Using DNA Data to Determine the Taxonomic Status of Ammopiptanthus Kamelinii in Kyrgyzstan (Thermopsideae, Leguminosae)

https://doi.org/10.11646/phytotaxa.360.2.2

Using DNA data to determine the taxonomic status of Ammopiptanthus kamelinii in Kyrgyzstan (Thermopsideae, Leguminosae)

WEI SHI1, 2, ZHIHAO SU1, 2, BORONG PAN1, 2 * & SHIXIN WU3 1Key Laboratory of Biogeography and Bioresources in Arid Land, Xinjiang Institute of Ecology and Geography, The Chinese Academy of Sciences, Xinjiang, 830011 Urumqi, China 2Turpan Eremophytes Botanic Garden, The Chinese Academy of Sciences, Xinjiang, 838008 Turpan, China 3Xinjiang Institute of Ecology and Geography, The Chinese Academy of Sciences, Xinjiang, 830011 Urumqi, China *Corresponding author ([email protected])

Abstract

The taxonomic status of Ammopiptanthus kamelinii has been unresolved because of numerous characters that make it similar to another closely related species, A. nanus. In this study, we set out to resolve the complex relationships among A. kamelinii using samples from three populations, with a total of 38 individuals. Phenotypic indices (plant height, canopy, and leaf characters) and DNA data (ITS 1–4 nrDNA markers, as well as trnH-psbA, trnL-trnF, and trnS-trnG cpDNA markers) were used to understand the controversial taxonomic status of A. kamelinii. The phenotypic characters of A. kamelinii did not show a significant difference from A. nanus, and the nrDNA data did not reflect any variability from A. nanus, but all the individuals of A. kamelinii in Kyrgyzstan showed two new haplotypes in the cpDNA. When the genetic data were combined, specimens of A. kamelinii clustered together with A. nanus; therefore, we have confirmed that A. kamelinii cannot be recognized as a separate species of A. nanus and should be merged with the latter species. The description of A. nanus is revised here.

Key words: Eudicots, Ammopiptanthus nanus, Tianshan Mountains, identification, molecular phylogenetic, morphology

Introduction

As the only broad-leaved evergreen angiosperm genus in the deserts of eastern Central Asia, Ammopiptanthus Cheng f. (Fabaceae) has been recognized as a tertiary relict genus (Fu 1992). Because of its habit and harsh living environment, Ammopiptanthus species have been of great interest for breeding research, and some genes have been cloned from A. mongolicus and A. nanus for their physiological characters. Specifically, the molybdenum cofactor sulfurase gene (Yu et al. 2015) and betaine aldehyde dehydrogenase (BADH) have been cloned from A. nanus (Yu et al. 2014). Additionally, a new dehydration responsive element binding protein (DREB), transcription factor gene (Li et al. 2015), cold-responsive gene (Gu & Cheng 2014), and cold-induced galactinol synthase gene (Gao et al. 2015, Song et al. 2013) in A. mongolicus have been cloned and identified. Due to the interest in genes cloned from this genus, it is important to clarify the species boundaries in order to better conserve the species richness. There are two resolved species-level taxa in Ammopiptanthus (Shi et al. 2017): A. mongolicus and A. nanus, although they have been merged into one species in the Flora of China (Wei & Lock 2010). Ammopiptanthus mongolicus and A. nanus were resolved as separate species based upon morphological, karyotypic, and DNA data (ITS 1–4 nrDNA markers, as well as trnH-psbA, trnL-trnF, and trnS-trnG cpDNA markers) (Shi et al. 2017). Lazkov (2006) described a new species in Kyrgyzstan, Ammopiptanthus kamelinii Lazkov, yet the species remains unresolved. The type specimen of A. kamelinii is not significantly distinct from A. nanus, although its type locality is outside the main range of A. nanus. Therefore, this study focuses on determining if A. kamelinii is in fact a new species. Ammopiptanthus species are found in stony and/or sandy desert habitats. The distribution of A. nanus is mainly in Ulugqat County in western Xinjiang Province, China, growing in a narrow altitudinal strip between 1,800 m and 2,800 m (Shi 2009, Su et al. 2016, Wei & Lock 2010, Zhang 2007). Both A. nanus and A. kamelinii are distributed in the central Tianshan Mountains (Lazkov 2006, Li 2011, Wei & Lock 2010) but in different mountain ranges (Fig. 1). The populations of A. nanus in Ulugqat County are mainly narrowly distributed in the Alay Mountains, while

