Research Article |
Corresponding author: Vazrick Nazari ( nvazrick@yahoo.com ) Academic editor: Harald Letsch
© 2020 Vazrick Nazari, Wolfgang ten Hagen.
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:
Nazari V, ten Hagen W (2020) Molecular taxonomy of Tomares hairstreaks (Lepidoptera, Lycaenidae, Theclinae). Deutsche Entomologische Zeitschrift 67(1): 19-33. https://doi.org/10.3897/dez.67.50252
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Tomares hairstreaks comprise about 10 species distributed from Europe and North Africa to Central Asia. The taxonomy of the genus is hampered by the absence of diagnostic characters by which specimens can be unambiguously assigned to species. Our investigation of morphology and DNA barcode variations within and between Tomares species shows that while well-defined species (T. ballus, T. mauritanicus, T. callimachus, T. desinens and T. fedtschenkoi) diverge, poorly characterized taxa (T. nogelii, T. nesimachus, T. dobrogensis, T. romanovi and T. telemachus) show very little to no differentiation in mtDNA. We reinstate Tomares callimachus spp. hafis (Kollar, 1849) as a valid subspecies (stat. rev.) and propose taxa telemachus Zhdanko, 2000 and uighurica Koçak, Seven & Kemal, 2000 as synonyms of T. romanovi and T. nogelii nogelii respectively (syn. nov.). We relegate Polyommatus epiphania Boisduval, 1848, recently revived as a valid subspecies of T. callimachus, back to synonymy under the latter, and reconsider the status of T. nogelii dobrogensis (Caradja, 1895) in the light of new molecular data. We use a nuclear gene (EF-1α) in addition to COI barcodes to reconstruct the phylogeny of the group.
biogeography, butterflies, DNA, hybridization, introgression, phylogenetics
Over the last decade, lycaenid butterflies have been a popular model group in studies of hybridization (
The ~10 species in Palaearctic hairstreak genus Tomares Rambur, 1840 (sensu
Among these, the closely related T. callimachus and T. desinens are both distinguished by the absence of orange coloration within the transverse bands on the underside of the hind wings (UNH). They both fly in sympatry in Azerbaijan and Iran (
The eastern species T. romanovi, often readily identifiable by its striking bluish-green UNH and the reduction or absence of maculae, is found from southeastern Turkey to the Kopet Dagh Mountains where it is sympatric with telemachus, a poorly described taxon based on undulated wing margins, light grey UNH and alleged differences in female genitalia, all variable characters interchangeable with the sympatric T. romanovi. Specimens with reduced green scales and prominent maculae on their UNH, approaching that of T. nogelii, occur also in Caucasus and southeastern Turkey.
The most difficult problem however concerns the taxonomic identity of the remaining three taxa, T. nogelii, T. nesimachus and T. dobrogensis. The issue has been addressed extensively in the past (
The documented variation and overlap of species characters and ranges between the taxa in the T. nogelii complex continues to be a serious problem in their interpretation. In their comprehensive investigation,
A total of 274 specimens representing all species and many subspecies of Tomares were sampled, of which 240 produced usable barcode sequences (Suppl. material
Two dry legs from each adult specimen were detached and stored in individual vials. The extraction of total genomic DNA, amplification and sequencing were performed in the Centre for Biodiversity Genomics (Guelph, Ontario, Canada) using previously described protocols (
The widespread mtDNA haplotype sharing observed among five species (T. nogelii, T. nesimachus, T. dobrogensis, T. romanovi, T. telemachus) did not help in resolving the long standing problem of species identities in this complex. To remedy this, we examined morphological characters and re-evaluated the taxonomic status and geographical boundaries of the available names under this complex specifically looking for cases of sympatry and synchrony. The problem of correct identification of specimens in this group however makes past records in the literature difficult to verify.
Dissections of male and female specimens of Tomares were carried out by WtH. Some of the dissected specimens were also included in the molecular analysis. Male and female genitalia were prepared using standard protocols and fixed in Euparal glycerin. Male genitalia were photographed in dorsal and ventral view. In a few cases, the aedeagus was damaged proximally. Female genitalia preparations included the last two tergites, but components often had to be fixed and photographed separately in dorsal view. Photographs were taken under a standardized condition and digitally processed. Females of T. telemachus and T. desinens were not dissected due to lack of sufficient material (Suppl. material
Neighbour-joining (NJ) trees for barcode data were constructed initially using the QUICKTREE algorithm (
Genitalia of both sexes in all Tomares species differed in size in accordance with the specimen wingspan. Female genitalia were relatively uniform, with triangular papillae anales, sclerotized ductus bursae and doctus seminalis, and round and membranous corpus bursae with no signa (Suppl. material
Right valvae in male genitalia of Tomares species. 1. T. mauritanicus GP76 (Morocco); 2. T. ballus GP77 (Morocco); 3. T. fedtschenkoi GP78 (Tajikstan); 4. T. desinens GP86 (Qazvin, Iran); 5. T. callimachus callimachus GP75 (Crimea); 6. T. callimachus hafis GP86 (Zanjan, Iran); 7. T. nesimachus GP84 (Damascus, Syria); 8. T. “telemachus” GP79 (Turkmenistan); 9. T. romanovi GP74 (Lorestan, Iran); 10. T. nogelii nogelii GP88 (Nevshehir, Turkey); 11. T. nogelii nogelii GP85 (Sivas, Turkey); 12. T. nogelii dobrogensis GP83 (Ukraine). All dissections and images by WtH.
