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Article

Molecular Phylogeny and Taxonomy of the Butterfly Subtribe Scolitantidina with Special Focus on the Genera Pseudophilotes, Glaucopsyche and Iolana (Lepidoptera, Lycaenidae)

by
Vladimir A. Lukhtanov
* and
Anastasia V. Gagarina
Department of Karyosystematics, Zoological Institute, Russian Academy of Sciences, Universitetskaya Nab. 1, 199034 Saint-Petersburg, Russia
*
Author to whom correspondence should be addressed.
Insects 2022, 13(12), 1110; https://doi.org/10.3390/insects13121110
Submission received: 4 November 2022 / Revised: 26 November 2022 / Accepted: 29 November 2022 / Published: 30 November 2022
(This article belongs to the Special Issue Systematics, Ecology and Evolution of Lepidoptera)
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Abstract

:

Simple Summary

The Palearctic butterfly genera Pseudophilotes, Glaucopsyche and Iolana have attracted the attention of many entomologists because their species are used as model objects for studying ecology and evolution. The genera have previously been the subjects of several taxonomic studies based on the analysis of their morphological and molecular characteristics, but none of these studies are based on complete species sampling. In our work, we used a set of mitochondrial and nuclear genes to reveal the phylogeny of these genera as well as the phylogeny of the subtribe Scolitantidina, to which these genera belong. In the genus Pseudophilotes, we identified 10 species including among them, the enigmatic Central Asian taxon P. panope, which has often been assigned to other genera. We clarified the taxonomic structure of the genus Glaucopsyche, which was found to consist of four subgenera. We confirm that the genus Iolana includes nine species distributed across the southwestern part of the Palearctic. The results obtained here will be important for the conservation of the Scolitantidina species, some of which are local and protected by national and international laws.

Abstract

The Palearctic blue butterfly genus Pseudophilotes Beuret, 1958 is not homogenous regarding the morphology of its genital structures. For this reason, some of its species have been considered to be representatives of other genera of the subtribe Scolitantidina (subfamily Polyommatinae). Here, we address these taxonomic problems by analyzing the phylogenetic relationships between the genera, subgenera, and species of this subtribe inferred via the analysis of five nuclear and two mitochondrial DNA sequences. We demonstrate that the enigmatic Asian species P. panope (Eversmann, 1851) belongs to the genus Pseudophilotes but not to Praephilotes Forster, 1938 or Palaeophilotes Forster, 1938 and does not represent the independent genus Inderskia Korshunov, 2000, as hypothesized previously. We synonymize P. svetlana Yakovlev, 2003 (syn. nov.) and P. marina Zhdanko, 2004 (syn. nov.) with P. panope. We demonstrate a deep genetic divergence between lineages that were previously considered as subspecies of the single species Iolana iolas (Ochsenheimer, 1816). As a result, we confirm the multispecies concept of the genus Iolana Bethune-Baker, 1914. We show that the Holarctic genus Glaucopsyche can be divided into four subgenera: Glaucopsyche Scudder, 1872 (=Shijimiaeoides Beuret, 1958), Apelles Hemming, 1931, Bajluana Korshunov and Ivonin, 1990, and Phaedrotes Scudder, 1876.

1. Introduction

The subtribe Scolitantidina Tutt, 1907 belongs to the tribe Polyommatini (subfamily Polyommatinae) and includes about 17–22 genera and about 85–100 described species distributed throughout the Holarctic and Oriental regions [1]. Eliot [2] recognized this group as a cluster of morphologically similar genera and called it “the Glaucopsyche section” (after the name of one of the genera in this group). Mattoni [3] treated it as the tribe Scolitantidini. Hesselbarth et al. [4] divided this group into the subtribes Scolitantidina and Glaucopsychina Hemming, 1931 within the tribe Polyommatini. Subsequent studies confirmed the monophyly of this group, but the division into the subtribes Scolitantidina and Glaucopsychina was not supported [5,6]. Over the past 50 years, this subtribe has been the subject of a series of taxonomic and phylogenetic studies based on the use of morphological and molecular markers [3,5,6,7,8,9]. Despite this, the phylogenetic position, taxonomic status (genus-subgenus-synonymy) and species diversity of some genera within the subtribe Scolitantidina have remained unclear. In particular, this applies to the genera Pseudophilotes, Glaucopshyche and Iolana.
The genus Pseudophilotes was found not to be homogenous regarding the morphology of its genital structures and larval food plants [10,11,12,13,14,15]. For this reason, some of its species have been considered to be representatives of other genera, including Rubrapterus [10,11], Inderskia [12], Praephilotes [13], and Palaeophilotes [14,15]. The genus Pseudophilotes includes somewhere between eight and twelve species that are distributed across the temperate zone of Eurasia from the Atlantic coast in the west to East Siberia (Yakutia) in the east, and are also found locally in North Africa and the Levant [16]. This genus has attracted the attention of numerous researchers, as some of its species have been used as models in ecological [17,18,19,20] and evolutionary studies [16,21], particularly in studies of insect–plant coevolution [22,23]. Nearly all species of the genus are considered endangered or threatened and are protected by international and/or national laws [24,25,26,27,28,29,30,31,32,33]. The genus Pseudophilotes has been the subject of several taxonomical studies based on analyses of its morphology [4,7,10,12,14,15,34,35,36] and molecular markers [6,9,16,20,21,37]. The morphological analyses revealed an unusually high level of male genitalia variations on both intra- and inter-specific levels, resulting in descriptions of several new taxa [10,12,14,34,35,36]. Available multilocus molecular studies have focused on particular species and species groups within the genus Pseudophilotes [16,20,21], but none of them are based on complete species sampling. In particular, no information on molecular markers is available for the Asian species P. panope, P. svetlana, and P. marina. The genus Glaucopshyche was revealed to be a paraphyletic entity in a phylogenomic study by Ugelvig et al. [6] and a monophyletic group in a whole-genome study by Zhang et al. [9]. However, both studies [6,9] were based on an incomplete sampling of nominal genera and did not include the taxon Bajluana Korshunov and Ivonin, 1990, which is based on the little-known and morphologically distinct species, Glaucopsyche argali.
The genus Iolana is distributed throughout countries surrounding the Mediterranean Sea, in the Levant, Iran, Central Asia, northern Pakistan and northern India. It is represented by a number of allopatric, clearly closely related, but morphologically well-differentiated taxa. These taxa are considered as (i) subspecies of the same species, (ii) representatives of the two species I. iolas and I. gigantea, or (iii) seven to nine independent species [4,13]. These three taxonomic hypotheses have never been tested using molecular markers.
Here, we address these taxonomic problems by analyzing the phylogenetic relationships between the species of the subtribe Scolitantidina inferred via an analysis of the nuclear genes ribosomal subunit 28S (28S), histone 3 (H3), elongation factor 1α (EF1α) and wingless (wg), the non-coding nuclear internal transcribed spacer 2 (ITS2), and two mitochondrial genes, cytochrome oxidase I and II (COI and COII).

