Corresponding author: Martin Wiemers (
Academic editor: A.Hausmann
The Palaearctic Grayling genus
Verovnik R, Wiemers M (2016) Species delimitation in the Grayling genus
Depending on which systematic order of classification is adhered to, the genus
The main reason for the extensive variation in phenotype can be linked with the specific ecological requirements of these butterflies. They are mostly petrophilous and limited to specific rock substrate to which they are perfectly adapted with their camouflaged underside wing pattern and cryptic coloration. Local adaptation to mimic the coloration of the rock substrate is, therefore, one of the main drivers for such large scale diversification (
Trying to resolve the systematics of this genus and its species delimitation has been thwarted by the fact that the genitalia of many
There has been no attempt to reconstruct the phylogeny of the genus or validate species status using molecular markers. Only the taxonomic position within subtribe
In order to resolve the relationship among cytochrome
With the aim of achieving consistency, we adopt the nomenclature of the most recent list of
Total genomic DNA was extracted from single legs, following the Mammalian tissue preparation protocol (GenElute Mammalian Genomic DNA miniprep kit from Sigma-Aldrich). For each sample a 657 bp fragment of the first subunit of the mitochondrial gene cytochrome
We used Bayesian inference to reconstruct a phylogenetic tree. To achieve more clarity the tree was constructed on a subset of samples including only unique haplotypes belonging to the same taxon. A hierarchical likelihood test was employed in order to test alternative models of evolution, using JModeltest v.0.1.1 (
No insertions or deletions were observed in the mitochondrial
Frequency distribution of pairwise intra- and interspecific p-distances of the
The calculated maximum connection for parsimony networks at the default 95% limit was 11 steps, and resulted in 9 separate networks within
Statistical Parsimony network of the ‘
Statistical Parsimony network of the ‘
Statistical Parsimony network of the ‘
The topology of the Bayesian Inference tree of all
Phylogeny of
This group, which forms a distinct network in the TCS analysis (Fig.
The ‘
The sister relationship of
It is important to note that the average genetic distance between two geographically separated subspecies,
In the TCS analysis, this group is split into 3 networks: a) the
The only two entirely Central Asian species available for analysis,
Within the ‘
Our study supports the monophyly of the genus
Another unexpected result is a deep split between
