Phytotaxa 450 (3): 246–256 ISSN 1179-3155 (print edition) https://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2020 Magnolia Press Article ISSN 1179-3163 (online edition)

https://doi.org/10.11646/phytotaxa.450.3.1

Allium schisticola, a new with peculiar morphological characters and an unexpected phylogenetic position

ALIREZA DOLATYARI1,4, HAMID MOAZZENI2,5*, SAEIDE HOSSEINI2,6, FRANK R. BLATTNER3,7 & REINHARD M. FRITSCH3,8 1 Bank, Iranian Biological Resource Center (IBRC), Research Complex of ACECR (Iranian Academic Center for Education, Culture & Research), Karaj, Iran. 2 FUMH herbarium, Department of Botany, Research Center for Plant Sciences, Ferdowsi University of Mashhad, P.O. Box 91779- 48974, Mashhad, Iran. 3 Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstrasse 3, D-06466 Seeland OT Gatersleben, Germany. 4 [email protected]; https://orcid.org/0000-0003-2866-1073 5 [email protected]; https://orcid.org/0000-0002-2406-2666 6 [email protected]; http://orcid.org/0000-0001-9367-5530 7 [email protected]; https://orcid.org/0000-0001-7731-7741 8 [email protected]; https://orcid.org/0000-0001-8207-7877 *Author for correspondence

Abstract

Allium schisticola is described from West Azarbaijan (Iran) as a new species. It is closely related to A. sabalense and A. sahandicum in having a similar flower color but differs by leaf, filament, and characters. We investigated the phylogenetic relationship of the new species based on sequences of the chloroplast trnL-trnF and nuclear ribosomal DNA internal transcribed spacer (ITS) regions in A. subg. Melanocrommyum compared with 109 accessions of this subgenus. Our results confirm its placement in A. subg. Melanocrommyum. However, the ITS tree showed that the new species should belong to A. sect. Melanocrommyum despite the fact that it represents many morphological characters of A. sect. Acanthoprason. Most peculiar for the new species are obtuse broadest near the tip (vs. very narrowly lanceolate up to triangular tepals in A. sect. Acanthoprason), with adaxially inconspicuous median vein (vs. conspicuous median vein in sect. Acanthoprason). The new species is diploid with a chromosome number of 2n = 2x = 16. Karyotype features and meiotic chromosomes behavior are presented for the new taxon. A detailed morphological description, illustrations, and a distribution map of the new species are given.

Keywords: Allium sabalense, Allium sahandicum, Iran, phylogeny, Acanthoprason, subg. Melanocrommyum

Introduction

The Allium Linnaeus (1753: 294) () with currently accepted 977 species (Govaerts et al. 2019) is one of the largest genera among the monocots. It is mainly distributed on the Northern Hemisphere from the to but adapted to diverse habitats (Fritsch 2012, Fritsch et al. 2010, Govaerts et al. 2019, Li et al. 2010). Most Allium species are perennial herbal geophytes characterized by tunicated , narrow to broad leaves, umbellate or head-like inflorescences, flowers mainly in pink, white and yellow; loculicidal capsules, rhomboidal or spheroidal, angled black seeds, and a specific odor and taste due to the presence of cysteine sulphoxides (Li et al. 2010). The genus has been classified into 15 subgenera and 85 sections (Friesen et al. 2006, Fritsch et al. 2010, Huang et al. 2014, Khassanov et al. 2011, Khassanov et al. 2013). Allium subg. Melanocrommyum (Webb & Berthelot 1848: 347) Rouy (1910: 378), with about 160 accepted species is the second largest subgenus of Allium (Fritsch 2016). Its members are naturally distributed from the Canary Islands to northwestern India, and most species occur in Asia Minor, Southwest Asia, and Central Asia (Fritsch 2016). Iran, with about 83 species (of over 155 Allium species growing naturally in Iran), belongs to the main center of diversity of this subgenus. Many members of this subgenus

