CompCytogen 1I7: | 13-128 (2023) COMPARATIVE *reerrervedorenacessiours doi: 10.3897/compcytogen. | 7.102830 Kas Cytogenetics https://compcytogen.pensoft.net International journal of Plant & Animal Cytogenetics, Karyosystematics, and Molecular Systematics More hidden diversity in a cryptic species complex: a new subspecies of Leptidea sinapis (Lepidoptera, Pieridae) from Northern Iran Vazrick Nazari!, Vladimir A. Lukhtanov’, Alireza Naderi?, Zdenek Faltynek Fric*, Vlad Dinca*’, Roger Vila’ | Department of Biology, University of Padova, Padova, Italy 2. Department of Karyosystematics, Zoological Institute of Russian Academy of Science, Universitetskaya nab. 1, 199034 St. Petersburg, Russia 3 National Natural History Museum & Genetic Resources, Tehran, Iran 4 Department of Biodiversity and Conservation Biology, Institute of Entomology, Biology Centre of the Czech Academy of Sciences, Ceské Budéjovice, Czech Republic § Ecology and Genetics Research Unit, University of Oulu, Oulu, Finland 6 Research Institute of the University of Bucharest (ICUB), University of Bucharest, Bucharest, Romania 7 Institut de Biologia Evolutiva (CSIC — Universitat Pompeu Fabra), Barcelona, Spain Corresponding author: Vazrick Nazari (nvazrick@yahoo.com) Academic editor: Nazar Shapoval | Received 1 March 2023 | Accepted 5 April 2023 | Published 4 May 2023 https://zoobank. org/ED37206B-54A 1-4DE4-849E-364E711D32FE Citation: Nazari V, Lukhtanov VA, Naderi A, Fric ZF, Dinca V, Vila R (2023) More hidden diversity in a cryptic species complex: a new subspecies of Leptidea sinapis (Lepidoptera, Pieridae) from Northern Iran. Comparative Cytogenetics 17: 113-128. https://doi.org/10.3897/compcytogen.17.102830 Abstract A new subspecies of Leptidea sinapis from Northern Iran, discovered by means of DNA barcoding, is described as Leptidea sinapis tabarestana ssp. nov. The new subspecies is allopatric with respect to other populations of L. sinapis and is genetically distinct, appearing as a well-supported sister clade to all other populations in COI-based phylogenetic reconstructions. Details on karyotype, genitalia, ecology and behaviour for the new subspecies are given and a biogeographical speciation scenario is proposed. Keywords allopatry, butterflies, DNA barcoding, Palearctic, taxonomy, Wood White Copyright Vazrick Nazari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 114 Vazrick Nazari et al. / Comparative Cytogenetics 17: 113-128 (2023) Introduction The cryptic diversity within the Leptidea sinapis (Linnaeus, 1758) complex progres- sively came to light in recent history (Réal 1988) with the discovery of differences in genitalic morphology (Lorkovi¢ 1993) and allozyme markers (Martin et al. 2003) between L. sinapis and L. reali Reissinger, 1989. It is considered one of the first docu- mented cases of cryptic species in Europe. Since then, numerous studies have revealed a plethora of new information on the mechanisms of speciation within this species complex (Mazel 2005; Bolshakov 2006; Friberg et al. 2008), including the presence of an additional widespread hidden taxon, L. juvernica Williams, 1946 (e.g. Dinca et al. 2011, 2013, 2021; Lukhtanov et al. 2011; Sichovd et al. 2015, 2016; Voda et al. 2015; Shtinkov et al. 2016; Talla et al. 2017, 2019a, b; Leal et al. 2018; Platania et al. 2020; Yoshido et al. 2020; Nasvall et al. 2021). Despite the explosion of interest in this group, many regions of Eurasia where Leptidea species occur are still not well sampled or studied. The new subspecies described in this paper was discovered accidentally in the course of a genetic investigation in order to determine whether any of the popula- tions of L. sinapis in Iran belong to the related cryptic species L. juvernica. Materials and methods Fourteen Iranian specimens from various disjunct populations in NW and N Iran were selected ex. coll. A. Naderi (Tehran) and W. ten Hagen (Germany) and their legs were submitted for DNA barcoding. Samples were processed in the Center for Biodiversity Genomics in Guelph, Ontario, Canada using standard protocols and LepF/LepR prim- ers, supplemented by failure-tracking