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Review Article
A new species of Ranunculus (Ranunculaceae) from Western Pamir-Alay, Uzbekistan
expand article infoNatalia V. Shchegoleva‡, Elena V. Nikitina§, Inom J. Juramurodov|, Andrei A. Zverev#, Orzimat T. Turginov|, Anvarbek M. Jabborov|, Ziyoviddin Yusupov¤«, Davron B. Dekhkonov¤, Tao Deng«, Hang Sun«
‡ Tomsk State University, Tomsk, Russia
§ Laboratory of Cadastre and Monitoring of Rare Plant Species, Institute of Botany of the Academy of Sciences of the Republic of Uzbekistan, Tashkent, Uzbekistan
| Laboratory Flora of Uzbekistan, Institute of Botany of the Academy of Sciences of the Republic of Uzbekistan, Tashkent, Uzbekistan
¶ University of Chinese Academy of Sciences, Beijing, China
# Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia
¤ Laboratory of Molecular Phylogeny and Biogeography, Institute of Botany of the Academy of Sciences of the Republic of Uzbekistan, Tashkent, Uzbekistan
« Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China

Corresponding author: Inom J. Juramurodov ( ijuramurodov@mail.ru )

Corresponding author: Hang Sun ( sunhang@mail.kib.ac.cn )

Academic editor: Marco Pellegrini
© 2022 Natalia V. Shchegoleva, Elena V. Nikitina, Inom J. Juramurodov, Andrei A. Zverev, Orzimat T. Turginov, Anvarbek M. Jabborov, Ziyoviddin Yusupov, Davron B. Dekhkonov, Tao Deng, Hang Sun.
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.
Citation: Shchegoleva NV, Nikitina EV, Juramurodov IJ, Zverev AA, Turginov OT, Jabborov AM, Yusupov Z, Dekhkonov DB, Deng T, Sun H (2022) A new species of Ranunculus (Ranunculaceae) from Western Pamir-Alay, Uzbekistan. PhytoKeys 193: 125-139. https://doi.org/10.3897/phytokeys.193.70757
Open Access

Abstract

New data on the phylogeny of four rare and endemic species of Ranunculus L. sect. Ranunculastrum DC. of western Pamir-Alai, one of which is new to science, have been obtained. Ranunculus tojibaevii sp. nov., from the Baysuntau Mountains, Western Hissar Range of Uzbekistan, is described. The new species is closely related to R. botschantzevii Ovcz., R. convexiusculus Kovalevsk. and R. alpigenus Kom., but differs in the blade of the radical leaves, which is rounded-reniform, segments 3–5-dissected, each 2–5-partite with elongated, rounded apical lobes. A phylogenetic analysis, using both the nuclear ribosomal internal transcribed spacer (ITS) and cpDNA (matK, rbcL, trnL-trnF), was informative in placing R. tojibaevii in context with its most closely-related species. Discussion on the geographic distribution, updated identification key, a detailed description, insights about its habitat and illustrations are provided.

Keywords

Endemic, Hissar Range, Pamir-Alay, phylogenetic analysis, Ranunculales, Ranunculastrum

Introduction

Ranunculus L., the largest genus in the Ranunculaceae Juss., includes ca. 600 genetically diverse species (Tamura 1995). The genus is distinguished by its high ecological-geographical diversity over a wide zonal spectrum ranging from the Arctic tundra through varied forests, steppes, deserts to exclusively aquatic habitats and high-altitude communities on nearly all continents (Paun et al. 2005). However, the main centres of speciation of Ranunculus are often in large mountain systems, where the formation of species is not only directly related to isolation, but also significantly depends on specific conditions of the highlands (Emadzade et al. 2015; Fernández Prieto et al. 2017; Shchegoleva 2018; Shchegoleva et al. 2020; Zverev et al. 2020).

More than 90 species of Ranunculus are distributed in Central Asia (Kovalevskaya 1972). Differentiation in the genus and the formation of locally endemic species are closely related to the history of the Tian Shan and Pamir-Alay Mountain formations. Here, more than half of the members of the genus are autochthonous representatives of the mountainous Central Asian flora, which arose in the process of regional adaptive diversification from ancient Mediterranean predecessors (Ovchinnikov 1971; Kamelin 1973). In the dry Central Asian seasonal climate, the features of these species are observed only in the short-term hydrothermal period of active vegetative growth.