Accepted by Alexander Sennikov: 22 Jun. 2018; published: 13 Jul. 2018 103 A. kamelinii in Kyrgyzstan is distributed in the Moldotau Mountains (Lazkov 2006, Li 2011). Compared with the narrow distribution of A. nanus in Ulugqat County, a compact area of A. kamelinii includes very few localities closely situated west of Lake Sonkel, between the Kekemeren, Jumgal, and Naryn Rivers. In contrast, A. mongolicus is mainly distributed in the Alxa Desert in Inner Mongolia, China, and extends north to southern Mongolia, where it is widely distributed (Zhang et al. 2006, Zhang 2007, Shi 2009, Li 2011, Ma et al. 2012, Shi et al. 2017). The taxonomy of A. kamelinii, which was added to the genus as a distinct species (Lazkov 2006, Shi et al. 2017), is problematic as its morphological characters and habitat range are very similar to A. nanus. Thus, the aim of this paper is to resolve the relationship of A. kamelinii within Ammopiptanthus, either supporting it as a distinct species or combining it with a previously resolved species.

FIGURE 1. The geographical distribution of A. nanus (Popov) S. H. Cheng (seven populations) and A. kamelinii Lazkov (three populations).

Materials and Methods

From August 11th to 15th, 2017, three populations including 38 individuals of A. kamelinii were found under the guidance of Prof. Lazkov, in the Moldotau Range of the Tianshan Mountains in Kyrgyzstan. Twelve or 13 individuals were collected per population. Fresh leaves from the 38 individuals were collected and dried in silica gel, and if a fruit was present on the individual, it was collected as well. The latitude, longitude, and altitude were recorded during sampling (Table 1), and the habitat of A. nanus in China was consistent with that described in previous studies (Table 2 in Shi et al. 2017; Table 2 in Su et al. 2016).

1. Phenotyping Calipers were used to measure leaf length (LL) and width (WL), and leaf size (LS, LS = LL×LW) and leaf shape (LSP, LSP = LL/LW) were calculated based on the LL and WL. Total plant height (H) and canopy coverage area (CA) were recorded for the aboveground portion of each individual. See the morphological indices and data listed in Table 3. Statistical analyses were performed using Excel 2010 and SPSS v.15.0 (SPSS Inc., Chicago, USA). Quartile plots were drawn using Origin 8.5 (Origin Lab. Wheeling, IL USA), and the results are shown in Fig. 2.