with mini-primers (mLepF and mLepR) (Hajib- abaei et al. 2006). Eleven additional samples from Javaherdeh (VLU396-VLU405, RVcoll10C196) sequenced in 2012 were later added to the dataset. The majority of these sequences were full length barcodes (658 bp). An additional specimen from Javaherdeh included later in our analysis (MR ZF 449) was isolated using the Geneaid Blood and Tissue kit and sequenced in the Czech Republic using RON-HCO primers, and thus only partially overlaps (420 bp) with the standard barcode region. Thirty-six new barcode sequences were submitted to GenBank (Accessions 0OQ359842—ONQ359877). In addi- tion to the sequences pertaining to the new taxon, a selection of 80 other samples from previous studies (Lukhtanov et al. 2011; Dinca et al. 2013; Shtinkov et al. 2016) repre- senting various haplotypes of L. sinapis and several other species of Leptidea was used to conduct the analyses in this study (Suppl. material 1). All records are publicly available in the BOLD dataset “DS-SINIRAN” (https://doi.org/10.5883/DS-SINIRAN). A Maximum Likelihood (ML) tree was generated with PHYML online (Guindon and Gascuel 2003) using the AIC criterion and 100 bootstrap replicates. The best-fit model selected by PHYML for the combined dataset (GTR + I+ J) was further cor- roborated by IQ-TREE (Nguyen et al. 2015), and parameters from this model were used to conduct a Bayesian analysis in MRBAYES 3.2.6 (Ronquist et al. 2012). The MCMC analysis was allowed to run for 10,000,000 generations until stationary was Leptidea sinapis tabarestana ssp. nov. 115 reached. Convergence of parameters after the exclusion of the burnin phase was tested using TRACER 1.7.1 (Rambaut et al. 2018). Trees were edited using FIGTREE 1.4.4 (Rambaut 2018). Genetic distances were calculated using the Maximum Composite Likelihood model in MEGA 11.0.8 (Tamura et al. 2021). A haplotype diagram only including L. sinapis, L. juvernica and L. reali was constructed in TCS 1.21 (Clement et al. 2000), with a 95% confidence limit for parsimony. Shorter barcode fragments or those with ambiguous bases were excluded from haplotype analyses. Male genitalia were examined following maceration in 10% potassium hydroxide (KOH) for 15 minutes at 95 °C, dissection and cleaning under a stereomicroscope and storage in tubes with glycerol. Male genitalia were photographed in a thin layer of 30% ethanol (without being pressed under a cover slip), using a Carl Zeiss Stemi 2000-C stereomicroscope equipped with a CMEX PRO-5 DC.5000p digital camera (RV) or a Leica DFC450 digital camera (ZFF). Care was taken to arrange the measured structures parallel to the focal plane of the stereomicroscope in order to minimize the measure- ment error. Measurements were performed based on digital photographs using the Ax- ioVision software (Carl Zeiss Microlmaging GmbH). Eight specimens were analysed and the dataset was combined with data from Dinca et al. (2011) (135 specimens). We measured three elements of the male genitalia: phallus length (PL), saccus length (SL) and vinculum width (VW), known to be the most informative for differentiat- ing Leptidea species (e.g. Dinca et al. 2011; Shtinkov et al. 2016) (Suppl. material 2). Bivariate scatterplots were generated using VW as a size variable (Shtinkov et al. 2016). Chromosome preparations were made for ten adult males representing the popula- tion from Javaherdeh (field codes VLU396-VLU405) and were processed as previously described (Vershinina and Lukhtanov 2010). Briefly, gonads were removed from the abdomen and placed into freshly prepared fixative (3:1; 96% ethanol and glacial acetic acid) directly after capturing the butterfly in the field. Testes were stored in the fixa- tive for 3-36 months at +4 °C. Then the gonads were stained in 2% acetic orcein for 30-60 days at +18—20 °C. Metaphase II (MII) and mitotic plates were examined us- ing the original two-phase method of chromosome analysis (Lukhtanov et al. 2020a). Abbreviation “ca” (circa) means that the count was made with approximation due to overlapping of some chromosomes or due to difficulties in distinguishing between