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Parnassius nebrodensis: A threatened but neglected Apollo butterfly species from Southern Europe (Lepidoptera: Papilionidae)

Authors:
  • N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences
  • Institute of Environmental Problems of the North, Ural Branch of Russian Academy of Sciences

Abstract and Figures

Recent multi-locus phylogenetic studies repeatedly showed that what was thought to be the Clouded Apollo butterfly Parnassius mnemosyne (Linnaeus, 1758) represents a cryptic species complex. This complex contains at least three distant species-level phylogenetic lineages. Here, we compile a set of morphology- and DNA-based evidences supporting the distinctiveness of two species in this group, i.e. P. mnemosyne s. str. and P. nebrodensis Turati, 1907 stat. rev. These species can be distinguished from each other based on a combination of diagnostic characters in the male genitalia structure, wing scale patterns, and the forewing venation. The species status of P. nebrodensis is supported based on unique nucleotide substitutions in the mitochondrial (COI, ND1, and ND5) and nuclear (Wg and EF-1a) genes. P. nebrodensis is endemic to the Western Mediterranean Region. This species shares a disjunctive range through the Pyrenees, Western and Central Alps, Apennines, and the Nebrodi and Madonie mountains on Sicily. Altogether 38 nominal taxa initially described as P. mnemosyne subspecies are considered here to be junior synonyms of P. nebrodensis. At first glance, P. nebrodensis can be assessed as an endangered species due to its restricted distribution, narrow range of habitats, and ongoing population decline. Isolated populations of this species scattered through mountain ranges need special management and conservation efforts.
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Ecologica Montenegrina, 40, 2021, 140-163
https://zoobank.org/urn:lsid:zoobank.org:pub:6EDA9875-78A1-4D77-B840-42FF417CFA80
Parnassius nebrodensis: A threatened but neglected Apollo butterfly
species from Southern Europe (Lepidoptera: Papilionidae)
IVAN N. BOLOTOV1, MIKHAIL Y. GOFAROV1, VYACHESLAV V. GORBACH2,
YULIA S. KOLOSOVA1, ALISA A. ZHELUDKOVA1, ALEXANDER V. KONDAKOV1
& VITALY M. SPITSYN1,*
1N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences,
Northern Dvina Emb. 23, 163000 Arkhangelsk, Russia
2Petrozavodsk State University, Lenina Av. 20, 185035 Petrozavodsk, Russia
*Corresponding author: spitsyn-v-m-91993@yandex.ru
Received 22 January 2021 Accepted by V. Pešić: 22 March 2021 Published online 23 March 2021.
Abstract
Recent multi-locus phylogenetic studies repeatedly showed that what was thought to be the Clouded Apollo butterfly
Parnassius mnemosyne (Linnaeus, 1758) represents a cryptic species complex. This complex contains at least three
distant species-level phylogenetic lineages. Here, we compile a set of morphology- and DNA-based evidences
supporting the distinctiveness of two species in this group, i.e. P. mnemosyne s. str. and P. nebrodensis Turati, 1907
stat. rev. These species can be distinguished from each other based on a combination of diagnostic characters in the
male genitalia structure, wing scale patterns, and the forewing venation. The species status of P. nebrodensis is
supported based on unique nucleotide substitutions in the mitochondrial (COI, ND1, and ND5) and nuclear (Wg and EF-
1a) genes. P. nebrodensis is endemic to the Western Mediterranean Region. This species shares a disjunctive range
through the Pyrenees, Western and Central Alps, Apennines, and the Nebrodi and Madonie mountains on Sicily.
Altogether 38 nominal taxa initially described as P. mnemosyne subspecies are considered here to be junior synonyms
of P. nebrodensis. At first glance, P. nebrodensis can be assessed as an endangered species due to its restricted
distribution, narrow range of habitats, and ongoing population decline. Isolated populations of this species scattered
through mountain ranges need special management and conservation efforts.
Key words: Apollo butterflies, cryptic species, phylogeny, Western Mediterranean Region, endangered species,
conservation.
Introduction
The Clouded Apollo Parnassius mnemosyne (Linnaeus, 1758) belongs to the subgenus Driopa Korshunov,
1988 [=the Mnemosyne species group] (Müller 1973; Ackery 1975; Weiss 1999; Korshunov 2002; Omoto et
al. 2004, 2009). All Driopa butterflies use Corydalis DC. and Dicentra Bernh. (Papaveraceae) taxa as larval
host plants (Korshunov 2002; Michel et al. 2008; Condamine 2018). Most species in this group are known to
Ecologica Montenegrina 40: 140-163 (2021)
This journal is available online at: www.biotaxa.org/em
http://dx.doi.org/10.37828/em.2021.40.13
BOLOTOV ET AL.
Ecologica Montenegrina, 40, 2021, 140-163 141
occur in Northern Asia, Central Asia, and North America (Weiss 1999; Korshunov 2002). In contrast, the
Clouded Apollo was thought to be a species widely distributed throughout Europe, the Urals, the Caucasus,
eastern Kazakhstan, and the Middle East, with a few records from Western Siberia (Weiss 1999; Korshunov
2002; Kudrna et al. 2011; Wiemers et al. 2018). The northernmost populations of this butterfly were
discovered in karst boreal landscapes of Northern European Russia between 65° and 66°N (Bolotov et al.
2013), while the southern boundary of the species’ range is situated in the Western Mediterranean Region
and the Middle East (Weiss 1999; Gratton et al. 2008; Kudrna et al. 2011). The Clouded Apollo was
considered an endangered species in Europe (Van Swaay and Warren 1999; Van Swaay et al. 2010, 2012). It
was shown that its population decline is largely associated with the cessation of traditional management
practices such as grazing and mowing at semi-natural grasslands and coppicing in woodlands (Väisänen and
Somerma 1985; Luoto et al. 2001; Descimon 2006; Van Swaay et al. 2012).
A variety of P. mnemosyne morphological forms was described as intraspecific taxa from various
parts of its broad range, including approximately 200 subspecies that were introduced based on minute
differences in wing markings pattern and size (Weiss 1999). Available taxonomic reviews of intraspecific
names linked to P. mnemosyne were based on morphological features alone (Leraut 1997; Weiss 1999; Mérit
and Mérit 2006). Weiss (1999) subdivided all the subspecies into two categories as follows: (1) ‘strong’
subspecies (i.e. the taxa that can clearly be distinguished from others using morphological features), and (2)
‘weak’ subspecies (i.e. those displaying less clear diagnostic features). It was shown that the wing markings
pattern in P. mnemosyne and related species is highly variable (Eisner 1968, 1971, 1974, 1976, 1978; Müller
1973; Weiss 1999). Ackery (1975) noted that the markings pattern could be applied as diagnostic features for
the species groups within the genus Parnassius Latreille, 1804 but cannot be used to separate species- and
subspecies-level taxa. It was found that the subspecies of P. mnemosyne are poorly correspond to the
population genetic structure (Descimon 1995) and to phylogeographic and phylogenetic patterns (Gratton
2006; Gratton et al. 2008; Michel et al. 2008). For example, the subspecies from Northern Europe are
generally less marked compared with those from southern regions (Weiss 1999). A long-term intraspecific
morphological variability of P. mnemosyne within a single locality can be linked to climatic fluctuations
(Eisner 1974).
A growing body of DNA-based research revealed that the Clouded Apollo shares a deep
phylogeographic structure, with three highly divergent lineages that could represent cryptic species (Gratton
2006; Gratton et al. 2008; Michel et al. 2008). In particular, the samples of P. mnemosyne collected in the
Western Mediterranean Region and Southern Anatolia and Iran were found to be distant from those sampled
through Northern and Eastern Europe and Central Asia (Figs 1-2). Recently, the existence of these species-
level clades was confirmed using multi-locus time-calibrated phylogenies and species delimitation modeling
(Condamine 2018; Condamine et al. 2018). It was shown that the Western Mediterranean lineage (=P.
mnemosyne sp.3 sensu Condamine, 2018) was isolated from other populations of P. mnemosyne since the
mid-Pliocene (Condamine 2018). Although the evaluation of cryptic species is a task of great importance to
resolve a broad array of scientific, conservation, and management issues (Bickford et al. 2007; Dincă et al.
2011, 2015; Platania et al. 2020a), these Clouded Apollo lineages are yet to be studied by means of
morphological and taxonomic approaches.
This paper (1) presents a taxonomic evaluation and morphological diagnosis for the Western
Mediterranean lineage of Parnassius mnemosyne species complex (=P. nebrodensis Turati, 1907 stat. rev.;
=P. mnemosyne sp.3 sensu Condamine, 2018); (2) illustrates the distribution of this species; and (3) revises a
number of P. mnemosyne subspecies, the type localities of which are situated within the range of P.
nebrodensis.
Materials and methods
Data sampling, and DNA amplification and sequencing
Samples of Parnassius species were studied in the collection of the Russian Museum of Biodiversity
Hotspots, N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian
Academy of Sciences, Arkhangelsk, Russia. The new mitochondrial cytochrome c oxidase subunit I (COI)
gene sequences were generated from a single leg of 48 specimens (Table 1) using the approaches of
Konopinski (2008) and Gratton (2006). The PCR mix contained approximately 200 ng of genomic DNA, 10
pmol of each primer, 200 μmol of each dNTP, 2.5 μl of PCR buffer (with 20 mmol MgCl2), 0.8 units Taq
PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE
142
DNA polymerase (SibEnzyme Ltd., Russia), and H2O was added for a final volume of 25 μl. The following
PCR conditions were used in the amplifications: 95°C (4 min), 32 cycles of 95°C (45 sec), 52°C (40 sec),
72°C (50 sec) and a final extension at 72°C (5 min). Additionally, 76 COI sequences were obtained from
NCBI’s GenBank, including two sequences of Parnassius simo Gray, 1853, a close relative of the subgenus
Driopa (see Omoto et al. 2004, 2009; Condamine et al. 2018), as outgroup (Table 1). The nuclear wingless
(Wg) gene fragment was amplified and sequenced from 10 specimens, including 3 specimens of Parnassius
nebrodensis and 4 specimens of P. mnemosyne s. str. (Table 2) The primers Lepwg1 and Lepwg2 were
applied for amplification (Brower and DeSalle 1998). The following PCR conditions were used in the
amplifications: 95 °C (5 min), 36 cycles of 95 °C (50 sec), 50 °C (50 sec), 72 °C (50 sec) and a final
extension at 72 °C (5 min). The resulting COI and Wg sequences were checked manually using Bioedit 7.1.9
(Hall 1999).
Figure 1. Ranges of taxa within the Parnassius mnemosyne species complex. (1) Range of P. nebrodensis stat. rev.
(data: Gratton 2006; Gratton et al. 2008; Michel et al. 2008; Todisco et al. 2010; Condamine et al. 2018; Litman et al.
2018; Dapporto et al. 2019; this study). (2) Approximate range of P. sp. ‘Middle East’ (data: Gratton 2006; Gratton et
al. 2008). (3) Range of P. mnemosyne s. str. (data: Weiss 1999; Gratton et al. 2008; Kudrna et al. 2011; Bolotov et al.
