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Ukrainian Journal of Ecology
Ukrainian Journal of Ecology, 2018, 8(1), 222-232 doi: 10.15421/2018_206
ORIGINAL ARTICLE
Notes on the molecular taxonomy of the Proclossiana eunomia complex (Lepidoptera, Nymphalidae: Argynnini):
analysis of DNA barcodes
R.V. Yakovlev1*, N.A. Shapoval2, G.N. Kuftina1, A.V. Kulak3, S.V. Kovalev4
1 Altai State University, Lenina pr. 61, Barnaul, 656049, Russia. 2Department of Karyosystematics, Zoological Institute of Russian Academy of Sciences, Universitetskaya nab. 1,
St. Petersburg, 199034, Russia. 3The Scientific and Practical Center for Animal Breeding of National Academy of Sciences of Belarus,
Academicheskaya str. 27, Minsk, 220072, Belarus. 4Parkovaya str. 38, Dolgoprudnyi, 141702, Russia. Corresponding author. E-mail: yakovlev_asu@mail.ru Submitted: 24.11.2017. Accepted: 15.01.2018
The Proclossiana eunomia (Esper, 1799) complex is currently composed of the several subspecies distributed throughout Palaearctic region and North America. Despite the fact that some of the taxa have differences in wing pattern and body size, previous assumptions on taxonomy not supported by molecular data. Therefore, the identity of certain populations of this complex has remained unclear and the taxonomic status of several recently described taxa is debated. Here, we provide insights into systematics of some Palaearctic members of this group using molecular approach, based on the analysis of the barcoding fragment of the COI gene taking into account known morphological differences.
Key words: mitochondrial DNA; haplotype mitochondrial network; molecular systematics; phylogeography; subspecies; taxonomic status.
Introduction
The Bog Fritillary, Proclossiana eunomia (Esper, 1799) was originally described from "Ostpreussen, Königsberg" (present day the Kaliningrad region). P. eunomia is a widespread boreo-alpine butterfly species, occupying wetland habitats. The species is local in temperate regions of western and eastern Europe (ranging from central France across southern Belgium, Germany, Poland, Austria, Czech Republic, Belarus, northern Ukraine to European part of Russia), very local in the Balkans (Serbia, Bulgaria), the Caucasus and Transcaucasia, northeastern Turkey (Kars), the Volga region, the Urals and the lowlands of Western Siberia. The butterfly is more common in Fennoscandia, Baltic countries and northwestern Russia, northern and eastern Siberia, the mountains of southern Siberia, southeastern Kazakhstan, northern and norhtwestern Mongolia, southeastern China (Heilongjiang province) and Sakhalin (Lukhtanov & Lukhtanov, 1994; Kudrna, 2002; Tuzov & Bozano, 2006; Lukhtanov et al., 2007; Lvovsky & Morgun, 2007; Tshikolovets et al., 2009a, b; Tshikolovets, 2011; Lang, 2012; Yakovlev, 2012; Toropov & Zhdanko, 2013), as well as in the Nearctic, ranging from Alaska and Labrador to Wisconsin and across the mountains to Colorado (Scott, 1986; Pyle, 2006). The taxonomic structure of this widespread species requires revision.
A number of taxa were described from Siberia: acidaiia Böber, 1809 (type locality - "de la Sibérie"); asiatica Staudinger, 1901 (type locality - "Sib.[erie] m.[eridionale] (Alt.[ai], Saj.[an], Kent.[ei], Amur)"); riamia Korshunov et Ivonin In Korshunov, 1998 (type locality - "Kamennyi ryam, near Kuznetskoe village, Chulym district, Novosibirsk region"); stromi Korshunov, 1998 (type locality -"W. Sayan, Pazaryk-Ergak-Tayga Range") and yakovlevi Korb, 1999 (type locality - "Altai, Ukok, Kudabaj river"). Recently, three subspecies of P. eunomia were described: P. eunomia ttelmena Gorbunov, 2007 In Gorbunov & Kosterin, 2007 from Kamchatka Peninsula; P. eunomia exspectata Morgun, 2011 and P. eunomia tenera Morgun, 2011 from the mountains of the Greater Caucasus and Armenia, respectively.