chromosome bivalents and trivalents. Images were edited in open source software GIMP 2.10.32 (The GIMP Development Team 2019) and Inkscape X11 (Inkscape Project 2020). Map was created using Simplemappr (Shorthouse 2010). Results None of the barcoded Iranian specimens belonged to L. juvernica. Specimens from the Iranian province of East Azerbaijan (Arasbaran) showed several haplotypes iden- tical to those of the common and widespread Eurasian L. sinapis; however, samples collected across the Alborz mountains from Talesh to NE Iran represented a unique and well-supported COI clade that appeared as sister to a weakly-supported clade con- taining all other L. sinapis (Figs 1, 2). A comparison of average uncorrected pairwise 116 Vazrick Nazari et al. / Comparative Cytogenetics 17: 113-128 (2023) Leptidea duponcheli JF512572 Iran Alborz 10km W Ghachsar Leptidea duponcheli LOWA-AR-21_ Armenia Khosrov Nature Reserve Leptidea duponcheli RVcolli0C240 Iran Hamadan Avaj Leptidea duponcheli JF512569 France Leptidea duponcheli ARP|-505a-001 Iran E Azerbaijan Arasbaran 1/99 Leptidea lactea JF512717 China Leptidea lactea JF512719 China 0.89/57 Leptidea jJactea JF512718 China 1/98 Leptidea morsei JF512618 Kazakhstan 1/100 Leptidea _ morsei HQ004591 Romania Leptidea morsei CSG08935 Russia Buryatiya Republic 0.96/98 eptidea morsei GU372563 South Korea Gangwon 199 Leptidea amurensis JF512620 Mongolia Leptidea amurensis FJ663713 Russia Altayskiy Kray Leptidea amurensis JF512622 China Leptidea amurensis NC 022686 China Leptidea amurensis FJ663710 Russia Buryatiya Republic 0.99/86 Leptidea amurensis KR363164 Japan eptidea amurensis KR363157 Japan Leptidea juvernica JF512716 Ireland 198 ve7 Leptidea juvernica GU655014 Semiany eptidea juvernica KC865982 Kazakhstan 0.68/51 0.9/61 Leptidea juvernica EF599643 Slovenia Leptidea juvernica EF599645 Slovenia Leptidea juvernica EF599640 Slovenia Leptidea juvernica JF512651 France Leptidea juvernica KC866126 Sweden Sep juvernica KC865949 Ital eptidea juvernica KC865980 Kazakhstan Leptidea juvernica HG969220 Russia Novosibirskaya Oblast eptidea juvernica KU355288 Bulgaria Leptidea juvernica JF512648 Kazakhstan [ Leptidea juvernica_HQ004596 Romania 0.85/66! Leptidea juvernica JF512649 Russia 0.88/77 Leptidea reali JF512704 Italy Leptidea reali JF512617 Spain Leptidea reali GU676645 Spain 0.69/61 Leptidea reali KG866117 Spain Leptidea reali JF512603 Spain Leptidea reali JF512616 Italy Leptidea reali JF512712 Italy 0.87/81 Leptidea sinapis ARPI-330d-001 388bp Iran Mazandaran Kojour 1/85 Leptidea sinapis DNAwthLeptidead02 Iran Ardabil-Astara Talysh Mts Leptidea sinapis ARPI-112j-001 611bp Iran Golestan Golestan forest Leptidea sinapis DNAwthLeptideaO03 Iran Ardabil-Astara Talysh Mts Leptidea sinapis ARPI-408-001 Iran Tehran Laloon 0.94/85 riences sinapis SN ae ee Mesandalan seiner, hes L. Sinapis eptidea sinapis a ran Mazandaran Javaherde samples. 0.97/81 Leptidea sinapis ARP|-524b-001 Iran Gilan Damash tabarestana ssp. n. Leptidea sinapis MR_ZF_449 420bp Iran Mazandaran Pol-e Zanguleh Leptidea sinapis DNAwthLeptidea009 Iran Mazandaran Mt. Samamus Leptidea sinapis DNAwthLeptidea00O1 Iran Ardabil-Astara Talysh Mts ; - Leptidea_sinapis DNAwthLeptidea008 Iran Mazandaran Mt. Samamus Leptidea sinapis MT260486 Bulgaria Leptidea sinapis MW502826 Spain 0.63/43 Leptidea sinapis KC866088 Bulgaria Leptidea sinapis DNAwthLeptidea012 Armenia W Dilijan Leptidea sinapis JF512597 Italy eptidea sinapis GU688515 Germany Leptidea sinapis DNAwthLeptidea006 Iran E Azarbaijan Arasbaran Leptidea sinapis MW502768 Spain Leptidea sinapis KC866005 Corsica Leptidea sinapis KC865994 Sardinia Leptidea sinapis KC866104 Macedonia Leptidea sinapis JF513025 Kazakhstan eptidea sinapis JF513027 Kazakhstan Leptidea sinapis KC866098 Macedonia Leptidea sinapis RVcoll11H081 Germany Leptidea sinapis HM393183 Austria Leptidea sinapis KC866101 Czech Republic Leptidea sinapis JF512697 Czech Republic eptidea sinapis JF512592 Romania Leptidea sinapis HG969226 Russia Novosibirskaya Oblast popiices sinapis Wonseies romania eptidea sinapis weden . . Leptidea sinapis HG969225 Russia Novosibirskaya