Despite a wide geographic coverage, various populations of T. ballus, T. mauritanicus and T. fedtschenkoi formed well-supported clusters with small internal variation. We observed a gap in DNA barcodes (1.00 ± 0.24%), as well as EF-1α sequences, between the “northern” (Kazakhstan, Ukraine, Russia and N. Azerbaijan) and “southern” (S. Azerbaijan, Armenia, Iran and Turkey) populations of T. callimachus. The disjunct Kazakh population of callimachus showed identical mtDNA haplotypes with specimens from Ukraine and southern Russia. Further subdivisions were evident within the southern cluster (Fig.
While average K2P distances between five Tomares taxa (ballus, mauritanicus, callimachus, desinens and fedtschenkoi) ranged between 1.6–3.0% (Table
Average K2P distances and standard deviation of COI barcodes between Tomares taxa.
ballus | mauritanicus | callimachus | desinens | fedtschenkoi | nogelii | nesimachus | dobrogensis | romanovi | telemachus | |
---|---|---|---|---|---|---|---|---|---|---|
ballus | 0.3 ± 0.2 | |||||||||
mauritanicus | 1.6 ± 0.2 | 0.2 ± 0.2 | ||||||||
callimachus | 3.0 ± 0.3 | 3.0 ± 0.2 | 0.6 ± 0.4 | |||||||
desinens | 2.5 ± 0.2 | 2.5 ± 0.1 | 2.4 ± 0.2 | 0.2 ± 0.2 | ||||||
fedtschenkoi | 2.4 ± 0.2 | 3.0 ± 0.2 | 2.5 ± 0.3 | 2.1 ± 0.2 | 0.2 ± 0.2 | |||||
nogelii | 2.2 ± 0.2 | 2.2 ± 0.2 | 2.3 ± 0.2 | 1.6 ± 0.1 | 2.4 ± 0.2 | 0.2 ± 0.2 | ||||
nesimachus | 2.2 ± 0.3 | 2.1 ± 0.2 | 2.3 ± 0.2 | 1.6 ± 0.2 | 2.3 ± 0.2 | 0.3 ± 0.3 | 0.3 ± 0.3 | |||
dobrogensis | 2.3 ± 0.3 | 2.2 ± 0.2 | 2.3 ± 0.3 | 1.7 ± 0.2 | 2.4 ± 0.3 | 0.4 ± 0.2 | 0.4 ± 0.3 | 0.5 ± 0.3 | ||
romanovi | 2.1 ± 0.3 | 2.0 ± 0.2 | 2.2 ± 0.2 | 1.5 ± 0.2 | 2.3 ± 0.2 | 0.4 ± 0.1 | 0.3 ± 0.2 | 0.5 ± 0.2 | 0.3 ± 0.2 | |
telemachus | 2.3 ± 0.3 | 2.2 ± 0.2 | 2.3 ± 0.2 | 1.6 ± 0.1 | 2.4 ± 0.2 | 0.3 ± 0.1 | 0.3 ± 0.2 | 0.4 ± 0.1 | 0.3 ± 0.1 | 0.2 ± 0.1 |
Summary of characters that show variation among taxa in the nogelii complex.