104 • Phytotaxa 360 (2) © 2018 Magnolia Press SHI ET AL. 1111~M-1120 N-1061~N-1070 (in TURP, collector: B. R. Pan) TURP, (in KU178944 M-1091~M-1100 KU178946KU178946 M-1131~M-1140 M-1141~M-1150 KU178943 MH499255 N-1096~N-1108 Lazkov, and GenBank accession numbers of Lazkov, KU178936 KU178942 N-1021~N-1030 KU178939 KU178940 KU178940 MH499254 A. kamelinii trnH–psbA trnL–trnFtrnH–psbA trnS–trnG KU178935 GenBank accession number Voucher ITS KU178933KU178933 KU178936KU178933 KU178936 KU178938KU178933 KU178936 KU178938 KU178944KU178933 KU178936 KU178938 KU178944KU178933 KU178936 KU178938 KU178944KU178933 KU178936 KU178938 KU178944 M-1001~M-1010 KU178933 KU178936 KU178938 KU178944 M-1011~M-1020 KU178933 KU178936 KU178938 KU178944 M-1021~M-1030 KU178933 KU178936 KU178938 KU178944 M-1031~M-1040 KU178936 KU178938 KU178944 M-1041~M-1050 KU178933 KU178938 KU178944 M-1051~M-1060 KU178933 KU178936 M-1061~M-1070 KU178933 KU178936 KU178938 M-1071~M-1080 KU178933 KU178936 KU178940 KU178944 M-1081~M-1090 KU178936 KU178941 KU178945KU178933 KU178941 KU178946 KU178936 M-1101~M-1110 KU178933 KU178941 M- KU178932 MF444199 M-1121~M-1130 KU178932 KU178934 MF444205KU178932 KU178934 KU178936 KU178944 KU178934 KU178936 KU178942KU178932 KU178942KU178932 KU178934KU178932 KU178934 KU178936 N-1001~N-1010 KU178932 KU178934 KU178936 KU178942 s.n. N-1011~N-1020 KU178934 KU178936 KU178942 KU178936 KU178942 KU178942 N-1031~N-1040 N-1041~N-1050 N-1051~N-1060 MH499251 MH499252MH499251 MH499252 MH499253MH499251 MH499253 MH499255 MH499252 MH499255 MH499253 N-1071~N-1082 N-1083~N-1095 (Popov) S.H. Cheng and § § § § § § § § § § § § § § § § § § § § § § § § § § A. nanus , (C) , (C) ‡ ‡ , 12(C) , 12(A) , 12(A) , 36(A) ‡ , 22(A) , 12(D) , 12(D) , 12(D) , 1(I) , 12(F) , 12(A) , 12(D) , 12(D) , 12(D) , 12(D) , 12(D) , 12(D) , 12(G) ‡ ‡ ‡ ‡ ‡ ‡ ‡ , 12(D) ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ , 11(A)1(C) , 21(D)3(E) , 8(G)4(H) , 34(A)2(B) ‡ ‡ , 10(G)2(H) ‡ ‡ ‡ , 13(b) , 13(b) † † , 1(a) , 12(b) † , 12(a) , 24(a) , 12(a) , 12(b) , 12(b) , 36(b) † , 22(b) , 12(a) , 12(a) , 12(a) , 12(a) , 12(a) , 12(a) , 12(a) , 12(a) , 12(b) † † † † † † , 24(a) † † † † † † † † † † † , 1 , 24(a) , 12(b) , 12(a) , 13 , 13 * † , 12(a) , 36(b) † † , 3 , 3 , 3 , 5 , 5 , 5 * * , 5 , 3 † † , 3 , 3 , 3 , 3 , 3 , 3 , 3 , 12 , 5 * * * * * * , 3 * * * * * * * * * * * * , 3 , 5 , 3 * , 3 , 5 * * * * phenotypes and genotypes (Maxim. ex Kom.) S.H. Cheng, 37.5° N 103.82° E 1921 m 3 37.5° N 103.84° E 1924 m 6 Ammopiptanthus mongolicus mountain southwest Location Latitude Longitude Elevation of individuals observed Number 23 2 Moldotau 3 Moldotau 41.71° N 74.35° E 41.94° N 1739 m 74.16° E 1693 m 12 12 67 Bayinkuluti Atushi 39.83° N 75.59° E 39.76° N 2109 m 76.39° E 2350 m 16 12 11 Bayinengeer15 40.24° N16 107.15° E Jingtai 1203 m (cultivated) Turpan 40.85° N 2 98.18° E – 80 m 1 1 1 Moldotau 41.71° N 74.34° E 1698 m 12 2345 Yinggen6 Rengenfeng7 Chulumiao8 Bianjing9 40.58° N 40.79° N Wuhai10 106.32° E 104.93° E Taole 41.69° N Jilantai m 1168 106.99° E 1330 m 42.08° N Dengkou12 1427 m Wulanbuhe 106.18° E 39.24° N14 10 926 m 106.82° E 6 38.43° N N 40.11° Beisi 24 40.49° N m 1108 106.57° E 40.05° N 105.69° E 106.86° E 106.78° E m 1168 Jingtai- 1078 m 1039 m 4 1091 m 3 2 18 6 38.98° N3 2 16 105.87° E 1762 m4 Kangsu5 Biaoertuokeyi 7 Heiziwei 39.5° N Tielieke 39.7° N 74.87° E 74.99° E 2551 m 39.8° N 2167 m 39.92° N 75.31° E 5 75.73° E 2395 m 11 2212 m 17 4 13 Jiergelang 37.99° N 105.25° E 1323 m 2 1 Yagan 41.63° N 103.22° E 1010 m 3 Number 1 Wuheshalu 39.66° N 74.75° E 2290 m 12 Population information of

