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New morphological and DNA evidence supports the existence of Calligonum jeminaicum Z. M. Mao (Calligoneae, Polygonaceae) in China
expand article infoWei Shi§, Pei-Liang Liu|, Jun Wen, Ying Feng, Borong Pan§
‡ Xinjiang Institute of Ecology and Geography, Urumqi, China
§ Turpan Eremophytes Botanic Garden, Chinese Academy of Sciences, Turpan, China
| Northwest University, Xi'an, China
¶ National Museum of Natural History, Smithsonian Institution, Washington, United States of America
Open Access

Abstract

Calligonum jeminaicum Z. M. Mao, a species regarded as endemic to China, was thought to be nonexistent owing to a lack of scientific records. The similarity of C. jeminaicum to C. mongolicum Turcz. warranted an investigation into the taxonomical relationship between these species. In this study, a naturally occurring population of C. jeminaicum was discovered and the taxonomical relationships of this species with C. mongolicum were resolved. Morphological traits, including fruit and flower characteristics, as well as nuclear (ETS, ITS) and chloroplast (psbA-trnH, ycf6-psbM, rpl32-trnL, rbcL, and trnL-F) DNA sequence data were studied to confirm the taxonomic status of C. jeminaicum. The nrDNA data (ITS1-2 and ETS) from C. jeminaicum reflected variability from the whole C. mongolicum complex, showing distinctive haplotypes in the Calligonum sect. Medusa Sosk. & Alexandr. The cpDNA data supplied similar evidence, showing unique branching in Bayesian and ML tree analyses. The specific status of C. jeminaicum is confirmed based on both morphological and molecular analyses. Here we present a revised description of C. jeminaicum along with its DNA barcode and discuss suggestions for the conservation of this species. Based on current evidence, this species was evaluated as Critically Endangered (CR) according to the IUCN criteria.

Keywords

Calligonum mongolicum complex, Central Asia, desert plant, IUCN, molecular phylogenetics, morphological traits

Introduction

Calligonum L. species are as ecologically important as some of the dominant shrubs and semi-shrubs in both active and inactive sand dunes in the African Sahara (Dhief et al. 2011, 2012) and the deserts of Central Asia (Losinskaya 1927; Bao and Grabovskaya-Borodina 2003; Amirabadi-zadeh et al. 2012). They are natural resources of tannins, food, medications, nectar, and antidotes (Liu et al. 2001; Badria et al. 2007; Askariyahromi et al. 2013; Essam et al. 2014). Calligonum is considered to be the only genus within Polygonaceae that contains C4 species (Pyankov et al. 2000) and displays rapid rates of evolution and diversification (Mabberly 1990). This accelerated differentiation process causes physiological (Su et al. 2005, 2013) and morphological (Mao and Pan 1986; Taia and Moussa 2011; Tao and Ren 2004) changes within these species that facilitate their tolerance of various extreme xeric conditions (Pyankov et al. 2000; Su and Zhao 2002). Thus, Calligonum species have been used as the major sand conservation species in northwestern China (Wang et al. 2014; Xie et al. 2014).

Calligonum jeminaicum Z. M. Mao was first described by Mao (1984) to be a local endemic species which only proliferated in the countryside near Jeminay in the northwest of the Gurbantunggut Desert (Mao 1984, 1992). It has been difficult to differentiate C. jeminaicum from C. mongolicum Turcz. owing to their similar morphological characteristics (Mao 1992; Bao and Grabovskaya-Borodina 2003). In addition, there has been no further record of this species to demonstrate its existence, leading to the question: does this endemic species actually exist? This question was resolved by specific field work in 2013 when a naturally occurring population with eight individuals of C. jeminaicum was found.