2013): (3a) confirmed range, and (3b) uncertain distribution. (4) Approximate boundary of P. mnemosyne s. str. and P.
sp. ‘Middle East’ ranges (data: Weiss 1999; Gratton 2006; Gratton et al. 2008; Kudrna et al. 2011; Bolotov et al. 2013).
(5) Type locality of P. nebrodensis stat. rev. in Italy [Sicily: “monti Nebrodi”] (Turati 1907). (6) Type locality of P.
mnemosyne s. str. in Finland (Honey and Scoble 2001, 2001a). (Map: Mikhail Y. Gofarov).
Morphological analyses
The dissection of the genitalia was performed using the standard methods for Lepidoptera (Bolotov et al.
2018). Each abdomen was macerated in a heated 10% KOH solution for 30 minutes. First, images of ventral
and lateral views of unmounted genitalia were obtained. Second, the genitalia of each specimen were
mounted on a glass slide with Histofluid® (Paul Marienfeld GmbH & Co., Germany), and its dorsal view was
photographed. Images of genitalia and wing scales were taken using a Leica M165C stereo microscope
BOLOTOV ET AL.
Ecologica Montenegrina, 40, 2021, 140-163 143
(Leica Microsystems GmbH, Germany). Images of the genitalia structures were taken with an AXIO
Zoom.V16 research microscope (Carl Zeiss, Germany). Images of specimens were taken with a Canon EOS
500D digital camera (Canon Inc., Tokyo, Japan). An image of the P. mnemosyne lectotype was obtained
from the Linnaeus’s Butterfly Type Specimens database of the Natural History Museum, London, UK
(Honey and Scoble 2001, 2001a).
Table 1. List of the COI sequences used in this study.
Species
Catalogue no.
Region
COI acc.
no.
In-group:
P. nebrodensis Turati, 1907 stat. rev.
RMBH G0327
France: Pyrenees, foothill
of Mt. Neouville
KF444468
P. nebrodensis Turati, 1907 stat. rev.
RMBH G0328
France: Pyrenees, foothill
of Mt. Neouville
MW460988
P. nebrodensis Turati, 1907 stat. rev.
RMBH G0329
France: Pyrenees, foothill
of Mt. Neouville
MW460989
P. nebrodensis Turati, 1907 stat. rev.
RMBH G0330
France: Pyrenees, foothill
of Mt. Neouville
MW460990
P. nebrodensis Turati, 1907 stat. rev.
RMBH G0331
France: Pyrenees, foothill
of Mt. Neouville
MW460991
P. nebrodensis Turati, 1907 stat. rev.
RMBH G0534
Spain: eastern Pyrenees
KX130690
P. nebrodensis Turati, 1907 stat. rev.
RVcoll.09-V684
Spain: Pyrenees,
Cerdanya
JF848003
P. nebrodensis Turati, 1907 stat. rev.
RVcoll.09-T076
Spain: Pyrenees, Lleida,
Val d'Aran
JF847984
P. nebrodensis Turati, 1907 stat. rev.
RVcoll.07-C024
France: Pyrenees, foothill
of Pic Petit de Segre
GU669633
P. nebrodensis Turati, 1907 stat. rev.
RVcoll.13-T947
Italy: Chieti, La
Maielletta
MN145197
P. nebrodensis Turati, 1907 stat. rev.
LEP-SS-00563
Italy: Calabria
MN144406
P. nebrodensis Turati, 1907 stat. rev.
11-I228
Italy: Calabria
MN144237
P. nebrodensis Turati, 1907 stat. rev.
RVcoll.11-I226
Italy: Calabria
MN143408
P. nebrodensis Turati, 1907 stat. rev.
RVcoll.11-I225
Italy: Calabria
MN141405
P. nebrodensis Turati, 1907 stat. rev.
14-V321
Italy: Avio, Trente
MN141943
P. nebrodensis Turati, 1907 stat. rev.
LEP-SS-00001
Italy: Calabria
MN143318
P. nebrodensis Turati, 1907 stat. rev.
15-C862
Italy: Calabria
MN143005
P. nebrodensis Turati, 1907 stat. rev.
LEP-SS-00002
Italy: Calabria
MN142317
P. nebrodensis Turati, 1907 stat. rev.
15-C867
Italy: Calabria
MN141765
P. nebrodensis Turati, 1907 stat. rev.
RVcoll.11-H952
Italy: Sicily
MN145369
P. nebrodensis Turati, 1907 stat. rev.
11-H953
Italy: Sicily
MN140069
P. nebrodensis Turati, 1907 stat. rev.
RVcoll.11-H737
Italy: Sicily
MN142921
P. nebrodensis Turati, 1907 stat. rev.
11-H738
Italy: Sicily
MN144890
P. nebrodensis Turati, 1907 stat. rev.
Pmne-ITNEB01
Italy: Nebrodi Mts., Sicily
GU947642
P. nebrodensis Turati, 1907 stat. rev.
GBIFCH-BOL
LEPAA_0273
Switzerland: Valais,
Martigny
MK186621
P. nebrodensis Turati, 1907 stat. rev.
GBIFCH-BOL
LEPAA_0229
Switzerland: Ticino,
Airolo
MK186623
P. nebrodensis Turati, 1907 stat. rev.
GBIFCH-BOL
LEPAA_0743
Switzerland: Graubunden,
Panix
MK186622
P. nebrodensis Turati, 1907 stat. rev.
Ch765
Switzerland
EU092983
P. nebrodensis Turati, 1907 stat. rev.
Fr525
France
EU092985
P. nebrodensis Turati, 1907 stat. rev.
Fr527
France
EU092986
P. nebrodensis Turati, 1907 stat. rev.
Fr528
France
EU092987
PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE
144
Species
Catalogue no.
Region
COI acc.
no.
P. nebrodensis Turati, 1907 stat. rev.
It639
Italy
EU092988
P. nebrodensis Turati, 1907 stat. rev.
Fr908
France
EU092989
P. nebrodensis Turati, 1907 stat. rev.
Fr911
France
EU092990
P. nebrodensis Turati, 1907 stat. rev.
Fr913
France
EU092991
P. nebrodensis Turati, 1907 stat. rev.
Sp916
Spain
EU092992
P. nebrodensis Turati, 1907 stat. rev.
Sp917
Spain
EU092993
P. nebrodensis Turati, 1907 stat. rev.
Sp921
Spain
EU092994
P. nebrodensis Turati, 1907 stat. rev.
Sp923
Spain
EU092995
P. nebrodensis Turati, 1907 stat. rev.
Sp924
Spain
EU092996
P. nebrodensis Turati, 1907 stat. rev.
It878
Italy
EU093015
P. nebrodensis Turati, 1907 stat. rev.
Ch892
Switzerland
EU093017
P. mnemosyne (Linnaeus, 1758) s.
str.
RMBH G0004
Belarus: Grodno Region
KF444471
P. mnemosyne (Linnaeus, 1758) s.
str.
RMBH G0221
Russia: Arkhangelsk
Region
KF444472
P. mnemosyne (Linnaeus, 1758) s.
str.
RMBH G0286
Russia: Arkhangelsk
Region
KF444473
P. mnemosyne (Linnaeus, 1758) s.
str.
RMBH G0280
Moldova
KF444470
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-532-4
Uzbekistan
KX130692
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-532-2
Uzbekistan
KX130691
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-332
Slovakia
KX130689
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-326
Russia: Middle Urals
KX130688
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-310
Russia: Nizhny Novgorod
Region
KX130687
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-321
Russia: Lipetsk Region
KX130686
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-275
Russia: Karelia
KX130685
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-274
Russia: Karelia
KX130684
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-271
Russia: Karelia
KX130683
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-266
Russia: Karelia
KX130682
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-269
Russia: Karelia
KX130681
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-537-2
Russia: Krasnodar Region
KX130680
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-537-1
Russia: Krasnodar Region
KX130679
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-285
Russia: Arkhangelsk
Region
KX130678
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-282
Moldova
KX130677
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-279
Moldova
KX130676
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-540
Kyrgyzstan
KX130675
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-653
Kyrgyzstan
KX130674
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-535
Iran
KX130673
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-538
Greece
KX130672
BOLOTOV ET AL.
Ecologica Montenegrina, 40, 2021, 140-163 145
Species
Catalogue no.
Region
COI acc.
no.
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-646
Armenia
KX130671
P. mnemosyne (Linnaeus, 1758) s.
str.
IEPN-644
Armenia
KX130670
P. mnemosyne (Linnaeus, 1758) s.
str.
Hu630
Hungary
EU092970
P. mnemosyne (Linnaeus, 1758) s.
str.
BH721
Bosnia
EU092972
P. mnemosyne (Linnaeus, 1758) s.
str.
At606
Austria
EU092977
P. mnemosyne (Linnaeus, 1758) s.
str.
At618
Austria
EU092978
P. mnemosyne (Linnaeus, 1758) s.
str.
At626
Austria
EU092981
P. mnemosyne (Linnaeus, 1758) s.
str.
Pmne-ITGAR05
Italy
EU836659
P. mnemosyne (Linnaeus, 1758) s.
str.
Pmne-GRKEL03
Greece
EU836665
P. mnemosyne (Linnaeus, 1758) s.
str.
Pmne-GRTAY01
Greece
EU836669
P. mnemosyne (Linnaeus, 1758) s.
str.
Pmne-LTKRE03
Lithuania
EU836676
P. mnemosyne (Linnaeus, 1758) s.
str.
Pmne-RUSAR01
Central European Russia
EU836682
P. sp. Middle East
W335
Turkey
AM231418
P. sp. Middle East
W280
Iran
AM231420
P. sp. Middle East
W311
Iran
AM231419
P. sp. Middle East
Tu1000
Turkey
EU093005
P. phoebus (Fabricius, 1793) [=P.
ariadne (Lederer, 1853)]
RMBH G0525
Russia: Altai Mts.
KX130693
P. phoebus (Fabricius, 1793) [=P.
ariadne (Lederer, 1853)]
N/A
N/A
EU093023
P. phoebus (Fabricius, 1793) [=P.
ariadne (Lederer, 1853)]
AC4-14
N/A
EF473794
P. phoebus (Fabricius, 1793) [=P.
ariadne (Lederer, 1853)]
W237
Russia: Altai Mts.
AM231429
P. phoebus (Fabricius, 1793) [=P.
ariadne (Lederer, 1853)]
Pari-KZTRB02
Kazakhstan: Tarbagatai
Mts.
GU947640
P. hoenei Schweitzer, 1912
RMBH G0003.1
Russia: Sakhalin Island
KF444469
P. hoenei Schweitzer, 1912
RMBH G0003.3
Russia: Sakhalin Island
MW460983
P. hoenei Schweitzer, 1912
RMBH G0003.4
Russia: Sakhalin Island
MW460984
P. hoenei Schweitzer, 1912
RMBH G0003.5
Russia: Sakhalin Island
MW460985
P. hoenei Schweitzer, 1912
RMBH G0003.6
Russia: Sakhalin Island
MW460986
P. hoenei Schweitzer, 1912
RMBH G0003.7
Russia: Sakhalin Island
MW460987
P. hoenei Schweitzer, 1912
RMBH G0629
Russia: Sakhalin Island
MW460995
P. hoenei Schweitzer, 1912
RMBH G0630
Russia: Sakhalin Island
MW460996
P. hoenei Schweitzer, 1912
RMBH G0631
Russia: Sakhalin Island
MW460997
P. hoenei Schweitzer, 1912
RMBH G0632
Russia: Sakhalin Island
MW460998
P. hoenei Schweitzer, 1912
RMBH G0633
Russia: Sakhalin Island
MW460999
P. hoenei Schweitzer, 1912
RMBH G0626
Russia: Kunashir Island
MW460992
P. hoenei Schweitzer, 1912
RMBH G0627
Russia: Kunashir Island
MW460993
P. hoenei Schweitzer, 1912
RMBH G0628
Russia: Kunashir Island
MW460994
P. hoenei Schweitzer, 1912
W218
Japan: Hokkaido
AM231427
P. hoenei Schweitzer, 1912
AC20-16
N/A
EF473801
PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE
146
Species
Catalogue no.