(Popov) A. kamelinii Lazkov A. mongolicus (Maxim. ex Kom.) S.H. Cheng Species Population A. nanus S.H. Cheng Number of individuals observed in leaf characters. ITS genotypes. Number of individuals observed in ITS genotype. Lowercase letters parentheses refer to different haplotypes. cpDNA haplotype. Uppercase letters in parentheses refer to different Number of individuals observed in cpDNA the DNA sequences used in this study. the DNA * Number of individuals observed in plant height and canopy area. TABLE 1. TABLE † ‡ §

UNTANGLING THE TAXONOMIC RELATIONSHIPS OF AMMOPIPTANTHUS KAMELINII Phytotaxa 360 (2) © 2018 Magnolia Press • 105 TABLE 2. Comparisons the phenotypic characters in both of the references and observation. height of form of leaves, its apex size of lateral veins number of edge of plants leaves conspicuous folioles legumes or not A. mongolicus References 1.5–2.0 base broadly cuneate to rounded, 2–3.5 × no Often 1, straight apex obtuse or acute 0.6–2 occasional 3 Observation 0.09–2.4 base broadly cuneate to rounded, no Often 1, straight apex obtuse or acute occasional 3 A. nanus References 0.4–0.7 rhombic-elliptic or broadly 1.5–4 × yes Often 3, constricted elliptic to broadly ovate, 1–2.4 occasional 1 and uneven apex obtuse, often mucronate Observation 0.05–1.32 rhombic-elliptic or broadly yes Often 3, constricted elliptic to broadly ovate, occasional 1 and uneven apex obtuse, often mucronate A. kamelinii References 0.5–0.8 elliptic to rhombic-elliptic, or 1.5–4 × yes Often 1, straight broadly lanceolate, apex and base 1–2.0 occasional 3 attenuate Observation 0.05–1.21 rhombic-elliptic or broadly yes Often 3, constricted elliptic to broadly ovate, apex occasional 1 and uneven obtuse, often mucronate

TABLE 3. The phenotypic characters in Ammopiptanthus nanus and A. kamelinii. SE of Lower Upper N total Mean Variance Min. Med. Max. mean 95% 95% A. nanus 78 0.65 0.03 0.58 0.72 0.09 0.05 0.69 1.32 The plant height (H) (m) A. kamelinii 100 0.76 0.04 0.26 0.35 0.14 0.10 0.79 1.44 The canopy area (CA) A. nanus 78 1.28 0.15 0.97 1.58 1.80 0.0020 0.99 6.43 (m2) A. kamelinii 100 1.64 0.11 1.43 1.84 1.08 0.0092 1.50 3.47 The length of leaves (LL) A. nanus 700 25.05 0.17 24.73 25.38 19.46 14.41 24.72 41.57 (mm) A. kamelinii 300 25.95 0.44 25.09 26.79 58.48 14.05 25.91 41.65 The width of leaves (LW) A. nanus 700 15.68 0.11 15.47 15.90 8.19 7.23 15.66 24.01 (mm) A. kamelinii 300 15.37 0.31 14.80 15.98 27.39 6.26 15.36 24.67 The size of leaves (LS) A. nanus 700 400.80 4.85 391.72 410.30 45.88 144.57 403.05 903.64 (mm×mm) A. kamelinii 300 442.89 8.50 427.70 459.50 43.87 200.16 445.15 696.20 The shape of leaves (LSP) A. nanus 700 1.62 0.10 1.60 1.64 0.34 0.96 1.62 3.72 (mm/mm) A. kamelinii 300 1.98 0.03 1.91 2.04 0.51 0.97 1.99 3.00

mm mm mm mm 45 Leaf length 25 Leaf width Leaf size 1000 40

35 20 800

30 600 15 25

400 20 10 15 200

10 5 0 Ulugqat Moldota Ulugqat Moldota cm cm Ulugqat Moldota mm/mm cm 180 4.0 Leaf shape Plant height 45000 Cannopy area 160 3.5 140