The rapid and complex evolutionary processes of Calligonum species have been reflected in their fruit morphology (Bao and Grabovskaya-Borodina 2003; Shi et al. 2009, 2016; Feng et al. 2010a; Soskov 2011). Fruit phenotype has been used as the key character to separate the whole Calligonum genus into four sections, namely sect. Calliphysa (Fish. & C. A. Mey.) Borszcz. (Fig. 1A), sect. Pterococcus (Pall.) Borszcz. (Fig. 1B), sect. Calligonum (Fig. 1C), and sect. Medusa Sosk. & Alexandr. (Fig. 1D). Members of sect. Calliphysa have membranous-saccate fruits, those of sect. Pterococcus have winged fruits, the fruits of sect. Medusa only show bristles without wings and membranes, and the fruits of sect. Calligonum show both wings and bristles but no membranes (Bao and Grabovskaya-Borodina 2003; Fig. 1). The most widely distributed species in Central Asia, C. mongolicum (sect. Medusa), has shown two karyotypes with different chromosome numbers (2n = 18 and 2n = 27) within the same population (Shi and Pan 2015); this species has heterogeneous phenotypes and forms a C. mongolicum complex with inter-crossed taxonomic relationships with other species in sect. Medusa (Soskov 2011; Shi et al. 2011, 2012; Shi and Pan 2015). Calligonum mongolicum has a large distribution area bordered by Xilinhot (Inner Mongolia) in the east, Kumul and Tutotu Basin (Xinjiang) in the west, Milan (Xinjiang) in the south, and Baitashan, Qitai, and Karamay (Xinjiang) in the north. The longitudinal range of C. mongolicum is about 30° (Pavlov 1936; Drobov 1953; Baitenov and Pavlov 1960; Sergievskaya 1961; Kovalevskaya 1971; Borodina 1989). The distribution range of C. jeminaicum lies within that of C. mongolicum (Mao 1992; Bao and Grabovskaya-Borodina 2003). The C. mongolicum complex has been the subject of several taxonomic studies, particularly those focused on species delimitation and identification (Feng et al. 2010b; Gulnur et al. 2010; Li et al. 2014; Shi et al. 2011, 2009, 2016). Both fruit and flower characteristics are used for distinguishing C. jeminaicum from C. mongolicum (Bao and Grabovskaya-Borodina 2003).

Figure 1. 

Fruit characters in the members of the four sections in Calligonum (A, sect. Calliphysa (Fisch. & C. A. Mey.) Borszcz.; B, sect. Pterococcus (Pall.) Borszcz.; C, sect. Calligonum ; D, sect. Medusa Sosk. & Alexandr.).

DNA analysis is regarded as one of the most important techniques to elucidate taxonomy (Kress et al. 2005; Hollingsworth et al. 2009). Previous studies have used Calligonum DNA data to resolve several conflicting taxonomic relationships, such as the use of RAPD markers to clarify the relationships of species in China (Ren et al. 2002), and the use of three chloroplast DNA markers (rbcL, matK, and trnL-F) to distinguish the Chinese species of Calligonum, although these conserved markers were not effective (Li et al. 2014). Additionally, cpDNA data have revealed the phylogeographic variation in different sections (Wen et al. 2015, 2016a, b), which was shown to be potentially valuable for DNA barcoding. ITS data have been used to effectively resolve taxonomical problems within the C. mongolicum complex (Shi et al. 2016, 2017). However, combined sequencing data from cpDNA and nrDNA have not been employed for clarifying the status of puzzling species in Calligonum. There is a need to further explore rapidly evolving DNA sequences that may be effective in resolving the taxonomic uncertainties in Calligonum.

In this study, nuclear ribosomal ITS and ETS sequences, together with five sets of cpDNA data (psbA-trnH, ycf6-psbM, rpl32-trnL, rbcL, and trnL-F) and the morphological characters, were used to confirm the existence of C. jeminaicum and clarify its relationship with C. mongolicum. We also suggest and discuss strategies for conserving C. jeminaicum.

Methods

Sample selection and species identification

All samples were collected from shoots of Calligonum individuals from Xinjiang, Qinghai, Inner Mongolia, Gansu, and Ningxia across the northwest of China during summer from 2006 to 2015 (Table 1).

Population information for C. mongolicum Turcz., C. jeminaicum Z. M. Mao and related species, and GenBank accession numbers of DNA sequences used in this study.