In their book V. and A. Lukhtanov (Lukhtanov & Lukhtanov, 1994) noted three subspecies for Siberia and east of Eurasia: ssp. eunomia (Middle and South Ural), ssp. ossianus Herbst, 1800 (Arctic and North Siberia) and ssp. asiatica Staudinger, 1901 (Middle and South Siberia, West Siberian Plain, Altai). Korshunov also noted these three subspecies for the region (Korshunov & Gorbunov, 1995) and additionally described two new subspecies: P. eunomia stromi and P. eunomia riamia (Korshunov, 1998). His interpretation of the distribution of P. eunomia subspecies is as follows: ssp. eunomia for the east Siberia to the Middle and Southern Trans-Urals; ssp. ossianus for the forest-tundra and the taiga zone of Asia, reaching north to Taimyr; ssp. acidaiia (= asiatica) for the mountains of southern Siberia. According to Korshunov this taxon distributed "further in the mountains of South Siberia, Yakutia and the north of the Far East" (Korshunov, 1998, p. 20). In this context it should be understood as east of the distribution area of ssp. stromi, and judging by the type locality - east of Todzha Village [Eastern Tuva]. Bogdanov generally follows the point of view proposed by Korshunov and Gorbunov (1995) specifying the distribution of ssp. ossiana as western Chukotka, Kamchatka, and the northern part of the Khabarovsk region. About the subspecies riamina (sic!) Bogdanov stated that: "its status requires a revision" (Bogdanov in Tuzov et al., 2000, p. 43). Gorbunov (2001) mentioned three subspecies for Siberia (ssp. eunomia, ssp. ossianus, ssp. acidaiia) and also established a new synonymy, but without providing any rationale: ossianus = riamia and acidaiia = asiatica = stromi = yakovlevi. Korb (2005) specifying the distribution of P. eunomia subspecies: ssp. acidaiia (mountains of South Siberia), ssp. asiatica (South and Middle Siberia, Amur, Ussuri regions and Sakhalin), ssp. eunomia (Central
area of European part, Caucasus) and ssp. ossianus (North of Eurasia, Far East and Kamchatka). Dubatolov et al. (2005) recognizes two subspecies for eastern Eurasia: ssp. ossianus (Chukotka, Magadan, Kamchatka, Khabarovsk, Jewish Autonomous region, Amur, Sakhalin, Yakutia, northern Transbaikal regions, ?NE China) and ssp. acidaiia (= asiatica) (Transbaikal, Sayans and Altai). Tuzov and Bozano (2006) accepted the following taxonomic structure of P. eunomia (here we omit western European and North American taxa): ssp. eunomia (central and southern Europe, Caucasus), ssp. ossianus (= riamia) (from northern Europe to western Chukotka, Kamchatka, Sakhalin and the Okhotsk Sea coast), ssp. acidaiia (= asiatica = stromi = yakovlevi) (from Altai and Sayan Mountains, southern Siberia to Amur and Ussuri regions). Korb and Bolshakov noted ssp. iteimena for Kamchatka and Chukotka; ssp. ossianus for the northern regions of Eurasia and partly middle part of European Russia (except for the distribution of ssp. iteimena); ssp. eunomia for Central Europe, Middle and Southern Ural and Caucasus; ssp. acidaiia for Southern Siberia, Amur, Primorsky krai and Sakhalin (Korb & Bolshakov, 2011); and indicated the subspecies described by Morgun (2011) from the Caucasus and Transcaucasia as valid (Korb & Bolshakov, 2016).
When examining large series of P. eunomia from different parts of southern Siberia, we have established that the distribution of this butterfly is essentially uninterrupted and all the characters indicated by various authors (small size, thin contrast pattern) fit within the limits of population variability.
Thus, contradictory taxonomic interpretations of intraspecific structure of P. eunomia exist in the literature. It should be noted, that correct taxonomic identification is often not possible based primarily on external morphological characters (Hajibabaei et al., 2006; Dinca et al., 2013; Shapoval & Lukhtanov, 2016). In contrast, the use of molecular markers (e.g. DNA barcodes) is becoming universally accepted as a useful and efficient tool for species identification, detecting previously unrecognized taxa and assisting in the resolution of taxonomic and phylogenetic problems (Hebert et al., 2004; Barrett & Hebert, 2005; Janzen et al., 2005; Hajibabaei et al., 2006; Smith et al., 2007; Velzen, Larsen & Bakker, 2009; Sourakov & Zakharov, 2011; Lukhtanov, Shapoval & Dantchenko, 2014), but see Brower (2006, 2010). It is particularly useful as an efficient start for taxonomic workflow (Kekkonen & Hebert, 2014). Yet, detailed phylogenetic studies are unavailable for P. eunomia. This paper addresses a DNA-based analysis of this taxon.
Material and methods
Specimens
Butterflies used for this analysis were collected in different regions of Russia, Belarus, Ukraine and Lithuania (Fig. 1). List of collected specimens with information including sampling localities is given in Table 1. DNA barcodes from a total of 37 individuals of P. eunomia were sequenced for this study. Additionally, 27 sequences of P. eunomia available from the GenBank and BOLD databases were included in our analysis. We used sequence of Coenonympha tuiiia eiwesi (Davenport, 1941) from Altai (GenBank accession number MG757148) as an outgroup to root the phylogram. Representatives of all principal members of the genus Boioria available from the GenBank database were included in our analysis to test the monophyly of P. eunomia and clarify phylogenetic relationships. The barcode analysis of P. eunomia complex involved 539 sequences.