3.0 120 30000

2.5 100

80 15000

Range 2.0 60 1.5 40 0 1.0 20

0.5 Ulugqat Moldota Ulugqat Moldota Ulugqat Moldota

FIGURE 2. Variation of taxonomic characters, including leaf traits (leaf length, width, form, and size), as well as plant height and canopy area in different populations of A. nanus (Popov) S. H. Cheng (seven populations) and A. kamelinii Lazkov (three populations).

106 • Phytotaxa 360 (2) © 2018 Magnolia Press SHI ET AL. Molecular phylogeny Total genomic DNA was extracted from dried leaf tissue using a modified CTAB method (Doyle et al. 1997, Rogers & Bendich 1985). The ITS region (ITS 1, 5.8S rDNA, ITS 2) was amplified and sequenced using primers ITS 1 and ITS 4 according to White et al. (1990), and the spacers trnH-psbA, trnS-trnG, and trnL-trnF were amplified and sequenced using the primers and protocols described in Sang et al. (1997), Shaw et al. (2005), and Taberlet et al. (1991), respectively. PCR products were purified using PCR Product Purification Kits from Shanghai SBS, Biotech Ltd., China. Sequencing reactions were conducted with the forward and reverse primers of the amplification reactions, using the DYEnamic ET Terminator Kit (Amersham Biosciences, Little Chalfont, Buckinghamshire, U.K.) with an ABIPRISM 3730 automatic DNA sequencer (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China). Electropherograms were edited and assembled using Sequencher 4.8 (Gene Codes, Ann Arbor, MI, USA). All new DNA data for the species was uploaded into Genbank, and the accession numbers for all samples are shown in Table 1. Sequences were initially aligned using ClustalX (Thompson et al. 1994) followed by manual adjustments using SeAl ver. 2.0 (Rambaut 2002). Phylogenetic analyses were conducted on each DNA region as well as on the combined nuclear and plastid data sets. Models were determined using jModelTest 2.1.4 (Darriba et al. 2012, Posada & Crandall 1998) for each dataset to determine the best fitting model of sequence evolution. Akaike information criterion (AIC) values were used to rank the best fit model for the Bayesian analyses. Phylogenetic relationships were inferred using Bayesian inference (BI) as implemented in MrBayes 3.2 (Ronquist et al. 2012). Maximum parsimony (MP) relationships were determined using PAUP version 4.0b10 (Swofford 2003). One cold and three incrementally heated Markov chain Monte Carlo (MCMC) chains were run for 2,000,000 generations. Trees were sampled every 100 generations. A partitioned Bayesian analysis of both the nuclear and the plastid datasets was also implemented by applying the previously determined models to each data partition (Brown & Lemmon 2007). For each dataset, MCMC runs were repeated twice to avoid spurious results. All Bayesian analyses produced split frequencies of less than 0.01, showing convergence between the paired runs. The first 2,000–5,000 trees were discarded as burn-in, and the remaining trees were used to construct majority-rule consensus trees. Furthermore, a network analysis was carried out considering the uncorrected p-distance between individuals and the same outgroup species from the Bayesian analyses, using SplitsTree 4.13.1 (Huson & Bryant 2006). Branch support was tested using bootstrapping (Felsenstein 1985) with 1,000 replicates.