Species Pop. No. (#, &)1 Location Latitude Longitude Elevation Gen-Bank accession number Voucher Number
ITS ETS psbA-trnH trnL-trnF ycf6-psbM rpl32-trnL rbcL
C. mongolicum 1(10, 4) Erjinaqi, Inner Mongolia 41°27.2’N, 100°26.3’E 1002m KU050846 KY316971 MN449309 MN449258 MN449070 MN449121 MN449172 C1101-C1110
KU050848 KY316973 MN449310 MN449259 MN449071 MN449122 MN449173
MN449311 MN449260 MN449072 MN449123 MN449174
MN449312 MN449261 MN449073 MN449124 MN449175
2 (10, 2) Hulishan, Inner Mongolia 41°58.3’N, 100°35.4’E 899m MN449220 MN449032 MN449313 MN449262 MN449074 MN449125 MN449176 C1111-C1120
MN449221 MN449033 MN449314 MN449263 MN449075 MN449126 MN449177
3 (10, 2) Qingtongxia, Ningxia 38°01.0’N, 105°55.9’E 1134m KU050847 KY316966 MN449315 MN449264 MN449076 MN449127 MN449178 C1121-C1130
KU050853 KY316970 MN449316 MN449265 MN449077 MN449128 MN449179
4 (10, 3) Mazongshan, Gansu 41°48.7’N, 098°42.4’E 12364m MN449222 MN449034 MN449317 MN449266 MN449078 MN449129 MN449180 C1145-C1154
MN449223 MN449035 MN449318 MN449267 MN449079 MN449130 MN449181
- - MN449319 MN449268 MN449080 MN449131 MN449182
5 (10, 2) Liuyuan, Gansu 43°20.5’N, 091°23.6’E 1273m KU050844 KY316963 MN449320 MN449269 - MN449132 MN449183 C1166-C1175
KU050845 KY316975 MN449321 MN449270 MN449081 MN449133 MN449184
6 (10, 3) Kelamayi, Xinjiang 47°19.6’N, 086°46.4’E 574m MN449224 MN449036 MN449322 MN449271 MN449082 MN449134 MN449185 C2101-C2110
MN449225 MN449037 MN449323 MN449272 MN449083 MN449135 MN449186
- MN449038 - - - - -
7 (10, 2) Wuerhe, Xinjiang 46°08.2’N, 086°12.9’E 415m KU050849 KY316969 MN449324 MN449273 MN449084 MN449136 MN449187 C2133-C2142
KU050850 KY316972 MN449325 MN449274 MN449085 MN449137 MN449188
8 (10, 4) Xinxinxia, Xinjiang 42°45.2’N, 095°28.7’E 1744m MN449226 MN449039 MN449326 MN449275 MN449086 MN449138 MN449189 C2165-C2174
MN449227 MN449040 MN449327 MN449276 MN449087 MN449139 MN449190
MN449228 MN449041 MN449328 MN449277 MN449088 MN449140 MN449191
MN449229 MN449042 MN449329 MN449278 MN449089 MN449141 MN449192
9 (10, 2) Qijiaojing, Xinjiang 43°35.3’N, 091°25.4’E 1142m KU050852 KY316960 MN449330 MN449279 MN449090 MN449142 MN449193 C2175-C2184
KU050841 MN449331 MN449280 MN449091 MN449143 MN449194
10 (10, 3) Hami1, Xinjiang 43°23.7’N, 091°32.5’E 1038m - - MN449290 MN449239 MN449051 MN449102 MN449153 C2011-C2020
KU050843 KY316962 MN449291 MN449240 MN449052 MN449103 MN449154
- MN449292 MN449241 MN449053 MN449104 MN449155
C. mongolicum 11 (10, 2) Hami2, Xinjiang 42°44.5’N, 093°55.5’E 812m MN449205 MN449019 MN449293 MN449242 MN449054 MN449105 MN449156 C2178-C2186
MN449206 MN449020 MN449294 MN449243 MN449055 MN449106 MN449157
12 (10, 3) Tashan, Xinjiang 45°01.7’N, 090°03.2’E 1018m MN449207 MN449021 MN449295 MN449244 MN449056 MN449107 MN449158 C2274-C2283
MN449208 - MN449296 MN449245 MN449057 MN449108 MN449159
MN449209 - MN449297 MN449246 MN449058 MN449109 MN449160
13 (10, 2) Chaidamu, Qinghai 39°09.7’N, 089°47.4’E 1680m MN449210 MN449022 MN449298 MN449247 MN449059 MN449110 MN449161 C0121-C0130
- - MN449299 MN449248 MN449060 MN449111 MN449162
14 (10, 3) Kumishi, Xinjiang 42°14.5’N, 088°13.4’E 919m MN449211 MN449023 MN449300 MN449249 MN449061 MN449112 MN449163 C0152-C0161
MN449212 MN449024 MN449301 MN449250 MN449062 MN449113 MN449164
MN449213 MN449025 MN449302 MN449251 MN449063 MN449114 MN449165
15 (10, 1) Heshuo, Xinjiang 42°16.9’N, 082°59.2’E 1105m MN449214 MN449026 MN449303 MN449252 MN449064 MN449115 MN449166 C0122-C0131
16 (10, 3) Mingfeng, Xinjiang 36°45.1’N, 082°59.3’E 1600m MN449215 MN449027 MN449304 MN449253 MN449065 MN449116 MN449167 C0174-C0184
MN449216 MN449028 MN449305 MN449254 MN449066 MN449117 MN449168
MN449217 MN449029 MN449306 MN449255 MN449067 MN449118 MN449169
17 (10, 2) Yutian, Xinjiang 36°45.2’N, 082°02.1’E 1648m MN449218 MN449030 MN449307 MN449256 MN449068 MN449119 MN449170 C0147-C0158
MN449219 MN449031 MN449308 MN449257 MN449069 MN449120 MN449171
C. jeminaicum 18 (8, 3) Jeminay, Xinjiang 47°19.3’N, 086°45.9’E 780m MN449232 MN449048 MN449334 MN449283 MN449094 MN449146 MN449197 C3225-C3233
MN449233 MN449049 MN449335 MN449284 MN449095 MN449147 MN449198
MN449234 MN449050 - - - - -
C. calliphysa 19 (0, 1) Mulei, Xinjiang 44°35.8’N, 090°39.7’E 574m KX186585 KY316976 MN449338 MN449287 MN449099 MN449150 MN449202 C0112-C0121
20 (0, 1) Qitai, Xinjiang 44°59.4’N, 089°57.5’E 540m KX186585 KY316976 MN449339 MN449288 MN449100 MN449151 MN449203 C2301-C2310
C. ebinuricum 21 (0, 3) Jinhe, Xinjiang 44°37.8’N, 083°11.1’E 370m MN449236 MN449045 MN449336 - MN449096 MN449148 MN449199 C1158-C1167
MN449237 MN449046 MN449337 MN449285 MN449097 MN449149 MN449200
MN449238 MN449047 - MN449286 MN449098 - MN449201
C. arborescens 22(0, 2) Huocheng, Xinjiang 44°4.58’N, 080°29.2’E 639m MN449230 MN449043 MN449332 MN449281 MN449092 MN449144 MN449195 C1168-C1177
MN449231 MN449044 MN449333 MN449282 MN449093 MN449145 MN449196
Pteroxygonum giraldii 23(0,1) Ningshan, Shaanxi 33°48.5’N, 108°39.7’E 1501m MN449235 - MN449340 MN449289 MN449101 MN449152 MN449204 P. L. Liu 431