246 Accepted by Lorenzo Peruzzi: 18 Jun. 2020; published: 30 Jun. 2020 can easily be recognized by having broad and flat leaves, rigid and most often strictly upright scapes of varying length and large fasciculate to globular inflorescences composed of many moderately small to large and often star-like flowers (Fritsch et al. 2010, Fritsch & Abbasi 2013). Allium subg. Melanocrommyum comprises ten sections in Iran, among which A. sect. Acanthoprason Wendelbo (1969: 27) with 25 accepted species is the largest section, morphologically recognized by relatively short peduncles and tepals forming a rigid spine-like median vein after anthesis (Fritsch & Abbasi 2009). Local people eat fresh or cooked young leaves of several members of A. sect. Acanthoprason (generally called “valak”). During a botanical excursion to northwest Iran, we found an Allium species growing on slate slopes. These specimens should belong to A. subg. Melanocrommyum, but we were not able to identify them using the keys and descriptions given in local Floras (Wendelbo 1971; Fritsch & Abbasi 2013, and references therein). They are described here as a new species.

TABLE 1. Voucher specimens, origin and GenBank accessions of taxa newly sequenced in the present study. All vouchers are kept in IBRC herbarium. IBRC No./ Taxon origin ITS trnL-trnF Herbarium no. IRAN. Zanjan: Zanjan towards Gilvan, 5 km Allium alamutense Razyfard, Zarre before Khanchaei road guard station, 2400 & R.M.Fritsch in Razyfard et al. P1010547/1616 - MT130434 m, 25 May 2014, Dolatyari, Ramezani & (2011: 353) Abdolahi IRAN. Ilam: Malekshahi, 5 km before Allium austroiranicum R.M.Fritsch Meimeh, 6 km on the road towards P1010320/1663 - MT130435 in Fritsch & Abbasi (2009: 26) Kabirkuh, 2200 m, 3 May 2014 Moazzeni, Dolatyari & Ramezani IRAN. Ilam: Ilam towards Eyvan, Chovar, Allium chrysantherum Boiss. & Reut. Reno forest park, 2 km from junction P1010382/1630 - MT130436 in Boissier (1882: 280). towards Park, 1500 m, 5 May 2014, Moazzeni, Dolatyari & Ramezani IRAN. Kermanshah: Eslam Abad-e Qarb Allium koelzii (Wendelbo 1966: 53) towards Mahidasht, Mersad strait, 1500 m, 6 P1010401/1607 - MT130437 Persson & Wendelbo (1979: 196) May 2014 Moazzeni, Dolatyari & Ramezani IRAN. West Azerbaijan: 61 km before Allium latifolium Jaub. & Spach Orumieh from Salmas, 1842 m, 6 June 2013, P1010022/1612 - MT130438 (1844: 2) Moazzeni, Dolatyari & Ramezani IRAN. Zanjan: Dandi towards Takht-e Allium mahneshanense Razyfard, Soleiman, NW mountain of lead and zinc Zarre & R.M.Fritsch in Razyfard et P1010506/1656 - MT130439 Anguran mine, 2850 m, 23 May 2014, al. (2011: 352) Dolatyari, Ramezani & Abdolahi IRAN. East Azerbaijan: S of Ahar, 3 km after Kaghalagh, Ghosh-e Dagh altitudes. Allium materculae Bordz. (1915: 73) P1009837/1834 - MT130440 2000 m, 2 June 2013, Moazzeni, Dolatyari & Ramezani Allium pseudohollandicum IRAN. Zanjan: Dandi towards Takab, 55 R.M.Fritsch in Fritsch & Abbasi km before Takab, 2500 m, 22 May 2014, P1010525/1638 - MT130441 (2013: 158) Dolatyari, Ramezani & Abdolahi IRAN. West Azerbaijan: 20 km from Khoy MT112908 Allium schisticola R.M. Fritsch, towards Qotor, 7 km towards Almalu- P1009984/3385 ‒ MT130443 Moazzeni & Dolatyari Hendowan villages, 1800 m, 5 June 2013, MT112917 Moazzeni, Dolatyari & Ramezani IRAN. Zanjan: Soltaniyeh, Arjin, N slope of Allium ubipetrense R.M.Fritsch in mountain W of sand mine, 2300 m, 22 May P1010476/1615 - MT130442 Fritsch & Abbasi (2009: 20) 2014, Dolatyari, Ramezani & Abdolahi

A NEW SPECIES OF ALLIUM SCHISTICOLA Phytotaxa 450 (3) © 2020 Magnolia Press • 247 FIGURE 1. Allium schisticola A–C: Cultivated plant at anthesis. D–F: in their natural habitat; D: Plant in the fruiting stage; E: Close-up of fruits; F: ; G: Seeds (Millimeter paper as background). All photos by Hamid Moazzeni.