DNA processing and sequencing
DNA extraction, amplification and further preparations for sequencing were held on the base of "Taxon" Research Resource Center and Department of Karyosystematics of the Zoological Institute of the Russian Academy of Sciences (St. Petersburg). DNA extraction from a single leg removed from the specimen was accomplished using the QIAamp DNA Investigator Kit (Qiagen, Netherlands) following the manufacturer's protocol. A fragment of the mitochondrial cytochrome oxidase I gene (COI ) was used as a mitochondrial molecular marker. Standard lepidopteran barcode primers (Hebert et al., 2004) were used for DNA amplification and resulted in a 658 bp fragment of the COI gene. The PCR amplifications were performed in a 50 pl reaction volume containing ca. 10-20 ng genomic DNA and 0.5 mM each of forward and reverse primer, 1 mM dNTPs, 10x PCR Buffer (0.01 mM Tris-HCl, 0.05 M KCl, 0.1% Triton X-100: pH 9.0), 1 unit Taq DNA Polymerase (Thermo Fisher Scientific, Lithuania), 5 mM MgCh. Amplification for COI gene fragment was carried out with the following conditions: initial denaturation at 94° C for 1 min, followed by 30 cycles of denaturation at 94° C for 45 s, annealing at 50° C for 45 s, and extension at 72° C for 1 min with a final extension at 72° C for 10 min. Amplified fragments were purified using GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Lithuania). Purification was carried out according to the manufacturer's protocol. Purified PCR product was used for direct sequencing.
Table 1. List of studied material (64 specimens). (*) - sequences obtained in the present study. (**) - sequence obtained in the present study and used as an outgroup._
GenBank/ BOLD number Taxon COI haplotype Locality Altitude Date Collectors/ References
MG757148** Coenonympha tullía elwesi - Russia, Altai Rep., Kosh-Agach distr., vicinity of Tabozhok vill. 2000 m 03.08.2016 R. Yakovlev
MG735633* Proclossiana eunomia ossiana Eu_ _07 Russia, Moscow reg., Sergiev Posad distr., SE Maloye Lake, Bat'kovskoye raised bog 135 m 08.06.2013 S. Kovalev
MG735634* Proclossiana eunomia ossiana Eu_ _01 Russia, Moscow reg., Sergiev Posad distr., vicinity of Bol'shoye Lake, Batkovskoye raised bog 130 m 28.05.2016 S. Kovalev
MG735635* Proclossiana eunomia ossiana Eu_ _01 Russia, Moscow reg., Shatursky distr., 1.7 km NW Tugolesye railway station 125 m 10.06.2016 S. Kovalev
MG735636* Proclossiana eunomia ossiana Eu_ _01 Russia, Moscow reg., Shatursky distr., 1.7 km NW Tugolesye railway station Russia, Yaroslavl reg., Pereslavsky distr., 1.1 km NE 125 m 10.06.2016 S. Kovalev
MG735637* Proclossiana eunomia ossiana Eu_ _04 Zhupeevo vill., Polovetsko-Kupanskoe (Bol'shoye) raised bog Russia, Yaroslavl reg., Pereslavsky distr., 1.1 km NE 170 m 04.06.2016 S. Kovalev
MG735638* Proclossiana eunomia ossiana Eu_ _07 Zhupeevo vill., Polovetsko-Kupanskoe (Bol'shoye) raised bog Russia, Yaroslavl reg., Pereslavsky distr., 2.6 km SE Kupanskoye vill., National Park «Plesheevo Lake» 170 m 04.06.2016 S. Kovalev
MG735639* Proclossiana eunomia eunomia Eu_ _01 140 m 13.06.2016 S. Kovalev
MG735640* Proclossiana eunomia eunomia Eu_ _01 Russia, Yaroslavl reg., Pereslavsky distr., 2.6 km SE Kupanskoye vill., National Park «Plesheevo Lake» 140 m 13.06.2016 S. Kovalev
MG735641* Proclossiana eunomia eunomia Eu_ _02 Russia, Moscow reg., Ruzsky distr., 3.6 km NE Novogorbovo village, State Reserve «Glubokoye Lake» 210 m 07.06.2015 S. Kovalev
MG735642* Proclossiana eunomia eunomia Eu_ _02 Russia, Moscow reg., Ruzsky distr., 3.6 km NE Novogorbovo village, State Reserve «Glubokoye Lake» 210 m 07.06.2015 S. Kovalev
MG735643* Proclossiana eunomia exspectata Eu_ _13 Russia, North Caucasus, Karachay-Cherkess Republic, Karachayevsky distr., Nature Reserve «Teberda», Nazylkol river valley 2450 m 23.07.2011 D. Morgun