The classical identification key was used to differentiate these species mainly based on fruit characteristics and geographic locations, and the C. mongolicum complex has been identified by its fruit characteristics (Mao 1992; Bao and Grabovskaya-Borodina 2003), primarily based on quantifiable differences in fruit and bristle size, such as fruit length (LF), fruit width (WF), bristle length (BS), bristle distance (BD), rib distance (RD), achene length (AL), achene width (AW), and fruit shape (FF) (Shi et al. 2012, 2016; Fig. 2A). The same fruit indices have been used to compare C. jeminaicum with C. mongolicum. The flower traits for differentiating between the two species were selected based on the identification key in “Flora of China” (Bao and Grabovskaya-Borodina 2003), including the shape of perianth segments (PS, broadly elliptic or ovate Fig. 2B), pedicel length (1–2 cm in C. jeminaicum and 2–4 cm in C. mongolicum: Fig. 2C), spreading or reflexed in fruit (PSF, Fig. 2D), and pedicel joint position (below or middle). The shape of perianth segments (Fig. 2B) and pedicel length (Fig. 2C) were used to make quantitative distinctions between C. jeminaicum and C. mongolicum.

Figure 2. 

Measurements of fruit characters (A) and flower traits (B form of perianth segments C pedicel), and the distinction of the form of perianth segments in Calligonum jeminaicum and C. mongolicum fruits (D).

Some species with distinctive fruit characters were used as references in the DNA data analysis: Calligonum calliphysa Bunge, which was previously named Calligonum junceum (Fisch. & C. A. Mey.) Litv. (Bao and Grabovskaya-Borodina 2003), is the only species in sect. Calliphysa, was selected as a representative species; Calligonum arborescens Litv. and Calligonum ebinuricum Ivanova ex Y. D. Soskov (sect. Medusa) were used for comparison because they are regarded as distinct from the C. mongolicum complex. The number of individuals used for morphological analysis and DNA extraction in each population and the accession numbers of some ITS and plastid marker sequences obtained from GenBank are given in Table 1.

Molecular protocols

For all the newly collected samples, total genomic DNA was extracted from fresh or silica gel dried leaves according to the protocol of Doyle and Doyle (1990) or the CTAB method of Doyle and Doyle (1990). The ribosomal DNA regions are known to be potentially problematic when inferring phylogeny (Alvarez and Wendel 2003). In this study, we followed the guidelines for obtaining reliable ITS sequences in plants proposed by Feliner and Rossello (2007). The ITS regions were amplified and sequenced using the previously described primers “ITS5a” and “ITS4” (Stanford et al. 2000). The ETS primers were designed by Shi et al. (2016): the forward primer ETScalli1: 5'-GTTACTTACACTCCCCACAACCCC-3' and the reverse primer 18SIGS: 5'-GAGACAAGCATATGACTACTGGCAGGATCAACCAG-3'. Primers and polymerase chain reaction (PCR) protocols used for the amplification of chloroplast psbA-trnH, ycf6-psbM, rpl32-trnL, trnL-F, and rbcL (the first part of the entire rbcL gene) were described in previous studies (Demesure et al. 1995; Small et al. 1998; Shaw et al. 2005, 2007; Falchi et al. 2009).