Oblast L. sinapis s. str. eptidea sinapis GU688533 Germany Leptidea sinapis RVcoll17E601 Spain Leptidea sinapis JF513034 France Leptidea sinapis KP871088 Spain Leptidea sinapis DNAwthLeptidea007 Georgia W Tiblisi Leptidea sinapis JF512693 Bulgaria Leptidea sinapis DNAwthLeptidea010 Daghestary Leptidea sinapis ARPI-456d-001 Iran E Azarbaijan Arasbaran Leptidea sinapis ARPI-400f-001 Iran E Azarbaijan Arasbaran Leptidea sinapis JF513047 Kazakhstan Leptidea sinapis DNAwthLeptidea011 Daghestan eptidea sinapis KC866100 Spain Leptidea sinapis MN139254 Ital Leptidea sinapis KC866089 Italy Leptidea sinapis GU675913 Spain Leptidea sinapis JF513046 Kazakhstan Leptidea sinapis DNAwthLeptidea005 Iran E Azarbaijan Arasbaran Leptidea sinapis DNAwthLeptidea004 Iran E Azarbaijan Arasbaran Leptidea sinapis GU675857 Spain 0.02 Leptidea sinapis KC866082 Spain ‘ 0.54/51 eptidea sinapis KC866097 France Figure |. Bayesian phylogeny of Leptidea COI barcodes. Node support values (Bayesian Posterior Probe- bilities / ML bootstrap) are shown only for supported nodes. All sequences are 658 bp in length unless indicated otherwise. distances between this new lineage and other Leptidea species showed that it is indeed genetically closer to L. sinapis (average: 0.74%; range: 0.42%—1.76%) and further from all the other Leptidea (Table 1). The genitalia of the eight specimens analysed belonging to the above-men- tioned COI lineage showed broad overlap with other specimens of L. sinapis and a certain degree of variability, despite their fairly restricted geographic origin (Fig. 3). Based on the three characters measured (PL, SL, VW), the male genitalia also indicated a close similarity to L. sinapis, with respect to which we did not notice any significant differences. Leptidea sinapis tabarestana ssp. nov. 117 L. juvernica L. sinapis s. str. ao & QYPn5 e) oO (e) e) iS oO L. reali Ce O i . a 2S L.s. tabarestana om ssp. n. t O e) e O—O0—9-9-09-9-9 00-00 Figure 2. TCS haplotype network for L. sinapis, L. reali and L. juvernica. Table |. Average uncorrected p-distances (in % of the COI barcoding region) and standard deviation between Leptidea taxa. L. duponcheli L.lactea LL. morsei L.amurensis L.juvernica L.reali L.s. sinapis L.s. tabarestana L. duponcheli (n=5) 0.27 £ 0.13 L. lactea (n=3) 5.80 + 0.13 0.00 + 0.00 L. morsei (n=4) 5.9440.26 2.3340.26 0.71 + 0.44 L. amurensis (n=7) 740+0.14 4.23+0.09 3.75+0.13 0.26+ 0.16 L, juvernica (n=15) 6.29 +£0.20 2.5140.16 3.39+0.24 3.97+0.15 0.30 40.13 L. reali (n=7) 5.35+0.16 2.33+0.13 2.96+0.25 3.79+0.15 1.75 +0.16 0.21 + 0.07 L. s. sinapis (n=44) 5.72+0.18 2.71+0.18 3.05+0.21 3.744£0.21 1.9740.21 0.9240.15 0.24+40.11 L. s. tabarestana(n=21) 5.69+0.18 2.76+0.16 2.78£0.25 4.0240.13 2.00£0.17 0.9640.19 0.74 + 0.20 0.02 + 0.05 Considering the allopatric distribution of the new taxon with respect to L. sinapis, its similar genitalia, and the fact that the new taxon appears to be genetically closer and phylogenetically sister to the rest of L. sinapis specimens, here we describe it as a new subspecies of L. sinapis: Leptidea sinapis ssp. tabarestana Nazari, Lukhtanov et Naderi, ssp. nov. https://zoobank.org/BED12A6B-C1D3-4897-8D40-A955333D6C7C Fig. 4a-i Type material. Holotype. 3 [white label] “330d= Mazandaran- E Kojour-/Kodir — 1000 m — 2.Jul.[20]10- / leg. A.R. Naderi”; [red label] “Holotype/ Leptidea sinapis tab- arestana / Nazari, Lukhtanov & Naderi 2023”. BOLD Sample ID: ARPI-330d-001; 118 Vazrick Nazari et al. / Comparative Cytogenetics 17: 113-128 (2023) SL/VW 1.70 1.90 2.10 2.30 2.50 2.70 2.90 PL/VW @ L. sinapis tabarestana L. sinapis @ L. reali L. juvernica Figure 3. Bivariate scatterplot based on male genitalia morphometry (phallus length, PL; saccus length, SL), using vinculum width (VW) as a size variable. L. s. tabarestana ssp. nov. overlaps broadly with L. s. sinapis s. str., however it is distinct from L. juvernica and L. reali. Inset: Male genitalia of L. sinapis tabarestana ssp. nov. (specimen MR ZF 449), showing the variables measured. Deposited in coll. National Natural History Museum & Genetic Resources of Depart- ment of