2. Materials and Methods

2.1. Taxon Sampling

According to Eliot [2], Mattoni [3], Ugelvig et al. [6], and Korshunov and Ivonin [36], the following genera should be included in the subtribe Scolitantidina (=Glaucopsyche section sensu Eliot, 1973):
Apelles Hemming, 1931 (Type-species [TS]: Polyommatus melanops Boisduval, [1828]);
Bajluana Korshunov & Ivonin, 1990 (TS: Lycaena argali Elwes, 1899);
Caerulea Forster, 1938 (TS: Lycaena coeligena coelestis Alpheraky, 1897);
Euphilotes Mattoni, [1978] (TS: Lycaena enoptes Boisduval, 1852);
Glaucopsyche Scudder, 1872 (TS: Polyommatus lygdamus Doubleday, 1841);
Inderskia Korshunov, 2000 (TS: Lycaena panope Eversmann, 1851);
Iolana Bethune-Baker, 1914 (TS: Lycaena iolas Ochsenheimer, 1816);
Maculinea van Ecke, 1915 (TS: Papilio alcon Denis & Schiffermüller, 1775);
Micropsyche Mattoni 1981 (TS: Micropsyche ariana Mattoni, 1981);
Otnjukovia Zhdanko [1997] (TS: Turanana tatjana Zhdanko, 1984);
Palaeophilotes Forster, 1938 (TS: Lycaena triphysina Staudinger, 1892);
Phaedrotes Scuder, 1876 (TS: Lycaena catalina Reakirt, 1866, currently a subspecies of Lycaena piasus Boisduval, 1852);
Phengaris Doherty, 1891 (TS: Lycaena atroguttata Oberthür, 1876);
Philotes Scudder, 1876 (TS: Lycaena regia Boisduval; 1869 currently subspecies of Lycaena sonorensis C. & R. Felder, [1865]);
Philotiella Mattoni, [1978] (TS: Lycaena speciosa H. Edwards, [1877]);
Praephilotes Forster, 1938 (TS: Lycaena anthracias Christoph, 1877);
Pseudophilotes Beuret, 1958 (TS: Papilio baton Bergsträsser, [1779]);
Rubrapterus Korshunov, 1987 (TS: Lycaena bavius Eversmann, 1832);
Scolitantides Hübner, 1819 (TS: Papilio battus Denis & Schiffermüller, 1775; currently a subspecies of S. orion Pallas 1771);
Shijimiaeoides Beuret, 1958 (TS: Lycaena barine Leech, 1893; currently subspecies of S. divina);
Sinia Forster, 1940 (TS: Glaucopsyche (Sinia) leechi Forster, 1940);
Subsolanoides Koiwaya, [1989] (TS: Subsolanoides nagata Koiwaya, 1981);
Turanana Bethune-Backer, 1916 (TS: Lycaena cytis Christoph, 1877).
For molecular analysis, we used representatives of all these nominal genera, except the very rare monotypic Central Asian genera Palaeophilotes, Micropsyche, Sinia and Subsolanoides. The species sampling included the type species for all studied nominal genera. For the genus Pseudophilotes, we analyzed representatives of all traditionally recognized species. The GenBank and/or BOLD accession numbers of the studied samples are presented in Table 1 and Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6. These accession numbers are searchable via GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 28 November 2022) and/or BOLD (https://boldsystems.org/index.php/Public_BINSearch?searchtype=records, accessed on 28 November 2022) sites that contain information about the sequences and vouchers.