The specific Sanger sequencing studies of the Calligonum mongolicum complex and other species were divided into two parts, with most experiments completed at the Smithsonian Institution in 2014, and additional data, particularly those concerning C. jeminaicum, being supplied by the Key Laboratory of Biogeography and Bioresource in Arid Land (KLBB), Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences. At the Smithsonian Institution, PCR amplification of DNA was performed using 10 ng of genomic DNA, 4 pmol of each primer, 0.5 U Taq polymerase (Bioline, Randolph, MA, USA), and 2.5 mM MgCl2 in a volume of 25 µL using a PTC-225 Peltier thermal cycler. The PCR cycling parameters were as follows: a 95 °C initial hot start for 5 min, 32 cycles of 94 °C for 30 s, primer-specific annealing (ITS and ETS: 55 °C for 60 s; the five cpDNA primers: 53 °C for 40 s), and 72 °C for 60 s, and a final extension of 72 °C for 10 min. At the Smithsonian Institute, the PCR products were isolated and purified using ExoSAP-IT (US Biological, Swampscott, MA, USA) and sequenced in both directions using the PCR primers. Cycle sequencing was carried out using an ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) with 5 ng of each primer, 1.5 µL of sequencing dilution buffer, and 1 µL of cycle sequencing mix in a 10 µL reaction volume. Cycle sequencing conditions comprised 30 cycles of 30 s denaturation (96 °C), 30 s annealing (50 °C), and 4 min elongation (60 °C). The sequencing products were separated on an ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA). At KLBB, the amplified products were purified using a PCR Product Purification Kit (Shanghai SBS, Biotech Ltd., China). Sequencing reactions were conducted with the forward and reverse PCR primers using the DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences, Little Chalfont, Buckinghamshire, U.K.) with an ABI PRISM 3730 automatic DNA sequencer (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China). Both strands of the DNA were sequenced with overlapping regions to ensure that each base was unambiguous. Electropherograms were assembled and consensus sequences were generated with Sequencher 4.5 (Gene Codes, Ann Arbor, MI, USA).

Phylogenetic and network analyses

Multiple sequence alignments were performed using MUSCLE in the Geneious v.10.0.6 platform (Kearse et al. 2012) using the default settings and manual adjustments. The phylogenetic tree reconstruction of the nrITS and ETS sequence alignment included 44 accessions: 35 newly generated nrITS sequences, 24 new ETS sequences, and nine ITS and 20 ETS sequences from GenBank (Table 1).

Phylogenetic analyses were conducted on both the nuclear and combined plastid datasets. The best-fit nucleotide substitution models for the ITS1, 5.8S, ITS2, ETS, psbA-trnH, ycf6-psbM, rpl32-trnL, trnL-F, and rbcL regions were determined separately using jModelTest (Darriba et al. 2012) and the Akaike information criterion (AIC) were used to rank the best-fit model for the Bayesian analyses.

Phylogenetic relationships were inferred using Bayesian inference (BI) as implemented in MrBayes v.3.2.5 (Ronquist and Huelsenbeck 2003) and the maximum likelihood (ML) analyses were accomplished with RAxML v.8.2 (Stamatakis 2014). Partitioned analyses of both the nuclear and plastid datasets were implemented by applying the previously determined models to each data partition (Brown and Lemmon 2007). The nuclear ITS dataset was partitioned into ITS1, 5.8S, and ITS2 partitions. For the concatenated plastid dataset, separate partitions were used for the psbA-trnH, ycf6-psbM, rpl32-trnL, trnL-F, and rbcL regions. 51 samples in Calligonum were selected as the ingroup and Pteroxygonum giraldii Dammer & Diels was selected as the outgroup. Two independent BI analyses with one cold and three incrementally heated Markov chain Monte Carlo (MCMC) chains were run for 10,000,000 generations, with trees sampled every 1,000 generations. All Bayesian analyses produced split frequencies of less than 0.01, indicating convergence between the paired runs. The first 2,500 trees were discarded as burn-in, and the remaining trees were used to construct a 50% majority-rule consensus tree and posterior probabilities (PP). In the ML analyses, rapid bootstrap analysis was performed with a random seed, 1,000 alternative runs, and the same partition scheme as was used in the Bayesian analysis. The model parameters for each partition of the dataset were optimized by RAxML with the GTRCAT command. Trees were visualized in FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). The ML bootstrap support values (BS) were labeled on the corresponding branches of the BI trees.

A network analysis was carried out with SplitsTree 4.13.1 (Huson and Bryant 2006) using the uncorrected p-distances between the C. mongolicum complex and C. jeminaicum species from the Bayesian analyses. Branch support was estimated using bootstrapping with 1,000 replicates (Felsenstein 1985).

