Skip to main content
NCBI home page
PhytoKeys logoLink to PhytoKeys
. 2017 Jan 16;(76):71–88. doi: 10.3897/phytokeys.76.10428

Reproductive biology and variation of nuclear ribosomal ITS and ETS sequences in the Calligonum mongolicum complex (Polygonaceae)

Wei Shi 1,2, Jun Wen 3, Yanfeng Zhao 1,2, Gabriel Johnson 3, Borong Pan 1,2
PMCID: PMC5301985  PMID: 28228687

Abstract Abstract

To explore the biosystematics of the Calligonum mongolicum complex (Polygonaceae), the flowering phenological period, breeding and pollination characters and seed set of the complex (Calligonum Mongolicum Turze, Calligonum chinense A. Los., Calligonum gobicum A. Los., Calligonum pumilum A. Los. and Calligonum zaidamense A. Los.) were documented in the Turpan Eremophyte Botanical Garden, China. The sequences of the nuclear ribosomal ITS and ETS region were employed to differentiate the Calligonum mongolicum complex and other species in sect. Medusae. The results showed species of the Calligonum mongolicum complex occupied overlapping flowering periods and had consistent pollination agents. Their breeding systems are all self-compatible, tend to be out-crossing and they interbreed amongst each other (out-crossing index, OCI = 4).The crosses within and amongst species had high seed sets (44 - 65%). Phylogenetic analyses of Calligonum sect. Medusae and the network analysis of nrDNA (ITS and ETS) in the complex suggest interbreeding amongst “species” within the complex and provide evidence for taxonomically merging the five species in the complex. The detected hybridisation, occurring within the complex, suggests the need to improve traditional methods of ex situ plant conservation in botanical gardens for maintaining genetic diversity of Calligonum within and amongst species from different geographic areas.

Keywords: Calligonum mongolicum complex, Phenology, Breeding System, Crossing experiments, Phylogeny, ETS, ITS

Introduction

Calligonum L. is widely distributed in Northern Africa, Southern Europe and Western and Central Asia (Bao and Alisa 2003). It is the only genus in Polygonaceae that contains C4 species (Pyankovet al. 2000) with rapid rates of evolution and diversification (Mabberley 2008). The taxonomy of this genus is complex (Xu 1998) and that of the Calligonum mongolicum Turcz. complex is especially difficult. Calligonum mongolicum Turcz. is widely distributed from Xilinhot-Inner Mongolia in the east, Kyzyl Kum Desert in Uzbekistan in the west, Milan in Xinjiang in the south, Baitashan, Qitai and Karamay in Xinjiang in the north, with a longitudinal range of about 30° (Pavlov 1936; Drobov 1953; Baitenov and Pavlov 1960; Sergievskaya 1961; Kovalevskaja 1971; Shi et al. 2011). Calligonum pumilum A. Los., Calligonum gobicum A. Los., Calligonum chinense A. Los., Calligonum alashanicum A. Los., Calligonum zaidamense A. Los. and Calligonum roborowskii A. Los. (1927) of the complex occur within the geographic range of Calligonum mongolicum (Losinskaja 1927; Bao and Grabovskaya-Borodina 2003). All of these more narrowly ranged species were merged into Calligonum mongolicum based on the variation of their fruit characters and the chromosome numbers (Soskov 1975a, 1975b). However, these species are currently recognised in the Flora of China treatment according to their fruit morphology (Bao and Grabovskaya-Borodina 2003; Mao et al. 1983). Nevertheless the fruits are overall similar, making it difficult to distinguish the species of the complex (Soskov 2011, Mao and Pan 1986, Shi et al. 2011; Table 1). Analyses of the reproductive biology of the complex are important for resolving the taxonomy and exploring the evolutionary processes (Stebbins 1950; Grant 1992, 1994; Oldfield 2009).

Table 1.

Differences in fruit characters among species of the Calligonum mongolicum complex according to the treatment in Flora of China, the monograph of Soskov (2011) and the observations by Shi et al. (2011). * NRR.