Results

Phenotyping The qualitative description of taxonomic characters of A. nanus and A. kamelinii showed no significant differences (Table 2). A comparison between the descriptions in Flora of China (Wei & Lock 2010) and our observations (shown in Table 2 and Figure 2), showed that the minimum canopy area (CA) of our sample of A. nanus individuals is 0.002 m2 and the maximum is 6.43 m2, while the minimum height is 0.05 m and the maximum is 1.32 m. For A. kamelinii, the minimum CA is 0.0092 m2 and the maximum CA is 3.47 m2, while the minimum height is 0.10 m and the maximum is 1.44 m. The differences between these characters are not significant (P = 0.061). The two species cannot be distinguished based upon only these two characters. Similarly, the measured leaf characters were also not sufficiently different to distinguish A. nanus and A. kamelinii. The LL of A. nanus (14.41–41.57 mm; 25.05 ± 0.17 mm) is slightly longer than that of A. kamelinii (14.05–41.65 mm; 25.95 ± 0.44 mm), but the difference is not significant (P = 0.12). Additionally, the leaf width for A. nanus (7.23–24.01 mm; 15.68 ± 0.11 mm) is a little larger than that of A. kamelinii (6.26–24.67 mm; 15.37 ± 0.31 mm; P = 0.22). The other two leaf characters, LS (P = 0.06) and LSP (P = 0.58) also are not significantly different between the two species, though the LS of A. nanus (400.80 ± 4.85 mm×mm) is smaller than that of A. kamelinii (442.89 ± 8.50 mm×mm), and the LSP of A. nanus (1.62 ± 0.10 mm) is narrower than that of A. kamelinii (1.98 ± 0.03 mm). Thus, the characters of leaves cannot distinguish between the two species (Table 3 and Fig. 2).

Molecular phylogeny Phylogenetic analyses were performed based on the combined nuclear and plastid datasets (ILD test, P = 0.084). No SNPs were identified in the nrDNA data from the 38 A. kamelinii samples, and all sequences aligned with A. nanus. The cpDNA markers (trnH-psbA, trnL-trnF, and trnS-trnG) and ITS 1–4 were combined to analyze the phylogenetic

UNTANGLING THE TAXONOMIC RELATIONSHIPS OF AMMOPIPTANTHUS KAMELINII Phytotaxa 360 (2) © 2018 Magnolia Press • 107 relationships among A. kamelinii, A. nanus, and A. mongolicus. Two new haplotypes (X & Y), which occurred in all the populations of A. kamelinii, were identified in the combined cpDNA dataset (Table 2). The tree topology of the MP and BI trees was the same, so only the BI tree is shown here (Fig. 3). Ammopiptanthus is independent with a monophyletic branch, the new haplotypes (X & Y, Fig. 3) are merged onto the same branch, and the relationships are well supported (100% bootstrap support and 1.00 posterior probability). The putative clades correspond to A. mongolicus (PP = 0.98, BI; PP = 99, MP) and A. nanus (PP = 0.99, BI; PP = 100, MP). Ammopiptanthus nanus and A. kamelinii did not form individual clades of each respective species; instead, the sampled accessions of the two species were intermixed. As shown in the network analysis in Fig. 4, the haplotypes (X & Y) of A. kamelinii and A. nanus were clustered in the same group with a bootstrap value of 99.6.

Baptisia australis

Thermopsis barbata 0.52/77 Anagyris foetida

Anagyris latifolia 1/100 Thermopsis alpina 0.66/77 Thermopsis lanceolata

1/100 Piptanthus nepalensis

Piptanthus concolor

0.72/ 50 Piptanthus laburnifolius

Sophora davidii 1/100 1/99 Sophora flavescens Sophora tomentosa

Ammopiptanthus kamelinii X 1/100 1/100 Ammopiptanthus nanus A Ammopiptanthus nanus C

Ammopiptanthus nanus B 1/100 Ammopiptanthus kamelinii Y

Ammopiptanthus mongolicus G

Ammopiptanthus mongolicus H 1/98 Ammopiptanthus mongolicus F

Ammopiptanthus mongolicus D

Ammopiptanthus mongolicus E

0.02 FIGURE 3. The majority-rule consensus tree from Bayesian inference and RAxML of Ammopiptanthus S. H. Cheng and other related species in Thermopsidae based on ITS 1-4 and three cpDNA sequences (trnH-psbA, trnL-trnF, and trnS-trnG). The numbers above the branches are the posterior probabilities.