Results

Phenotyping

The descriptions of the shape of perianth segments in fruit (PSF) and the pedicel joint position (below or middle) used to distinguish between the two species were qualitatively compared. The shape of perianth segments in fruit differs between the two species: spreading in the fruit of C. mongolicum, but reflexed in that of C. jeminaicum (Fig. 2D).

The morphological differences between C. mongolicum and C. jeminaicum focus primarily on their fruit and flower characteristics. Compared with the ambiguous characters in C. mongolicum, these taxonomical characters of C. jeminaicum were clearer and more stable. Quantitative comparisons of the fruit traits (Fig. 2A), the perianth segment shape (broadly elliptic or ovate, identified by the value of the length of the perianth segments/width of the perianth segments: Fig. 2B), and the pedicel length (Fig. 2C) were made between the two species (Fig. 3). Although some fruit characters appeared simultaneously in the two species and led to difficulty in distinguishing C. jeminaicum from C. mongolicum, the shape of perianth segments in fruit could be regarded as an effective character for their identification (Fig. 2D).

The quantifiable morphological characters in both fruits and flowers were compared between the two species. The fruit of C. mongolicum (0.106–1.880 cm; 1.134 ± 0.284 cm) was significantly (P = 0.026) longer than that of C. jeminaicum (0.415–0.649 cm; 0.432 ± 0.44 mm). Additionally, the fruit width (FW) for C. mongolicum (0.226–1.742 cm; 0.923 ± 0.347 cm) was larger than that of C. jeminaicum (0.348–0.508 cm; 0.428 ± 0.113 cm; P = 0.017). The bristle length of C. jeminaicum (0.372 ± 0.020 cm) was significantly shorter (P = 0.06) than that of C. mongolicum (0.312 ± 0.121 cm), and the bristle distance (0.077 ± 0.006 cm) and rib distance (0.087 ± 0.004 cm) of C. jeminaicum were significantly smaller than those of C. mongolicum (bristle distance 0.131 ± 0.032 cm, P = 0.01; rib distance 0.105 ± 0.032 cm, P = 0.02). Significant differences were also detected in achene length (0.823 ± 0.146 cm in C. mongolicum and 0.195 ± 0.105 cm in C. jeminaicum, P = 0.00) and achene width (0.359 ± 0.089 cm in C. mongolicum and 0.333 ± 0.004 cm in C. jeminaicum, P = 0.00) (Fig. 3), although the difference in achene width was small. The fruit shape, as the key character, was substantially different between the two species (P = 0.000), with the subglobose fruit of C. jeminaicum (1.048 ± 0.467 cm/cm) being much more rounded than the broadly ellipsoid fruit of C. mongolicum (1.357 ± 0.442 cm/cm). Thus, the fruit characteristics could be used to distinguish between the two species (Fig. 3). Both the pedicel length (P = 0.00) and the form of perianth segments (P = 0.01) of the two species showed significant differences. The pedicel length of C. jeminaicum (0.313 ± 0.004 cm) was much longer than that of C. mongolicum (0.219 ± 0.03 cm). The shape of perianth segments for C. jeminaicum (1.222 ± 0.167 cm/cm) was broader than that of C. mongolicum (2.544 ± 1.799 cm/cm) (Fig. 3).

Figure 3. 

Quantitative comparisons of fruit and flower characters in Calligonum mongolicum and C. jeminaicum.

Molecular phylogeny

The aligned matrix of 44 accessions of the combined nrITS and ETS sequences comprised 807 bp that did not include any abnormal SNPs or unreasonable sequences according to the Phi test (P = 0.0321). The best-fit substitution models were GTR+G for ETS (nucleotide frequencies A: 0.200803 C: 0.329510 G: 0.295074 T: 0.174613) and GTR+I+G for nrITS (nucleotide frequencies A: 0.163227 C: 0.337699 G: 0.352720 T: 0.146353) based on the jModelTest (Darriba et al. 2012) results. The GTR+G model was selected for the ML analyses of the aligned matrix of nrDNA.

The two phylogenetic tree reconstruction methods, BI and ML, produced consistent topologies. However, the nuclear and the chloroplast data were analyzed separately to reconstruct the phylogenetic relationships among C. jeminaicum, the C. mongolicum complex, and other species in Calligonum because obviously different topologies based on the nuclear (Fig. 4, 5) and the chloroplast (Fig. 6) data were found. In the nrDNA data, no single nucleotide polymorphism (SNP) was identified among the C. jeminaicum samples, but the species from the C. mongolicum complex showed heterogeneity and did not form a single clade (Fig. 4). The populations of the C. mongolicum complex, C. arborescens, C. calliphysa, and C. jeminaicum, were distributed within the same broad geographic region. The three individuals of C. ebinuricum, which had specific fruit characteristics that were different from the C. mongolicum complex, formed an independent clade (Fig. 4). Interestingly, the p-distance among the Calligonum taxa for the ITS and ETS regions reached 11.364% between species C. arborescens and C. calliphysa. The p-distance was as high as 22.54% between C. ebinuricum and the C. mongolicum complex group, which reflects their interspecific differentiation. Consistent results were obtained in the ML analysis in the same phylogenetic tree for nrDNA, conforming the C. mongolicum complex and C. jeminaicum independently (Fig. 4, PP = 1, BS = 98%).