MG735644* Proclossiana eunomia exspectata Eu_ _12 Russia, North Caucasus, Karachay-Cherkess Republic, Karachayevsky distr., Nature Reserve «Teberda», Nazylkol river valley 2450 m 23.07.2011 D. Morgun
MG735645* Proclossiana eunomia exspectata Eu_ _12 Russia, North Caucasus, Karachay-Cherkess Republic, Karachayevsky distr., Nature Reserve «Teberda», Nazylkol river valley 2450 m 23.07.2011 D. Morgun
MG735646* Proclossiana eunomia exspectata Eu_ _13 Russia, North Caucasus, Karachay-Cherkess Republic, Karachayevsky distr., Nature Reserve «Teberda», Nazylkol river valley 2450 m 23.07.2011 D. Morgun
MG735651* Proclossiana eunomia exspectata Eu_ _13 Russia, North Caucasus, Karachay-Cherkess Republic, Karachayevsky distr., Nature Reserve «Teberda», Nazylkol river valley 2450 m 21.07.2011 D. Morgun
MG735647* Proclossiana eunomia acidalia Eu_ _22 Russia, Buryatia, Tunkinsky distr., Mondy vill., Huluigasha Mts. 1800 m 2430.06.2016 A. Kosarev
MG735648* Proclossiana eunomia acidalia Eu_ _24 Russia, Buryatia, Tunkinsky distr., Mondy vill., Huluigasha Mts. 1800 m 2430.06.2016 A. Kosarev
MG735649* Proclossiana eunomia riamia Eu_ _21 Russia, Novosibirsk reg., Chulym distr., Kamennyi ryam 16.06.2005 A. Kareev
MG735650* Proclossiana eunomia riamia Eu_ _19 Russia, Novosibirsk reg., Chulym distr., Kamennyi ryam 16.06.2005 A. Kareev
MG735652* Proclossiana eunomia riamia Eu_ _20 Russia, Omsk reg., Krutinsky distr., 44 km NW Krutinka vill., 5 km SW Gulyai-Pole vill. 0708.06.2016 S. Knyazev
MG735653* Proclossiana eunomia riamia Eu_ _20 Russia, Omsk reg., Krutinsky distr., 44 km NW Krutinka vill., 5 km SW Gulyai-Pole vill. 0708.06.2016 S. Knyazev
MG735654* Proclossiana eunomia ossiana Eu_ _01 Russia, Karelia, Kondopoga reg., Nature Reserve «Kivach», bog Blizkoe 31.05.2016 V. Gorbach
MG735655* Proclossiana eunomia ossiana Eu_ _01 Russia, Karelia, Kondopoga reg., Nature Reserve «Kivach», bog Blizkoe 31.05.2016 V. Gorbach
MG735656* Proclossiana eunomia Eu_ _06 Ukraine, near Kiev, Belichi vill., tract Lyubka 02.06.2011 I. Plyusch
MG735657* Proclossiana eunomia Eu_ _06 Ukraine, near Kiev, Belichi vill., tract Lyubka 02.06.2011 I. Plyusch
MG735658* Proclossiana eunomia eunomia Eu_ _01 Ukraine, Rivne reg., Nature Reserve «Rivne», near vill. Bel'sk 22.06.2005 I. Plyusch
MG735659* Proclossiana eunomia eunomia Eu_ _05 Russia, South Ural, Chelyabinsk reg., near Ozersk 15.06.2015 S. Rybalkin
MG735660* Proclossiana eunomia eunomia Eu_ _05 Russia, South Ural, Chelyabinsk reg., near Ozersk 15.06.2015 S. Rybalkin
MG735661* Proclossiana eunomia eunomia Eu_ _01 Lithuania, Vilnius reg., Daubeni vill. 06.06.2015 D. Mikalauskas
MG735662* Proclossiana eunomia eunomia Eu_ _01 Lithuania, Vilnius reg., Daubeni vill. 06.06.2015 D. Mikalauskas
MG735663* Proclossiana eunomia eunomia Eu_ _01 Belarus, Minsk reg., Nature reserve «Talka» 18.06.2016 A. Kulak
MG735664* Proclossiana eunomia eunomia Eu_ _03 Belarus, Minsk reg., Nature reserve «Talka» 18.06.2016 A. Kulak
MG735665* Proclossiana eunomia eunomia Eu_ _03 Belarus, on the border of Minsk and Vitebsk reg., Biosphere Reserve «Berezinsky» 20.06.2016 A. Kulak
MG735666* Proclossiana eunomia eunomia Eu_ _01 Belarus, on the border of Minsk and Vitebsk reg., Biosphere Reserve «Berezinsky» 20.06.2016 A. Kulak
MG735667* Proclossiana eunomia itelmena Eu_ _18 Russia, Kamchatka p-la, Bistrinskiy distr., Esso vill., Sredinniy r., Oligende mts. 1100 m 0920.07.2005 D. Goshko
MG735668* Proclossiana eunomia itelmena Eu_ _17 Russia, Kamchatka p-la, Bistrinskiy distr., Esso vill., Sredinniy r., Oligende mts. 1100 m 0920.07.2005 D. Goshko
MG735669* Proclossiana eunomia itelmena Eu_ _17 Russia, Kamchatka p-la, Bistrinskiy distr., Esso vill., Sredinniy r., Oligende mts. 1100 m 0920.07.2005 D. Goshko
JF415682 Proclossiana eunomia Eu_ _10 Germany, Bavaria, Oberbayern reg., Koenigsdorf, Weitfilz 600 m 03.06.2005 Huasmann et al., 2011