Fruit morphology Calligonum mongolicum Calligonum pumilum (syn. Calligonum rubescens) Calligonum chinense (syn. Calligonum litwinowii Drob.) Calligonum gobicum (syn. Calligonum litwinowii Drob.) Calligonum zaidamense (syn. Calligonum litwinowii Drob.)
Flora of China Soskov (2011) Shi et al. (2011) Flora of China Soskov (2011) Shi et al. (2011) Flora of China Soskov (2011) Shi et al. (2011) Flora of China Soskov (2011) Shi et al. (2011) Flora of China Soskov (2011) Shi et al. (2011)
Fruit length (mm) 8–12 8–12 5–15 7–12 12–22 mm 5–17 10–15 9–12 8–13 11–18 9–12 10–12 10–17 9–12 11–18
Seta length (mm) – 3.5–5 1–5 – (3)5–8(10) mm 1–5 – 3.5–5 2–7 – 3.5–5 2–4 – 3.5–5 3–6
NRR* 2 or 3 (1)2(3) 2 or 3 1 (2)3 1 or 2 3 2 or 3 2 or 3 2 2 or 3 2 2 2 or 3 2
Ribs flat or elevated prominent or not flat prominent or not – elevated prominent or not flat little elevated flat flat little elevated flat flat little elevated flat
Seta texture & branching soft, thin, 2 or 3 -branched soft, thin, 2-branched soft, thin, 2, 3 or 4 branched soft, thin, 2 or 3-branched soft, 3–4 -branched soft, thin, 2, 3 or 4 branched thick, stiff, 2 or 3 branched thick, 3 or 4 branched thick, stiff thick, breakable,
2-branched		 thick, 3 or 4 branched thick, breakable thick, breakable,
2-branched		 thick, 3 or 4 branched thick, breakable
Seta distance (mm) – moderately dense 0.2–2 – 0.7–1 1–2 – 0.5–1.2 0.5–2 – 0.5–1.2 0.1–1.8 – 0.5–1.2 1.2–2.3
Nutlet length (mm) – 9–10 5–10 – 7–10 5–12 8–11 6–10 1.5–9.2 – 6–10 6.7–8.2 – 6–10 7.2–12
Nutlet width (mm) – 2.8–3 2–6 – 3–3.5 2–5 3–5 4–5 3.6–9.8 – 4–5 3.0–4.1 – 4–5 3.1–7.2
Nutlet coiled or not and its form not coiled, ellipsoid not coiled coiled or not coiled, ovoid coiled coiled or not coiled, ellipsoid coiled coiled, ellipsoid not coiled, oblong coiled not coiled, ellipsoid not coiled, broadly ovoid or ellipsoid coiled not coiled, broadly ovoid or ellipsoid
Open in a new tab

Studies on the reproductive biology of Calligonum are rare. Kang et al. (2011) assessed information from four taxa (Calligonum calliphysa Bunge, Calligonum rubicundum Bge., Calligonum densum Borszcz and Calligonum ebinuricum Ivanova) which were selected from each section (four sections in Calligonum) and revealed that all the investigated species were self-compatible but there was no hybridisation amongst them. A few examples of hybridisation were mentioned such as between Calligonum dubjanskyi Litv. and Calligonum bubuyri B. Fedtsch. ex Pavl., between Calligonum acanthopterum and Calligonum leucocladum and between Calligonum acanthopterum Borszcz. and Calligonum leucocladum (Schrenk) Bunge (Soskov 1975b). These reported hybrids occurred between species within a section, including sect. Peterococus and sect. Medusae. The taxonomic relationships of the genus have been tested by the applications of several molecular techniques, such as the RAPD markers (Ren et al. 2002) and other chloroplast DNA markers (trnL-F, matK, atpB-rbcL, psbA-trnH, psbK-psbl and rbcL) (Tavakkoli et al. 2010; Sanchez et al. 2011; Abdurahman and Sabirhazi 2012; Sun and Zhang 2012; Li et al. 2014), but the markers employed so far have been inefficient for resolving the taxonomic problems in Calligonum. It was expected that reproductive biology and faster-evolving nuclear DNA sequences (Sang 2002; Zimmer and Wen 2012) might shed some light on the taxonomy of the genus.