Ranunculus tojibaevii was first discovered in 2013 on the Baysuntau Highlands (Khodzha-Gurgur-ata Mountain) on the south-western spur of the Hissar Range (Pamir-Alay). The populations were detected again in 2019 while working on the Flora of Uzbekistan Project (Sennikov et al. 2016). It should be noted that the flora of the Western Pamir-Alay is characterised by a high taxonomic diversity (Kamelin 1973; Vasilchenko and Vasileva 1985; Tojibaev et al. 2016; Makhmudjanov et al. 2019; Yusupov et al. 2020).

The morphological features indicated that the unknown plants belonged to R. subg. Ranunculus sect. Ranunculastrum (Hörandl and Emadzade 2012; Baltisberger and Hörandl 2016). The main differences between sect. Ranunculastrum and other sections of Ranunculus are the presence of a triangular beak equal to or longer than the achene body, a receptacle completely glabrous, a taproot partly tuberous and a mostly elongate fruit (Hörandl and Emadzade 2012).

The unknown plants closely resembled R. botschantzevii Ovcz. (Ovchinnikov 1941) and R. convexiusculus Kovalevsk. (Kovalevskaya 1972), as well as R. alpigenus Kom. (Komarov 1896) in their main morphological features. The molecular results presented here also clearly substantiated its independent taxonomic status. In this study, we present a morphological description of these plants, which we named Ranunculus tojibaevii Schegol. & Turginov. Figures showing its features, a map of its distribution, taxonomy and an identification key separating it from the most closely-related species are also provided.

Materials and methods

Morphological observations and measurements of R. tojibaevii were made on two populations; in total, 34 individuals were compared. Voucher specimens have been deposited in the National Herbarium of Uzbekistan – TASH (Tashkent, Uzbekistan). Additionally, two closely-related species, R. convexiusculus and R. botschantzevii, from the same territory and high-altitude regions were examined. Herbarium specimens at TASH, TAD, LE, FRU, AA, MW, LE and TK were also compared. Studies of closely related species were performed during field expeditions to Uzbekistan, Tajikistan and Kyrgyzstan, from 2017–2021 (Shchegoleva et al. 2020). The distribution map was generated in ESRI ArcGIS 10 software using GPS coordinates (www.esri.com). The conservation status was established, based on IUCN Criteria (IUCN 2019).

Molecular methods

DNA extraction, amplification and sequencing

DNA isolation was performed using a Plant Genomic DNA Kit (TIENGEN Biotech, Beijing, China) according to the manufacturer’s protocol. CTAB extraction protocol with some modifications was used to extract genomic DNA from herbarium specimens of R. alpigenus (Doyle and Doyle 1987).

Selected nuclear DNA regions ITS1-ITS2 (for herbarium specimen R. alpigenus) and ITS1-ITS4; plastid DNA regions matK, rbcL and trnL-F were amplified on a thermal cycler (BioRad) using the 2X PCR Taq Plus MasterMix with dye (Applied Biological Materials Inc., Canada). Amplification of the DNA regions was carried out by using primers of the forward and reverse primer sets (TsingKe, China) (Table 1).

Table 1.
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Primers used in this study.

Primer name Sequences (forward / reverse) DNA fragment size, bp Primer source
ITS1-18S ITS4-26S 5’-TCCGTAGGTGAACCTGCGG-3’ 5’-TCCTCCGCTTATTGATATGC-3’ ~ 700 bp White et al. (1990)
ITS1 5’-TCCGTAGGTGAACCTGCGG -3’ ~ 650 bp White et al. (1990)
ITS2 5’-GCTGCGTTCTTCATCGATGC-3’
matK-390F matK-1326R 5’-CGATCTATTCATTCAATATTTC-3’ 5’-TCTAGCACACGAAAGTCGAAGT-3’ ~ 900 bp Cuenoud et. al. (2002)
trnL-F_F trnL-F_R 5’-CGAAATCGGTAGACGCTACG -3’ 5’-ATTTGAACTGGTGACACGAG-3’ ~ 900 bp Taberlet et al. (1991)
rbcLaF rbcLaR 5’-ATGTCACCACAAACAGAGACTAAAGC-3’ 5’-GTAAAATCAAGTCCACCRCG-3’ ~ 600 bp Kress and Erickson (2007)