Discussion

The conservation of Ammopiptanthus species is a major concern as habitats are shrinking due to climate change and because of their limited initial range as endemic species. Ammopiptanthus mongolicum and A. nanus have been categorized as endangered (Ge et al. 2005, Wu & Wu 1996), because their reproduction in natural habitats is unfavorable. Thus, it was important to determine the taxonomic relationships of A. kamelinii with the rest of the genus to propose more effective conservation measures.

To better understand the taxonomy of A. kamelinii, we employed molecular systematic and morphological techniques, both of which supported the merger of A. kamelinii into A. nanus. Plant height and canopy for both species were similar, and the leaf form index showed that the leaves of A. nanus and A. kamelinii are both suborbicular, while those of A. mongolicus are subulate. The shape and the size of the leaves in both of A. nanus and A. kamelinii are similar (Table 3 and Fig. 2), differing from those of A. mongolicus. In addition, the pods, which are pubescent, have been observed for the first time (Table 3), and pubescent pods are considered common characters among all Ammopiptanthus species. These observations are consistent with previous descriptions by Li & Yan (2011) and Lazkov (2006). Other taxonomic features (Li & Yan, 2011; Table 1) were not analyzed in this paper, but may be useful as descriptive morphological characters in the future. A previous study on A. kamelinii and A. mongolicus by Lazkov (2006) described the leaflet number of the first species as usually one, occasionally three, and the legume margin as straight. However, in our study, the leaflet number of A. kamelinii was usually three, occasionally one, and the legume margin was constricted and uneven, similar to A. nanus (Table 2; Fig. 2). There is another difference in the pubescence of pods, which types were described and identified by Lazkov (2006) as a unique character in A. kamelinii, differing from A. mongolicus and A. nanus. The difference in pubescence (longer and lax in A. nanus, short and dense in A. kamelinii) is not profound and is not necessarily indicative of taxonomic difference at the rank of species. Furthermore, we have resolved the taxonomic status of A. kamelinii. The ITS sequences provided evidence to explore the taxonomic relationships in Ammopiptanthus within a geographic region, and the region may be considered a DNA barcode for the genus (Shi et al. 2017). All 38 individuals of A. kamelinii had the same ITS 1–4 haplotype (HA) as in A. nanus. The Bayesian analysis of the combined sequences of A. nanus and A. kamelinii (H-a) resulted to these species belonging to a well-supported clade, with well-supported differentiation from A. mongolicum (B) (Figs. 3 & 4). The ITS data from this study shows that A. nanus and A. kamelinii should be combined into one species. Our work, together with previous studies carried out with polymorphic cpDNA markers (Shi et al. 2017, Su et al. 2016, Zhang et al. 2015a), has shown higher values of genetic diversity in A. nanus (Su et al. 2016), and two new haplotypes (H-X & H-Y) were identified in the cpDNA in the three populations of A. kamelinii, which adds to the overall genetic diversity of A. nanus. Both of the two new haplotypes (X & Y) are well-supported along the same branch with the haplotypes (A, B & C), and are separated from the five other haplotypes (H-D to H-H) of A. mongolicum.