2.2. DNA Studies

The nuclear DNA sequences 28S, ITS2, EF1-a and wg were obtained from the department of Karyosystematics (Zoological Institute RAS, St. Petersburg) using the primers and protocols described in [16]. Standard COI barcodes (partial sequences of the cytochrome c oxidase subunit I gene) were obtained from the Canadian Centre for DNA Barcoding (CCDB, Biodiversity Institute of Ontario, University of Guelph) using their standard high-throughput protocol described by deWaard et al. [38]. The pictures, and collection data of these specimens have been deposited and can be freely downloaded from the BOLD Public Data Portal (http://www.boldsystems.org/index.php/databases, accessed on 28 November 2022). Information about the obtained sequences is presented in Table 1.
For the analyses we used our own sequences as well as published sequences (nuclear sequences 28S, H3, EF1-a, wingless, and ITS2 and mitochondrial genes COI and COII) extracted from GenBank [6,9,16,20,21,39,40,41,42,43] (Table 2). The GenBank/BOLD/museum accession numbers of the analyzed sequences are presented in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6. Two taxa (Lampides boeticus and Phylaria cyara) belonging to the Lampides and Phylaria sections sensu Eliot, 1973 were used to root the tree. The nuclear ribosomal 28S rRNA gene fragment and the nuclear ITS2 sequences were aligned with the software MAFFT v7.245, using the iterative refinement method G-INS-i [44] via the MAFFT online server (http://mafft.cbrc.jp/alignment/server/, accessed on 28 November 2022). As the ITS2 region consists of highly variable sections, its alignment remained partly ambiguous. We therefore used the software Aliscore v.2.0 (The Leibniz Institute for the Analysis of Biodiversity, Bonn, Germany) [45] to identify the ambiguously aligned or randomly similar sections within the ITS2 alignment as described previously [16]. Other sequences were aligned using BioEdit software [46] and were edited manually. Nucleotide substitution models for each dataset were estimated based on the Bayesian information criterion using jModeltest, version 2 [47]. The best fitting models were as follows: GTR + G + I for 28S, GTR + G + I for COI, GTR + G for H3, K2 + G + I for EF1a, K2 + G for wg, GTR + G + I for COII and K2 + G for ITS2.
The Bayesian analyses (Bayes inference, BI) were performed for each individual data set (28S, COI, H3, EF1-a, wg, COII, and ITS2) using the program MrBayes 3.2 [48] and the best fitting models. Two runs of 10,000,000 generations with four chains (one cold and three heated) were performed. The consensus of the obtained trees was visualized using FigTree 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 28 November 2022). These analyses revealed no significant gene tree–species tree conflicts in the data. Then, the genes were concatenated. In doing so, we were based on the evidence that combining multiple mitochondrial DNA barcodes with multilocus nuclear data for representative major taxa can significantly improve the resolution of phylogenetic analysis [49]. The concatenated alignment is presented in the Supplementary Materials (Table S1). The BI analysis of the concatenation 28S + COI + H3 + EF1-a + wg + COII + ITS2 was performed using partition of the data by gene.

2.3. Genus and Subgenus Concepts

We have previously argued that a genus-rank taxon must meet four criteria: (1) monophyly, (2) morphological discreteness, (3) conformity to a certain evolutionary age interval, and (4) conformity to historical nomenclatural traditions (stability and preservation of traditionally recognized taxa) [50]. While the first, second, and fourth criteria seem to be almost universally accepted, the use of criterion three (correspondence of the genus to a certain evolutionary age) is less obvious, and the evolutionary ages of traditionally accepted genera in different groups of living organisms vary greatly. Therefore, in this paper, we used three parameters as a genus criterion. Two of them are obligatory: (1) the monophyly and (2) the morphological discreteness from other genera. One was optional (the group was traditionally considered as a genus). We interpreted the existence of reasonable doubts about the monophyly of a genus as being in favor of dividing the group into two or more undoubtedly monophyletic entities.
As a subgenus, we considered a lineage that was also monophyletic and morphologically discrete but for which there was no tradition to consider it as a genus. Usually such a lineage (subgenus) in combination with other lineages (subgenera) forms a traditionally accepted genus. An additional (though not obligatory) reason for giving a lineage the status of a subgenus was the presence of a previously described available name for it.

2.4. Methodology of Molecular Taxonomy: Genomics, Phylogenomics, DNA-barcoding, and Mixed (Phylogenomics + Barcoding) Approaches

We live in a time when works based on genome-wide data are beginning to appear in insect taxonomy [e.g., 8,9], but at the same time, articles based on multilocus data (phylogenomic approach) [e.g., 6,16,20] or on single mitochondrial gene COI (DNA-barcoding approach) [39,40] still dominate. It seems to us that in between these methodologies is the mixed approach proposed by Talavera et al. [49] who demonstrated that DNA barcodes combined with multilocus data of representative taxa could generate reliable, higher-level phylogenies. This approach is indispensable for poorly studied groups and allows us to combine the suitable length of concatenated sequences for representative (“skeleton”) taxa with the completeness of species sampling.