Figure 4. 

Bayesian inference tree of the concatenated nuclear ITS and ETS sequence data showing Calligonum jeminaicum and its congeners. Bayesian posterior probabilities and maximum likelihood bootstrap support values are given above the branches.

Figure 5. 

Neighbor-net analyses based on uncorrected p-distances of the nuclear ITS and ETS sequence data. Numbers indicate bootstrap values over 1,000 replicates.

The neighbor-net constructed for the C. mongolicum complex and C. jeminaicum using the ITS and ETS sequences (Fig. 5) also did not support a single clade for the C. mongolicum complex. The three C. jeminaicum samples formed a separate branch from other groups, which is distant from the entire C. mongolicum complex, with a bootstrap support value of 94.9%.

Independent phylogenetic trees were reconstructed based on the concatenated plastid dataset, including the psbA-trnH, ycf6-psbM, rpl32-trnL, trnL-F, and rbcL regions, using the BI and ML methods. The tree topologies of the BI and ML trees were identical, and only the BI tree is shown (Fig. 6). A new haplotype (X), which occurred in all the C. jeminaicum individuals, was identified in the combined cpDNA dataset. The distribution of the C. mongolicum complex within the cpDNA tree could be separated into five to six regions that appear to reflect their geographical distribution. The first branch included sequences from six populations of the C. mongolicum complex (3, 4, 5, 9, 10, and 11) that were distributed in the west and northeastern regions of the Tengger Desert, where C. arborescens and C. calliphysa occurred sympatrically with these six populations. The second independent branch included sequences from four populations (14, 15, 16 and 17) from the Taklimakan Desert. The third independent branch included sequences from three populations (6, 7, and 8) from the Gurbantunggut Desert in the east of Xinjiang. Populations 12 and 13 comprised C. mongolicum complex samples from the Qaidam Desert that were distributed sympatrically with C. ebinuricum. Population 1 was the most phylogenetically distant from other populations, perhaps owing to its geographic isolation in the extreme north of Inner Mongolia. However, the new haplotype X of C. jeminaicum was separated from the above-mentioned branches of the C. mongolicum complex with strong support (Fig. 6, PP = 1, BS = 100%). Meanwhile, the other reference species of Calligonum (C. ebinuricum, C. arborescens, and C. calliphysa) did not form their own separate branches, but were interspersed within branches of the C. mongolicum complex (Fig. 6).

Figure 6. 

Bayesian inference tree of the concatenated plastid DNA sequence data (psbA-trnH, ycf6-psbM, rpl32-trnL, rbcL, and trnL-F) showing Calligonum jeminaicum and its congeners. Bayesian posterior probabilities and maximum likelihood bootstrap support values are given above the branches.

Discussion

600 species names are known in Calligonum, but only 90 of these were recognized (Pavlov 1936; Baitenov and Pavlov 1960; Sergievskaya 1961; Drobov 1953; Kovalevskaya 1971; Liu and Yong 1985). Most of the new names occurring in Calligonum were subsequently ignored or merged into existing names (Pavlov 1936; Kovalevskaya 1971; Bao and Grabovskaya-Borodina 2003). Different taxonomists have controversial opinions on species delimitations in Calligonum (Soskov 2011; Zhang 2007; Sabirhazi et al. 2010; Abdurahman et al. 2012; Shi et al. 2016, 2017). For example, C. rubescens was treated as an independent species (Soskov 2011) by merging three species, C. pumilum, C. alashanicum, and C. jeminaicum. The taxonomical relationships of C. pumilum, C. alashanicum, and C. mongolicum have been clarified, with C. pumilum and C. alashanicum being merged into C. mongolicum (Shi et al. 2009). Additionally, C. rubescens was treated as a synonym of C. mongolicum (Shi et al. 2016). The relationship between C. jeminaicum and C. mongolicum was analyzed in the present study.

The morphological identification system, which has been used in the C. mongolicum complex (Shi et al. 2009), was employed here for phenotypic discrimination. Our results demonstrated that the fruit characters, which were confusing among members of the C. mongolicum complex, in addition to flower characteristics, can be used to distinguish C. jeminaicum from the C. mongolicum complex by statistical analysis. C. jeminaicum could be identified as a good species based on its morphology (Figs 2, 3).