UNTANGLING THE TAXONOMIC RELATIONSHIPS OF AMMOPIPTANTHUS KAMELINII Phytotaxa 360 (2) © 2018 Magnolia Press • 109 In comparison with A. mongolicum, A. nanus has a narrow distribution in the Alay Mountains and Moldotau Mountains, which are both part of the the Tianshan mountain range (Lazkov 2006, Su et al. 2016), with a narrow range of habitats in high mountain regions. The distribution of the genetic variation of A. nanus in the Tianshan Mountains was consistent with the expected signatures, illustrating the wide distribution of a star-phylogeny pattern (Comes & Kadereit 1998, Hewitt 2000). We found that haplotype A was widespread across every sampled location of the region/group, and rare haplotypes B and C were limited to single populations of the valleys in the western Tianshan Mountains (Su et al. 2016). The new haplotypes X and Y also reflected a similar situation with haplotypes B and C in another valley of the western Tianshan Mountains. This genetic structure is usually considered to be influenced by the climate oscillations that have occurred since the late Quaternary (Hewitt 2004). There was a great deal of glaciation that developed at high altitudes of the Tianshan Mountains during the glaciation, and the extent of the glacial area varied in response to the alternation of glaciation and interglaciation (Shi et al. 1999). During the interglacial period, environmental conditions became warmer and, with the increasing temperature, a greater amount of run-off from melting glacial ice created a wetter environment in some valleys (Williams et al. 1993). The improved habitats were more suitable for recovery, thus the species would have thrived and expanded outwards. A previously proposed hypothesis speculated that A. nanus might have originated from a founder population containing only a small fraction of the genetic variation present in its progenitor species, and it might have experienced founder events or bottlenecks over its evolutionary history (Chen et al. 2009, Ge et al. 2005). Our study agreed with this hypothesis, as the variant of haplotype A might have advanced with the range expansion of the refugia, reaching a high frequency, and spreading to other suitable habitats in valleys of the western Tianshan Mountains, such as haplotypes X and Y. The maintenance of genetic diversity is a critical issue in the conservation of threatened species (Frankel 1983). Genetic drift and inbreeding are the most concerning genetic factors leading to a loss of genetic diversity in species with small population sizes and fragmented distribution patterns, sometimes pushing them into ‘‘extinction vortices’’ (Lande 1998). The populations of A. kamelinii should be added to A. nanus to conserve the relatively high amounts of the genetic diversity and rare genetic variations. Below, we formally merge A. kamelinii into A. nanus and make some minor revisions to the Flora of China treatments (Wei & Lock 2010):

Ammopiptanthus nanus (Popov) S.H.Cheng (1959: 1384). Basionym:—Piptanthus nanus Popov (1931: 1). Type:— CHINA. Xinjiang: Kashi, Kisyl-Ssu supra urbem et Tjian-Schan Centrali: rupestribus et delivibus lapidosis, 4 August 1929 (fr.), M. Popov 28 (holotype LE!). = Ammopiptanthus kamelinii Lazkov (2006: 135). Type:—KYRGYZSTAN. Tian-Schan Centralis, jugum Moldotau, in declivio generali boreali, vallis fl. Akpol (in systemate fl. Kokomeren) in declivia schistosa, 1720 m. s. m., 25 July 1981, S. Ikonnikov & G. Ladygina [Herbarium Florae Rossicae 7626] (holotype LE, isotypes LE etc.). Evergreen shrubs, 0.5–0.8 (–1+) m tall; bark yellowish brown. Stems terete, weakly ridged, gray-puberulent at first, glabrescent. Leaves 3-foliolate, occasionally 1-foliolate; stipules small, triangular, adnate to petiole, silvery tomentose; petiole 4–15 mm; leaflets broadly elliptic to broadly ovate, 1.5–4 × 0.6–2.4 cm, densely silvery tomentose on both surfaces, lateral veins conspicuous, base broadly cuneate to rounded, apex obtuse, often mucronate. Flowers 4 to 15, in short, dense terminal racemes; bracts ovate, 5–6 mm, deciduous; pedicels ca. 1 cm, subglabrous, with 2 bracteoles at midpoint. Calyx 5–7 mm. Corolla yellow, ca. 2 cm, petals long-clawed. Ovary stipitate, glabrous. Legume linear- oblong, 3–8 × 1–2 cm, flat, apex acute to obtuse, margin constricted and uneven; stipe 8–10 mm. Seeds 2 to 5, orbicular-reniform, ca. 6 mm diam. Vernacular name:—xiao sha dong qing, ai sha dong qing. Phenology:—Ammopiptanthus nanus flowers from April through June and fruits from May through August. Distribution and habitat:—Ammopiptanthus nanus is only found in gravel hillsides in Kashi, Xinjiang Province, China, and also in Moldotau Mts., Kyrgyzstan.

Acknowledgements

This research was financed by the National Natural Science Foundation of China (Project No. 41271070), the Special Service Project of the Chinese Academy of Sciences (Project No. TSS-2015-014-FW-4-1), and the Natural Science Foundation of Xinjiang (Project No. 2017D01A82). We are grateful to Prof. G. Lazkov (Laboratory of Flora, Institute for Biology and Soil Studies, Academy of Sciences, Kyrghyzstan), who provided guidance in searching for samples of

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