Character | nogelii, dobrogensis | nesimachus | romanovi, telemachus |
---|---|---|---|
collection dates | 25 April–30 May | 5 April–31 May | 15 April–31 May |
elevation (m) | 85–2075 | 250–2000 | 600–1300 |
habitat | hygric habitats | xeric rocky habitats with sparse vegetation | usually xeric rocky habitats with sparse vegetation; rarely other |
zoogeographic zone | Pontomediterranean – Armenian | Syrian – Palaeoeremic | Iranian – Caspian |
larval host plant (primary, secondary) | Astragalus, Asteracantha | Astracantha, Astragalus | Astragalus |
orange patch on UPF | absent in 40% of specimens | always present | always present |
dark patch at the tip of UPF | continuous along costal and outer margins | nearly triangular | continuous along costal and outer margins |
submarginal black spots on UPF | connected, forming an undulated dark band | variable; usually a series of disjunct spots, sometimes connected to form a deeply serrated band | connected, forming an undulated or serrated dark band |
marginal black border on UPF | always wide, equally or wider than costal border | always narrow | always wide, equally or wider than costal border |
orange patch on UPH | reduced or absent in nearly 30% of specimens, if present always narrow and nearly rectangular | always present, wide, nearly rectangular basally, with both sides of the angle more or less equal in length | always present, variable in size and shape |
UNH pattern (see Suppl. material |
usually gray-brown with prominent maculae | usually gray-brown with prominent maculae | usually uniform bluish-green with no maculae; varies in peripheral populations |
needle-shape spine in male genitalia (see Fig. |
long | short | long |
TCS Haplotype Network of the nogelii complex. Colors indicate morphological identifications (red = nogelii, blue = dobrogensis, orange = nesimachus, green = romanovi, yellow = telemachus). The most common haplotype (large circle) comprises central and eastern Turkish individuals of nogelii, ‘nesimachus’ and ‘dobrogensis’, as well as a single nesimachus from Israel.
Distribution of taxa in the nogelii complex. Shapes represent morphological identifications (□ = nogelii, ∆ = nesimachus, ○ = romanovi), colors represent COI barcode haplotypes (red = nogelii haplotypes, orange = nesimachus haplotypes, green = romanovi haplotypes). Sites with shared or more than one haplotypes are circled. Records in gray are concatenated from literature. Approximate taxon boundaries are inferred from represented haplotypes. For haplotype network, see Figure
Our phylogenetic reconstruction of combined sequence data strongly supports monophyly of Tomares and five species within the genus (ballus, mauritanicus, fedtchenkoi, callimachus and desinens). However, throughout all analyses, the taxa nogelii, nesimachus, romanovi, dobrogensis and telemachus formed a well-supported clade, within which they were paraphyletic with respect to each other (Fig.
Bayesian phylogeny of selected Tomares sequences based on combined data (COI + EF-1α). Values above branches are bootstrap support obtained under Parsimony and Likelihood criteria for each node, and values below branches are Bayesian posterior probabilities. Images: 1) ballus wth013 Morocco, 2) ballus wth055 Spain, 3) mauritanicus wth017 Morocco, 4) fedtchenkoi wth020 Kyrgyzstan, 5) callimachus callimachus wth051 Azerbaijan, 6) callimachus hafis wth053 Iran, 7) desinens wth042 Iran, 8) dobrogensis wth080 Crimea, 9) nogelii zma153 Turkey, 10) nesimachus wth065 Syria, 11) romanovi obscura zma161 Turkey, 12) romanovi cachetinus zma146 Azerbaijan, 13) romanovi romanovi wth010 Armenia, 14) telemachus wth005 Turkmenistan.
No fossils of Tomares are known, and the only fossil attributable to Theclinae is a geologically very young larva (
While morphology and DNA barcodes unequivocally demonstrate separate species status for T. ballus, T. mauritanicus and T. fedtchenkoi, they do not support recognition of subspecies within them. Separating populations into subspecies in the highly variable T. mauritanicus has been dismissed before (
The split in the range of T. callimachus, supported by both COI and EF-1α genes, suggests a long period of lack of genetic exchange between the northern and the southern populations. The male genitalia in southern populations show a distinctly narrow and needle-shaped spine that is very different from the northern group (Fig.
The remaining five taxa (nogelii, nesimachus, dobrogensis, romanovi and telemachus) form a clade of closely-related haplotypes with no apparent distinction between taxa. The concordance between mitochondrial COI and nuclear EF-1α genes rules out selective sweeps caused by endosymbiotic bacteria (
While Oberthür’s original (1893) description and illustration of nesimachus from “Akbès” (Hatay, southern Turkey) matched very well with our examined material from southern Turkey and the Levant, the central and eastern Turkish specimens generally matched better with T. nogelii. We did not detect presence of any of the ‘nesimachus’ haplotypes among central and eastern Turkish populations, where various ‘ecotypes’ of nogelii all share a different haplotype. We did not find character combinations proposed by
All other records of nogelii, nesimachus and dobrogensis from central and eastern Turkey represent various populations of T. nogelii ssp. nogelii with different larval hosts that share a common, widespread haplotype across central to northeastern Turkey (Fig.