DNA data are used as key evidence for taxonomical conclusions, and can also reveal the systematics among species or genera (Alvarez and Wendel 2003; Feliner and Rossello 2007). Molecular analyses of both nrDNA ITS and cpDNA sequence data (trnL-F, matK, atpB-rbcL, psbA-trnH, psbK-psbL, and rbcL) fail to fully elucidate the taxonomical relationships within Calligonum (Tavakkoli et al. 2010; Sanchez et al. 2011; Sun and Zhang 2012; Li et al. 2014; Gouja et al. 2014), but some minor and reasonable taxonomical discrepancies among the controversial species group were resolved by combining the morphological and DNA data, for example, within the C. mongolicum complex (Shi et al. 2009, 2016, 2017) and between C. trifarium and C. ebinuricum (Abdurahman et al. 2012). The nrDNA tree, which combined nrITS and ETS data, suggested a lack of phylogenetic structure within the C. mongolicum complex, but it can be used to distinguish uncontested species in sect. Medusa, such as C. arich, C. ebinuricum, and C. taklimakanense (Shi et al. 2016). In the present study, C. jeminaicum formed a separate branch based on the nrITS and ETS data (Figs 4, 5), which is not consistent with the past or present occurrence of hybridization or interbreeding of C. jeminaicum with the C. mongolicum. Meanwhile, the cpDNA data were employed to confirm the taxonomic relationship of the C. mongolicum complex with C. jeminaicum. A new cpDNA haplotype (X) was identified in C. jeminaicum, and its separation from other haplotypes of the C. mongolicum complex and other species in sect. Medusa was well supported (Fig. 6). A high level of genetic diversity was also found in previous studies based on polymorphic cpDNA markers in the sect. Medusa (Wen et al. 2016b), especially in the C. mongolicum complex. The cpDNA information also revealed that the distributional ranges of some species in the C. mongolicum complex were geographically close or adjacent to each other (Figs 6). The distribution of genetic variation of the C. mongolicum complex in the Gurbantunggut Desert was consistent with its geographical signal, and the network analysis illustrated that genetic relationships in Calligonum formed a mesh pattern (Fig. 5). Compared to C. mongolicum, C. jeminaicum has a very narrow distribution with only one known population in the northwest of the Gurbantunggut Desert, which is also within the main distribution region of C. mongolicum (Mao and Pan 1986). It has been proposed that C. jeminaicum may contain only a small fraction of the total genetic variation present in its progenitor species in ancient Middle Asia (Sergievskaya 1961; Badria et al. 2007). This may have expanded the range of these xerophytes and allowed them to spread to other suitable habitats in the Jeminay area.

As an accepted name, C. jeminaicum has been confirmed as an endemic species which is found only within a relict area in the northwest of the Gurbantunggut Desert. C. jeminaicum has been on the brink of extinction over the past 40 years owing to the habitat of the only population being near the roads and the small number of individuals. Although the plants observed appeared to be healthy, the conservation of this plant species with an extremely small population (PSESP) (Wade et al. 2016) should receive appropriate attention in the future. As a result of a new policy framework, several national- and regional-level conservation strategies for China’s PSESPs are being implemented (Yang et al. 2015). For many of these species (Ren et al. 2012; Wang et al. 2017), the extinction of a population is irreversible; therefore, recognizing the immediate importance of these risk factors and understanding their interactions are crucial for developing future conservation plans (Volis 2016). The in situ conservation of the genetic diversity of C. jeminaicum for the long-term survival of this species requires a new management strategy that considers its reproductive biology and the future potential of hybridization/interbreeding. In the ex situ conservation of C. jeminaicum, special efforts are needed to ensure the isolation of genetic resources.

Since Calligonum jeminaicum is accepted as an independent species based on our new evidence; the threatened status of this species can be evaluated according to the International Union for Conservation of Nature (IUCN) Red List categories and criteria (IUCN 2012). This species was first collected by Zumei Mao together with Borong Pan from a single site near Jeminay, Xinjiang, China in the year 1979. It was described as a new species to science in 1984 (Mao 1984). Pan searched for this species in the original site and the surrounding area in 2008 but failed to find it. The first author (Wei Shi) searched for it again in 2013 in the Jeminay area and only a population with 8 mature (fruiting) individuals was found. No seeding or young individual was found in this population. No other collection or report of this species is available. Thus we evaluated Calligonum jeminaicum as Critically Endangered (CR) according to criteria D “Population size estimated to number fewer than 50 mature individuals” (IUCN 2012).

Acknowledgments

This research was financed by the Natural Science Foundation of Xinjiang (Project No. 2017D01A82). We thank the CAS Research Center for Ecology and Environment of Central Asia support for part of this work, as well as the literary editing activities supplied by the subject editor Alexander Sukhorukov.

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