248 • Phytotaxa 450 (3) © 2020 Magnolia Press DOLATYARI ET AL. FIGURE 2. Close relatives of the new species. A–B: Allium cardiostemon, A: Habit, B: Inflorescences; C–D: A. sabalense, C: Habit, D: Close-up of a flower. E–G: A. sahandicum, E: Close-up of a flower, F–G: Habit. All photos by Hamid Moazzeni.

Material and methods

Source of material:—Living plants of the new Allium species and other members of A. subg. Melanocrommyum

A NEW SPECIES OF ALLIUM SCHISTICOLA Phytotaxa 450 (3) © 2020 Magnolia Press • 249 were collected during fieldwork and were planted in the research farm of Iranian Biological Resource Center (IBRC). Several herbarium specimens were also prepared for further morphological investigation. All the specimens were deposited at IBRC herbarium. DNA Isolation, Amplification, and Sequencing:—Total genomic DNA was extracted from silica dried materials of 20 specimens (10 individuals of the new species and 10 species of A. subg. Melanocrommyum) using the 2× cetyltrimethylammonium bromide (CTAB) extraction protocol (Doyle & Doyle 1987). Voucher information and GenBank accession numbers for all newly included specimens are listed in Table 1. The nuclear ribosomal DNA internal transcribed spacer (ITS) and the plastid trnL-trnF regions were amplified and sequenced for ten individuals of the new species and ten endemic species from A. subg. Melanocrommyum for trnL-trnF (for more details see Table 1). For ITS, ITS-1 and ITS-2 were amplified separately using amplification primers (Blattner 1999; for ITS-1, the primers ITS-A and ITS-C, for ITS-2 the primers ITS-Dmel and ITS-B) following Gurushidze et al. (2008). The trnL together with the trnL–trnF intergenic spacer was amplified using the primers of Taberlet et al. (1991) as described in Gurushidze et al. (2010). Amplification products for both markers were purified using PEG (Joly et al. 2006). Sequencing was performed by BGI (Hong Kong) using the PCR primers. Sequences were trimmed, assembled, and aligned using Geneious version 6.1.2 (https://www.geneious.com). The newly generated sequences for ten accessions of the new species and ten closely related species were submitted to GenBank (accession numbers MT112908 to MT112917 for ITS and MT130434 to MT130443 for trnL-trnF, see Table 1). The new ITS sequences corresponding to the new species were added to the ITS matrix from Fritsch & Abbasi (2013: 198-202). For trnL-trnF, all sequences of A. subg. Melanocrommyum were obtained from GenBank. Allium kujukense Vvedensky (1923: 124) and A. oreophilum Meyer (1831: 37) served as outgroups (Fritsch & Abbasi 2013). The sequences were aligned using MAFFT Plug-in in Geneious with the default setting. The best substitution model for each alignment was selected using jModelTest v.2.1.4 (Darriba et al. 2012). The GTR + G + I and GTR + G models were determined as the best-fit model for nuclear and chloroplast markers, respectively. Phylogenetic Analyses of Sequence Data:—Phylogenetic analyses were performed using maximum likelihood (ML) and Bayesian inference (BI). The ML analysis was carried out using the IQ-TREE web server (http://iqtree.cibiv. univie.ac.at/) using 1,000 bootstrap replicates, obtained by the ultrafast bootstrap approximation (UFBoot) (Minh et al. 2013). Bayesian inference (BI) of the individual was performed using MrBayes v.3.1.2 (Huelsenbeck & Ronquist 2001) at the CIPRES portal in San Diego, CA, USA (http://www.phylo.org/index.php/portal/) with default prior settings, for 20 million MCMC generations. The quality of the analysis was checked by comparing likelihood values and parameter estimates from different runs in Tracer v.1.6 (http://tree.bio.ed.ac.uk/software/tracer/), and the initial 25% of the trees were discarded as burn-in. The remaining trees were summarized in a 50% majority-rule consensus tree. Karyological studies:—The method of mitosis metaphase spread preparation, the equipment and the software used for taking images and measuring chromosomes, and the calculated karyotype parameters were just the same as in Dolatyari et al. (2018). For meiosis analysis, we followed Dolatyari & Saeidi Mehrvarz (2017) with minor modifications. The bulbs of the investigated taxon were transferred to the research farm. The year after the young inflorescences of eight plants were fixed twice, each time 12 hours, in Carnoy’s solution. The fixed materials were immediately used for squash preparations. In this Allium group, each inflorescence represents one individual, so it was possible to infer separately for each plant the meiotic images and data of normal pollen mother cells (PMCs) and to calculate the percentage of irregularities. For this species, we did not test the fertility percentage of pollen grains.