A split between
Given the high resolution of the basal clades within the
– Incomplete lineage sorting: recent speciation could result in unresolved relationships among these closely related species; however, well-defined species borders in terms of constant wing pattern differentiation coupled with broad overlaps in species ranges challenges this hypothesis.
– Recent gene flow: gene flow between closely related taxa is a known phenomenon (
– Pseudogenes or
The most enigmatic taxon among the '
Although we are aware of the pitfalls of using single gene trees in the interpretation of phylogenetic patterns (
We would like to express our gratitude to Wolfgang Eckweiler for his identification of several specimens from voucher photos housed in the BOLD database and we thank Evgeny V. Zakharov, Vlad Dinca and Axel Hausmann for their agreement to use unpublished DNA sequences from their projects in the BOLD database. We are thankful to our colleagues Tarkan Soyhan, Filip Franeta, Dubi Benyamini and Joseph Verhulst for providing additional samples of
List of samples of the genus
ID | GenBank | Species | Location | Lat | Long | Date | Legit |
---|---|---|---|---|---|---|---|
LA16 |
|
|
Baboshtice, Körce, Albania |
|
|
11.vii.2012 | Rudi Verovnik |
LA17 |
|
|
Baboshtice, Körce, Albania |
|
|
11.vii.2012 | Rudi Verovnik |
LA19 |
|
|
Devoll Gorge, Körce, Albania |
|
|
10.vii.2012 | Rudi Verovnik |
LA24 |
|
|
Pletvar Pass, Prilep, Macedonia |
|
|
14.vii.2010 | Rudi Verovnik |
LA28 |
|
|
Sivas, Turkey |
|
|
22.vii.2009 | Tarkan Soyhan |
LA29 |
|
|
Eskişehir, Turkey |
|
|
16.vi.2007 | Tarkan Soyhan |
LA75 |
|
|
Galičica Pass, Macedonia |
|
|
30.vii.2013 | Filip Franeta |
LA76 |
|
|
Falakro Mt., Greece |
|
|
7.vii.2013 | Filip Franeta |
LA77 |
|
|
Katara Pass, Metsova, Greece |
|
|
22.vii.2012 | Filip Franeta |
LA78 |
|
|
Granitis, Drama,Greece |
|
|
27.vii.2013 | Rudi Verovnik |
LA79 |
|
|
Katara Pass, Metsova, Greece |
|
|
26.vii.2013 | Rudi Verovnik |
LA80 |
|
|
Drenovë, Korcë, Albania |
|
|
21.vii.2013 | Rudi Verovnik |
LA81 |
|
|
Drenovë, Korcë, Albania |
|
|
21.vii.2013 | Rudi Verovnik |
LA82 |
|
|
Mt. Hermon, Israel |
|
|
2013 | Dubi Benyamini |
LA83 |
|
|
Mt. Hermon, Israel |
|
|
2013 | Dubi Benyamini |
LA84 |
|
|
Pletvar Pass, Prilep, Macedonia |
|
|
2013 | Filip Franeta |
LA85 |
|
|
Pletvar Pass, Prilep, Macedonia |
|
|
2013 | Filip Franeta |
LA86 |
|
|
Veles, Topolka, Macedonia |
|
|
2010 | Filip Franeta |
LA87 |
|
|
Mt. Parnassos, Greece |
|
|
2010 | Filip Franeta |
LA88 |
|
|
Drenovë, Korcë, Albania |
|
|
2013 | Filip Franeta |
LA89 |
|
|
Mt. Aladaglar, Turkey |
|
|
2006 | Filip Franeta |
LA90 |
|
|