Region
COI acc.
no.
P. hoenei Schweitzer, 1912
N/A
N/A
EU093019
P. stubbendorfii Menetries, 1849
RMBH G0634
Russia: Primorye
MW461000
P. stubbendorfii Menetries, 1849
AC5-2
South Korea
EF473800
P. stubbendorfii Menetries, 1849
N/A
South Korea
GU372549
P. stubbendorfii Menetries, 1849
SWCN09-5002
South Korea
GU696036
P. stubbendorfii Menetries, 1849
005-LOWA-815
Russia: Altai
FJ663912
P. stubbendorfii Menetries, 1849
2005-LOWA-153
Russia: Altai
FJ663914
P. eversmanni Ménétriés, [1850]
AC23-68
N/A
EF473797
P. eversmanni Ménétriés, [1850]
AC1-14
N/A
EF473796
P. eversmanni Ménétriés, [1850]
KWP_Ento_3722
2
USA: Alaska
KU875779
P. eversmanni Ménétriés, [1850]
2005-LOWA-107
Russia: Altai
FJ663893
P. eversmanni Ménétriés, [1850]
W251
Russia: Amur Region
AM231430
P. orleans Oberthür, 1890
W212
China
AM231433
P. orleans Oberthür, 1890
AC10-5
N/A
EF473802
P. orleans Oberthür, 1890
GGS01
China
MH518694
P. orleans Oberthür, 1890
PO-2011-59CI
China
JQ922045
P. nordmanni Ménétries in
Simashko, 1850
AC20-5
N/A
EF473799
P. nordmanni Ménétries in
Simashko, 1850
W226
Russia: Krasnodar Region
AM231432
P. nordmanni Ménétries in
Simashko, 1850
Pnor-RUKRS01
Russia: Krasnodar Region
GU947641
P. glacialis Butler, 1866
TS28
China
MH518684
P. glacialis Butler, 1866
SS26
China
MH518656
P. glacialis Butler, 1866
NTS01
China
MH518546
P. glacialis Butler, 1866
LJS20
China
MH518454
P. glacialis Butler, 1866
NTS29
China
MH518574
P. clodius Ménétriés, 1857
N/A
Western North America
MK947428
P. clodius Ménétriés, 1857
N/A
Western North America
MK947427
P. clodius Ménétriés, 1857
N/A
Western North America
MK947426
P. clodius Ménétriés, 1857
N/A
Western North America
MK947425
P. clodius Ménétriés, 1857
AC4-5
N/A
EF473795
Outgroup:
P. simo Gray, [1853]
AC4-12
N/A
EF473815
P. simo Gray, [1853]
AC13-4
N/A
EF473813
N/A not available. Parnassius ariadne (Lederer, 1853) is treated here as a synonym of P. phoebus (Fabricius, 1793) (see Hanus and
Theye 2010 for explanation).
Phylogenetic analyses and species delimitation
The alignments of the COI and Wg sequence data sets were performed using the MUSCLE algorithm
implemented in MEGA7 (Kumar et al. 2016). For the phylogenetic analyses, we used a dataset with 116
unique haplotypes selected from 135 COI sequences (Table 1) with an online fasta sequence toolbox (FaBox
v. 1.5; Villesen 2007). The maximum likelihood phylogenetic analyses were carried out with IQ-TREE v.
1.6.12 (Nguyen et al. 2015) through an online web server (http://iqtree.cibiv.univie.ac.at) (Trifinopoulos et
al. 2016). The best-fit evolutionary model was selected for each codon separately using Model Finder based
BOLOTOV ET AL.
Ecologica Montenegrina, 40, 2021, 140-163 147
on Bayesian Information Criterion (BIC) (Kalyaanamoorthy et al. 2017). Bootstrap support (BS) values were
estimated by means of an ultrafast bootstrap (UFBoot2) approach (Hoang et al. 2018). The Bayesian
phylogeny was calculated with MrBayes v. 3.2.7a (Ronquist et al. 2012) in the San Diego Supercomputer
Center, USA through CIPRES Science Gateway (Miller et al. 2010). The MrBayes analyses were carried out
in two separate runs of 20,000,000 generations, each with four Markov chains, one cold and three heated
(temperature set at 0.1). Trees were sampled every 1000th generation. The first 15% of trees were discarded
as burn-in, and the majority rule consensus phylogeny was calculated from the remaining trees. The GTR+G
substitution model was applied to each codon position.
The consensus COI phylogeny inferred from the W-IQ-TREE analyses was used as an input tree for
the mPTP species-delimitation model of Kapli et al. (2017) via online mPTP server (http://mptp.h-its.org).
The mPTP approach seems to be the most accurate tool to delineate species-level clades within large
phylogenies (Kapli et al. 2017). This model returns more general Molecular Operational Taxonomic Units
(MOTUs) compared with other species-delimitation methods (Kapli et al. 2017; Aksenova et al. 2019).
Uncorrected p-distances between COI sequences were calculated using MEGA7 software (Kumar et al.
2016).
Table 2. Nucleotide substitutions between Parnassius species (subgenus Driopa) based on the nuclear Wg gene
fragment.
Species
Region
GenBank
acc. No.
Nucleotide position
20
33
47
50
56
77
120
146
161
173
218
230
233
275
290
329
338
*P. nebrodensis
stat. rev.
France:
Pyrenees
MW468409
A/T
C
C/T
T
A
T
A
G
G
A
C
C
C
G/T
A/C
C
T
*P. nebrodensis
stat. rev.
France:
Pyrenees
MW468414
A/T
C
C/T
T
A
T
A
G
G
A
C
C
C
G/T
A/C
C
T
*P. nebrodensis
stat. rev.
France:
Pyrenees
MW468410
A/T
C
C/T
T
A
T
A
G
G
A
C
C
C
G/T
A/C
C
T
*P. mnemosyne s.
str.
Belarus:
Grodno
MW468412
A
C
T
T
A
T
A
G
G
A
C
C
C
G
C
C
T
*P. mnemosyne s.
str.
Belarus:
Grodno
MW468413
A
C
T
T
A
T
A
G
G
A
C
C
C
G
C
C
T
*P. mnemosyne s.
str.
Russia:
Arkhangels
k Region
MW468407
A
C
T
T
A
T
C
G
G
A
C
T
C
G
C
C
T
*P. mnemosyne s.
str.
Russia:
Arkhangels
k Region
MW468408
A
C
T
T
A
T
C
G
G
A
C
T
C
G
C
C
T
*P. phoebus
Russia:
Altai Mts.
MW468411
A
T
T
T
A
T
A
A
G
A
T
C
C
G
C
T
C
*P. hoenei
Russia:
Sakhalin
Isl.
MW468405
A
C
T
T
G
T
A
G
G
G
C
C
A/C
G
C
C
C
*P. hoenei
Russia:
Sakhalin
Isl.
MW468406
A
C
T
T
G
T
A
G
G
G
C
C
A/C
G
C
C
C
P. clodius
USA
FJ756879
A
C
T
C
G
C
A
G
A
A
C
C
C
G
C
C
C
*New sequences generated under this study. Parnassius ariadne (Lederer, 1853) is treated here as a synonym of P. phoebus
(Fabricius, 1793) (see Hanus and Theye 2010 for explanation).
Range mapping
The distribution maps of Parnassius mnemosyne species complex and P. nebrodensis stat. rev. were
compiled using ESRI ArcGIS 10 based on published sources (Weiss 1999; Gratton 2006; Gratton et al. 2008;
Michel et al. 2008; Todisco et al. 2010; Kudrna et al. 2011; Bolotov et al. 2013; Condamine et al. 2018;
Litman et al. 2018; Dapporto et al. 2019) and our data. The type localities of subspecies were digitized based
on original descriptions and general works (Eisner 1968, 1971, 1974, 1978; Ackery 1975; Weiss 1999).
PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE
148
Figure 2. Range of Parnassius nebrodensis Turati, 1907 stat. rev. and a narrow contact zone between allopatric ranges
of this species and P. mnemosyne (Linnaeus, 1758) s. str. based on DNA sequence data. (1) Range of P. nebrodensis
(data: Gratton 2006; Gratton et al. 2008; Michel et al. 2008; Todisco et al. 2010; Condamine et al. 2018; Litman et al.
2018; Dapporto et al. 2019; this study). (2) Occurrences of P. nebrodensis: (2a) Pyrenees (sample used for
morphological and molecular analyses in this study), and (2b) other areas (data: Gratton 2006; Gratton et al. 2008). (3)
Type locality of P. nebrodensis in Italy [Sicily: “monti Nebrodi”] (Turati 1907). (4) Type localities of nominal taxa that
were described as P. mnemosyne subspecies but are being considered as synonyms of P. nebrodensis under this study
(see Table 5 for detail). (5) A southern fragment of P. mnemosyne s. str. range adjoining the contact zone with P.
nebrodensis (data: Weiss 1999; Gratton et al. 2008; Kudrna et al. 2011): (5a) confirmed range, and (5b) uncertain
distribution. (6) A contact zone between P. nebrodensis and P. mnemosyne s. str. ranges. (Map: Mikhail Y. Gofarov).
Results and discussion
Phylogenetic divergence between Parnassius (Driopa) taxa
Both COI maximum likelihood and Bayesian phylogenies returned similar topology with 11 MOTUs: P.
nebrodensis Turati, 1907 stat. rev., P. mnemosyne (Linnaeus, 1758) s. str., P. sp. ‘Middle East’, P. phoebus
(Fabricius, 1793) [=P. ariadne (Lederer, 1853)], P. stubbendorfii Menetries, 1849, P. hoenei Schweitzer,
1912, P. glacialis Butler, 1866, P. eversmanni Ménétriés, [1850], P. orleans Oberthür, 1890, P. nordmanni
Ménétries in Simashko, 1850, and P. clodius Ménétriés, 1857 (Figs 1-3). These MOTUs were supported by
mPTP species-delimitation model of Kapli et al. (2017). Interestingly, P. phoebus was found to be more
closely related to P. mnemosyne s. str. compared with P. nebrodensis (Fig. 3), as it was shown previously
(Condamine et al. 2018).
Mean uncorrected COI p-distances among the delineated MOTUs ranged from 2.57% (between P.
clodius and P. eversmanni) to 8.47% (between P. orleans and P. glacialis) (Table 3). In almost all the
MOTUs, maximum intraspecific distances were lower than minimum interspecific sequence divergence,
except for Parnassius sp. ‘Middle East’ (2.71% vs 3.80%, respectively) (Table 4). P. mnemosyne s. str. and
P. nebrodensis were separated by a 3.94% mean distance that is larger than that between well-defined
BOLOTOV ET AL.