Description of the new species

Allium schisticola R.M. Fritsch, Moazzeni & Dolatyari, sp. nov. (Figs. 1 & 3)

Morphologically, most similar to A. schisticola are A. sabalense R.M. Fritsch in Fritsch & Abbasi (2013: 106) and A. sahandicum R.M. Fritsch in Fritsch & Abbasi (2013: 108). Allium schisticola resembles A. sahandicum and A. sabalense in having almost pink flowers (Fig. 2). However, the new species differs by the number of leaves (3–4(–5) vs. 1–2 ), narrower leaves (0.8–2 cm ) vs. broader leaves (1.5–5 cm ), longer pedicels (15–20 mm) vs. 8–10(–15) mm and 1.5–4 cm in A. sahandicum and in A. sabalense, respectively. Moreover, tepals of the new species are obtuse and broad near the tip with adaxially inconspicuous median vein (Fig. 1B) while the tepals of A. sahandicum are subacute (Fig. 2E) and rounded in A. sabalense (Fig. 2D). Furthermore, A. schisticola has larger filaments (as long as tepals) vs. shorter (2/3–4/5 as long as tepals) in A. sahandicum and (1/2–2/3 as long as tepals) in A. sabalense.

250 • Phytotaxa 450 (3) © 2020 Magnolia Press DOLATYARI ET AL. FIGURE 3. Distribution of Allium schisticola and its close allies in Iran and neighbouring countries. A. schisticola (yellow star), A. sabalense (green squares), A. sahandicum (red dots).

Type:—IRAN. West Azerbaijan: 20 km from Khoy towards Qotor, 7 km towards Almalu-Hendowan villages, 1800 m, 38° 29’ 16”N, 44° 41’ 30”E, 5 June 2013, Moazzeni, Dolatyari & Ramezani P1009984 (holotype, FUMH!, isotype: IBRC!, and cultivated in the research farm of IBRC).

Bulbs ovoid, 2–3 cm long and in diameter; outer tunics grayish-brown. Scape ± flexuous, terete, smooth, 4–8 cm long, 2–3 mm in diameter, glaucous, purple suffused. Leaves 3–4(–5), glaucous green, laminae lanceolate, initially rather flat lying on the soil later arcuately ascending and recurved to the soil, strongly canaliculate, shortly arcuately tapering into the hooded apex; margin smooth, 10–25 cm long, 0.8–2 cm broad. Spathe membranous, split into 3 ovate-acuminate, ± patent valves; pale yellowish-brown with inconspicuous veins. Inflorescence initially very dense and broadly fasciculate, later semi-globose and rather loose, many-flowered; 4–6 cm in diameter. Pedicels thick, stiff, straight, 15–20 mm long, green to purplish flushed fading towards the base. Flowers funnel-shaped star-like. Tepals oblong-linear, obtuse, broadest near the tip with inside inconspicuous median vein, 6–7 mm long and 2 mm wide (outer tepals slightly narrower than the inner ones), pink later fading to white with inconspicuous darker purple median vein. Filaments as long as the tepals, fleshy, basally triangular and 1 mm connate at base and adnate to the tepals, pinkish throughout or slightly whitish. Anthers oblong, about 1 mm long, purple. Pollen yellow. Ovary short cylindrical-globose triangular, surface smooth and dull green, ca. 2 mm long and in diameter; nectary ducts lead in nose-like transversal pockets near the base. Style cylindrical, 2–3 mm long, pinkish. undivided, white. Capsule depressed globose triangular, ca. 3 mm long and 3–4 mm in diam., greenish; valves widely opening, broadly elliptic

A NEW SPECIES OF ALLIUM SCHISTICOLA Phytotaxa 450 (3) © 2020 Magnolia Press • 251 with 3 shallow longitudinal furrows. Seeds 1 per locule, sector-like drop-shaped, concave-convex, concave side finely tuberculate without or with a few irregular ledges surrounded by sharp marginal ledges, convex side irregularly reticulate lacunose, 2.5–3.5 mm long, 2–2.5 mm wide, 1 mm thick, dull black.