JF415681 Proclossiana eunomia Eu_ _11 Germany, Bavaria, Oberbayern reg., Bad Toelz-Wolfratshausen, Zellwies 600 m 21.05.2009 Huasmann et al., 2011
JX034677 Proclossiana eunomia Eu_ _10 Germany, Bavaria, Schwaben reg., Ostallgaeu, Lamerdingen, Kleinkitzighofen 600 m 10.06.2004 Mutanen et al., 2012
GU655024 Proclossiana eunomia Eu 11 Germany, Bavaria, Oberbayern reg., Landsberg am Lech, 708 m 14.06.2009 Huasmann
Diessen am Ammersee, Vilgertshofener Forst et al., 2011
KX044586 Proclossiana eunomia Eu_ _10 Germany, Bavaria, FFB, Maisach 14.05.2011 Mutanen et al., 2016
FJ664012 Proclossiana eunomia Eu_ _01 Russia, Saint-Petersburg reg., Oranienbaum, Tamengont 22.06.2001 Lukhtanov et al., 2009
FJ664014 Proclossiana eunomia Eu_ _01 Russia, Pskovskaya obl., Sebezh distr., Osyno 01.07.2001 Lukhtanov et al., 2009
FJ664013 Proclossiana eunomia Eu_ _01 Russia, Pskovskaya obl., Sebezh distr., Osyno 01.07.2001 Lukhtanov et al., 2009
GU676265 Proclossiana eunomia Eu_ _16 Spain, Cantabria, Brana Vieja 1408 m 04.07.2008 Dincä et al., 2015
GU676266 Proclossiana eunomia Eu_ _16 Spain, Cantabria, Brana Vieja 1408 m 04.07.2008 Dincä et al., 2015
GU676267 Proclossiana eunomia Eu_ _16 Spain, Cantabria, Brana Vieja 1408 m 04.07.2008 Dincä et al., 2015
GU676268 Proclossiana eunomia Eu_ _16 Spain, Cantabria, Brana Vieja 1408 m 04.07.2008 Dincä et al., 2015
HM901618 Proclossiana eunomia Eu_ _14 Spain, Catalonia, Girona reg., Meranges 1555m 29.06.2007 Dincä et al., 2015
HM901612 Proclossiana eunomia Eu_ _14 Spain, Catalonia, Girona reg., Meranges 1555 m 29.06.2007 Dincä et al., 2015
GU677019 Proclossiana eunomia Eu_ _14 Spain, Catalonia, Girona reg., Meranges 1555 m 29.06.2007 Dincä et al., 2015
JF853660 Proclossiana eunomia Eu_ _22 Finland, Ostrobottnia borealis pars australis, Kiiminki 01.01.2005 Mutanen et al., 2012
HM875824 Proclossiana eunomia Eu_ _22 Finland, Ostrobottnia borealis pars australis, Kempele 11.06.2009 Mutanen et al., 2012
HM875823 Proclossiana eunomia Eu_ _23 Finland, Ostrobottnia borealis pars australis, Kempele 11.06.2009 Mutanen et al., 2012
PHLAI555-13 Proclossiana eunomia Eu_ _08 Austria, Tirol, Nordtirol reg., Reither Moor/ Seefeld S 1178 m 19.06.2004 Huemer & Hebert, 2016
PHLAI572-13 Proclossiana eunomia Eu_ _10 Austria, Tirol, Nordtirol reg., Gasthaus Stefansbruecke -Innsbruck S 700 m 04.06.2004 Huemer & Hebert, 2016
KM572223 Proclossiana eunomia Eu_ _10 Austria, Vorarlberg, Im Moos/ Bizau W 650 m 20.06.2013 Huemer et al., 2014
GU676336 Proclossiana eunomia Eu_ _15 Andorra, Refugi de Riu dels Orris, per sota del (vall del Madriu), Escaldes-Engordany 2180 m 19.07.2008 Dincä et al., 2015
PHLAI571-13 Proclossiana eunomia Eu_ _09 Italy, South Tyrol, Suedtirol reg., Fischerwiesen, Sankt Valentin auf der Heide 1450 m 12.06.2007 Huemer & Hebert, 2016
PHLAI570-13 Proclossiana eunomia Eu_ _09 Italy, South Tyrol, Südtirol reg., Reschen N 12.06.2007 Huemer & Hebert, 2016
KX047880 Proclossiana eunomia Eu_ _22 Norway, Nord-Trondelag, Lierne reg., Jakthuset, Nordli 02.07.2008 Mutanen et al., 2016
Phylogenetic reconstruction
The sequences were edited and aligned using CHROMAS 2.4.3 (http://www.technelysium.com.au/), Geneious 8.1.6 (Kearse et al., 2012), and BioEdit 7.0.3 (Hall, 2011) software. The alignment was unambiguous, as all the sequences were of equal length and included no insertions/deletions. Primer sequences were cropped. A Bayesian approach for estimating phylogeny was used. Bayesian analyses were performed using the program MrBayes 3.2 (Ronquist et al., 2012), with the nucleotide substitution model GTR+G+I as suggested by jModelTest (Posada, 2008). TRACER, v1.4 was used for summarizing the results of Bayesian phylogenetic analyses (http://beast.bio.ed.ac.uk/Tracer). A median-joining haplotype network was built using PopART (Clement et al., 2002). Genetic distances were calculated for COI gene using MEGA v7.0.14 (Kumar, Stecher & Tamura, 2016).