Ecologica Montenegrina, 40, 2021, 140-163 149
species such as P. clodius vs P. eversmanni (2.57%), and P. mnemosyne s. str. vs P. phoebus (3.84%) (Table
3). Furthermore, our comparison of the Wg gene sequences revealed that all the studied species share 2-4
unique nucleotide substitutions, although P. nebrodensis has double peaks at the four informative sites
(Table 2).
In summary, our novel results align with earlier studies that underscored P. nebrodensis [=P.
mnemosyne sp.3] as a separate cryptic species based on the COI, NADH dehydrogenase 1 (ND1), and NADH
dehydrogenase 5 (ND5) gene fragments (Gratton 2006; Michel et al. 2008; Condamine et al. 2018).
Furthermore, Gratton (2006) showed that the nuclear elongation factor-1 alpha (EF-1a) gene indicates the
complete isolation of the Western Mediterranean haplogroup (i.e. P. nebrodensis) and P. mnemosyne s. str.
and that any evidence of nuclear admixture between these taxa does not occur.
Table 3. Mean uncorrected p-distances (%) between Parnassius species (subgenus Driopa) based on the mitochondrial
COI gene fragment.
Species
ParNeb
ParMne
ParMEs
ParPho
ParStu
ParHoe
ParGla
ParEve
ParOrl
ParNor
P. nebrodensis
stat. rev.
[ParNeb]
P. mnemosyne s.
str. [ParMne]
3.94
P. sp. Middle
East [ParMEs]
4.10
2.71
P. phoebus
[ParPho]
4.38
3.84
4.32
P. stubbendorfii
[ParStu]
5.78
6.36
6.51
6.44
P. hoenei
[ParHoe]
4.83
4.78
5.19
4.72
3.60
P. glacialis
[ParGla]
8.46
8.33
8.08
8.41
5.63
6.78
P. eversmanni
[ParEve]
5.05
4.98
5.68
4.96
4.50
4.27
6.92
P. orleans
[ParOrl]
6.92
7.09
7.59
6.68
6.34
5.20
8.47
4.67
P. nordmanni
[ParNor]
6.92
6.37
6.85
6.40
6.34
5.86
8.39
5.65
6.85
P. clodius
[ParClo]
4.69
4.54
5.04
4.61
4.82
4.06
6.89
2.57
5.06
5.45
Parnassius ariadne (Lederer, 1853) is treated here as a synonym of P. phoebus (Fabricius, 1793) (see Hanus and Theye 2010 for
explanation).
Differential diagnosis of Parnassius nebrodensis
P. nebrodensis and P. mnemosyne s. str. cannot clearly be separated based on markings pattern alone,
although the first species is usually darker, with stronger black markings on the wings, and a broader black
spraying along veins distally (Fig. 4). However, several morphological features, outlined below, can help to
distinguish these taxa.
(1) Forewing venation (Fig. 5). P. nebrodensis shares an acute angle between veins R4 and R5 (ca.
30-50º in both sexes), while this angle is more obtuse in P. mnemosyne s. str. (ca. 60-70º in both sexes). This
difference is caused by the degree of proximal bending of the vein R4: weak in P. nebrodensis vs strong in P.
mnemosyne s. str. (see Fig. 5). Additionally, the endpoints of veins between R3 and Cu2 are shifted to the
forewing apex in both sexes of P. nebrodensis compared with P. mnemosyne. The veins R3 and R4 in P.
nebrodensis male are almost parallel, while in P. mnemosyne s. str. male vein R4 is slightly curved, and is
lopsided to the forewing tornus (see Fig. 5). Finally, the female’s forewing cell of P. nebrodensis shares an
elongate median veinlet, crossing the discocellular veinlet, prolonging to the postdical area, and finishing at
the middle of the vein M1. This median veinlet is clearly distinct in the forewing markings pattern (Figs. 5
and 6B). Contrastingly, in P. mnemosyne s. str. female this median veinlet is short and indistinct (Figs. 5, 6C,
and 6D).
PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE
150
Figure 3. Maximum likelihood phylogeny of Parnassius (subgenus Driopa) taxa based on the COI haplotype
sequences (Table 1). The red stars indicate the species-level clades supported by mPTP species-delimitation model. The
name of each sequence contains the following information: GenBank accession number | sampling region. An asterisk
indicates Parnassius nebrodensis sequence from the type locality (Nebrodi, Sicily). The black numbers near branches
indicate bootstrap support values of IQ-TREE/BPP of MrBayes. Outgroup is not shown.
BOLOTOV ET AL.
Ecologica Montenegrina, 40, 2021, 140-163 151
(2) Forewing scale patterns (Fig. 6). In P. nebrodensis female, black diamond-shaped scales in the
discocellular black spot are similar by size and shape to those in the blackish postdiscal band (Fig. 6B). In P.
nebrodensis male, similar pattern occurs in the black postdiscal spot (if present) and in the space between
veins Sc and R2 (Fig. 6E-F). Contrastingly, black scales at the blackish postdiscal band in P. mnemosyne s.
str. female, and those at the black postdiscal spot and space between veins Sc and R2 in this species’ male are
considerably smaller, with elongate, piliform shape (Fig. 6C-D, 6G-H).
(3) Male genitalia structure (Fig. 7). P. nebrodensis shares a smaller and weakly sclerotized uncus;
the uncus arms are triangular and straight, with narrow and sharp apex and a broad base (Fig. 7A3, 7A5,
7B1, and 7B3). Apical process of the P. nebrodensis valva is rounded at the end (Fig. 7A6 and 7B4).
Aedeagus is slightly shorter (1.4 times longer than the valva’s length) (Fig. 7A1, 7A2, 7A4, and 7B2) than
that in P. mnemosyne s. str. In contrast, P. mnemosyne s. str. shares a larger and strongly sclerotized uncus;
the uncus arms are narrower at the base, with broader and thicker apex (Fig. 7C3, 7C5, 7D1, and 7D3).
Apical process of the valva is flattened at the end (Fig. 7C6 and 7D4). Aedeagus is slightly longer (1.5 times
longer than the valva’s length) (Fig. 7C1, 7C2, 7C4, and 7D2) than that in P. nebrodensis.
Table 4. Inter- and intraspecific genetic divergence of Parnassius species (subgenus Driopa) based on the
mitochondrial COI gene fragment.
Species
Number of
sequences
Minimum
interspecific
sequence
divergence (%)
Maximum
intraspecific
sequence
divergence (%)
Mean
intraspecific
sequence
divergence (%)
P. nebrodensis stat. rev.
42
3.94
2.22
0.98
P. mnemosyne s. str.
36
2.71
1.86
0.60
P. sp. Middle East
4
2.71
3.80
2.29
P. phoebus [=P. ariadne]
5
3.84
0.53
0.25
P. stubbendorfii
6
3.60
0.88
0.63
P. hoenei
17
3.60
0.91
0.37
P. glacialis
5
5.63
0.77
0.44
P. eversmanni
5
2.57
0.48
0.20
P. orleans
4
4.67
0.48
0.49
P. nordmanni
3
5.45
0.00
0.00
P. clodius
5
2.57
1.78
1.19
Parnassius ariadne (Lederer, 1853) is treated here as a synonym of P. phoebus (Fabricius, 1793) (see Hanus and Theye 2010 for
explanation).
A brief morphological re-description of Parnassius nebrodensis
Male external morphology and markings (Figs 4A, 5A, 6E-F). Forewing length is 2831 mm. Antenna dark-
brown, club black, fusiform, with narrower terminal segments (apiculus). Eye brown, surrounded by greyish-
black scales. Frons and top of the head with mixed grey and black hairs. Palpus with mixed grey and black
hairs on each segment. Thorax black with long yellowish-grey hairs. Legs black with grey hairs. Abdomen
black with greyish-white hairs dorsally, and with yellowish-grey hairs ventrally. Upperside of the forewing
with milk-white ground color and black veins. The base of the forewing black. Cell with two rectangular (or
oval) black spots. Apical and subapical areas darkened. Black diamond-shaped scales in the black spot at the
end of the cell are similar in size and shape to the scales in the black postdiscal spot (if present) and in the
space between veins Sc and R2. Hindwing with the same ground color and black veins as those on the
forewing; basal area and dorsum black, with dense greyish-black hairs. Unclear black spot in the basal part
of the space between veins M1 and M2. The distal ends of veins usually marked with black. Underside of
both wings with the same markings pattern as that on the upperside. Male genitalia (Figs 7A1-A6, 7B1-B4).
Typical Driopa pattern. Uncus deeply bifurcated and weakly sclerotized, its two arms are triangular and
straight (running parallel to each other), with narrow and sharp apex. Apical process of the valva rounded
distally, with a curved thick spur. Aedeagus slender, 1.4 times longer than the length of the valva, broadened
at the base. Saccus cylindrical, with a rounded apex, short, as long as 1/5 of the general length of the
genitalia.
PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE
152
Figure 4. Dorsal view of Parnassius nebrodensis Turati, 1907 stat. rev. and P. mnemosyne (Linnaeus, 1758) s. str.
specimens [coll. RMBH]. (A) Male of P. nebrodensis [France: Pyrenees, foothill of Mt. Neouville]. (B) Female of P.
nebrodensis [the same locality]. (С) Male of P. mnemosyne [Republic of Belarus: 5 km E from Ruda Yavorskaya
village]. (D) Female of P. mnemosyne [the same locality]. (Photos: Artem A. Frolov).
Female external morphology and markings (Figs 4B, 5B, and 6B). Forewing length is 2930 mm. Antenna,
eye, frons, top of the head, palpus, thorax, legs, and abdomen with the same patterns as those in male.
Upperside of the forewing with milk-white ground color and black veins. The base of the forewing blackish.
Cell with two rectangular (or oval) black spots. The postdiscal area with a transverse blackish band and an
unclear blackish spot. Marginal area of the forewing strongly darkened. Black diamond shape scales in the
black spot at the end of the forewing’s cell are identical to those forming the blackish postdiscal band.
Hindwing with the same ground color and black markings along veins as those on the forewing. Basal area
and dorsum black, with dense greyish-black hairs. The basal part of the space between veins M1 and M2 with
a large black spot, which merges with black markings in the basal area and along the dorsum of the
hindwing. The costal margin with a rounded black spot. Underside of both wings with the same markings
pattern as that on the upperside. Female genitalia (Fig. 7E). Typical Driopa pattern. Ductus bursae thick,
with a small funnel-shaped extension at the connection with membranous corpus bursae.
Distribution and biology of Parnassius nebrodensis
Species within the P. mnemosyne species complex share largely allopatric ranges (Fig. 1). In particular, P.
nebrodensis is endemic to the Western Mediterranean mountain ranges, i.e. the Pyrenees, Western and
Central Alps, Apennines, and Nebrodi and Madonie on Sicily (Fig. 2). Habitats of this species are situated
within the altitudinal belt of 900 to 2200 m (Dannehl 1929; Eisner 1968; Napolitano and Descimon 1994;
Descimon 1995, 2006; Weiss 1999; Gratton 2006). In Italy, P. nebrodensis mostly occurs at the edges
between deciduous forest and meadows, and flies from late May to mid-July (Gratton 2006). Corydalis
BOLOTOV ET AL.