FIGURE 4. Majority rule consensus tree of Bayesian analysis of ITS sequences showing the phylogenetic position of Allium schisticola. Bayesian posterior probabilities and bootstrap values derived from a Maximum-Likelihood analysis are given above and below branches, respectively. This analysis is based on the sequence data set of 109 accessions of A. subg. Melanocrommyum.

Distribution:—Endemic to NW Iran (West Azarbaijan). Phenology:—Flowering in May, fruiting in July. Etymology:—The epithet “schisticola” refers to the habitat of the new species which is growing on slate slopes. Distribution and habitat:—A. schisticola is a local endemic to NW Iran (Fig. 3) where it grows in open habitats on fine-grained shale-type sedimentary rock slopes rich in clay. IUCN Red List category:—The new species is known so far only from the type location at elevations above 1800

252 • Phytotaxa 450 (3) © 2020 Magnolia Press DOLATYARI ET AL. m, and its distribution is very narrow both altitudinally and geographically. Unfortunately, the area is highly under grazing and destruction pressure. Therefore, the risk of local extinction of the type location is rather high. For these reasons, A. schisticola is here assessed as Critically Endangered (CR, B1+2ab(iii)) according to IUCN Red List criteria (IUCN 2001).

FIGURE 5. Mitotic and meiotic images of Allium schisticola. a) mitotic metaphase plate, b) idiogram, c) diplotene substage of prophase I showing eight bivalents, d) metaphase I showing 7II and 2I (arrowheads), e) anaphase I showing 8 – 8 segregation, f) telophase I showing lagged chromosomes (arrow) and chromatin bridge (arrowhead). Scale bars = 5μm. Arrows and arrowheads indicate irregularities.

Phylogenetic placement of the new species:—The phylogenetic analyses for both markers support the placement of the new species in A. subg. Melanocrommyum (Fig. 4; ITS: pp. 0.97, ML bootstrap 90%, trnL-trnF: pp. 1.0, ML bootstrap 94%), but with a position as a monophyletic (pp. 0.97, ML bootstrap 90% in ITS tree) in A. sect. Melanocrommyum rather than in A. sect. Acanthoprason in the ITS tree (Fig. 4). Allium schisticola, in the ITS tree, is sister to A. cardiostemon (pp. 1.0; ML bootstrap 56%), a morphologically rather different species characterized by a slender habit and deep brownish-purple flowers (Fig. 2A & B) emitting an unpleasant odor. This genetic similarity is unexpected. An uncommon feature is obtuse tepals broadest near the tip (vs. very narrowly lanceolate up to triangular tepals dominating in A. sect. Acanthoprason), with adaxially inconspicuous median veins (vs. conspicuous median veins in A. sect. Acanthoprason). However, although the trnL-trnF sequences presented an unresolved tree (Appendix1), the position in A. subg. Melanocrommyum (pp. 0.99; ML bootstrap 94%) and a close relationship with A. akaka S.G.Gmel. ex Schult. & Schult.f. in Römer & Schultes (1830: 1132) (pp. 0.86; ML bootstrap 81%) of A. sect. Acanthoprason is confirmed. Reasons for the discrepancy between the A. sect. Acanthoprason-like morphological appearance of A. schisticola and the results of the molecular marker regions suggesting a phylogenetic position within A. sect. Melanocrommyum could be a result of an old hybridization event that resulted in introgression of A. sect. Melanocrommyum rDNA in an Acanthoprason . The specific mode of the evolution of the rDNA cistron, known as concerted evolution, could easily explain the presence of only ‘foreign’ ITS sequences. As homogenization can work into maternal or paternal direction the outcome of hybridization might be completely maternal-derived rDNA, completely paternal-derived rDNA, simultaneous presence of both rDNA types (for younger hybrids only), or a mosaic