Results and Discussion
-!<^j/Vocto5$i(wia eunomia
Boloria selene Boloria perryi
Boloria thore 091 'u Boloria selenis
rUf
Boloria oscarus
Boloria chariclea Boloria titania —Boloria freija
Boloria euphrosyne
i|—Boloria frigga I 1 Boloria improba
-Boloria polaris
-k^J Boloria gong
■ Coenonympha tullia ehvesi
The phylogenetic analysis based on the mitochondrial COI marker
(Fig. 2) recovered all P. eunomia sequences in a highly supported __j—Lwori»™teMs
clade (Bayesian posterior probability = 1). Within P. eunomia clade, vm^Boioriahegemone
no distinct intraspecific sublineages were recovered and monophyly was not retrieved for any described subspecies, indicating a close evolutionary relationship among all the individuals of this species (Fig. 3). Specimens from Novosibirsk and Omsk region, identified as P. eunomia riamia was found to be paraphyletic with respect to other P. eunomia individuals. In general, P. eunomia appeared to be genetically homogenous with respect to COI (the average genetic distance among all studied specimens was 0.41%±0.11%), which is also reflected in the COI haplotype network (Fig. 4). Haplotype network analysis of a dataset of 64 specimens revealed 24 haplotypes, which however, cannot be clustered in specific haplogroups. Each haplotype differs from neighboring haplotype not more than by two nucleotide substitutions. The analysis shows that there is no significant difference in the range of inter- and intrapopulation COI sequence divergence. Thus,
Fig. 2. The phylogenetic tree of the Boloria and Proclossiana species based on analysis of the cytochrome oxidase subunit I (COI) gene from 539 specimens. Numbers at nodes indicate Bayesian posterior probability. Branches with Bayesian posterior probability values >0,5 are shown. Scale bar = 0.6 substitutions per position. Proclossiana eunomia cluster highlighted in orange.
composition of each haplotype does not always reflect current taxonomic interpretations, the particular geographical areas and geographical distribution of butterflies. Unlike COIgene sequences, by which P. eunomia displayed remarkable genetic homogeneity given the geographical area covered, different populations do often demonstrate prominent morphological differences in wing
pattern, wing shape and body size (Figs. 5-6).
Boloria dia
rGBLAB32413 P. eunomia Germany
— GBLAC36813 P. eunomia Germany
— JF415682 P. eunomia Germany
— GBLAB78013 P. eunomia Germany
-JF415681 P. eunomia Germany
-GU655024 P. eunomia Germany
— JX034677 P. eunomia Germany
— KX044586 P. eunomia Germany
— PHLAI57213 P. eunomia Austria
— KM572223 P. eunomia Austria -PHLAI55513 P. eunomia Austria
-PHLAI57113 P. eunomia Italy -PHLAI57013 P. eunomia Italy
— MG735643 P. eunomia exspectata Russia, Caucasus
— MG735646 P. eunomia exspectata Russia, Caucasus ■ MG735651 P. eunomia exspectata Russia, Caucasus
-MG735644 P. eunomia exspectata Russia, Caucasus
—MG735645 P. eunomia exspectata Russia, Caucasus —MG735669 P. eunomia itelmena Russia, Kamchatka
— MG735668 P. eunomia itelmena Russia, Kamchatka
— MG735667 P. eunomia itelmena Russia, Kamchatka
— FJ664012 P. eunomia Russia, St. Petersburg -FJ664014 P. eunomia Russia, Pskov
FJ664013 P. eunomia Russia, Pskov
— MG735634 P. eunomia ossiana Russia, Moscow MG735635 P. eunomia ossiana Russia, Moscow
— MG735636 P. eunomia ossiana Russia, Moscow MG735637 P. eunomia ossiana Russia, Yaroslavl'
— MG735639 P. eunomia Russia, Yaroslavl'
— MG735640 P. eunomia Russia, Yaroslavl' MG735641 P. eunomia Russia, Moscow MG735642 P. eunomia Russia, Moscow
— MG735654 P. eunomia ossiana Russia, Karelia
— MG735655 P. eunomia ossiana Russia, Karelia MG735656 P. eunomia Ukraine MG735657 P. eunomia Ukraine
— MG735658 P. eunomia Ukraine
-MG735659 P. eunomia Russia, South Ural
— MG735660 P. eunomia Russia, South Ural
MG735661 P. eunomia Lithuania -MG735662 P. eunomia Lithuania
— MG735663 P. eunomia Belarus MG735664 P. eunomia Belarus MG735665 P. eunomia Belarus
MG735666 P. eunomia Belarus
— MG735633 P. eunomia ossiana Russia, Moscow
— MG735638 P. eunomia ossiana Russia, Yaroslavl -HM901618 P. eunomia Spain
HM901612 P. eunomia Spain GU677019 P. eunomia Spain