3. Results

3.1. Mitochondrial Tree

Phylogenetic trees based exclusively on mitochondrial genes performed poorly, resulting in numerous polytomies (Figure 1). However, there were nodes that had good support. Thus, within the genus Glaucopsyche, the clade Glaucopsyche lycormas + Shijimiaeoides divina was identified. The genus Pseudophilotes was monophyletic and divided into two subgenera Pseudophilotes sensu stricto and Rubrapterus. The P. panope complex of the genus Pseudophilotes (P. panope + P. marina + P. svetlana) was monophyletic and isolated from other species of the genus. Pseudophilotes abencerragus and P. barbagiae were sister species.

3.2. Nuclear Tree

On the nuclear tree (Figure 2), the clades representing the genera Turanana, Phengaris + Caerulea, and Glaucopsyche (including Shijimiaeoides divina) received good support. The genus Pseudophilotes was found to be monophyletic and divided into two subgenera Pseudophilotes sensu stricto and Rubrapterus. The subgenus Rubrapterus was found to include two monophyletic species: P. (R.) fatma and P. (R.) bavius. The nuclear data supported the monophyly of the subgenus Pseudophilotes; however, within this subgenus the phylogeny was not resolved. Only three species of the subgenus Pseudophilotes were found to be supported monophyletic entities (P. abencerragus, P. marina and P. barbagiae).

3.3. Concatenated Tree

No serious topology conflict was found between the mitochondrial and nuclear trees. Therefore, the mitochondrial and nuclear data were combined resulting in a mixed matrix [49], in which both the DNA barcodes of multiple species and specimens and the multilocus data of representative taxa were represented (Table S1). This led to a noticeable increase in the resolution of the resulting phylogram (Figure 3). The following genera and suprageneric groups were identified as monophyletic: Phengaris (including Maculinea), Phengaris + Caerulea, Philotiella, Euphilotes, Philotiella + Euphilotes (Figure 3) Turanana (including Otnjukovia), Pseudophilotes (Figure 4), Turanana + Pseudophilotes (Figure 3), Glaucopsyche (Figure 5), Iolana (Figure 6), Praephilotes, Phaedrotes, and Scolitantides (Figure 3).