The Calligonum mongolicum complex is almost exclusively diploids with 2n (2x) = 18, except Calligonum roborowskii with 2n (4x) = 36 (Wen et al. 2016), although a polyploid count was reported as 2n (3x) = 27 (Shi et al. 2013) in an individual of Calligonum mongolicum. The situation is markedly different in other species of the Calligonum sect. Medusae which are polyploids with the most frequent chromosome number 2n (4x or 6x) = 36 or 54 (Wang and Yang 1985; Wang and Guan 1986; Shi and Pan 2015). The above chromosomal data indicate the significant role of polyploidy in the evolution of the sect. Medusae of Calligonum. The flowering phenology, characters of breeding systems and pollination and fruit set of the Calligonum mongolicum complex (Calligonum mongolicum, Calligonum pumilum, Calligonum chinense, Calligonum alashanicum and Calligonum zaidamense) have been documented by the authors, leaving out the tetraploid Calligonum roborowskii (see also Wen et al. 2016). The phylogeny of Calligonum sect. Medusae has been reconstructed using nuclear ribosomal markers (ITS and ETS). The new data will be used to discuss the taxonomic implications of the species complex and the conservation strategy of Calligonum in botanical gardens.

Materials and methods

Five species of the Calligonum mongolicum complex (Calligonum mongolicum, Calligonum pumilum, Calligonum chinense, Calligonum alashanicum and Calligonum zaidamense) were selected by the authors, leaving out the tetraploid Calligonum roborowskii. These selected species were brought to (TEBG) from their natural habitats during 2011 to 2013 and were planted in the germplasm garden of Calligonum (Table 2, Qi and Pan 2010; Shi et al. 2013).

Table 2.

Voucher information for the samples used in the study.

Species Pop. individuals (flowers in an individual) Location Num. in DNA analysis Coordinates
ITS ETS
Calligonum mongolicum M1 3(25) Erlianhaote, Neimeng, China E112°03' N43°45' 898 m M1–2 KU050839 KY316968
M1–3 KU050840 KY316961
M2 3(25) Qingtongxia, Ninxia, China E105°55' N38°01' 1134 m M2–1 KU050847 KY316966
M2–2 KU050853 KY316970
M3 3(25) Erjinaqi, Inner Mongolia China E100°26' N41°27' 1002 m M3–1 KU050846 KY316971
M3–2 KU050848 KY316973
M3–3 KU050838 KY316979
M4 3(30) Wuerhe, Kelamayi, Xinjiang, China E 85°45’ N 46° 9’ 521 m M4–1 KU050849 KY316969
M4–3 KU050850 KY316972
Calligonum pumilum P1 3(50) Hami, Xinjiang, China E091°32 N43°23' 1038 m P1–1 KU050851 KY316974
P1–2 KU050852 *
P1–3 KU050841 KY316960
P2 3(25) Hami, Xinjiang, China E091°23' N43°20' 1273 m P2–3 KU050843 KY316962
P3 3(25) Liuyuan, Gansu, China E095°28' N95°28' 1744 m P3–1 KU050844 KY316963
P3–2 KU050845 KY316975
Calligonum chinense C1 3(100) Zhangye, Gansu, China E100°18' N39°28' 1458 m C1–2 KY316981 KY316977
Calligonum gobicum G1 3(100) Mingqing, Gansu, China E102°52' N38°34' 1369 m – – –
Calligonum alashanicum A1 3(100) Erjinaqi, Inner Mongolia China E100°27' N41°43' 969.8 m A1–2 KY316980 KY316967
Calligonum zaidamense Z1 3(100) Zhangye, Gansu, China E100°18' N39°03' 1458 m Z1–1 KY316982 KY316978
Z1–2 KY316983 KY316965
Calligonum calliphysa 1 Calligonum calliphysa KX186585 KY316976
Calligonum arich 6 KC585438 –
KC585446 –
KC585445 –
KC585444 –
KC585477 –
AB542775 –
Calligonum comosum 2 Calligonum comosum KC585417 –
KC585430 –
Calligonum caput-medusae 1 JB187106 –
Calligonum ebinuricum 1 Calligonum ebinuricum JQ731664 –
Calligonum ebinuricum 1 Calligonum ebinuricum JQ731665 –
Calligonum ebinuricum 1 Calligonum ebinuricum JQ731663 –
Calligonum molle 1 GQ206245 –
Calligonum crinitum 1 AB542776 –
Calligonum junceum 1 Calligonum junceum GQ206243 –
Calligonum junceum 1 Calligonum junceum AB542774 –
Calligonum junceum 1 Calligonum junceum JX987230 –
Calligonum polygonoides 1 AB542776 –
Calligonum mongolicum 1 Calligonum mongolicum JX259384 –
Calligonum mongolicum 1 Calligonum mongolicum JX259385 –
Calligonum roborowskii 1 Calligonum roborowskii JX259386 –
Calligonum roborowskii 1 Calligonum roborowskii JX259387 –
Calligonum takemakanense 1 Calligonum takemakanense JX259390 –
Calligonum persicum 1 Calligonum persicum AB542777 –
Open in a new tab