Mt. Aladaglar, Turkey |
|
|
2006 | Filip Franeta |
LA92 |
|
|
Mt. Iti, Greece |
|
|
1999 | Filip Franeta |
LA94 |
|
|
Drenovë, Korcë, Albania |
|
|
2013 | Filip Franeta |
LA95 |
|
|
Devoll Gorge, Körce, Albania |
|
|
2013 | Filip Franeta |
LA97 |
|
|
Mersin, Turkey |
|
|
12.vii.2010 | Tarkan Soyhan |
LA124 |
|
|
Saabo Digur La, Ladakh, India |
|
|
15.vii.2013 | Joseph Verhulst |
BPAL1699–12 |
|
Azerbaijan: near Shamkir, 1300 m |
|
|
31.vii.2011 | Tikhonov V. | |
BPAL1700–12 |
|
Azerbaijan: near Shamkir, 1300 m |
|
|
31.vii.2011 | Tikhonov V. | |
BPAL1703–12 |
|
Russia: North Ossetia-Alania, rv. Ardon, Skasan, 1850 m |
|
|
12.viii.2011 | Tikhonov V. | |
BPAL2136–13 |
|
Tajikistan: Khodra-Mumin Mnt. | 26.v.2001 | A. Petrov | |||
BPAL2137–13 |
|
Tajikistan: Khodra-Mumin Mnt. | 26.v.2001 | A. Petrov | |||
BPAL2138–13 |
|
Iran: Char Mahall-o-Bahtiyari, Sahr-e-Kord, 2000 m | 28.v.2002 | P. Hofmann | |||
BPAL2139–13 |
|
Iran: Kerman, Kuh-e-Madvar, 5 km S Jowzan, 2400–2600 m | 24.v.2002 | P. Hofmann | |||
BPAL2140–13 |
|
Iran: Kerman, Kuh-e-Segoch, Mahan Pass, 2400–2600 m | 21.v.2002 | P. Hofmann | |||
BPAL2141–13 |
|
Iran: Azarbayjan-e-Sharqi, N Taran, Kuh-e-Sabalan, 2900–3000 m | 10.vii.2001 | Westphal | |||
BPAL2142–13 |
|
Iran: Azarbayjan-e-Sharqi, N Taran, Kuh-e-Sabalan, 2900–3000 m | 10.vii.2001 | Westphal | |||
BPAL2145–13 |
|
China: Xinjiang, Tian Shan, Borohoro Shan, 40 km SSW Kytun, 1850–2050 m |
|
|
08.vii.2006 | Grieshuber | |
BPAL2147–13 |
|
Iran: Kerman, Kuh-e-Madvar, 5 km S Jowzan, 2200–2400 m | 28.v.1999 | P. Hofmann | |||
BPAL2152–13 |
|
Iran: Khorasan, Kopet Dagh, 15 km E Emam Qoli, N Quchan, 2100–2200 m | 19.vi.2001 | P. Hofmann | |||
BPAL2153–13 |
|
Iran: Khorasan, Kopet Dagh, Qoucan, 1800 m | 13.vii.2000 | Hacz-Köszegi | |||
BPAL2154–13 |
|
Iran: Khorasan, Kopet Dagh, Qoucan, 1800 m | 14.vii.2000 | Hacz-Köszegi | |||
BPAL2155–13 |
|
Iran: Khorasan, Kopet Dagh, Qoucan, 1800 m | 15.vii.2000 | Hacz-Köszegi | |||
BPAL2156–13 |
|
Iran: Tehran, Elburs, Tuchal, 2400–2600 m | 16.vi.2001 | P. Hofmann | |||
BPAL2158–13 |
|
Iran: Tehran, Elburs, Tuchal, 2400–2600 m | 16.vi.2001 | P. Hofmann | |||
BPAL2159–13 |
|
Iran: Tehran, Elburs, Tuchal, 2400–2600 m | 16.vi.2001 | P. Hofmann | |||
BPAL2160–13 |
|
Turkey: Artvin, Kilickaya, 1100–1200 m | 01.vi.1998 | P. Hofmann | |||
BPAL2162–13 |
|
Turkey: Erzurum, Dikmen, SW Üzundere, 1300 m | 16.vii.1998 | P. Hofmann | |||
BPAL2172–13 |
|
Turkey: Bayburt, 5 km N Bayburt, 1500 m | 10.vii.1998 | P. Hofmann | |||
BPAL2173–13 |
|
Turkey: Erzincan, 5 km SE Caglayan, 1400 m | 08.vii.1998 | P. Hofmann | |||
BPAL2174–13 |
|