4. Discussion

Our analysis revealed four supported main lineages within the subtribe Scolitantidina: (1) Phengaris + Caerulea; (2) Euphilotes + Philotiella; (3) Pseudophilotes + Turanana, and (4) Scolitantides + Philotes + Praephilotes + Iolana + Glaucopsyche. This result is consistent with the previous molecular data [6] but does not support the division of the studied group into the subtribes Scolitantidina and Glaucopsychina [4]. Within lineage (1) we found a pattern that was previously [5,6,7,51] described: the nominal genus Maculinea was nested within the genus Phengaris. The genus Phengaris (including Maculinea) was a sister of Caerulea. Within lineage (2), the sublineages Euphilotes and Philotiella were found to be closely related and weakly differentiated taxa. Euphilotes and Philotiella were described by Mattoni as two distinct genera [3]. Zhang et al. [8] downgraded Philotiella to the rank of a subgenus of Euphilotes because their COI barcodes differed by only 3.3%. Our data also showed that Philotiella was better treated as a subgenus than a genus.
Lineage (3) included two sister genera: Turanana and Pseudophilotes (6, 9, our data). Phylogenomic data for Otnjukovia [5,6] and genomic data for Micropsyche [9] demonstrated that these taxa were junior subjective synonyms of Turanana.
The genus Pseudophilotes is divided into two subgenera: Pseudophilotes sensu stricto and Rubrapterus. Within the subgenus Pseudophilotes, one of the most controversial points is the phylogenetic position of the species P. barbagiae, endemic to Sardinia. In the work of Todisco et al. [37] and Bartoňová et al. [21], it was shown that, according to mitochondrial data, this was a sister species of P. abencerragus, which is distributed across North Africa, the Iberian Peninsula, and the Levant. At the same time, according to the combined nuclear–mitochondrial data [16], P. barbagiae was found to be included in the same clade as the European species P. panoptes and P. baton. Our analysis, as well as the data of Wiemers et al. [52], tends to support the sister relationship between P. barbagiae and P. abencerragus. The position of P. barbagiae on the phylogenetic tree is essential for deciding whether the species originated from Africa or from Europe, but it should be recognized that this issue has not yet been resolved. In the situation of apparent mitonuclear discordance, genome-wide data may be needed to resolve this problem.
Pseudophilotes panope, described by E. Eversmann from NW Kazakhstan, is one of the rarest and most enigmatic species of the subtribe Scolitantidina. Researchers previously attributed it to the genera Pseudophilotes, Praephilotes, Paleophilotes, Inderskia, or considered it as a species whose genus was unknown [3,53]. The obtained nuclear and mitochondrial molecular data indicated the undoubted proximity of this taxon to species of the subgenus Pseudophilotes (Pseudophilotes), resulting in the synonymy: Pseudophilotes Beuret, 1958 (=Inderskia Korshunov, 2000, syn. nov.). Pseudophilotes panope has long been known in western Kazakhstan [15] and has only recently been found in eastern Kazakhstan (described as Paleophilotes [sic] marina Zhdanko, 2004) and Mongolia (described as Pseudophilotes svetlana Yakovlev, 2003). A detailed analysis of the external morphology, male genitalia and ecological preferences of populations belonging to P. panope, P. marina and P. svetlana was carried out by Morgun [15]. This author concluded that “all populations are the forms of one species with slightly different phenotypes, which may be due to adaptation (e.g., color, type of soil in inhabited biotopes, altitude above sea level)”. Tshikolovets et al. [53,54] downgraded P. marina and P. svetlana to subspecies of P. panope. Our study revealed identical DNA barcodes in the populations from west and east Kazakhstan and Mongolia. Based on this, we propose a synonymy: P. panope Eversmann, 1851 (=svetlana Yakovlev, 2003, syn. nov.; =marina Zhdanko, 2004, syn. nov.).
An interesting feature of P. panope is its monophagy on Astragalus lasiophillus Ledebur (Fabaceae), whereas caterpillars of other species of the genus are predominantly associated with the plants of the family Lamiaceae [3,4,5,6,7,8,14,15,16,17,18,19,20,21,22]. Association with Astragalus lasiophillus has been also confirmed by us for the east Kazakhstan population of the species via observation of oviposition (Figure 7). A possible clue to this unusual feature is that another species of the genus, P. abencerragus, can also facultatively feed on plants of the family Fabaceae [22]. Feeding on legumes (Fabaceae) is probably an ancestral trait of Polyommatini butterflies [22]; this trait was either lost in the Pseudophilotes lineage but reappeared in P. panope as a reversion, or it was maintained in P. panope when the ancestor of the remaining Pseudophilotes switched to Lamiaceae.
Our study demonstrated that within the subgenus Pseudophilotes, only three species, P. panope, P. abencerragus, and P. barbagiae, were clearly differentiated with respect to DNA barcodes and other studied molecular markers (Figure 1, Figure 2, Figure 3 and Figure 4). As for the species complex P. baton, P. panoptes, P. vicrama, P. sinaicus, and P. jacuticus, as noted earlier, they share the same or similar DNA barcodes (Figure 4) despite their morphological differences [21]. With the data available, it is impossible to decide whether this complex represents completely separated species with secondary contacts, stages of an incomplete speciation, or a single polymorphic species [21]. In our opinion, in accordance with the principle of nomenclatural stability and preservation of traditionally recognized taxa, P. baton, P. panoptes, P. vicrama, P. sinaicus, and P. jacuticus should be interpreted as species until further evidence is obtained in favor of or against their species status. In any case, we must state that molecular (based on DNA barcodes) identification of the species P. baton, P. panoptes, P. vicrama, P. sinaicus, and P. jacuticus seems to be problematic.
Within the Scolitantides–Glaucopsyche lineage (3), the genus Glaucopsyche was revealed in our study to be a paraphyletic group, with the species Glaucopsyche piasus forming a separate cluster on the tree (Figure 3). However, support for major basal branches within the Scolitantides–Glaucopsyche lineage was low in our study; therefore, the identified paraphyly of the genus Glaucopsyche cannot be considered proven. The genus Glaucopshyche was revealed as a paraphyletic entity in a phylogenomic study by Ugelvig et al. [6] and as a monophyletic group in a whole-genome study by Zhang et al. [9]. The later authors revealed a closer relationship between Glaucopsyche piasus (subgenus Phaedrotes) and other Glaucopsyche species than with Iolana, Praephilotes, Scolitantides, and Philotes. Our data showed that the genus Glaucopsyche also included three additional sublineages, which together formed a monophyletic unity. These three lineages can be interpreted as the subgenera Glaucopsyche sensu stricto, Apelles Hemming, 1931, and Bajluana Korshunov and Ivonin, 1990.
Within these three later subgenera, Bajluana was the most differentiated with respect to male genitalia [36,55]. The subgenus Bajluana included one species, Glaucopsyche (Bajluana) argali, which is endemic to the Altai and Saur-Tarbagatai Mts. Four groups of populations of this species are known: (1) the nominotypical subspecies (G. argali argali, mountains surrounding the Chuya steppe in the Russian Altai), (2) subspecies argali chingiz Churkin, 2005 (the southern part of the Mongolian Altai, (3) subspecies argali arkhar Lukhtanov, 1990 (the Saur, Tarbagatai, and Monrak mountains in Kazakhstan) and (4) the southern part of the Kurchum range in the Kazakhstan Altai (Salkyn-Cheku mountain). The analysis of the DNA barcodes showed that despite the geographical isolation, the first, third, and fourth groups of populations were similar to each other. For the second group of populations, molecular data are not yet available.
Shijimiaiodes divina is traditionally assigned to the independent genus Shijimiaiodes (and sometimes also to the genus Sinia by mistake, see [6]). However, molecular data point to its closeness to the core species of the subgenus Glaucopsyche (Glaucopsyche). Morphologically, this species is also similar to the typical Glaucopsyche, especially to G. lycormas [56], which differs in the presence of yellow or reddish spots on the underside of the hindwings. It is obvious that the presence/absence of these yellow or reddish spots is a highly variable characteristic within the subfamily Polyommatinae even on an intra-specific level [13]. Therefore, we support the opinion [9] on the synonymy of Glaucopsyche Scudder, 1872 (=Shijimiaeoides Beuret, 1958).
The subgenus Glaucopsyche also includes two little-known species from Central Asia: G. charybdis and G. laetifica. Both species inhabit near-water biotopes (riverbanks) in the desert zone, and their caterpillars are associated with licorice (Glycyrrhiza) (Fabaceae) [57]. The species are allopatric. Glaucopsyche charybdis is found in the basins of the Amu Darya, Zeravshan and Syr Darya (Fergana Valley) rivers. Glaucopsyche laetifica is found in the basin of the river Ili and in the downstream of the Syr-Darya River. Glaucopsyche charybdis (hind wing underside is gray-brown) and G. laetifica (hind wing underside is blue-green) are morphologically well distinguishable, but their DNA barcodes turned out to be similar. From the Dzhungarian Alatau Mts in eastern Kazakhstan, the morph G. alexis var. aeruginosa is known, resembling G. laetifica in color. The DNA barcode data showed that the var. aeruginosa was a color variant of G. alexis and was not conspecific with G. laetifica.
The monophyly of the genus Iolana and deep molecular differentiation of its species were revealed. This supports the multi-species concept of this genus [58,59] rather than a mono-species (I. iolas [4]) or two-species (I. iolas and O. gigantea [60]) system. There are no molecular data for two species of this genus (I. gilgitica and I. arjanica) but judging by the degree of morphological differentiation of their genitalia (59), they are good taxa of the species level. A deep differentiation between the African and Iberian populations attributed to I. debilitata was revealed. Perhaps they also represent different species.