Environment, Tehran, Iran. Paratypes. Gilan: 14 Damash, 1200m, 26.III.2021, leg. et coll. A.R. Naderi (ARPI-524b-001, AR# 254); 192 Khoshkab, rd. Siyahkal-Deylaman, 02.VII.1990, leg. et coll. Harandi. Ardabil/Gilan: 244 19 Paf Ardabil-Astara (Pafhohe, W Tunnel), 1600m, 10.V.2010, W. ten Hagen. Tehran: 19 Laloon, 2000-2200 m, 30.VII.2013, leg. et coll. ALR. Naderi (ARPI-408-001, AR# 186). Mazandaran: 14 Chalus road, Yush road, 40 km from Pole Zangooleh, 2400 m, 4.VII.1997, leg. & coll. A.R. Naderi (AR# 58); 24'4 Galanderoud, 1000 m, 13.VII.07, leg. & coll. A.R. Naderi; 14 Si- ahkal, 03.VII.1990, leg. et coll. Harandi; 14 Pol-e Zanguleh — Baladeh Rd, W of Mi- nak, 36.2254°N, 51.58409°E, 15.V.2016, leg. & coll. Z. F. Fric, Biology Centre CAS, Institute of Entomology (IECA) (MR ZF 449); 104.3 Javaherdeh (Jirkooh), 36.866, 50.506, 24.VII.2011, leg. V. Lukhtanov & N. Shapoval, in Institut de Biologia Evo- lutiva (CSIC-UPF), Butterfly Diversity and Evolution Lab (VLU396-VLU405); 4333, 1299 ibid, in coll. Zoological Institute of Russian Academy of Sciences; Leptidea sinapis tabarestana ssp. nov. 119 330d=Mavzandaran-E. Pica Kodir-1000m-2. Jul. 10- leg. A-R.Naderi Figure 4. Adults a-i L. sinapis ssp. tabarestana j-o L. sinapis ssp. sinapis a—c holotype Mazandaran: Kojur (¢ ARPI-330d-001) d Golestan: Golestan forest (4 ARPI-112j-001) e Gilan: Damash (4' ARPI- 524b-001) f Tehran: Laloon (2 ARPI-408-001) g Mazandaran: Javaherdeh (4 MR ZF 449) h, i Ardabil/ Gilan: Talesh (69 DNAwthLeptidea001-2) j, k Iran: E. Azerbaijan prov.: Kaleybar (4 DNAwthLep- tidea006, 9-004) I, m Iran: E. Azerbaijan prov.: Arasbaran (2 ARPI-479a [not barcoded], d' ARPI- 456d-001) n, o Russia: Daghestan Republic (4 DNAwthLeptidea010-11). Scale bar: 20 mm. 14, Javaherdeh (Samamus Mt.), 14.VIII.2010, leg. V.V. Tshikolovets, in Institut de Biologia Evolutiva (CSIC-UPF), Butterfly Diversity and Evolution Lab (RV- coll10C196); 14 Samamus Mt., 15 km S Ramsar, 1350 m, 8.VIII.2003, leg. W. ten Hagen; 14 Samamus Mt., S Rudbar, N Javaherdeh, 1500 m, 21.VI.2006, leg. W. ten Hagen; 14 Samamus Mt., 2800 m, 29.V.2009, leg. et coll. Harandi. Golestan: 14 Golestan Forest, 800-1000 m, 13.V.2001, leg. & coll. A.R. Naderi (112j, AR# 185). 120 Vazrick Nazari et al. / Comparative Cytogenetics 17: 113-128 (2023) Description. Male (Fig. 4a, b, d, e, g, h). Length of forewing 16-21 mm; ground colour pure white. First generation forewing upperside with a rectangular grey-black apical patch, veins v3 and v4 under this patch often covered with dark scales near the outer margin; forewing discal cell covered in grey scales that extend faintly along the costa towards the apex; a small dark patch near the base at the Inner margin. Hindwing upper- side veins near the base of the wing covered with dark scales, otherwise without any other markings; the dark scales of the underneath show through. Forewing underside ground colour white with light yellowish-greenish tint at the apex, along the costa and at the dis- cal cell except for a yellowish discoidal spot not covered in grey scales; all veins except v2 covered with dark scales at the outer half of the wing. Hindwing underside ground colour greenish-yellow covered in sparse grey scales; discal cell and space s5 lighter and covered in fewer dark scales; a faint postdiscal band broken into two sections: a costal S- shaped part and a lower postdiscal section in the form of a slightly curved streak. Second generation similar but grey scales on the underside highly reduced, sometimes completely absent. Female (Fig. 4f, i). Length of forewing 19-23 mm; similar to male but bigger, forewing apex more rounded; apical dark patch highly reduced, sometimes absent. Male genitalia (Fig. 3 inset). Based on the eight dissections examined, the male genitalia appear similar to that of the nominotypical sinapis. The three elements of the male genitalia (phallus length, saccus length and vinculum width) measured for L. s. tabarestana ssp. nov. (PL: 1.47£0.07, SL: 0.6040.06, VW: 0.71£0.04, n=8) were comparable to those of the nominotypical L. sinapis (PL: 1.60£0.08, SL: 0.6340.04, VW: 0.79£0.05, n=48) (Suppl. material 