Leptidea sinapis ssp. tabarestana Nazari, Lukhtanov et Naderi, 2023

Nazari, Vazrick, Lukhtanov, Vladimir A., Naderi, Alireza, Fric, Zdenek Faltynek, Dincă, Vlad & Vila, Roger, 2023, More hidden diversity in a cryptic species complex: a new subspecies of Leptidea sinapis (Lepidoptera, Pieridae) from Northern Iran, Comparative Cytogenetics 17, pp. 113-128 : 113

Leptidea sinapis ssp. tabarestana Nazari, Lukhtanov et Naderi ssp. nov.

Fig. 4a-i

Type material.

Holotype. ♂ [white label] "330d= Mazandaran- E Kojour-/Kodir - 1000 m - 2.Jul.[20]10- / leg. A.R. Naderi"; [red label] "Holotype/ Leptidea sinapis tabarestana / Nazari, Lukhtanov & Naderi 2023". BOLD Sample ID: ARPI-330d-001; Deposited in coll. National Natural History Museum & Genetic Resources of Department of Environment, Tehran, Iran.

Paratypes. Gilan: 1♂ Damash, 1200m, 26.III.2021, leg. et coll. A.R. Naderi (ARPI-524b-001, AR# 254); 1♀ Khoshkab, rd. Siyahkal-Deylaman, 02.VII.1990, leg. et coll. Harandi. Ardabil/Gilan: 2♂♂ 1♀ Paß Ardabil-Astara ( Paßhöhe, W Tunnel), 1600m, 10.V.2010, W. ten Hagen. Tehran: 1♀ Laloon, 2000-2200 m, 30.VIII.2013, leg. et coll. A.R. Naderi (ARPI-408-001, AR# 186). Mazandaran: 1♂ Chalus road, Yush road, 40 km from Pole Zangooleh, 2400 m, 4.VII.1997, leg. & coll. A.R. Naderi (AR# 58); 2♂♂ Galanderoud, 1000 m, 13.VII.07, leg. & coll. A.R. Naderi; 1♂ Siahkal, 03.VII.1990, leg. et coll. Harandi; 1♂ Pol-e Zanguleh - Baladeh Rd, W of Minak, 36.2254°N, 51.58409°E, 15.V.2016, leg. & coll. Z. F. Fric, Biology Centre CAS, Institute of Entomology (IECA) (MR ZF 449); 10♂♂ Javaherdeh (Jirkooh), 36.866, 50.506, 24.VII.2011, leg. V. Lukhtanov & N. Shapoval, in Institut de Biologia Evolutiva (CSIC-UPF), Butterfly Diversity and Evolution Lab (VLU396-VLU405); 43♂♂, 12♀♀ ibid, in coll. Zoological Institute of Russian Academy of Sciences; 1♂, Javaherdeh (Samamus Mt.), 14.VIII.2010, leg. V.V. Tshikolovets, in Institut de Biologia Evolutiva (CSIC-UPF), Butterfly Diversity and Evolution Lab (RVcoll10C196); 1♂ Samamus Mt., 15 km S Ramsar, 1350 m, 8.VIII.2003, leg. W. ten Hagen; 1♂ Samamus Mt., S Rudbar, N Javaherdeh, 1500 m, 21.VI.2006, leg. W. ten Hagen; 1♂ Samamus Mt., 2800 m, 29.V.2009, leg. et coll. Harandi. Golestan: 1♂ Golestan Forest, 800-1000 m, 13.V.2001, leg. & coll. A.R. Naderi (112j, AR# 185).

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 upperside 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 discal 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 Leptidea 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: 0.60 ± 0.06, VW: 0.71 ± 0.04, n=8) were comparable to those of the nominotypical L. sinapis (PL: 1.60 ± 0.08, SL: 0.63 ± 0.04, VW: 0.79 ± 0.05, n=48) (Suppl. material 2).

Diagnosis.

Morphologically inseparable from the nominotypical L. sinapis , however the new taxon is distinguishable from it only by COI barcodes. Unlike ssp. Leptidea sinapis 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:AAA6298), 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 metaphase 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 2n=ca 58. An exact diploid number could not be determined due to numerous overlaps or contacts of chromosomes (Fig. 5 ).

The MI metaphase cells were not found in the studied individuals; however, MII 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 chromosomal 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 Iranian Talesh; however, the karyotype of the population from Yardimli in Republic of Azerbaijan’s Talysh region was studied previously ( Lukhtanov 1992). The latter population (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, Gilan, 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. Leptidea sinapis 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. Leptidea sinapis tabarestana .

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. Leptidea sinapis 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 adrastiodes (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. Leptidea sinapis tabarestana displays any host plant preferences different from the rest of populations of ssp. Leptidea sinapis sinapis .

Figures

Figure 1. Bayesian phylogeny of Leptidea COI barcodes. Node support values (Bayesian Posterior Probebilities / ML bootstrap) are shown only for supported nodes. All sequences are 658 bp in length unless indicated otherwise.

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.

Figure 4. Adults a-i L. sinapis ssp. tabarestana j-o L. sinapis ssp. sinapis a-c holotype Mazandaran: Kojur (♂ ARPI- 330 d- 001) d Golestan: Golestan forest (♂ ARPI- 112 j- 001) e Gilan: Damash (♂ ARPI- 524 b- 001) f Tehran: Laloon (♀ ARPI- 408 - 001) g Mazandaran: Javaherdeh (♂ MR ZF 449) h, i Ardabil / Gilan: Talesh (♂ ♀ DNAwthLeptidea 001 - 2) j, k Iran: E. Azerbaijan prov.: Kaleybar (♂ DNAwthLeptidea 006, ♀ - 004) l, m Iran: E. Azerbaijan prov.: Arasbaran (♀ ARPI- 479 a [not barcoded], ♂ ARPI- 456 d- 001) n, o Russia: Daghestan Republic (♂ ♀ DNAwthLeptidea 010 - 11). Scale bar: 20 mm.

Figure 5. Karyotype of Leptidea sinapis tabarestana ssp. nov. a, b mitotic cell demonstrating ca 58 chromosomes (sample VLU 396) c, d MII plate demonstrating 29 entities, 22 entities were interpreted as bivalents (shown by blue dots on Fig. 5 d) and 7 entities were interpreted as trivalents (shown by green dots on Fig. 4 d). Scale bar: 10 μm.

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.

Figure 7. Leptidea sinapis tabarestana ssp. nov. a adult b, c habitat in Iran, Mazandaran Prov., Javaherdeh (Jirkooh), 24. VII. 2011. Photos: V. Lukhtanov.