To obtain sequences of the genes of interest, PCR amplification was carried out according to the following parameters (except R. alpigenus): for ITS1-ITS4, initial denaturation for 3 min at 94 °C, followed by 35 amplification cycles: 30 s at 94 °C, 30 s at 50–54 °C, 1 min at 72 °C; elongation 7 min at 72 °C; for matK– an initial denaturation for 3 min 94 °C, followed by 35 amplification cycles: 30 s 94 °C, 1 min 51 °C, 1 min 72 °C; final extension 10 min at 72 °C; for rbcL, an initial denaturation for 4 min 95 °C, followed by 34 amplification cycles: 1 min 94 °C, 1 min 50 °C, 1 min 72 °C; final extension 10 min at 72 °C; for trnL-F, an initial denaturation for 3 min 94 °C, followed by 32 amplification cycles: 45 s 94 °C, 45 s 50 °C, 1 min 72 °C; final extension 8 min at 72 °C.

PCR amplification for R. alpigenus was performed for ITS1-ITS2, with the following programme: initial denaturation at 94 °C/5 min; 35 amplification cycles at 94 °C/30 s, at 54 °C/30 s, at 72 °C/ 45 s; elongation at 72 °C/7 min; for rbcL, 94 °C/4 min, 34 cycles: 94 °C/30 s, 54 °C/ 45 s, 72 °C/45 s; final extension at 72 °C/10 min.

Taxon sampling

To determine the taxonomic status and systematic position of R. tojibaevii, we sampled 24 species of Ranunculus. New nDNA (ITS) and cpDNA intergenic spacers (matK, rbcL, trnL-trnF) sequences for nine species were generated. We also used available sequences of 15 Ranunculus species from GenBank (www.ncbi.nlm.nih.gov/Genbank) (Table 2). We used Ranunculus subgenus Auricomus as the outgroup (Hörandl and Emadzade 2012; Almerekova et al. 2020).

Table 2.
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Accession numbers of samples used for phylogenetic analyses of Ranunculus (* newly-generated sequences).

Species GenBank accession number
ITS matK rbcL trnL-F
Ranunculus acris AY680167 AY954199 HQ590232 –
R. alpigenus* OM283824 – OM287560 –
R. arvensis HQ650550 HQ650551 MK925091 –
R. auricomus FM242803 FM242739 JN893758 –
R. botschantzevii* MW540744 MW748677 MW748685 MW748693
R. convexiusculus* MW540743 MW748676 MW748684 MW748692
R. flammula AY680185 AY954204 MK526480 –
R. glaberrimus KP687273 JF509974 MG247649 –
R. inamoenus KP687279 KP687302 MG249011 –
R. japonicus EU591982 AY954200 MH657741 DQ410744
R. leptorrhynchus* MW737444 MW748673 MW748681 MW748689
R. linearilobus* MW737445 MW748674 MW748682 MW748690
R. lingua AY680184 AY954206 JN892742 –
R. muricatus DQ410718 AY954191 HM850296 DQ410740
R. paucidentatus* MW540747 MW748679 MW748687 MW748695
R. pygmaeus KP687287 KP687310 KC483860 –
R. regelianus* MW737446 MW748675 MW748683 MW748691
R. repens MT271835 HM565166 MK925397 EU382995
R. sceleratus MT271836 GU257993 AB517148 DQ410746
R. sulphureus JF509969 JF509983 KC483870 –
R. talassicus* MW540748 MW748680 MW748688 MW748696
R. tojibaevii* MW540745 MW748678 MW748686 MW748694
R. tojibaevii* OM278385 OM287558 OM287559 –
R. trichophyllus KC620483 AY954133 L08766 –
R. turneri FM242817 FM242741 MG249550 –