2). Diagnosis. Morphologically inseparable from the nominotypical L. sinapis, how- ever the new taxon is distinguishable from it only by COI barcodes. Unlike ssp. sinapis, which in Iran (East Azerbaijan province) is strictly limited to humid and damp forests or clearings, the new subspecies is found primarily in semi-humid or even semi-dry mountainous habitats. Etymology. The subspecies name is a reference to “Tabarestan”, the medieval name for the mountainous regions south of the Caspian coast in northern Iran and roughly corresponding to the modern-day province of Mazandaran, the type locality of L. s. tabarestana ssp. nov. DNA barcode analysis. The COI barcodes of L. s. tabarestana ssp. nov. fall within the Barcode Identification Number (BIN) of L. sinapis (BOLD:AAAG6298), however they form a unique and distinct cluster that is on average 0.74% (range: 0.42%-—1.76%) distant from all other L. sinapis (Fig. 1). Uncorrected p-distances are smaller than those between L. sinapis and L. reali (0.92%) or between L. sinapis and L. juvernica (1.97%) (Table 1). Since the topology of ML and Bayesian trees were similar, only the Bayesian tree is shown with ML bootstrap values plotted on the supported nodes. In both trees, the L. s. tabarestana ssp. nov. clade appeared as sister to all other L. sinapis samples with strong support (Fig. 1). Karyotype. Of the 10 specimens studied, only two samples demonstrated meta- phase plates suitable for counting the number of chromosomes. Such a low proportion of adult males with dividing cells is a common phenomenon in the genus Leptidea and has been noted previously (Lukhtanov et al. 2011). In the sample VLU396, in mitotic cells, the diploid number of chromosomes was determined to be approximately Leptidea sinapis tabarestana ssp. nov. 121 2n=ca 58. An exact diploid number could not be determined due to numerous over- laps or contacts of chromosomes (Fig. 5). The MI metaphase cells were not found in the studied individuals; however, MI metaphase plates were found in the sample VLU405. The MII plates demonstrated clear traces of the phenomenon for which we previously used the term inverted meiosis (Lukhtanov et al. 2018; 2020a, b). In this type of meiosis, heterozygosity for chro- mosomal fusions/fissions leads to the very specific chromosomal structures at the MII stage, when heterozygotes retain a configuration similar to that of trivalents. Such trivalent-like structures were observed at the MII stage in the sample U405 (shown in green in Fig. 5d). The number of such trivalent-like structures reached 7, while the total number of chromosome entities was n = 29. If these elements are interpreted as trivalents, then the diploid number can be estimated as 2n = 65. If these elements are bivalents, then the diploid chromosome number is 2n = 58. Thus, the preliminary haploid number of chromosomes can be estimated as n = 29-33. Previously, a chromosome cline was found in L. sinapis, within which the diploid chromosome number gradually decreases from 2n = 106 in Spain to 2n = 56 in Sweden and in eastern Kazakhstan (Lukhtanov et al. 2011, 2018). Thus, the studied population from Mazandaran, Iran has an oriental variant of karyotype, that is, with a relatively low number of chromosomes. We were not able to study the karyotype from the Ira- nian Talesh; however, the karyotype of the population from Yardimli in Republic of Azerbaijan's Talysh region was studied previously (Lukhtanov 1992). The latter popula- tion (Azerbaijani Talysh) demonstrated variation in the haploid chromosome number from n = 28 to n = 34 (Lukhtanov 1992), thus, similar to the Mazandaran population. Distribution and ecology. So far, the presence of L. s. tabarestana ssp. nov. has been confirmed by DNA evidence only in northern Iran, in provinces of Ardabil, Gi- lan, Mazandaran, Tehran and Golestan (Fig. 6). Specimens from the Talysh mountains in Republic of Azerbaijan, across the border from Iranian Talesh region, show the same karyotype and possibly belong to ssp. tabarestana, however this remains to be further confirmed by DNA sequencing. In Turkmenistan, even though reports