— GU676336 P. eunomia Andorra GU676265 P. eunomia Spain GU676267 P. eunomia Spain GU676266 P. eunomia Spain GU676268 P. eunomia Spain
— IF853660 P. eunomia Finland
— HM875824 P. eunomia Finland HM875823 P. eunomia Finland
— KX047880 P. eunomia Norway
— MG735647 P. eunomia acidalia Russia, Buryatiya
— MG735648 P. eunomia acidalia Russia, Buryatiya - MG735649 P. eunomia riamia Russia, Novosibirsk
" MG735650 P. eunomia riamia Russia, Novosibirsk
iMG735652 P. eunomia riamia Russia, Omsk „ IS
0.96*- MG735653 P. eunomia riamia Russia, Omsk
■ Coenonympha tullía elwesi
0,5
Fig. 3. Consensus Bayesian tree of the Prociossiana eunomia complex inferred from COI sequences. Numbers at nodes indicate Bayesian posterior probability. Branches with Bayesian posterior probability values >0,8 are shown. Scale bar = 0.5 substitutions per position
Prociossiana eunomia itelmena
Prociossiana eunomia itelmena was described on the basis of slight morphological differences in wing shape and wing coloration (the pattern on the upperside is thinner than in ssp. acidaiia, the marginal spots on the hindwing underside are silver) (Gorbunov & Kosterin, 2007). Three specimens of P.. eunomia itelmena, collected at the type locality (Kamchatka, Esso Village) form two haplotypes: Eu_17 and Eu_18 (Fig. 4). These haplotypes differ by one nucleotide substitution within the 628 bp region of the COI gene. At the same time, haplotype Eu_17 of P. eunomia itelmena differs by only one nucleotide substitution from haplotypes Eu_16, which comprise specimens of P. eunomia ossiana from Spain (Cantabria) and Eu_07, which consists of two specimens collected in Moscow region and Yaroslavl region.
Proclossiana eunomia exspectata
Proclossiana eunomia exspectata was described as a separate subspecies on the basis of male genitalia characters (structure and shape of the valva and its costal process), the external morphological characters and the ecological preferences (Morgun, 2011). Five specimens of P. eunomia exspectata, collected at the type locality (Russia, North Caucasus, vicinity of Teberda, Nazylkol River valley) form two haplotypes: Eu_12 and Eu_13 (Fig. 4). These haplotypes differ by one nucleotide substitution within the 628 bp region of the COIgene. Haplotype Eu_13 of P. eunomia exspectata differs by two nucleotide substitutions from haplotypes Eu_16, which comprise specimens of P. eunomia ossiana from Spain (Cantabria) and Eu_07, which consists of two specimens collected in Moscow region and Yaroslavl region.
Proclossiana eunomia riamia
Of the Asian range of the distribution, the most externally distinguished subspecies is Proclossiana eunomia riamia. P. eunomia riamia was described as a separate subspecies on the basis of larger wingspan and wing coloration (very dark and contrast background of the upperside of the wings, in particular, totally black marginal area on the hindwings; brick-red background of the hindwing underside; silver pale elements on the hindwing underside) (Korshunov, 1998). P. eunomia riamia found only associated with raised bogs (the so-called, ryams) (Fig. 7) in the south of West-Siberian Plain. Raised bogs, isolated from other mesophilic communities, significantly differ from the surrounding forest-steppe in flora (Valutskiy, 2011) and fauna of Papilionoidea (Knyazev, 2009; Ivonin et al., 2013; Yakovlev & Kareev, 2013). It was of special interest to study P. eunomia riamia, since it represents a remote population, separated by more than 300-350 km from the closest known locality of P.eunomia in Altai Republic. Four specimens of P. eunomia riamia collected at the type locality and Omsk region form three haplotypes: Eu_19, Eu_20, Eu_21. These haplotypes differ by 1-2 nucleotide substitutions within the 628 bp region of the COI gene. Haplotypes of P. eunomia riamia differs by at least two nucleotide substitutions from neighboring haplotype Eu_16 (Fig. 4). Furthermore, P. eunomia riamia specimens have single fixed substitution (C^T) at position 42 of the COI gene, which never occurred in other studied representatives of P. eunomia and form a separated paraphyletic lineage in Bayesian inference tree.