Phylogenetic analyses

Sequence alignments were performed using ClustalW (Thompson et al. 2002) as implemented in MEGA X software (Kumar et al. 2018). The best partitioning scheme for the combined dataset contained two partitions: the ITS data; and the three plastid sequences data (matK, rbcL, trnL-trnF). Phylogenetic reconstruction was first conducted separately, based on the nuclear and the plastid data. Visual inspection determined that differences between the nuclear and the plastid trees were solely due to resolved/collapsed clades. No topological incongruence with a high support value (posterior probabilities and bootstrap percentages) was found. To further test whether the nuclear and plastid data could be combined for phylogenetic reconstruction, the incongruence length difference (ILD, Farris et al. 1995) test was conducted in PAUP* 4.0a169 (current) by using only the informative sites, heuristic search, tree-bisection-reconnection (TBR) branch-swapping algorithm, simple addition sequence and 1,000 replicates. The ILD test between the nuclear and the plastid data found p = 0.322, indicating insignificant support for incongruence between the two datasets. Therefore, the nuclear and the plastid sequences were combined into one dataset for phylogenetic analyses using SequenceMatrix software (Vaidya et al. 2011).

Phylogenetic trees were reconstructed using Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian Inference (BI). For ML, we employed raxmlGUI 2.0 (Edler et al. 2020), with 1,000 bootstrap replicates and, for BI, we used MrBayes v.3.1.2 (Huelsenbeck and Ronquist 2001) with 10,000,000 generations with random trees sampled every 1,000 generations. In the latter analysis, after discarding the first 25% trees as burn-in, a 50% majority-rule consensus tree was constructed from the remaining trees to estimate Posterior Probabilities (PP). For analyses, a model of nucleotide substitution was selected, based on the Akaike Information Criterion (AIC) using jModelTest2 on XSEDE (www.phylo.org). Phylogenetic analyses were also performed with the MP method using PAUP* 4.0a169. The MP bootstrap analysis was performed with heuristic search, TBR branch-swapping, 1,000 bootstrap replicates, random addition sequence with ten replicates, a maximum of 1,000 trees saved per round. Trees were visualised in FigTree v.1.4.0 (Rambaut 2012).

Results and discussion

The phylogenetic tree, based on the nuclear and plastid sequences (Fig. 1), showed that R. tojibaevii is sister to R. convexiusculus and R. botschantzevii with high support values PP = 1, MP = 94% and ML = 94%. Ranunculus tojibaevii, R. convexiusculus and R. botschantzevii formed a clade with well supported values (PP = 0.8, MP = 71% and ML = 64%).

Figure 1. 

Bayesian tree based on combined nuclear (ITS) and plastid (matK, rbcL, trnL-trnF) sequence data showing phylogenetic position of Ranunculus tojibaevii Schegol. & Turginov in R. sect. Ranunculastrum. Bayesian Posterior Probability (PP) / Maximum Parsimony (MP) is given on each branch, respectively; Maximum Likelihood (ML) is below branches. The classification is according to Hörandl and Emadzade (2012). * denotes the Ranunculus species analysed in this study. The new species is highlighted in bold.

The results of the phylogenetic analysis were similar to the results of Hörandl and Emadzade (2012) and Almerekova et al. (2020). Species of R. sect. Ranunculastrum are of particular interest. In our case, these native Asian species are mainly limited to the foothills and mountains of Central Asia (southern Kazakhstan, Uzbekistan, Kyrgyzstan, Tajikistan and Turkmenistan), as well as to the mountains of Afghanistan, Iran and Pakistan.

It is obvious that R. sect. Ranunculastrum in Central Asia is heterogeneous. The species forming sub-clusters in this section differ in their area of distribution, altitude confinement and time of origin, thereby confirming the neoendemic nature and origin of R. tojibaevii.

Taxonomy

Ranunculus tojibaevii Schegol. & Turginov, sp. nov.

urn:lsid:ipni.org:names:77296907-1
Figs 2, 3

Diagnosis

Similar to R. botschantzevii, R. convexiusculus and R. alpigenus morphologically, but differing in the rounded-reniform radical leaves dissected into 3–5 segments, each 2–5-partite into elongated lobes rounded at the apex (Fig. 4). Ranunculus tojibaevii differs from R. alpigenus in having fewer levels of leaf blade dissection. It differs from R. convexiusculus in the dissection of the radical leaf blades, larger flowers and having somewhat white, bristle-like hairs on the root collar and also from R. botschantzevii by the rounded apical lobes of the basal leaves and more xeromorphic habit.