5. Taxonomic Conclusions

We propose the following taxonomic arrangement of the subtribe Scolitantidina Tutt, 1907
Subtribe Scolitantidina Tutt, 1907 (= Glaucopsychina Hemming, 1931)
Genus Euphilotes Mattoni, [1978]
  Subgenus Euphilotes (Euphilotes) Mattoni, [1978]
  Subgenus Euphilotes (Philotiella Mattoni, [1978])
GenusPhengaris Doherty, 1891 (=Maculinea van Ecke, 1915)
GenusCaerulea Forster, 1938
GenusGlaucopsyche Scudder, 1872
   Subgenus Glaucopsyche (Glaucopsyche) Scudder, 1872 (=Shijimiaeoides Beuret, 1958)
   Subgenus Glaucopsyche (Apelles Hemming, 1931)
   Subgenus Glaucopsyche (Bajluana Korshunov & Ivonin, 1990)
   Subgenus Glaucopsyche (Phaedrotes Scudder, 1876)
Genus Iolana Bethune-Baker, 1914
Genus Praephilotes Forster, 1938
Genus Palaeophilotes Forster, 1938 (no molecular data available)
Genus Scolitantides Hübner, 1819
Genus Turanana Bethune-Backer, 1916 (= Otnjukovia Zhdanko, [1997]; = Micropsyche Mattoni, 1981)
Genus Pseudophilotes Beuret, 1958
   Subgenus Pseudophilotes (Pseudophilotes) Beuret, 1958 (=Inderskia Korshunov, 2000, syn. nov.)
   Subgenus Pseudophilotes (Rubrapterus Korshunov, 1987)
Genus Sinia Forster, 1940 (no molecular data available)
Genus Subsolanoides Koiwaya, [1989] (no molecular data available)
We propose the following taxonomic arrangement of the genera Pseudophilotes Beuret, 1958 and Iolana Bethune-Baker, 1914
Genus Pseudophilotes Beuret, 1958
  Subgenus Pseudophilotes (Pseudophilotes Beuret, 1958) (=Inderskia Korshunov, 2000, syn. nov.)
P. (P.) panope (Eversmann, 1851) (=svetlana Yakovlev, 2003, syn. nov.; (=marina Zhdanko, 2004, syn. nov.)
P. (P.) abencerragus (Pierret, 1837)
P. (P.) barbagiae De Prins & Poorten, 1982
P. (P.) panoptes (Hübner, [1813])
P. (P.) baton (Bergsträsser, [1779])
P. (P.) vicrama (Moore, 1865)
P. (P.) jacuticus Korshunov and Viidalepp, 1980
P. (P.) sinaicus Nakamura, 1975
   P. (P.) sinaicus sinaicus Nakamura, 1975
   P. (P.) sinaicus jordanicus Benyamini, 2000 (no molecular data available)
   Subgenus Pseudophilotes (Rubrapterus Korshunov, 1987)
P. (R.) bavius (Eversmann, 1832)
P. (R.) fatma (Oberthür, 1890)
Genus Iolana Bethune-Baker, 1914
I. iolas (Ochsenheimer, 1816)
I. debilitata (Schultz, 1905)
  I. debilitata debilitata (Schultz, 1905)
  I. debilitata farriolsi de Sagarra, 1930
I. lessei Bernardi, 1964
I. alfierii Wiltshire, 1948
I. arjanica Rose, 1979 (no molecular data available)
I. kermani Dumont, 2004
I. andreasi (Sheljuzhko, 1919)
I. gilgitica (Tytler, 1926) (no molecular data available)
I. gigantea (Grum-Grshimailo, 1885)

Supplementary Materials

The following supporting information can be downloaded at: www.mdpi.com/article/10.3390/insects13121110/s1, Table S1: The concatenated alignment (28S + COI + H3+ EF1-a + wg + COII + ITS2).