Ecologica Montenegrina, 40, 2021, 140-163 153
solida (L.) Clairv. is considered its key larval host plant (Descimon 1995). The biology and habitat
preference of P. nebrodensis in France were described by several authors (Descimon 1995, 2006; Mérit and
Mérit 2006).
Figure 5. Forewing venation of Parnassius nebrodensis Turati, 1907 stat. rev. and P. mnemosyne (Linnaeus, 1758) s.
str. (A) Male of P. nebrodensis [France: Pyrenees, foothill of Mt. Neouville]. (B) Female of P. nebrodensis [the same
locality]. (C) Male of P. mnemosyne [Moldova]. (D) Female of P. mnemosyne [Linnaeus’s lectotype, Finland; redrawn
after Honey and Scoble 2001a]. Red arrows indicate diagnostic features of the two species (see differential diagnosis for
detail). (Graphics: Ivan N. Bolotov).
Taxonomic issues related to Parnassius nebrodensis
More than 40 P. mnemosyne subspecies were described from the West Mediterranean Region (Turati 1907;
Verity 1907; Fruhstorfer 1908; Pagenstecher 1911; Dannehl 1929; Ackery 1973; Eisner 1958, 1968, 1971,
1974, 1976, 1978; Sala and Bollino 1992; Weiss 1999; Beccaloni et al. 2003) (Table 5). The oldest names
introduced from this region are as follows: P. mnemosyne nebrodensis Turati, 1907 (Sicily, the type locality
(TL): “monti Nebrodi”), P. m. pyraenaeca Turati, 1907 (France, TL: “Gèdre, negli alti Pirenei”), and P. m.
pyrenaica Verity, 1907 (France, TL: “Pyrenees Orientalis”). The last two names were found to be primary
homonyms of P. apollo pyrenaica Harcourt, 1896 (Fruhstorfer 1908). Fruhstorfer (1908) assumed that the
work of Turati (1907) was published in 1908. However, the numbers of 1–3 of the “Naturalista Siciliano”’s
volume 20 containing Turati’s “Nuove forme di Lepidotteri” were issued in 1907. Further revisions also
fixed this year (Eisner 1978; Weiss 1999; Beccaloni et al. 2003).
Hence, P. nebrodensis is considered here as a valid name for the Western Mediterranean lineage of
P. mnemosyne species complex (=P. mnemosyne sp.3 sensu Condamine, 2018). Altogether 38 subspecies of
P. mnemosyne described from this region are considered here to be synonyms of P. nebrodensis based on the
PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE
154
Figure 6. Forewing scales of Parnassius nebrodensis Turati, 1907 stat. rev. and P. mnemosyne (Linnaeus, 1758) s. str.
[coll. RMBH]. (A) Position of the enlarged fragment with scale patterns at the forewing. (B) Female of P. nebrodensis
[France]. (C) Female of P. mnemosyne [Moldova]. (D) Female of P. mnemosyne [Northern European Russia]. (E) Male
of P. nebrodensis [France]. (F) Male of P. nebrodensis [France]. (G) Male of P. mnemosyne [Moldova]. (H) Male of P.
mnemosyne [Northern European Russia]. Red and blue arrows indicate diagnostic features of each species for the
female and male, respectively (see differential diagnosis for detail). (Graphics and photos: Ivan N. Bolotov).
BOLOTOV ET AL.
Ecologica Montenegrina, 40, 2021, 140-163 155
Figure 7. Male and female genitalia of Parnassius nebrodensis Turati, 1907 stat. rev. and P. mnemosyne (Linnaeus,
1758) s. str. [coll. RMBH]. (A) Male genitalia of P. nebrodensis [France; slide RMBH G0328]: (A1) lateral view, (A2)
ventral view, (A3) dorsal view, (A4) aedeagus, (A5) uncus, and (A6) apical process of the valva. (B) Male genitalia of
P. nebrodensis [France; slide RMBH G0330]: (B1) dorsal view, (B2) aedeagus, (B3) uncus, and (B4) apical process of
the valva. (C) Male genitalia of P. mnemosyne [Moldova; slide RMBH G0279]: (C1) lateral view, (C2) ventral view,
(C3) dorsal view, (C4) aedeagus, (C5) uncus, and (C6) apical process of the valva. (D) Male genitalia of P. mnemosyne
[Belarus; slide RMBH G0004]: (D1) dorsal view, (D2) aedeagus, (D3) uncus, and D4) apical process of the valva. (E)
Female genitalia of P. nebrodensis [France; slide RMBH G0331]. (F) Female genitalia of P. mnemosyne [Moldova;
slide RMBH G0280]. (G) Female genitalia of P. mnemosyne [Belarus; slide RMBH G0005]. Black arrows indicate
diagnostic features of the two species (see differential diagnosis for detail). (Photos: Artem A. Frolov).
PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE
156
type localities and available molecular data (Table 5 and Fig. 2). We did not revise several nominal taxa that
can also belong to P. nebrodensis but whose type localities are situated within a narrow contact zone
between the first species and P. mnemosyne s. str. in the Alps. These taxa are as follows: P. mnemosyne
adamellicus Kunz, 1922, P. mnemosyne ausonicus Bryk, 1912, P. mnemosyne benacensis Dürck, 1922, P.
mnemosyne cuneifer Fruhstorfer, 1908, P. mnemosyne lessinicus Dannehl, 1933, P. mnemosyne venetus
Wagner, 1910 (see Weiss 1999). Their status and taxonomic affinities should be determined in the future
using newly collected topotypes. Ackery (1973) listed subspecies P. mnemosyne esperi Bryk, 1922 (TL:
“Mt. Superga, Piémont, Italien”) but earlier Eisner (1966) showed that the type locality of this taxon is
erroneous, as the type series was collected in Württemberg, Germany.
The intraspecific taxonomy of P. nebrodensis and P. mnemosyne s. str. needs further revision using
morphological and molecular analyses of topotypes. As for P. nebrodensis, there are at least two more or less
divergent COI subclades within this species: (1) Sicily and southern Italy (Calabria), and (2) the Pyrenees,
and the Italian Peninsula with Central and Western Alps (Gratton 2006; this study: Fig. 3). At first glance,
these haplotype groups can correspond to subspecies-level taxa. For instance, the Sicilian populations might
have evolved in isolation during approximately 400 Ka, and could represent a specific evolutionary unit
(Gratton 2006).
Table 5. Subspecies of Parnassius mnemosyne (Linnaeus, 1758) synonymized with P. nebrodensis Turati, 1907 stat.
rev. in this study.
No
*
Taxa**
Country
Type locality
Coordinates of the type
locality***
Latitude
Longitude
1
P. mnemosyne aldinae Nardelli &
Giandolfo, 1991 syn. nov.
Italy
Sicily: eastern Nebrodi Mts, Cesaro,
Villa Miraglia
37°52' N
14°41' E
2
P. mnemosyne arollaensis Eisner, 1938 syn.
nov.
Switzerland
Arolla, Wallis
46°01′ N
07°29′ E
3
P. mnemosyne calabricus Turati, 1911 syn.
nov.
Italy
Calabria: Aspromonte
38°10′ N
16°00′ E
4
P. mnemosyne cassiensis Stépi, 1909 syn.
nov.
France
Ste. Baume Mt., near Marseille
43°19′ N
05°45′ E
5
P. mnemosyne cayollensis Dujardin, 1967
syn. nov.
France
Southern Alps: Cayolle Pass
44°15' N
06°45' E
6
P. mnemosyne ceuzensis Eisner, 1957 syn.
nov.
France
Céüse Pass, south of Grenoble
44°31′ N
05°56′ E
7
P. mnemosyne constantinii Turati, 1919
syn. nov.
Italy
Apennines: Lago Santo, S.
Pelligrino
44°24′ N
10°00′ E
8
P. mnemosyne cosenzaensis Eisner, 1978
syn. nov.
Italy
Lago di Ampollino, Provinz
Cosenza
39°12′ N
16°38′ E
9
P. mnemosyne costarum Bryk, 1922 syn.
nov.
Italy
Roccaraso, Valle de Petrella,
Caserta
41°51′ N
14°05′ E
10
P. mnemosyne dinianus Fruhstorfer, 1908
syn. nov.
France
Digne
44°06′ N
06°14′ E
11
P. mnemosyne ekplektus Rütimeyer, 1968
syn. nov.
Switzerland
Bern: Bundalp, Kiental
46°32′ N
07°46′ E
12
P. mnemosyne euaquilensis Bryk & Eisner,
1932 syn. nov.
Italy
Abruzzi: Gran Sasso
42°28′ N
13°33′ E
13
P. mnemosyne eucomitis Bryk & Eisner,
1932
Italy
Abruzzi: Maiella
42°03′ N
14°03′ E
14
P. mnemosyne excelsus Verity, 1911 syn.
nov.
France
French Alps: Mt Cenis
45°15′ N
06°54′ E
15
P. mnemosyne fruhstorferi Turati, 1909 syn.
nov.
Italy
Italia centr., Mt Autore
41°57′ N
13°12′ E
16
P. mnemosyne gallicus Bryk & Eisner, 1930
syn. nov.
France
Savoyen: Bonnéval-sur-Arc
45°22′ N
07°03′ E
17
P. mnemosyne guccinii Sala & Bollino,
1992 syn. nov.
Italy
Apennines: Passo della Cisa
44°28′ N
09°56′ E
18
P. mnemosyne hunti Dujardin, 1968 syn.
nov.
France
Southern Alps: St. Barnabé
43°48′ N
07°02′ E
19
P. mnemosyne matuta Bryk, 1922 syn. nov.
France
Mte. Authion bei Sospel
43°53′ N
07°27′ E
20
P. mnemosyne mixtus Fruhstorfer, 1922
syn. nov.
Switzerland
Binn (also syntypes from Berisal
and Lötschental)
46°22′ N
08°11′ E
BOLOTOV ET AL.
Ecologica Montenegrina, 40, 2021, 140-163 157
No
*
Taxa**
Country
Type locality
Coordinates of the type
locality***
Latitude
Longitude
21
P. mnemosyne montdorensis Kolar, 1943
syn. nov.
France
Northern Massif Central: Mont Dore
(Puy-de-Dôme)
45°34′ N
02°48′ E
22
P. mnemosyne ozalensis Bustillo & de
Aizpurua, 1977 syn. nov.
Spain
Northern Spain: Selva de Oza,
Hecho (Huesca)
42°51′ N
00°42′ W
23
P. mnemosyne parmenides Fruhstorfer,
1908 syn. nov.
France
Alpes maritimes
43°50′ N
07°10′ E
24
P. mnemosyne phaiohyalinus Rütimeyer,
1968 syn. nov.
Switzerland
Bern: Unteres, Urbachtal, südlich
von Innertkirchen
46°26′ N
08°13′ E
25
P. mnemosyne puschlavensis Eisner, 1758
syn. nov.
Switzerland
Le Prese, Puschlav
46°18′ N
10°05′ E
26
P. mnemosyne rencurelensis Vergely &
Willien, 1972 syn. nov.
France
Mt. Noir, Vercors
43°27′ N
02°20′ E
27
P. mnemosyne republicanus Peebles &
Bryk, 1931 syn. nov.