Figure 2. 

Ranunculus tojibaevii Schegol. & Turginov (Shchegoleva, Jabborov, Turginov, holotype TASH-003743).

Type

Uzbekistan. Hissar Ridge, Baysuntau, Khodzha-Gurgur-ata Mountains, vicinity of the Village Gumatag, 38°22.2888'N, 67°21.0834'E, 2482 m a.s.l., 30 May 2019, N. Shchegoleva, A. Jabborov, O. Turginov (holotype: TASH-003743; isotypes: TASH-003748, TASH-003749, TASH-003750, TK-002339).

Figure 3. 

Ranunculus tojibaevii Schegol. & Turginov. Habitat (A flowering B fruiting) and flower (C). Scale bar: 1 cm.

Description

Herbs perennial. Roots dimorphic, some roots subulate, up to 0.5 mm thick, storage roots palmately-thickened, ca. 2.5 mm thick; root collar with milk-white bristle-like hairs. Stems 10–12(–15) cm tall, up to 2 mm diam., erect, branched, pubescent with white curly hairs, 1–3-flowered. Leaves dimorphic, radical leaves 2–3, 1.6–2 × 1.5–2.2 cm, blade rounded-reniform, 3–5-dissected, segments 2–5-partite, elongate, lobes apically rounded; cauline leaves 1–2, petiole short, slender, blade trisected, lobes 0.6–0.9 × 0.1–0.2 cm, oblong-lanceolate. Flowers 1.6–2.4 cm diam., sepals 0.4–0.6 mm long, ovate-concave, sparsely white pubescent; petals 0.9–1.2 cm long, well-developed, ovate, apex rounded. Infructescence globose-ovoid; receptacle oblongoid, glabrous; achenes 1–1.8 mm long, with white bristle-like hairs; beak hamate-curved.

Figure 4. 

Series of basal leaves in related species A Ranunculus tojibaevii (from the holotype) B R. alpigenus C R. botschantzevii D R. convexiusculus. Scale bar: 1 cm.

Specimen seen (paratype)

Uzbekistan. Pamir-Alay, South-western spurs of the Hissar Ridge, Baysuntau, vicinity of the Village Gumatag, amongst the stones, 4 June 2013, O. Turginov (TASH-003754).

Phenology

Flowering in May. Fruiting in May and June.

Distribution

Ranunculus tojibaevii is distributed in the Khodzha-Gurgur-ata of the Baysuntau Mountains area of Hissar Ridge (Fig. 5). The closely-related R. convexiusculus is endemic to Central Asia and R. botschantzevii is endemic to the Western Pamir-Alay.

Figure 5. 

Distribution of Ranunculus tojibaevii, R. alpigenus, R. botschantzevii and R. convexiusculus.

Habitat and plant associations

Ranunculus tojibaevii is rupicolous on southern and western exposed limestone outcrops and in cracks and crevices of large boulders at 2,450–2,500 m. a.s.l. The region is alpine and rather xerophytic. The common taxonomic composition of phytocenosеs includes Cerasus amygdaliflora Nevski (Rosaceae), Corydalis ledebouriana Kar. & Kir. (Papaveraceae), Cousinia regelii C.Winkl. (Asteraceae), Eremurus regelii Vved. (Asphodelaceae), Gagea gymnopoda Vved. (Liliaceae), Iris khassanovii Tojibaev & Turginov, I. parvula (Vved.) T.Hall & Seisums, I. stolonifera Maxim. (all Iridaceae), Rheum maximowiczii Losinsk. (Polygonaceae), Tulipa lanata Regel (Liliaceae) and Ziziphora pamiroalaica Juz. (Lamiaceae).

Conservation status

Ranunculus tojibaevii is a local, narrowly distributed endemic, represented by two or three individuals per m2 within an area of < 500 m2. It should therefore be assigned the status EN (Endangered), Criteria B1 ab(i, ii, iii)+ B2 ab(i, ii, iii), following the IUCN Standards and Petitions Committee (IUCN 2019).