Author Contributions

Project design, V.A.L.; conceptualization, V.A.L.; methodology, V.A.L., A.V.G.; PCR amplification and sequencing, A.V.G.; molecular analysis, V.A.L., A.V.G.; writing—original draft preparation, V.A.L., A.V.G.; writing—review and editing, V.A.L., A.V.G.; figure preparation, V.A.L. Collecting material, V.A.L., A.V.G. All authors have read and agreed to the published version of the manuscript.

Funding

The study was supported by the Russian Science Foundation Grant No 19-14-00202 (Continuation) to the Zoological Institute of the Russian Academy of Sciences (St. Petersburg, Russian Federation).

Data Availability Statement

All the analyzed DNA sequences are available via the GenBank links provided (Table 1).

Acknowledgments

We thank A.G.Lukhtanov for help in collecting P. panope in Kazakhstan, E.A.Pazhenkova for the sequence of P. bavius from Bashkortastan, Russia, R. Vila for the sequences of I. debilitata and I. lessei, Gerardo Lamas for his valuable advice on publications containing information about North American species of the subtribe Scolitantidina.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, and interpretation of data; in the writing of the manuscript; and in the decision to publish the results.

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Figure 1. BI tree of the subtribe Scolitantidina based on mitochondrial genes (COI +COII dataset). Posterior probabilities are indicated at the nodes.
Figure 1. BI tree of the subtribe Scolitantidina based on mitochondrial genes (COI +COII dataset). Posterior probabilities are indicated at the nodes.
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Figure 2. BI tree of the subtribe Scolitantidina based on nuclear sequences (28S + H3 + EF1-a + wingless + ITS2 dataset). Posterior probabilities are indicated at the nodes.
Figure 2. BI tree of the subtribe Scolitantidina based on nuclear sequences (28S + H3 + EF1-a + wingless + ITS2 dataset). Posterior probabilities are indicated at the nodes.
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Figure 3. BI of the subtribe Scolitantidina based on concatenation of nuclear and mitochondrial genes (28S + H3 + EF1-a + wingless + ITS2 + COI + COII dataset). Posterior probabilities are indicated at the nodes. (1), (2), (3), and (4) are the four supported main lineages within the subtribe Scolitantidina.
Figure 3. BI of the subtribe Scolitantidina based on concatenation of nuclear and mitochondrial genes (28S + H3 + EF1-a + wingless + ITS2 + COI + COII dataset). Posterior probabilities are indicated at the nodes. (1), (2), (3), and (4) are the four supported main lineages within the subtribe Scolitantidina.
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Figure 4. Fragment of the concatenated (28S + H3 + EF1-a + wingless + ITS2 + COI + COII) BI tree. The subgenus Pseudophilotes (Pseudophilotes) (=Inderskia, syn. nov.) is shown. Posterior probabilities are indicated at the nodes.
Figure 4. Fragment of the concatenated (28S + H3 + EF1-a + wingless + ITS2 + COI + COII) BI tree. The subgenus Pseudophilotes (Pseudophilotes) (=Inderskia, syn. nov.) is shown. Posterior probabilities are indicated at the nodes.
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Figure 5. Fragment of the concatenated (28S + H3 + EF1-a + wingless + ITS2 + COI + COII) BI tree. The genus Glaucopsyche is shown. A is the subgenus Apelles. Posterior probabilities are indicated at the nodes.
Figure 5. Fragment of the concatenated (28S + H3 + EF1-a + wingless + ITS2 + COI + COII) BI tree. The genus Glaucopsyche is shown. A is the subgenus Apelles. Posterior probabilities are indicated at the nodes.
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Figure 6. Fragment of the concatenated (28S + H3 + EF1-a + wingless + ITS2 + COI + COII) BI tree. The genus Iolana is shown. Posterior probabilities are indicated at the nodes.
Figure 6. Fragment of the concatenated (28S + H3 + EF1-a + wingless + ITS2 + COI + COII) BI tree. The genus Iolana is shown. Posterior probabilities are indicated at the nodes.
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Figure 7. Habitat (a) and larval foodplant (Astragalus lasiophillus) (b) of P. panope in Dzhungarian Alatau, E. Kazakhstan. Photo: V.Lukhtanov.