Turkey: Gümüshane, Demirkaynak, 13 km SW Torul, 1100 m | 06.vii.1998 | P. Hofmann | |||
BPAL2175–13 |
|
Turkey: Bayburt, 5 km N Bayburt, 1500 m | 10.vii.1998 | P. Hofmann | |||
BPAL2176–13 |
|
Turkey: Erzincan, 5 km SE Caglayan, 1400 m | 08.vii.1998 | P. Hofmann | |||
BPAL2177–13 |
|
Turkey: Erzurum, road Bayburt-Ispir, Laleli, 1300–1400 m | 11.vii.1998 | P. Hofmann | |||
BPAL2178–13 |
|
Iran: Char Mahall-o-Bahtiyari, Sahr-e-Kord, 2000 m | 28.v.2002 | P. Hofmann | |||
BPAL2179–13 |
|
Iran: Kerman, Kuh-e-Madvar, 5 km S Jowzan, 2400–2600 m | 24.v.2002 | P. Hofmann | |||
BPAL2180–13 |
|
Iran: Kerman, Kuh-e-Madvar, 5 km S Jowzan, 2400–2600 m | 24.v.2002 | P. Hofmann | |||
BPAL2181–13 |
|
Iran: Khorasan, Kopet Dagh, 15 km E Emam Qoli, N Quchan, 2100–2200 m | 19.vi.2001 | P. Hofmann | |||
BPAL2182–13 |
|
Iran: Tehran, Elburs, 15 km NE Firuzkuh pass, 1300–2400 m | 24.vii.2000 | P. Hofmann | |||
BPAL2183–13 |
|
Iran: Mazandaran, Khosh-Yeylaq, 65 km NE Shahrud, 2000–2100 m | 23.vi.2001 | P. Hofmann | |||
BPAL2185–13 |
|
Iran: Khorasan, Kopet Dagh, 15 km E Emam Qoli, N Quchan, 2100–2200 m | 19.vi.2001 | P. Hofmann | |||
BPAL2245–13 |
|
Israel | 22.vi.2013 | V.A.Lukhtanov & A.V.Novikova | |||
BPAL2246–13 |
|
Israel | 22.vi.2013 | V.A.Lukhtanov & A.V.Novikova | |||
BPAL2247–13 |
|
Israel | 22.vi.2013 | V.A.Lukhtanov & A.V.Novikova | |||
BPAL2281–14 |
|
Syria: Bloudan, 1500 m | 16.vii.1999 | A, Salk | |||
BPAL2282–14 |
|
Syria: Bloudan, 1500 m | 16.vii.1999 | A, Salk | |||
BPAL2692–14 |
|
Israel | 03.vii.2014 | V.Lukhtanov & A. Novikova | |||
BPAL2701–14 |
|
Israel | 03.vii.2014 | V.Lukhtanov & A. Novikova | |||
BPAL2702–14 |
|
Israel | 03.vii.2014 | V.Lukhtanov & A. Novikova | |||
BPAL2728–14 |
|
Israel | 04.vii.2014 | V.Lukhtanov | |||
BPAL2731–14 |
|
Israel | 04.vii.2014 | V.Lukhtanov | |||
EULEP451–14 |
|
Ukraine | 11.vii.2007 | local collector | |||
EULEP452–14 |
|
Ukraine | 11.vii.2007 | local collector | |||
EULEP453–14 |
|
Ukraine | 11.vii.2007 | local collector | |||
EULEP487–14 |
|
Russia |
|
|
23.vii.1998 | K. Nupponen | |
EULEP488–14 |
|
Russia |
|
|
14.vii.1998 | K. Nupponen | |
EZHBA660–07 |
|
Russia |
|
|
17.vii.2000 | Oleg Kosterin | |
EZHBA661–07 |
|
Russia |
|
|
17.vii.2000 | Oleg Kosterin | |
EZHBA662–07 |
|
Russia |
|
|
17.vii.2000 | Oleg Kosterin | |
EZHBA899–07 |
|
Russia |
|
|
30.vi.2004 | Oleg Kosterin | |
EZHBA900–07 |
|
Russia |
|
|
30.vi.2004 | Oleg Kosterin | |
EZROM089–08 |
|
|
Romania: Transylvania: Suatu |
|
|
16.viii.2006 | Dinca Vlad |
EZROM848–08 |
|
|
Romania: Transylvania: Suatu |
|
|
16.viii.2006 | Dinca Vlad |
EZSPM470–09 |
|
|
Spain: Granada: San Juan (Sierra Nevada) |
|
|
16.vii.2009 | Dinca V. |