Spain
Hispania, Maladetta, Val de l'Esera
42°13′ N
00°21′ E
28
P. mnemosyne rogervarleti Eisner &
Epstein, 1968 syn. nov.
Switzerland
Val Lavizarra, Ticino
46°26′ N
08°40′ E
29
P. mnemosyne romanus Garavaglia, 1940
syn. nov.
Italy
Abruzzi: Terminillo
42°29′ N
13°00′ E
30
P. mnemosyne sbordonii Eisner & Racheli,
1971 syn. nov.
Italy
Mt Vulture
40°57′ N
15°38′ E
31
P. mnemosyne schawerdae Bryk, 1922 syn.
nov.
Italy
Apennines: Mti Sibillini, Mt
Nerone, Passo la Calla
43°51′ N
11°45′ E
32
P. mnemosyne symphorus Fruhstorfer, 1910
syn. nov.
Italy
Macugnaga
45°58′ N
07°58′ E
33
P. mnemosyne temora Fruhstorfer, 1922
syn. nov.
Switzerland
Glarus: Lake Talalpsee
47°06′ N
09°08′ E
34
P. mnemosyne tergestus Fruhstorfer, 1910
syn. nov.
Switzerland
Kanton Uri, Umgebung von Erstfeld
46°53′ N
08°38′ E
35
P. mnemosyne thebaida Fruhstorfer, 1922
syn. nov.
Switzerland
Val Maggina am Nordfuss des Mt.
Camogh
46°08′ N
09°04′ E
36
P. mnemosyne turatii Fruhstorfer, 1908 syn.
nov.
France
Central Pyrénées: Gedre, Cauterets
42°53′ N
00°07′ W
37
P. mnemosyne vernetanus Fruhstorfer, 1908
syn. nov.
France
Pyrénées Orientales
42°30′ N
02°45′ E
38
P. mnemosyne vivaricus Bernardi & Viette,
1961 syn. nov.
France
Southern Massif Central: Chambon
Forest
45°56′ N
00°41′ E
*The numbers of localities are correspond to their numbers on the map (Fig. 2). **Taxonomic names and localities are given based
on Weiss (1999). ***The majority of coordinates are rather approximate (±5 km or even more) due to the vague type localities in old
references, being linked to a vast region.
Conclusion
Recent multi-locus phylogenetic studies revealed that P. mnemosyne sensu lato represents a complex of
cryptic species that contains P. mnemosyne s. str. and two additional species-level taxa P. mnemosyne sp.2
from the Middle East and P. mnemosyne sp.3 from Southern Europe (Condamine 2018; Condamine et al.
2018). In respect to the high levels of genetic divergence and weak morphological differences, the P.
mnemosyne species complex is similar to a group of cryptic taxa discovered within the genus Leptidea
Billberg, 1820 (Pieridae) (Dincă et al. 2011). The latter complex contains three species: Leptidea sinapis
(Linnaeus, 1758), L. reali (Reissinger, 1989), and L. juvernica (Williams, 1946). These morphologically
cryptic white-wood butterfly taxa can reliably be identified by means of either DNA sequences or
karyological data (Dincă et al. 2011; Lehtonen et al. 2017; Talla et al. 2019). Additional remarkable
examples of cryptic species were recently discovered within temperate and tropical butterflies such as
Muschampia proto (Ochsenheimer, 1808) (Hesperiidae), Lasiommata spp. (Nymphalidae), Polyommatus
valiabadi (Rose & Schurian, 1977), and Rhamma spp. (Lycaenidae) (Lukhtanov et al. 2015; Prieto et al.
2019; Platania et al. 2020b; Hinojosa et al. 2021).
Here, we show that P. mnemosyne sp.3 sensu Condamine (2018) and Condamine et al. (2018)
belongs to P. nebrodensis. It is a distinct species and can be distinguished from P. mnemosyne s. str. based
PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE
158
on a combination of morphological features such as the forewing venation, forewing scales pattern, and the
male genitalia structure. Phylogenetically, it is the most distant taxon among the P. mnemosyne species
complex. This species complex contains not less than three additional species: P. mnemosyne s. str., P.
phoebus [=P. ariadne], and P. sp. ‘Middle East’ (Omoto et al. 2004; Gratton 2006; Gratton et al. 2008;
Michel et al. 2008; Condamine 2018; Condamine et al. 2018). The status of Parnassius sp. ‘Middle East’
(=P. mnemosyne sp.2) from southern Anatolia and Iran (Gratton 2006; Gratton et al. 2008; Condamine 2018;
Condamine et al. 2018) remains unclear, although it may belong to a separate cryptic species. However, the
discussion on the taxonomic placement of this taxon is beyond the scope of this study, and will be published
elsewhere. The P. stubbendorfii species complex represents another example of a clade combining allopatric
cryptic taxa among the subgenus Driopa, and it contains P. stubbendorfii, P. hoenei, and P. glacialis (e.g.
Yagi et al. 2011; Condamine et al. 2018; this study). It should be noted that subspecies-level taxa in
Parnassius usually share much lower genetic distances (e.g. Yakovlev et al. 2020).
Although Gratton (2006) assumed that the wing pattern’s differentiation could trigger a pre copula
mechanism of reproductive isolation between P. nebrodensis and P. mnemosyne s. str., we show that these
taxa share clear differences in the male genitalia structure, most likely precluding their interbreeding at the
contact zonas of secondary sympatry (e.g. in northeastern Italy: see Figs 1-2). Michel et al. (2008) suggested
a possibility of interspecific hybridization events between these clades, while Gratton (2006) revealed the
lack of nuclear exchange between them using the EF-1a gene fragment. The latter conclusion was supported
by our research based on the the Wg gene. In summary, the species status of P. nebrodensis is confirmed on
the basis of five genes: COI, ND1, ND5, Wg, and EF-1a (Gratton 2006; Gratton et al. 2008; Michel et al.
2008; Condamine 2018; Condamine et al. 2018; this study).
P. nebrodensis is known to occur disjunctively throughout the Western Mediterranean mountain
ranges (Spain, France, Andorra, Italy, and southeastern Switzerland). This region houses a plethora of
regional endemic species, and shares the maximum level of butterfly endemism in Europe (Kudrna et al.
2011). Among papilionids, Zerynthia cassandra (Geyer, [1830]) and Z. rumina (Linnaeus, 1767) are
characteristic examples of taxa, the local ranges of which are confined to certain parts of the Western
Mediterranean Region (Kudrna et al. 2011; Zinetti et al. 2013; Camerini et al. 2018). Interestingly, P.
nebrodensis reveals the high levels of intraspecific genetic variability in the COI gene. This unusual genetic
pattern could be driven by a strong metapopulation structure at the regional scale, with a number of local
populations having a larger or smaller degree of isolation from others, e.g. in southern France (Napolitano et
al. 1988; Napolitano and Descimon 1994; Descimon 1995, 1996) and Italy (Gratton 2006).
It should be noted that the distribution of P. nebrodensis is restricted compared with that of P.
mnemosyne s. str. It occurs within a narrow range of mid- and high-altitude habitats, with several populations
being threatened or declining due to human activities (Descimon 1995, 2006; Gratton 2006; Mérit and Mérit
2006). Moreover, rapid climate warming may reduce the altitudinal belt suitable to Parnassius butterflies at
the southern edge of Europe (Descimon 2006). Hence, P. nebrodensis may be considered an endangered
species due to its limited distribution (Figs 1-2), narrow range of habitats, and continuing population decline,
although this tentative assessment must be clarified in the future. This species contains a variety of more or
less isolated mountain populations, and special management and conservation efforts are urgently needed to
prevent further fragmentation of its disjunctive range.
Acknowledgements
This study was partly supported by the Ministry of Science and Higher Education of the Russian Federation
(projects АААА-А17-117033010132-2 to Y.S.K. and V.M.S., and АААА-А18-118012390161-9 to
M.Y.G.), and Russian Foundation for Basic Research (project 18-44-292001 to I.N.B. and A.V.K.). We are
grateful to Dr. Oleg S. Pokrovsky and Dr. Lyudmila S. Shirokova (Toulouse, France), and to Artem A.
Frolov (Moscow, Russia) for their help during this study.
References
Ackery, P. R. (1973) A list of the type specimens of Parnassius (Lepidoptera: Papilionidae) in the British
Museum (Natural History). Bulletin of the British Museum of Natural History (Entomology), 29, 1
35.
BOLOTOV ET AL.
Ecologica Montenegrina, 40, 2021, 140-163 159
Ackery, P. R. (1975) A guide to the genera and species of Parnassiinae (Lepidoptera: Papilionidae). Bulletin
of the British Museum of Natural History (Entomology), 31(4), 71105.
Aksenova, O. V., Bolotov, I. N., Gofarov, M. Y., Kondakov, A. V., Vinarski, M. V., Bespalaya, Y. V.,
Kolosova, Y. S., Palatov, D. M., Sokolova, S. E., Spitsyn, V. M., Tomilova, A. A., Travina, O. V. &
Vikhrev, I. V. (2018) Species richness, molecular taxonomy and biogeography of the radicine pond
snails (Gastropoda: Lymnaeidae) in the Old World. Scientific Reports, 8, 11199.
https://doi.org/10.1038/s41598-018-29451-1
Beccaloni, G., Scoble, M., Kitching, I., Simonsen, T., Robinson, G., Pitkin, B., Hine, A. & Lyal, C. (Eds.)
(2003) The Global Lepidoptera Names Index (LepIndex). The Natural History Museum, London,
UK. Available from: https://www.nhm.ac.uk/our-science/data/lepindex/lepindex/ (accessed 15
January 2020).
Bickford, D., Lohman, D. J., Sodhi, N. S., Ng, P. K., Meier, R., Winker, K., Ingram, K. K. & Das, I. (2007)
Cryptic species as a window on diversity and conservation. Trends in Ecology & Evolution, 22(3),
148155. https://doi.org/10.1016/j.tree.2006.11.004
Bolotov, I. N., Gofarov, M. Y., Rykov, A. M., Frolov, A. A. & Kogut, Y. I. (2013) Northern boundary of the
range of the Clouded Apollo butterfly Parnassius mnemosyne (L.) (Papilionidae): climate influence
or degradation of larval host plants? Nota lepidopterologica, 36 (1), 1933.
Bolotov, I. N., Kondakov, A. V. & Spitsyn, V. M. (2018) A review of tiger moths (Lepidoptera: Erebidae:
Arctiinae: Arctiini) from Flores Island, Lesser Sunda Archipelago, with description of a new species
and new subspecies. Ecologica Montenegrina, 16, 115. https://doi.org/10.37828/em.2018.16.1
Brower, A. V. Z. & DeSalle, R. (1998) Patterns of mitochondrial versus nuclear DNA sequence divergence
among nymphalid butterflies: the utility of wingless as a source of characters for phylogenetic
inference. Insect Molecular Biology, 7(1), 7382. https://doi.org/10.1046/j.1365-2583.1998.71052.x
Camerini, G., Groppali, R. & Minerbi, T. (2018) Observations on the ecology of the endangered butterfly
Zerynthia cassandra in a protected area of Northern Italy. Journal of Insect Conservation, 22, 4149.
https://doi.org/10.1007/s10841-017-0036-6
Condamine, F. L. (2018) Limited by the roof of the World: Mountain radiations of Apollo swallowtails
controlled by diversity-dependence processes. Biology Letters, 14(3), 20170622.
https://doi.org/10.1098/rsbl.2017.0622
Condamine, F. L., Rolland, J., Höhna, S., Sperling, F. A. & Sanmartín, I. (2018) Testing the role of the Red
Queen and Court Jester as drivers of the macroevolution of Apollo butterflies. Systematic Biology,
67(6), 940964. https://doi.org/10.1093/sysbio/syy009
Dannehl, F. (1929) Neue Formen und geographische Rassen aus meinen Ausbeuten und Erwerbungen der
letzten Jahre. Mitteilungen der Münchner Entomologischen Gesellschaft, 19 (59), 97116.