Notes

Ranunculus tojibaevii differs from closely-related species by its habitat on well-heated limestone outcrops, as well as in cracks and crevices of large boulders at ca. 2,500 m a.s.l., which is atypical of related species. Ranunculus convexiusculus is on clayey-stony soil, less often on slopes of fine earth, at 2,000–2,600 m a.s.l. Ranunculus botschantzevii is hygrophilous in wet mountain meadows with melting snow and on slopes of fine clayey soil at 2,400–3,500 m a.s.l. Ranunculus alpigenus grows on slopes of fine soil of the alpine belt at 2,800–4,000 m a.s.l. All these species are endemic to the western Pamir-Alay. The vicariant species to R. alpigenus is R. badachschanicus Ovcz. & Koch. from the western Pamirs.

Etymology

Ranunculus tojibaevii is named after Komiljon Tojibaev, a leading botanist, professor and academician from Uzbekistan who actively promotes the botanical sciences in Central Asia.

Table 3.
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Comparison of R. tojibaevii, R. botschantzevii, R. convexiusculus and R. alpigenus.

R. tojibaevii R. botschantzevii R. convexiusculus R. alpigenus
Blade of radical leaves rounded-ovate, 3–5-dissected, segments further 2–5-dissected, lobules elongated, rounded at apex triangular-reniform, 3–5-partite, segments narrowly cuneate, unequally and subacutely dentate reniform, dissected 1/3 to nearly 1/2 of its length, lobes broad incised-dentate broadly ovate, dissected into pinnatipartite segments, segments tripartite, terminal lobules oblong
Sepals narrowly elliptic, concave, less than half as long as petals, with long reclinate hairs elliptic, concave, some shorter than the petals, with sparse, long reclinate hairs elliptic, concave, half as long as petals, with short reclinate spreading hairs elliptic, concave, with scattered hairs
Petals oblong-obovate, greenish-yellow, base cuneate, margin undulate obovate, bright yellow, becoming dark when dry, base broadly cuneate, margin undulate very broadly ovate, golden yellow, base cuneate, margin slightly undulate oblong-ovate, yellow-green, base narrowly cuneate, marginundulate
Achenes 1.0–1.8 mm long, asymmetrically ovate, slightly convex, with semi-appressed hairs 2.2–2.5 mm long, oblong, slightly laterally compressed, with appressed hairs 1.8–2.5 mm long, oblong, slightly convex, with appressed hairs 1.5–2.0 mm long, asymmetrically obovate, laterally compressed, with scattered not appressed hairs

Key to Ranunculus tojibaevii and similar species (Table 3)

1 Blades of basal leaves broadly ovate, dissected into pinnatipartite segments, with tripartite-oblong terminal lobules R. alpigenus
– Radical leaves 3–5-dissected or lobed-incised 2
2 Blade of radical leaves 1/3 or nearly 1/2 unequally partite into broad incised-dentate lobes R. convexiusculus
– Blade of radical leaves 3–5-dissected 3
3 Blade of basal leaves triangular-reniform, 3–5-dissected almost to the base, wedge-shaped segments, unequally sharp-toothed R. botschantzevii
– Blade of radical leaves is round-reniform, 3–5-dissected, each section divided into 2–5 elongated lobules; apex of lobules rounded R. tojibaevii

Acknowledgements

This study was supported by the International Partnership Program of the Chinese Academy of Sciences (151853KYSB20180009), the framework of the Second Tibetan Plateau Scientific Expedition and Research (STEP) program (2019QZKK0502), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA20050203), the Key Projects of the Joint Fund of the National Natural Science Foundation of China (U1802232), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2019382), Yunnan Young & Elite Talents Project (YNWR-QNBJ-2019-033), the Chinese Academy of Sciences “Light of West China” Program, the state research project “Taxonomic revision of polymorphic plant families of the flora of Uzbekistan” (FZ-20200929321), the State Programs for 2021–2025 years “Grid mapping of the flora of Uzbekistan” and “The creation of DNA bank and barcoding of endemic plants of Uzbekistan”, as well as in accordance with the current State Assignment of Central Siberian Botanical Garden at Siberian Branch of Russian Academy of Sciences (AAAA-A21-121011290026-9).

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