of L. sinapis from the Kopet Dagh mountains are as of yet unconfirmed (Tshikolovets 1998), these also likely belong to ssp. tabarestana. Figure 5. Karyotype of Leptidea sinapis tabarestana ssp. nov. a, b mitotic cell demonstrating ca 58 chro- mosomes (sample VLU396) c, d MII plate demonstrating 29 entities, 22 entities were interpreted as bivalents (shown by blue dots on Fig. 5d) and 7 entities were interpreted as trivalents (shown by green dots on Fig. 4d). Scale bar: 10 um. 122 Vazrick Nazari et al. / Comparative Cytogenetics 17: 113-128 (2023) i." & ots Ete Ry, Mea ete fea 5 % Figure 6. Distribution of Leptidea sinapis in E Turkey, S Caucasus and N Iran. Black dots: barcoded L. s. sinapis; red dots: barcoded L. s. tabarestana ssp. nov.; blue dot: karyotyped sample from Yardamli in Republic of Azerbaijan's Talysh region (most likely L. s. tabarestana ssp. nov.); white dots: non-barcoded material, data obtained from literature or personal collections. In the Iranian Talesh mountains, L. s. tabarestana ssp. nov. occurs approximately 100 km from the closest population of the nominotypical L. sinapis in Arasbaran region. The habitat of ssp. tabarestana is in the Alborz forest belt, in humid meadows, forest river banks, forest clearings, and sometimes gardens at mountain steppes from 1000 to 2000 m above sea level. Adults fly mostly in undisturbed or lightly-grazed habitats with lush of green vegetation (Fig. 7). The accompanying species include Ochlodes hyrcana (Christoph, 1893), Pieris napi mazandarana Eitschberger, 1987, Lasiommata adrasti- odes (Bienert, [1870]), and Maniola jurtina (Linnaeus, 1758). It is normally found in two (or maybe three) generations, from April at lower altitudes to the end of September at higher altitudes. The early stages of L. s. tabarestana ssp. nov. are unknown, however adults are often seen near Lathyrus plants (AN, personal observation). Even though the larval host plant is likely among the herbaceous Fabaceae of the genera Lathyrus, Vicia, Lotus etc., it is as of yet unrecorded and thus it is unclear if ssp. tabarestana displays any host plant preferences different from the rest of populations of ssp. sinapis. Discussion Reissinger (1989) recognized twelve subspecies of L. sinapis across its range, including reali and juvernica, both of which were later confirmed as separate species (Dinca et al. 2011, 2013). Since then, this complex has taken a central stage in efforts to understand the mechanisms of cryptic speciation in butterflies, and thus the idea of the existence of subspecies within L. sinapis seems to have slowly faded away. Modern taxonomic treatments of the group (e.g., Bozano et al. 2016) regard all populations of L. sinapis Leptidea sinapis tabarestana ssp. nov. 125 Figure 7. Leptidea sinapis tabarestana ssp. nov. a adult b, c habitat in Iran, Mazandaran Prov., Javaherdeh Jirkooh), 24.VII.2011. Photos: V. Lukhtanov. from Europe to NW Mongolia as a single entity, corresponding to the nominotypical subspecies. It occasionally flies in sympatry with closely related and extremely similar species of Leptidea across its range and can be separated from them only by DNA se- quencing and analysis of karyotype or genitalia. In a similar vein, in this study we found no single external morphological charac- ter or combination of characters that could reliably separate L. s. tabarestana ssp. nov. from the nominotypical L. sinapis. Individual variation in morphology observed with- in L. s. tabarestana ssp. nov. is not unexpected, as similar variation can also be seen in L. s. sinapis, as well as other species within the genus. In the Arasbaran mountains in NW Iran, where the nominotypical L. sinapis is found, individuals flying in colder slopes at high altitudes (1700—2000 m) tend to be smaller and darker, while those found in warmer forests at lower altitudes (1200-1400 m) are usually larger in size and have a lighter complexion. Recent studies have estimated the age of the most recent common ancestor (MRCA) of L. sinapis at 1.5 mya, and for MRCA of sinapist+juvernica at 3 mya (Talla et al. 2017). The subsequent dispersal of L. sinapis