Dapporto, L., Cini, A., Vodă, R., Dincă, V., Wiemers, M., Menchetti, M., Magini, G., Talavera, G., Shreeve,
T., Bonelli, S., Casacci, L. P., Balletto, E., Scalercio, S. & Vila, R. (2019) Integrating three
comprehensive data sets shows that mitochondrial DNA variation is linked to species traits and
paleogeographic events in European butterflies. Molecular Ecology Resources, 19(6), 16231636.
https://doi.org/10.1111/1755-0998.13059
Descimon, H. (1995) La conservation des Parnassius en France: aspects zoogéographiques, écologiques,
démographiques et génétiques. Rapport d'études de l'OPIE, 1, 154. http://www.insectes.xyz/re-
parnass.htm
Descimon, H. (2006). La conservation des Parnassius de France. Situation en 1995 et situation en 2006, 11
ans après. Bulletin des Lépidoptéristes Parisiens, 15 (33), 3455.
Dincă, V., Lukhtanov, V., Talavera, G. & Vila, R. (2011) Unexpected layers of cryptic diversity in wood
white Leptidea butterflies. Nature Communications, 2, 324. https://doi.org/10.1038/ncomms1329
Dincă, V., Montagud, S., Talavera, G., Hernández-Roldán, J., Munguira, M. L., García-Barros, E., Hebert, P.
D. N. & Vila, R. (2015) DNA barcode reference library for Iberian butterflies enables a continental-
scale preview of potential cryptic diversity. Scientific Reports, 5, 12395.
https://doi.org/10.1038/srep12395
Eisner, C. (1958) Parnassiana Nova XVIII. Varia. Zoologische Mededelingen, 34(1), 13.
Eisner, C. (1966) Parnassiidae-Typen in der Sammlung J. C. Eisner. Zoologische Verhandelingen, 81(1), 1
81.
Eisner, C. (1968) Parnassiana Nova XLIII. Nachträgliche Betrachtungen zu der Revision der Subfamilie
Parnassiinae (Fortsetzung 16). Zoologische Mededelingen, 43(2), 917.
PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE
160
Eisner, C. (1971) Parnassiana Nova XLVI. Nachträgliche Betrachtungen zu der Revision der Subfamilie
Parnassiinae (Fortsetzung 19). Zoologische Mededelingen, 45(6), 8790.
Eisner, C. (1974) Parnassiana Nova XLIX. Die Arten und Unterarten der Baroniidae, Teinopalpidae und
Parnassiidae (Erster Teil) (Lepidoptera). Zoologische Verhandelingen, 135, 396.
Eisner, C. (1976) Parnassiana Nova XLIX. Die Arten und Unterarten der Baroniidae, Teinopalpidae und
Parnassiidae (Zweiter Teil) (Lepidoptera). Zoologische Verhandelingen, 146, 99259.
Eisner, C. (1978) Parnassiana Nova LIV. Dr. S. Wagener's Bemerkungen zu den Parnassus-Formendes
Apennin aus Geografisch-Ökologischer Sicht. Zoologische Mededelingen, 53(21), 237243.
Fruhstorfer, H. (1908) Neue Parnassier aus der mnemosyne-Gruppe. Internationale Entomologische
Zeitschrift, 3, 1718.
Gratton, P. (2006) Phylogeography and conservation genetics of Parnassius mnemosyne L., 1758
(Lepidoptera, Papilionidae). Doctoral dissertation. Tor Vergata University of Rome, Italy, 102 pp.
Gratton, P., Konopiński, M. K. & Sbordoni, V. (2008) Pleistocene evolutionary history of the Clouded
Apollo (Parnassius mnemosyne): genetic signatures of climate cycles and a ‘time-dependent’
mitochondrial substitution rate. Molecular Ecology, 19, 42484262. https://doi.org/10.1111/j.1365-
294X.2008.03901.x
Hall, T. A. (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for
Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 9598.
Hanus J. & Theye M.-L. (2010) Parnassius phoebus (Fabricius, 1793), a misidentified species (Lepidoptera:
Papilionidae). Nachrichten des Entomologischen Vereins Apollo, 31(12), 784.
Hinojosa, J. C., Dapporto, L., Brockmann, E., Dincă, V., Tikhonov, V., Grishin, N., Lukhtanov, V. A. &
Vila, R. (2021) Overlooked cryptic diversity in Muschampia (Lepidoptera: Hesperiidae) adds two
species to the European butterfly fauna. Zoological Journal of the Linnean Society, zlaa171.
https://doi.org/10.1093/zoolinnean/zlaa171
Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q. & Vinh, L. S. (2017) UFBoot2: Improving the
ultrafast bootstrap approximation. Molecular Biology and Evolution, 35(2), 518522.
https://doi.org/10.1093/molbev/msx281
Honey, M. R. & Scoble, M. J. (2001) Linnaeus's Butterfly Type Specimens. The Natural History Museum,
London, UK. Available from: http://www.nhm.ac.uk/research-curation/research/projects/linntypes
(accessed 06 July 2013)
Honey, M. R. & Scoble, M. J. (2001a) Linnaeus's butterflies (Lepidoptera: Papilionoidea and Hesperioidea).
Zoological Journal of the Linnaean Society, 132, 277399. https://doi.org/10.1006/zjls.2001.0265
Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. (2017) ModelFinder:
Fast model selection for accurate phylogenetic estimates. Nature Methods, 14, 587589.
https://doi.org/10.1038/nmeth.4285
Kapli, P., Lutteropp, S., Zhang, J., Kobert, K., Pavlidis, P., Stamatakis, A. & Flouri, T. (2017) Multi-rate
Poisson tree processes for single-locus species delimitation under maximum likelihood and Markov
chain Monte Carlo. Bioinformatics, 33(11), 16301638.
https://doi.org/10.1093/bioinformatics/btx025
Kim, M., Wan, X., Kim, M. J., Jeong, H. C., Ahn, N.-H., Kim, K.-G., Han, Y. S. & Kim, I. (2010)
Phylogenetic relationships of true butterflies (Lepidoptera: Papilionoidea) inferred from COI, 16S
rRNA and EF- sequences. Molecules and Cells, 30, 409425. https://doi.org/10.1007/s10059-010-
0141-9
Konopinski, M. K. (2008) A set of primers conserved in genus Parnassius (Lepidoptera, Papilionidae) for
amplification and sequencing of 1016 bp fragment of cytochrome oxidase subunit I from museum
specimens. Molecular Ecology Resources, 8, 675677. https://doi.org/10.1111/j.1471-
8286.2007.02045.x
Korshunov, Y. P. (2002) Butterflies of the Northern Asia [In Russian]. KMK Scientific Press Ltd., Moscow,
424 pp.
Kudrna, O., Harpke, A., Lux, K., Pennerstorfer, J., Schweiger, O., Settele, J. & Wiemers, M. (2011)
Distribution Atlas of butterflies in the Europe. Gesellschaft fur Schmetterlingschutz, Halle, 576 pp.
Kumar, S., Stecher, G. & Tamura, K. (2016) MEGA7: molecular evolutionary genetics analysis version 7.0
for bigger datasets. Molecular Biology and Evolution, 33(7), 18701874.
https://doi.org/10.1093/molbev/msw054
BOLOTOV ET AL.
Ecologica Montenegrina, 40, 2021, 140-163 161
Lehtonen, S., Lehtonen, I., Teräs, A., Varrela, J., Virta, P. & Vesterinen, E. J. (2017) DNA barcoding reveals
widespread occurrence of Leptidea juvernica (Lepidoptera: Pieridae) in southern Finland.
Entomologisk Tidskrift, 138, 151159.
Leraut, P. J. A. (1997) Liste systématique et synonymique des Lépidoptères de France, Belgique et Corse
(deuxième édition). Alexanor Supplément, Paris, 526 pp.
Litman, J., Chittaro, Y., Birrer, S., Praz, C., Wermeille, E., Fluri, M., Stalling, T., Schmid, S., Wyler, S. &
Gonseth, Y. (2018) A DNA barcode reference library for Swiss butterflies and forester moths as a
tool for species identification, systematics and conservation. PLoS ONE, 13(12), e0208639.
https://doi.org/10.1371/journal.pone.0208639
Lukhtanov, V. A., Dantchenko, A. V., Vishnevskaya, M. S. & Saifitdinova, A. F. (2015) Detecting cryptic
species in sympatry and allopatry: analysis of hidden diversity in Polyommatus (Agrodiaetus)
butterflies (Lepidoptera: Lycaenidae). Biological Journal of the Linnean Society, 116(2), 468485.
https://doi.org/10.1111/bij.12596
Lukhtanov, V. A., Sourakov, A., Zakharov, E. V. & Hebert, P. D. (2009) DNA barcoding Central Asian
butterflies: increasing geographical dimension does not significantly reduce the success of species
identification. Molecular Ecology Resources, 9(5), 13021310. https://doi.org/10.1111/j.1755-
0998.2009.02577.x
Luoto, M., Kuussaari, M., Rita, H., Salminen, J. & von Bonsdorff, T. (2001) Determinants of distribution
and abundance in the Clouded Apollo butterfly: a landscape ecological approach. Ecography, 24,
601617. https://doi.org/10.1111/j.1600-0587.2001.tb00494.x
Mérit, X. & Mérit, V. (2006) Contribution à la connaissance des sous-espèces françaises de Parnassius
(Driopa) mnemosyne (Linnaeus, 1758) (Lepidoptera, Papilionidae). Systématique, biologie et
implications pour la conservation de l’espèce. Bulletin des Lépidoptéristes Parisiens, 15 (33), 27.
Michel, F., Rebourg, C., Cosson, E. & Descimon, H. (2008) Molecular phylogeny of Parnassiinae butterflies
(Lepidoptera: Papilionidae) based on the sequences of four mitochondrial DNA segments. Annales
de la Société entomologique de France (N.S.), 44(1), 136.
https://doi.org/10.1080/00379271.2008.10697541
Miller, M. A., Pfeiffer, W. & Schwartz, T. (2010) Creating the CIPRES Science Gateway for inference of
large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop
(GCE), New Orleans, 18.
Müller, A. (1973) Die mnemosyne-Gruppe der Gattung Parnassius Latreille unter Berücksichtigung neuer
Schuppenmerkmale ihrer Arten (Lep. Parnassiidae). Deutsche Entomologische Zeitschrift, 20, (1-3),
211276.