Figure 7. Habitat (a) and larval foodplant (Astragalus lasiophillus) (b) of P. panope in Dzhungarian Alatau, E. Kazakhstan. Photo: V.Lukhtanov.
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Table
Table 1. List of the studied samples and obtained sequences.
Table 1. List of the studied samples and obtained sequences.
SpeciesBOLD/Field IDGeneBankGeneCountryLocality
Glaucopsyche alexis
var. aeruginosa		BPALB161-16OP712325COIIsraelHermon
BPALB162-16OP712326 IsraelHermon
BPAL2627-14 OP712327 IsraelBeit Jan
BPAL2522-14OP712328 IsraelNahal Trivon
BPAL3276-16OP712334 KazakhstanDzhungarian Alatau, Kolbai
BPAL3274-16 OP712332 KazakhstanKolbai
BPAL3275-16OP712333 KazakhstanKolbai
BPAL3408-16OP712338 KazakhstanKyzylagash
Glaucopsyche argaliLOWAM265-11OP712339COIKazakhstanKurtshum Mts, Salkyn-Cheku
LOWAM268-11OP712340 KazakhstanSalkyn-Cheku
LOWAM267-11OP712341 KazakhstanSalkyn-Cheku
LOWAM266-11 OP712342 KazakhstanSalkyn-Cheku
Glaucopsyche laetificaBPAL3283-16 OP712335COIKazakhstanIli valley, Koktal
BPAL3284-16 OP712336 KazakhstanKoktal
BPAL3285-16OP712337 KazakhstanKoktal
Glaucopsyche melanopsBPAL3540-16 OP712329COIMoroccoAgadir 30.90 N 7.24 W
BPAL3541-16OP712330 MoroccoAgadir
BPAL3546-16OP712331 MoroccoAgadir
Iolana alfieriiBPAL2358-14 OP712343COIIsraelAvdat
BPAL2524-14OP712348 IsraelAvdat
BPAL2525-14OP712349 IsraelAvdat
BPAL2902-15OP712350 IsraelHar-A-Negev
BPAL2359-14OP712352 IsraelAvdat
Iolana andreasiLOWAM286-11 OP712351COIIranShahkuh
Iolana andreasi khayyamiBPAL2452-14OP712346COIIranTehran, Polur
BPAL2453-14 OP712347 IranTehran, Polur
Iolana kermaniBPAL2450-14OP712344COIIranKerman, Kuh-e-Segoh
BPAL2451-14OP712345 IranKerman
Praephilotes anthraciasBPAL3279-16 OP712323COIKazakhstanMatai
BPAL3280-16OP712324 KazakhstanMatai
Pseudophilotes abencerragusBPALB525-18OP644300COIIsrael
BPALB526-18OP644301 Israel
BPALB527-18OP644302 Israel
BPALB528-18OP644303 Israel
BPAL3567-16OP644314 Morocco32.5853 N 6.05611 W
Pseudophilotes baviusBPALB030-16 OP644305COIRussiaBashkortostan, 54.89 N 53.646 E
Pseudophilotes panopeL2-14OP679877ITS2KazakhstanKoibyn
L2-15OP679878 KazakhstanKoibyn
L2-16OP679879 KazakhstanKoibyn
L2-14OP681138WinglessKazakhstanKoibyn
L2-14OP681135EF1αKazakhstanKoibyn
L2-15OP681136 KazakhstanKoibyn
L2-16OP681137 KazakhstanKoibyn
L2-14OP67897228SKazakhstanKoibyn
L2-15OP678973 KazakhstanKoibyn
L2-16OP678974 KazakhstanKoibyn
BPALB512-18OP644294COIKazakhstanKoibyn
BPALB513-18OP644295 KazakhstanKoibyn
BPALB514-18OP644296 KazakhstanKoibyn
BPALB515-18OP644297 KazakhstanKoibyn
BPALB516-18 OP644298 KazakhstanKoibyn
BPALB517-18OP644299 KazakhstanKoibyn
BPALB601-19OP644310 Kazakhstan25 km NE Atyrau
BPALB602-19OP644311 Kazakhstan21 km NE Atyrau
BPALB603-19 OP644312 MongoliaHovd, Arshantyn Nuru
BPAL3287-16OP644315 KazakhstanKoibyn
Pseudophilotes vicramaBPALB553-18 OP644304COIIsrael
BPALB284-17 OP644306 Tajikistan
BPALB331-17 OP644307 Tajikistan
BPALB359-17 OP644308 Tajikistan
BPALB470-17OP644309 Israel
Pseudophilotes jacuticusBPALB605-19OP644313COIRussiaYakutia, Yakutsk
Table
Table 2. Fragments of DNA sequences used for phylogenetic analysis.
Table 2. Fragments of DNA sequences used for phylogenetic analysis.
SequenceTotal Length, bpNumber of
Variable Sites		Number of
Parsimony Informative Sites
COI1497454353
COII679184116
EF1a1161238157
H33275742
ITS244910481
wg36912067
28S8209365
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Lukhtanov, V.A.; Gagarina, A.V. Molecular Phylogeny and Taxonomy of the Butterfly Subtribe Scolitantidina with Special Focus on the Genera Pseudophilotes, Glaucopsyche and Iolana (Lepidoptera, Lycaenidae). Insects 2022, 13, 1110. https://doi.org/10.3390/insects13121110

AMA Style

Lukhtanov VA, Gagarina AV. Molecular Phylogeny and Taxonomy of the Butterfly Subtribe Scolitantidina with Special Focus on the Genera Pseudophilotes, Glaucopsyche and Iolana (Lepidoptera, Lycaenidae). Insects. 2022; 13(12):1110. https://doi.org/10.3390/insects13121110

Chicago/Turabian Style

Lukhtanov, Vladimir A., and Anastasia V. Gagarina. 2022. "Molecular Phylogeny and Taxonomy of the Butterfly Subtribe Scolitantidina with Special Focus on the Genera Pseudophilotes, Glaucopsyche and Iolana (Lepidoptera, Lycaenidae)" Insects 13, no. 12: 1110. https://doi.org/10.3390/insects13121110

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