eastward however appears to have occurred much later, either before or after the Last Glacial Maximum (LGM) (24,000 to 17,000 years ago) (Lukhtanov et al. 2011). During the Pleistocene, dense forests covered the entire northern Iran, from the northwest (Azerbaijan province) across the Alborz mountains 124 Vazrick Nazari et al. / Comparative Cytogenetics 17: 113-128 (2023) and extending further into the northeast (Kopet Dagh); However, since the Last Glacial Maximum (LGM; 21 kya), the Alborz mountain range has been nearly entirely isolated from all other regions surrounding it. Subsequent decline in forest cover resulted in isolated refugia in parts of southern Caucasus as well as in northern Iran (Yousefi et al. 2015; Asadi et al. 2018; Parvizi et al. 2018; Liu et al. 2019; Saberi-Pirooz et al. 2020). With the likely extinction of intervening populations, the range of many butterflies adapted to this habitat — including the ancestral L. sinapis— became fragmented, result- ing in the geographic and genetic isolation of L. s. tabarestana ssp. nov. Presence of Wolbachia endosymbionts affecting mtDNA in Leptidea has been not- ed previously (e.g. Solovyev et al. 2015) and we cannot rule out that this may have had an effect on our results. Further studies are needed to confirm the presence of L. s. tabarestana ssp. nov. in the Republic of Azerbaijan and in Turkmenistan. Potential sympatric occurrence of the two entities in the intervening areas in NW Iran needs to be investigated. If the two are found to co-occur sympatrically and synchronically without geneflow, or other new information (e.g., karyotype, nuDNA, morphology etc.) comes to light that clearly signals the two taxa to be distinct at species level, the taxon tabarestana may be raised as bona species. Acknowledgements We thank Wolfgang ten Hagen (Mémlingen, Germany), Amir-Hossein Harandi (Esfa- han, Iran), Payam Zehzad (Tehran, Iran) and Vadim V. Tshikolovets (Pardubice, Czech Republic and Kyiv, Ukraine) for providing specimens or information on the new subspe- cies, and Nazar Shapoval (St. Petersburg, Russia) for help in collecting the samples from Javaherdeh. Hossein Rajaei (Stuttgart, Germany) kindly provided georeferenced distri- bution data for the Iranian L. sinapis. The specimen MR ZF 449 was barcoded by Michal Rindos, the genitalia were dissected by Petr Heiman and the photographs were taken by Nikolai Ignatev. We thank Nikolay Shtinkov, Anatoly Krupitsky, and two anonymous re- viewers whose helpful comments greatly improved the manuscript. Vladimir Lukhtanov was supported by the Russian Science Foundation (RSF 19-14-00202 Continuation) (analysis of karyotypes) and by the State Research Project No. 122031100272-3 (collect- ing the material). 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Heredity 125: 138-154. https://doi.org/10.1038/ s41437-020-0325-9 Yousefi M, Ahmadi M, Nourani E, Behrooz R, Rajabizadeh M, Geniez P, Kaboli M (2015) Upward altitudinal shifts in habitat suitability of Mountain Vipers since the Last Glacial Maximum. PLoS ONE 10(9): e0138087. https://doi.org/10.1371/journal.pone.0 138087 128 Vazrick Nazari et al. / Comparative Cytogenetics 17: 113-128 (2023) Supplementary material | Material examined and GenBank accessions Authors: Vazrick Nazari, Vladimir A. Lukhtanov, Alireza Naderi, Zdenek Faltynek Fric, Vlad Dinca, Roger Vila Data type: excel file Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/compcytogen. 17.102830.suppl1 Supplementary material 2 Length of male genitalia components (phallus, saccus, vinculum) and ratios (PL/VW, SL/VW) among Leptidea specimens measured in this study Authors: Vazrick Nazari, Vladimir A. Lukhtanov, Alireza Naderi, Zdenek Faltynek Fric, Vlad Dinca, Roger Vila Data type: excel file Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/compcytogen. 17.102830.suppl2 ORCID Vazrick Nazari https://orcid.org/0000-0001-9064-8959 Vladimir A. Lukhtanov https://orcid.org/0000-0003-2856-2075 Zdenek Faltynek Fric https://orcid.org/0000-0002-36 11-8022 Vlad Dinca https://orcid.org/0000-0003-1791-2148 Roger Vila https://orcid.org/0000-0002-2447-4388