Collection of phenological information

Phenological information of the Calligonum species was collected from field investigations. The phenological observations were made once every two days during the growing period, according to the method of the Chinese Phenological Observation Standard (Zhu and Wan 1973). The investigated flowering phenological periods included flower bud appearance, beginning of flowering, flower blooming, end of flowering and fruit maturity. The starting date of a species’ growing period was expressed in the day of year (calculated from 1 January of the current year and thereafter).

Five plants from each species in the field were randomly selected to document the flowering phenology and they were observed every day in the blooming and fruiting periods from 2011 to 2013.

Pollen morphology

(SEM) was used to document the micromorphology of pollen. Samples were dehydrated and were then placed on aluminium stubs using double-sided adhesive tape and sputter coated with gold in a Hitachi E-1010 Ion Sputter Coater, following Wen and Nowicke (1999). The materials were subsequently observed and photographed under a Hitachi S-4800 scanning electron microscope. Pollen sizes from both polar view (P) and equatorial view (E) were measured using 10 grains of each sample.

Controlled crossing experiments and observations on fruit and seed sets

The breeding systems of the Calligonum mongolicum complex were examined by a hand-pollination test. More than 1600 buds were marked and bagged before opening during the period 2011 to 2013. Each flower of an individual plant was randomly assigned to one of the following treatments with each treatment, except hybridisation, including about 30 flowers in each taxon: i) autonomous pollination: no treatment but just bagging to test self-pollination naturally; ii) selfing: test for self-compatibility by bagging and undertaking pollination from the same flower; iii) geitonogamous selfing: emasculation, bagging and pollination in the same individual but using different flowers, to test for self-compatibility; iv) crossing: emasculation, bagging and pollination from another individual that was located more than 2m from the recipient v) apomixis: emasculation, bagging but no pollen; vi) natural pollination: emasculation, no bagging; vii) autonomous pollination via geitonogamy: bagging the whole branch; viii) hybridisation: emasculation and cross-pollinations with four other species, each species included 100 flowers. The stigma receptivity time was about 12 hours; and the pollen viability was about 12-24 hours (XS Kang, W Shi and BR Pan, unpublished data).

DNA extraction, amplification and sequencing

Nineteen (19) individuals of six species, Calligonum mongolicum, Calligonum pumilum, Calligonum chinense, Calligonum alashanicum, Calligonum zaidamense and Calligonum calliphysa were sequenced and 24 ITS sequences of Calligonum from GenBank were downloaded (Table 2). Young green branches of each species were collected from natural populations in China (Table 2). The samples were collected from adult individuals with green healthy branches (with no signs of parasitism or of drought stress). They were dried in silica gel and kept in a freezer at -25 °C. Voucher specimens of the studied material were deposited in the (XJBI).