Napolitano, M. & Descimon, H. (1994) Genetic structure of French populations of the mountain butterfly
Parnassius mnemosyne L. (Lepidoptera: Papilionidae). Biological Journal of the Linnean Society,
53, 325344. https://doi.org/10.1111/j.1095-8312.1994.tb01016.x
Napolitano, M., Geiger, H. J. & Descimon, H. (1988) Structure démographique et génétique de quatre
populations provençales de Parnassius mnemosyne (L.) (Lepidoptera Papilionidae): isolement et
polymorphisme dans des populations “menaces”. Genetics Selection Evolution, 20, 5162.
Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. (2015) IQ-TREE: A fast and effective
stochastic algorithm for estimating maximum likelihood phylogenies. Molecular Biology and
Evolution, 32, 268274. https://doi.org/10.1093/molbev/msu300
Omoto, K., Katoh, T., Chichvarkhin, A. & Yagi, T. (2004) Molecular systematics and evolution of the
“Apollo” butterflies of the genus Parnassius (Lepidoptera: Papilionidae) based on mitochondrial
DNA sequence data. Gene, 326, 141147. https://doi.org/10.1016/j.gene.2003.10.020
Omoto, K., Yonezawa T. & Shinkawa T. (2009) Molecular systematics and evolution of the recently
discovered “Parnassian” butterfly (Parnassius davydovi Churkin, 2006) and its allied species
(Lepidoptera, Papilionidae). Gene, 441, 8088. https://doi.org/10.1016/j.gene.2008.10.030
Pagenstecher A. (1911) Über die Geschichte, das Vorkommen und die Erscheinungsweise von Parnassius
mnemosyne L. Jahrbücher des Nassauischen Vereins für Naturkunde, 64, 261310.
Platania, L., Menchetti, M., Dincă, V., Corbella, C., Kay‐Lavelle, I., Vila, R., Wiemers, M., Schweiger, O. &
Dapporto, L. (2020a) Assigning occurrence data to cryptic taxa improves climatic niche assessments:
Biodecrypt, a new tool tested on European butterflies. Global Ecology and Biogeography, 29(10),
18521865. https://doi.org/10.1111/geb.13154
PARNASSIUS NEBRODENSIS - A NEGLECTED APOLLO BUTTERFLY SPECIES FROM SOUTHERN EUROPE
162
Platania, L., Vodă, R., Dincă, V., Talavera, G., Vila, R., & Dapporto, L. (2020b) Integrative analyses on
Western Palearctic Lasiommata reveal a mosaic of nascent butterfly species. Journal of Zoological
Systematics and Evolutionary Research. https://doi.org/10.1111/jzs.12356
Prieto, C., Nuñez, R., & Hausmann, A. (2019). Molecular species delimitation in the genus Rhamma
Johnson, 1992 (Lepidoptera: Lycaenidae, Theclinae). Mitochondrial DNA Part A, 30(1), 101117.
https://doi.org/10.1080/24701394.2018.1462348
Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Höhna, S., Larget, B., Liu, L.,
Suchard, M. A. & Huelsenbeck, J. P. (2012) MrBayes 3.2: Efficient Bayesian Phylogenetic Inference
and Model Choice Across a Large Model Space. Systematic Biology, 61(3), 539542.
https://doi.org/10.1093/sysbio/sys029
Sala, G. & Bollino, M. (1992) A new subspecies of Parnassius mnemosyne L. from Tosco-Emilian
Apennines and considerations about populations of the same range (Lepidoptera, Papilionidae).
Atalanta, 23 (12), 123126.
Sikes, D. S., Bowser, M., Morton, J. M., Bickford, C., Meierotto, S. & Hildebrandt, K. (2017) Building a
DNA barcode library of Alaska’s non-marine arthropods. Genome, 60(3), 248259.
https://doi.org/10.1139/gen-2015-0203
Talla, V., Johansson, A., Dincă, V., Vila, R., Friberg, M., Wiklund, C. & Backström, N. (2019) Lack of gene
flow: narrow and dispersed differentiation islands in a triplet of Leptidea butterfly species.
Molecular Ecology, 28(16), 37563770. https://doi.org/10.1111/mec.15188
Tao, R., Xu, C., Wang, Y., Sun, X., Li, C., Ma, J., Hao, J. & Yang, Q. (2020) Spatiotemporal differentiation
of Alpine butterfly Parnassius glacialis (Papilionidae: Parnassiinae) in China: Evidence from
mitochondrial DNA and nuclear single nucleotide polymorphisms. Genes, 11(2), 188.
https://doi.org/10.3390/genes11020188
Todisco, V., Gratton, P., Cesaroni, D. & Sbordoni, V. (2010) Phylogeography of Parnassius apollo: hints on
taxonomy and conservation of a vulnerable glacial butterfly invader. Biological Journal of the
Linnean Society, 101(1), 169183. https://doi.org/10.1111/j.1095-8312.2010.01476.x
Trifinopoulos, J., Nguyen, L. T., von Haeseler, A. & Minh, B. Q. (2016) W-IQ-TREE: a fast online
phylogenetic tool for maximum likelihood analysis. Nucleic Acids Research, 44 (W1), W232W235.
https://doi.org/10.1093/nar/gkw256
Turati, C. E. (1907) Nuove forme di Lepidotteri. Naturalista Siciliano, 20 (13), 148.
Väisänen, R. & Somerma, P. (1985) The status of Parnassius mnemosyne (Lepidoptera: Papilionidae) in
Finland. Notulae Entomologicae, 65, 109118.
Van Swaay, C. A. M. & Warren, M. S. (1999) Red Data Book of European Butterflies (Rhopalocera). Nature
and Environment, No. 99. Council of Europe Publishing, Strasbourg, 259 pp.
Van Swaay, C. A. M., Cuttelod, A., Collins, S., Maes, D., Munguira, M. L., Šašić, M., Settele, J., Verovnik,
R., Verstrael, T., Warren, M., Wiemers, M. & Wynhoff, I. (2010) European Red List of Butterflies.
Publications Office of the European Union, Luxembourg, 47 pp.
Van Swaay, C., Collins , S., Dušej, G., Maes, D., Munguira M. L., Rakosy, L., Ryrholm , N., Šašić, M.,
Settele, J. , Thomas , J. A., Verovnik , R., Verstrael , T., Warren, M., Wiemers, M. & Wynhoff, I.
(2012) Dos and Don'ts for butterflies of the Habitats Directive of the European Union. Nature
Conservation, 1, 73153.
Verity, R. (1907) Rhopalocera Palaearctica, 1, 77124.
Villesen P. (2007) FaBox: an online toolbox for fasta sequences. Molecular Ecology Notes, 7, 965968.
https://doi.org/10.1111/j.1471-8286.2007.01821.x
Weiss, J. C. (1999) The Parnassiinae of the World. Part 3. Hillside Books, Canterbury, pp. 137235.
Wiemers, M., Balletto, E., Dincă, V., Fric, Z. F., Lamas, G., Lukhtanov, V., Munguira, M. L., van Swaay, C.
A. M., Vila, R., Vliegenthart, A., Wahlberg, N. & Verovnik, R. (2018) An updated checklist of the
European butterflies (Lepidoptera, Papilionoidea). ZooKeys, 811, 945.
https://doi.org/10.3897/zookeys.811.28712
Yagi, T., Katoh, T., Chichvarkhin, A., Shinkawa, T. & Omoto, K. (2001) Molecular phylogeny of butterflies
Parnassius glacialis and P. stubbendorfii at various localities in East Asia. Genes & Genetic
Systems, 76(4), 229234. https://doi.org/10.1266/ggs.76.229
Yakovlev, R. V., Shapoval, N. A., Bakhaev, Y. I., Kuftina, G. N. & Khramov, B. A. (2020) A new
subspecies of Parnassius arcticus (Eisner, 1968) from the Momsky Range (Yakutia, Russia). Acta
Biologica Sibirica, 6, 93105. https://doi.org/10.3897/abs.6.e55925
BOLOTOV ET AL.
Ecologica Montenegrina, 40, 2021, 140-163 163
Zaman, K., Hubert, M. K. & Schoville, S. D. (2019) Testing the role of ecological selection on colour pattern
variation in the butterfly Parnassius clodius. Molecular Ecology, 28(23), 50865102.
https://doi.org/10.1111/mec.15279
Zinetti, F., Dapporto, L., Vovlas, A., Chelazzi, G., Bonelli, S., Balletto, E. & Ciofi, C. (2013) When the rule
becomes the exception. No evidence of gene flow between two Zerynthia cryptic butterflies suggests
the emergence of a new model group. PLoS ONE, 8(6), e65746.
https://doi.org/10.1371/journal.pone.0065746
... Recently Bolotov et al. (2021) separated the populations of Parnassius mnemosyne (Linnaeus, 1758) from Spain, France, Switzerland and Italy as a distinct species based on morphological and molecular characters. They applied the oldest published name among the populations of the separate species, Parnassius nebrodensis Turati, 1907[sic], as the name for the new species but this name is unavailable under the ICZN Code (1999) because it was described as an aberration and is thus infrasubspecific. ...
... Of these names, at least four or five belong to the South West European species, and since all of these names have equal priority, it is necessary to apply the First Reviser Principle under Code article 24.2 in order to choose the valid name for the new species. Bolotov et al. (2021) listed dinianus, parmenides, turatii, and vernetanus as definitely belonging to the new species, and also stated that the specific identity of cuneifer from the 'contact zone' is uncertain. All five of these names were published as subspecies of Parnassius mnemosyne by Fruhstorfer on 18 May 1908, as well as silesiacus, mesoleucus, demaculatus and tubulus. ...
... Thus, the name nebrodensis is attributable to Rothschild, 1918 and has the new combination of Parnassius turatii nebrodensis Rothschild, 1918. Bolotov et al. (2021) effectively synonymized all populations of the new species under Parnassius nebrodensis without any subspecies as per their Table 5. It is beyond the scope of this publication to determine the validity of the various populations at subspecies level, other than the necessity of reinstating the Sicilian subspecies, nebrodensis Rothschild, 1918, above. ...
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Genome‐scans in recently separated species can inform on molecular mechanisms and evolutionary processes driving divergence. Large‐scale polymorphism data from multiple species pairs are also key to investigate the repeatability of divergence – if radiations tend to show parallel responses to similar selection pressures and/or underlying molecular forces. Here we used whole genome re‐sequencing data from six wood white (Leptidea sp.) butterfly populations, representing three closely related species with karyomorph variation, to infer the species' demographic history and characterize patterns of genomic diversity and differentiation. The analyses supported previously established species relationships and there was no evidence for post‐divergence gene flow. We identified significant intraspecific genetic structure, in particular between karyomorph extremes in the wood white (L. sinapis) – a species with a remarkable chromosome number cline across the distribution range. The genomic landscapes of differentiation were erratic and outlier regions were narrow and dispersed. Highly differentiated (FST) regions generally had low genetic diversity (θπ), but increased absolute divergence (DXY) and excess of rare frequency variants (low Tajima's D). A minority of differentiation peaks were shared across species and population comparisons. However, highly differentiated regions contained genes with overrepresented functions related to metabolism, response to stimulus and cellular processes, indicating recurrent directional selection on a specific set of traits in all comparisons. In contrast to the majority of genome‐scans in recently diverged lineages, our data suggest that divergence landscapes in Leptidea have been shaped by directional selection and genetic drift rather than stable recombination landscapes and/or introgression. This article is protected by copyright. All rights reserved.