Hybridization is not rare in butterflies, and any slight overlap in morphology, behaviour and ecology are likely to allow it to occur (
For additional synonymy, see
Tomares ballus (Fabricius, 1787)
Distribution. Southwest France to southern Spain and Portugal, Gibraltar, Morocco, Algeria, north Libya, south Tunisia and north Egypt.
Larval host. Lotus hispidus, Boujeania hispida (?), Anthyllis vulneraria, A. cyticoides, Heliatheum sp. and Medicago sp. in Spain (
Tomares mauritanicus (Lucas, 1849)
Distribution. Algeria and Morocco.
Larval host. Hedysarum pallidum, Hippocrepis multisiliquosa, H. minor, Astragalus epiglottis, and A. pentaglottis (
Tomares callimachus (Eversmann, 1848)
ssp. callimachus (Eversmann, 1848)
= Polyommatus epiphania Boisduval, 1848 stat. rev.
Distribution. From Ukraine to Central Asia and N Azerbaijan.
Larval host. Recorded on a number of Astragalus species from Alatau Mountains and NW Kazakhstan to South Russia, Crimea and Georgia: Astragalus leptostachys, A. macropterus, A. physodes, A. suprapilosus, A. utriger and A. vulpinus, as well as Hedysarum candidum in Crimea and Onobrychis radiate in Georgia (
ssp. hafis (Kollar, 1849) stat. rev.
Distribution. Lesser Caucasus, Armenia, south and southeast Turkey, north Iraq, west, southwest, north and northeast Iran to Kopet Dagh.
Larval host. Not recorded. The record of Astragalus physodes from “Kulp” (Diyarbakir, Turkey) by
ssp. huertasae Tshikilovets & Pagès, 2016
Distribution. Pakistan: Baluchistan.
Larval host. Not recorded.
Tomares desinens Nekrutenko & Effendi, 1980
Distribution. Southeast Azerbaijan, east Turkey (Van), north and northwest Iran.
Larval host. Not recorded.
Tomares fedtschenkoi (Erschoff, 1874)
Distribution. South Turkmenistan, Uzbekistan, Kyrgyzstan, south Kazakhstan and Tajikistan. Records from Afghanistan and Pakistan are questionable (
Larval host. Astragalus chlorodontus and Astragalus agameticus (
Tomares nogelii (Herrich-Schäffer, [1851])
ssp. nogelii (Herrich-Schäffer, [1851])
=uighurica Koçak, Seven and Kemal in Koçak, 2000 syn. nov.
Distribution. Northeast to central Anatolia, and south to the Levant.
Larval host. Asteracantha spp. (early fliers); Astragalus ponticus and A. micropterus (late fliers) in Turkey (
ssp. dobrogensis (Caradja, 1895)
Distribution. Romania, Crimea, Ukraine. Does not occur in Turkey.
Larval host. Astragalus ponticus in Ukraine and Romania (
Tomares nesimachus (Oberthür, 1893)
Distribution. Southern Turkey (Mersin, Adana, Hatay to Mardin) to Lebanon, Israel and Jordan.
Larval host. Astracantha spp. (Oorschot and Wagner 2000); Astragalus macrocarpus in Israel and Jordan (
Tomares romanovi (Christoph, 1882)
= Tomares telemachus Zhdanko in
Distribution. East Turkey, Georgia, Armenia, Azerbaijan, Iran, and Kopet Dagh range in Turkmenistan.
Larval host. Astragalus finitimus in Kopet Dagh and in Armenia (Yerevan)(
We thank Harry van Oorschot † (Amsterdam, the Netherlands), Andree Salk (Berlin, Germany), Sergej Churkin (Moscow, Russia), Vadim Tshikolovets (Kiev, Ukraine), Giancristoforo Bozano (Milano, Italy), Tomasso Racheli (Rome, Italy), Valentin Tikhonov (Pyatigorsk, Russia), Gerardo Lamas (Lima, Peru), and Martin Lödl (Natural History Museum Vienna, Austria) for providing specimens, and Mihai Stănescu, Mario Ramos González and Harald Letsch for their reviews and helpful comments. This research was supported through funding to the Canadian Barcode of Life Network from Genome Canada (through the Ontario Genomics Institute), NSERC and other sponsors listed at http://www.bolnet.ca. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
SI 1. Material examined and Genbank accessions.
Data type: Microsoft Excel Spreadsheet
SI 2. Male and female genitalia dissections of Tomares species.
Data type: pdf document
SI 3. Androconia, forewing upperside and hindwing underside in select Tomares species.
Data type: pdf document
SI 4. Phylogenetic trees resulting from Maximum Parsimony (MP, PAUP) and Maximum Likelihood (ML, PHYML) analyses of COI, EF-1a and Combined datasets with bootstrap support values.
Data type: pdf document