EZSPN732–09 |
|
|
Spain: Granada: Laguna Seca, Hueneja |
|
|
18.vii.2008 | S. Montagud , J. A. Garcia-Alama & J. Garcia |
EZSPN733–09 |
|
|
Spain: Granada: Laguna Seca, Hueneja |
|
|
18.vii.2008 | S. Montagud , J. A. Garcia-Alama & J. Garcia |
EZSPN735–09 |
|
|
Spain: Granada: Laguna Seca, Hueneja |
|
|
18.vii.2008 | S. Montagud , J. A. Garcia-Alama & J. Garcia |
EZSPN736–09 |
|
|
Spain: Granada: Laguna Seca, Hueneja |
|
|
18.vii.2008 | S. Montagud , J. A. Garcia-Alama & J. Garcia |
EZSPN791–09 |
|
|
Spain: Granada: North-East Granada province |
|
|
23.vii.2008 | Gil, Felipe |
GWOSF831–10 |
|
|
Cyprus |
|
|
05.vi.2010 | M. Seizmair |
IRANB276–08 |
|
Iran |
|
|
29.vii.2002 | Vazrick Nazari | |
IRANB278–08 |
|
Iran |
|
|
29.vii.2002 | Vazrick Nazari | |
IRANB279–08 |
|
Iran |
|
|
22.vi.2001 | Vazrick Nazari | |
IRANB285–08 |
|
Iran |
|
|
16.viii.2000 | Vazrick Nazari | |
IRANB292–08 |
|
Iran |
|
|
01.vii.2001 | Vazrick Nazari | |
LOWA019–06 |
|
|
Kazakhstan: Tienschan: Kurdai Pass |
|
|
11.vi.2000 | V.Lukhtanov |
LOWA021–06 |
|
|
Kazakhstan: Tienschan: Kurdai Pass |
|
|
11.vi.2000 | V.Lukhtanov |
LOWA022–06 |
|
|
Kazakhstan: Tienschan: Kurdai Pass |
|
|
11.vi.2000 | V.Lukhtanov |
LOWA024–06 |
|
|
Kazakhstan: Tienschan: Kurdai Pass |
|
|
11.vi.2000 | V.Lukhtanov |
LOWA150–06 |
|
|
Russia |
|
|
07.vii.1999 | V.Lukhtanov |
LOWA315–06 |
|
|
Kazakhstan: Ust-Kamenogorsk Region: Kendyrlik |
|
|
14.vii.1997 | V. Lukhtanov |
LOWA316–06 |
|
|
Kazakhstan: Ust-Kamenogorsk Region: Kendyrlik |
|
|
14.vii.1997 | V. Lukhtanov |
LOWA516–06 |
|
|
Kyrgyzstan: Gultcha distr.: Chiitala |
|
|
29.vii.1995 | V. Lukhtanov |
LOWA517–06 |
|
|
Kyrgyzstan: Gultcha distr.: Chiitala |
|
|
29.vii.1995 | V. Lukhtanov |
LOWA608–06 |
|
|
Uzbekistan: Kashkardarinskaya obl.: Tamshush |
|
|
20.vi.1994 | V. Lukhtanov |
LOWA680–06 |
|
|
Kazakhstan: Dzhambulskaya obl.: Kurdai Pass |
|
|
28.vi.1993 | V. Lukhtanov |
LOWA681–06 |
|
|
Kazakhstan: Dzhambulskaya obl.: Kurdai Pass |
|
|
28.vi.1993 | V. Lukhtanov |
LOWA787–06 |
|
|
Kazakhstan |
|
|
22.vi.1997 | V. Lukhtanov |
LOWA788–06 |
|
|
Kazakhstan |
|
|
22.vi.1997 | V. Lukhtanov |
LOWAB040–07 |
|
Armenia |
|
|
Andrei Sourakov | ||
LOWAB041–07 |
|
Armenia |
|
|
Andrei Sourakov | ||
LOWAB046–07 |
|
Armenia |
|
|
Andrei Sourakov | ||
LOWAB046–07 |
|
Armenia |
|
|
Andrei Sourakov | ||
LOWAB047–07 |
|
Armenia |
|
|
Andrei Sourakov | ||
LOWAB048–07 |
|
Armenia |
|
|
Andrei Sourakov | ||
WMB1212–13 |
|
Morocco |
|
|
01.vii.2011 | Vila, R., Dinca, V. & Voda, R. | |
WMB1213–13 |
|
Morocco |
|
|
01.vii.2011 | Vila, R., Dinca, V. & Voda, R. | |
WMB2163–13 |
|
Morocco |
|
|
15.vii.2012 | Tarrier, Michel |