A NEW SPECIES OF ALLIUM SCHISTICOLA Phytotaxa 450 (3) © 2020 Magnolia Press • 253 pattern in the rDNA combining parts of both parental sequence types. A final answer regarding the occurrence of hybridization is not possible with the markers we employed here but would need additional genome-wide sequence data to detect or exclude the presence of traces of introgressed genomic material. Chromosome number and karyotype features:—Our data shows that this is a diploid species with a chromosome base number of x = 8. Its karyotype is symmetrical in chromosomes length and centromere position (Table 2 & Figs 5 a & b). 2n = 16 is the prevalent chromosome number in A. subg. Melanocrommyum (Dolatyari et al. 2018; Friesen et al. 2006; Fritsch & Astanova 1998). The karyotype features of the new species are very similar to the karyotypes of the studied members of A. subg. Melanocrommyum (Dolatyari et al. 2018). Its chromosome complement includes 14 non-satellited chromosomes with centromeres at median positions and two sub-median satellited chromosomes of ‘type P’ that are the smallest chromosomes (Dolatyari et al. 2018; Table 2, Fig. 5a & b). A detailed comparison of the calculated asymmetric parameters for the new species (Table 2) with the data of tables 2a & 2b published by Dolatyari et al. (2018) pointed to Allium egorovae Agababian & Oganesian (2000: 95), A. iranshahrii R.M.Fritsch in Fritsch & Abbasi (2013: 69), and A. akaka subsp. akaka as karyologically most similar species.

TABLE 2. Chromosome number and karyotype features of the studied species (Abbreviations: x Basic Chromosome Number; 2n Somatic Chromosome Number; Sat. Chr. No. Number of Satellited Chromosomes; THL Total Haploid Length;

MCA Mean Centromeric Asymmetry; CVCL Coefficient of Variation of Chromosome Length; CVCI Coefficient of Variation of Centromeric Index, m Median centromere chromosome, smsatP submedian satellited chromosome of type P).

Taxon x 2n Karyotype formula Sat. Chr. No. THL MCA CVCL CVCI

Allium schisticola 8 16 14m + 2smsatP 2 91.43 19.19 11.67 7.31

TABLE 3. Meiotic characterization of the studied accession.

Chromosome Mean chiasmata % Irregularities in % Irregularities No. of examined Plants configurations in frequency per cell anaphase I – telophase I in anaphase II-telophase II Metaphase I

6 8II, 7II + 2I 16.91 6.03 Not calculated

Male meiosis and chromosome behavior:—The inflorescence structure (-like cymes which include many flowers at different developmental stages) and the large chromosome size of Allium species enable researcher to easily examine the behavior of chromosomes at cycles I and II of meiotic division. We examined the young inflorescences of eight plants, but finally, meiotic results were attained for six individuals (Table 3). The gametic chromosome number of the studied material is n = 8. We found predominantly eight bivalents at prophase and metaphase I (Fig. 5c), but in some cells seven bivalents and two univalents (Fig. 4d) were also observed. The anthers of one flower of the second examined plant showed various chromosome configurations at metaphase I (uni- to multivalents). The chiasma frequency was estimated for 12 cells at diakinesis and metaphase I. The mean chiasmata frequency per each cell was 16.91 which means that each bivalent was associated by at least two chiasmata prior to segregation at anaphase I. The segregation of homologous chromosomes is mostly normally (8 - 8) at anaphase I - telophase I (Fig. 5e)/ anaphase II - telophase II, but abnormal meiocytes also were detected (Fig. 5f). Lagged chromosomes, chromatin bridges, and micronuclei were rarely observed abnormalities. Altogether 646 PMCs at anaphase I - telophase I stages were investigated, of which, on average, 6.03% of the cells showed one or two of the aberrations mentioned above. The percent of irregularities varied from 3.03% to 8.85% between the examined individuals. The meiotic chromosome behavior of the new species is very similar to meiosis in the studied members of A. subg. Melanocrommyum (A. Dolatyari, unpublished data).

Acknowledgements

We are grateful to Maryam Behroozian and Khadijeh Motahari (Ferdowsi University of Mashhad) for preparing the geographical map. We also thank Hossein Ramezani for his assistance in field surveys.

254 • Phytotaxa 450 (3) © 2020 Magnolia Press DOLATYARI ET AL. References

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