Fig. 4. Haplotype network based on COI barcodes. Every mutation is marked with a bar; number of mutations separating haplotypes is given in parentheses. The circle size is proportional to the number of samples represented.
Proclossiana eunomia acidalia
Proclossiana eunomia acidaiia inhabits the taiga zone of the Urals and Siberia (up to western Transbaikal). The subspecies can be distinguished by thin strokes on the upperside of the wings, and light yellow (without silvery and pearly elements) pattern of the underside of the wings. Two specimens from Buryatia, identified as P. eunomia acidalia form two haplotypes (Eu_24 and Eu_22), separated by one nucleotide substitution. One of these specimens (GenBank accession number MG735647) share COI haplotype with the samples of P. eunomia from Norway and Finland (Fig. 4, haplotype Eu_22).
To conclude, our results reveals little congruence between previous interpretations of the variously recognized subspecies of P. eunomia based mainly on geographical distribution and/or relatively minor morphological characters with molecular data (COIgene) obtained. We revealed high level of similarity in COI barcodes between all studied populations of P. eunomia. Thus, molecular data provide evidence to support a monotypic species hypothesis rather than the recognition of several subspecific forms, with the exception of P. eunomia riamia. P. eunomia riamia has noticeable morphological differences in wing pattern, accompanied by distinct position on the phylogenetic tree and presence of fixed nucleotide substitution, which does not allow us to synonymize this butterfly to any other subspecies of P. eunomia.
Fig. 5. Prociossiana eunomia, adult male specimens: upperside (right) and underside (left). a) P. e. eunomia, Ukraine, near Kiev, Belichi vill., tract Lyubka, 2.06.2011, leg. I. Pljustch; b) P. e. ossiana, Norway, Finnmark, Gargia, 16.06.1992, leg. I. Pljustch; c) P. e. acidalia (topotype of yakovlev!), Russia, Altai, Ukok plateau, Mai-Pak, 2300 m, 29.06.1997, leg. R. Yakovlev; d) P. e. acidalia, Mongolia, Hovd aimak, Bulgan-gol basin, middle stream of Ulyasutaj-Gol river, 2500-3000 m, 25-26.06.2004, leg. R. Yakovlev & D. Ryzhkov; e) P. e. acidalia (topotype of stromi), Russia, S. Tuva, 20 km N Samagaltaj, 1200 m, 17.06.2001, leg. R. Yakovlev.
Fig. 6. Proclossiana eunomia, adult male specimens: upperside (right) and underside (left). a) P. e. acidalia, Russia, Buryatia, Tunkinski distr., Mondy vill., Khulugaisha Mt., 1800 m, 24-30.06.2016, leg. A. Kosarev; b) P. e. acidalia, Russia, Chelyabinsk reg., Katav-Ivanovsk env., 12.06.2007, leg. V. Zurilina; c) P. e. itelmena, Russia, Kamchatka, Esso vill., 600 m, 12.07.2003, ex coll. D. Goshko; d) P. e. riamia, Russia, Omsk reg., Krutinsky distr., 44 km NW Krutinka vill., 5 km SW Gulyai-Pole vill., 7.06.2016, leg. S. Knyazev; e) P. e. riamia, Russia, Novosibirsk reg., Chulym distr., near Kuznetsky vill., Kammeny Ryam, 19.06.2006, leg. V. Ivonin.
Fig. 7. Habitat of P. eunomia riamia. a) Novosibirsk reg., Gus'kovsky Ryam (photo by V. Ivonin); b) Omsk reg., "Orlovskoe zajmistche" Ryam, 5 km SW Gulyaj Pole village (photo by S. Knyazev)
Acknowledgements
The authors are grateful to Prof. Boris Kondratieff (Fort Colins) for language editing. We thank S. Knyazev (Omsk), V. Dubatolov, V. Ivonin, A. Kareev (Novosibirsk), V. Gorbach (Petrozavodsk), A. Kosarev (Irkutsk), I. Pljustch (Kiev) for providing samples of specimens, important for this study. The financial support for this study was provided by the grant No. 16-54-00118 Be^_a from the Russian Foundation for Basic Research and grant No. B16P-159 from Belorussian Foundation for Basic Research. The work was performed using equipment of the "Taxon" Research Resource Center (http://www.ckp-rf.ru/ckp/3038/?sphrase_id=8879024) of the Zoological Institute of the Russian Academy of Sciences (St. Petersburg).
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Citation:
Yakovlev, R. V., Shapoval, N. A., Kuftina, G. N., Kulak, A.V., Kovalev, S.V. (2018). Notes on the molecular taxonomy of the Proclossiana eunomia complex (Lepidoptera, Nymphalidae: Argynnini): analysis of DNA barcodes. Ukrainian Journal of Ecology, <9[1), 222-232. | (QE^^^MI Thi^ work is licensed under a Creative Commons Attribution 4.0. License
