. We confirmed the existence of two distinct lineages, one of which is described here as C. basistrigella. Its status as a distinct species is supported by morphology, nuclear DNA (histone H3 gene) and by mtDNA (COI-DNA barcodes), although shared haplotypes of the latter suggest introgression or incomplete lineage sorting.
Species delineation with DNA barcodes. In Lepidoptera, although authors generally reject the use of a threshold to delineate species, an empirical 2% (K2P) intraspecific distance value has often been proposed, pragmatically, as indicating “deep divergence” suggestive of potential overlooked or cryptic diversity (
Contact zone. We found that Callisto basistrigella occurs in sympatry with C. coffeella in the Carnic Alps, Leitnertal, 2150 m (East Tyrol, Austria) and Sappada 1600-1800 m (Italy), without evidence of admixture in this area. The two cases of nuclear/mitochondrial discordance revealed suggests possible genetic introgression between the two species. Further sampling and the use of fast evolving markers will be needed to investigate the course of a putative contact zone as well as the extent of gene flow between the two species.
Biogeography and speciation. The distribution of C. basistrigella as currently known is shared by several other endemic Lepidoptera. The south-eastern Alps is considered as one of the major areas of endemism in the region (
Allopatric isolation during the last glacial period is probably the main process by which C. basistrigella and C. coffeella diverged. Indeed, as many other cold-adapted LepidopteraC. coffeella populations may have had a wide distribution in the periglacial tundra belts during the last glacial period. With increasing temperatures during the last interglacial period, C. coffeella may have moved northwards while southern populations moved up in altitude in the Alps (
Our results highlight the need to carry out additional intraspecific studies looking at patterns of both morphological and genetic variability within species across their ranges, which can reveal overlooked diversity and new species (
We are grateful to Marko Mutanen (Oulu, Finland), Peter Lichtmannecker (Adlkofen, Germany) and Andreas Segerer (Munich, Germany) for kindly giving us an access to unpublished barcode data of Callisto; to Jurij Rekelj (Kranj, Slovenia), Jurate and Willy De Prins (London, UK), Stanislav Gomboc (Kranj, Slovenia), Melania Massaro and Paolo Pantini (Bergamo, Italy), Jürg Schmid (Illanz, Switzerland), Christian Wieser (Klagenfurt, Austria) and Josef Wimmer (Steyr, Austria) for sending specimens for DNA barcoding; Stefan Heim (Innsbruck, Austria) for photographs of the adults and genitalia; Christelle Robinet (Orléans, France) for help with mapping; Emmanuelle Magnoux and Beatrice Courtial (Orléans, France) for assistance in the molecular analyses; Vladimir Shishov (Krasnoyarsk, Russia) for providing valuable comments on statistics.
We acknowledge the team at the Biodiversity Institute of Ontario, University of Guelph, Ontario, Canada for their assistance in the production of DNA barcodes. Funding for DNA barcoding was partly provided by the government of Canada through Genome Canada and the Ontario Genomics Institute in support of the International Barcode of Life project, and by NSERC. Sequence analysis was enabled by a grant from the government of Canada through Genome Canada and the Ontario Genomics Institute in support of the International Barcode of Life Project. Our work was also aided by the BOLD informatics platform whose development is funded by the Ontario Ministry of Economic Development and Innovation. Peter Huemer gratefully acknowledges funding from inatura Erlebnis Naturschau (Dornbirn, Austria) and the Promotion of Educational Policies, University and Research Department of the Autonomous Province of Bolzano – South Tyrol for support of the project “Genetic biodiversity archive – DNA barcoding of Lepidoptera of the central Alpine region (South, East and North Tyrol)”. Natalia Kirichenko was supported by a fellowship of LE STUDIUM®, France.
We thank two anonymous reviewers and the editor, Erik van Nieukerken, for their valuable comments on the manuscript.
List of studied specimens of Callisto coffeella and C. basistrigella and collection data.
Data type: collection data / voucher depository / genetic code.
Explanation note: The list of 135 examined specimens of Callisto coffeella and C. basistrigella sp. n., their collection data (country, locality, GPS coordinates, collection date and collector name) and depository data (museum or private collection) are provided in the supplementary table S1. All specimens have been studied morphologically; the barcoded samples are supplied with sample ID, process ID, GenBank COI and GenBank H3 (if nuclear gene histone H3 was analyzed).