Total genomic DNAs were extracted from fresh or silica gel dried assimilating branches following the protocol of Doyle and Doyle (1990). In this study, the protocols were followed for obtaining ITS sequences in plants by Wen and Zimmer (1996), Stanford et al. (2000) and Feliner and Rossello (2007). The ETS primers were newly designed for the study with the forward primer ETScalli1: 5’-GTTACTTACACTCCCCACAACCCC-3’ and the reverse primer as18SIGS: 5’-GAGACAAGCATATGACTACTGGCAGGATCAACCAG-3’. The DNA amplifications via a (PCR) were 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: 95 °C initial heating for 5 min, 40 cycles of 94 °C for 30s, 55°C for 45s for ITS (60°C for 40s for ETS) and 72°C for 60s and 72 °C for 10 min for final extension. The PCR products were purified using EXO-SapIT (US Biological, Swampscott, MA, USA) and sequenced in both directions using PCR primers. The ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) was carried out for cycle sequencing with mixing in a 10 µL reaction volume including 5 ng of primer, 1.5 µL of sequencing dilution buffer and 1 µL of cycle sequencing. The conditions were as follows: 35 cycles of 96 °C for 30s denaturation, 50 °C for 30s annealing and 60 °C for 4 min elongation. An ABI 3730xl DNA analyser (Applied Biosystems, Foster City, CA, USA) was used for separating the sequencing products. Both strands of DNA with overlapping regions ensured that each base was double-checked. We assembled the electropherograms and generated the consensus with Sequencher 4.5 (GeneCodes, Ann Arbor, MI, USA).

Sequences were initially aligned using MUSCLE 3.8.31 (Edgar 2004), followed by manual adjustments using GENEIOUS 8.1.2 (Kearse et al. 2012). The newly generated sequences from the 20 samples of Calligonum were deposited in GenBank (Table 2). The jModeltest 2.1.7 (Posada 2008, Darriba et al. 2012) was used to show the best-fit model of sequence evolution for each data. The Bayesian inferences were run according to the model chosen by the (AIC) method. Phylogenetic relationships were inferred using both (ML) in RAxML (Stamatakis 2006) and (BI) in MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). Bayesian analyses were conducted using the combined ITS and ETS data sets, using partitions of the respective models from the jModeltest. The ML analyses also used the partition of the two markers. The bootstrap analysis (Felsenstein 1985) was executed with 1000 replicates, with a maximum of 100 trees saved per replicate. The Bayesian inference was run with 2,000,000 generations and the (MCMC) run had one cold and three incrementally heated chains. For each dataset, all Bayesian analyses produced split frequencies of less than 0.01 and convergence between the paired MCMC runs were repeated twice to avoid spurious results. The remaining trees were used to construct majority-rule consensus trees after discarding the first 2000-5000 trees as burn-in before stationary conditions were established. A neighbour-net analysis was conducted using the uncorrected p-distance between individuals and the programme SplitsTree 4.13.1 (Huson and Bryant 2006). Branch support was tested using bootstrapping with 1000 replicates.

Results

Phenological data

The bisexual flowers occur in groups of two to four in assimilating branches of the Calligonum species. The perianth has five tepals, which are green or red with a broad white margin abaxially, ovate, unequal and persistent in fruits. The flower has 12-18 stamens and the filaments are connate at the base. The pollen presentation pattern is gradual and, when pollen is viable, the stigmas also have receptivity (no dichogamy) (BR Pan, unpublished data).

The five Calligonum species flower from mid-April to mid-May in the field. The duration of Calligonum mongolicum and Calligonum gobicum for flowering was generally from mid-April to early May, whereas that of Calligonum pumilum, Calligonum chinense and Calligonum zaidamense was from late April to mid-May; individual species of Calligonum mongolicum continued to flower sporadically until late May. Thus the blooming period was similar for Calligonum both in field and in TEBG (Figure 1).

Figure 1.

Figure 1.

Open in a new tab

The phenological phases of the Calligonum mongolicum complex. 1 Calligonum mongolicum 2 Calligonum chinense 3 Calligonum gobicum 4 Calligonum pumilum and 5 Calligonum zaidamense.

The blooming periods of the complex overlapped and the percentage overlap was about 80–100% (Figure 1). The peak flowering periods of Calligonum mongolicum complex occurred at the same time in early May. Although flowering was generally ending in early May, flowering in some individuals of Calligonum mongolicum was still at its peak until mid-May.

Floral visitors

The major pollinators for collecting pollen and nectar were Apismellifera L. and Halictus sp., both of which collected pollen in pollen baskets on their third legs and, occasionally, pollen also adhered to their chests and then contacted with the stigmas whilst feeding. These species frequently visited nearby flowers on the same plant individual and frequent visits on the same flowers were also undertaken. Other recorded species were nectar thieves including some flies (Lasiopticus sp., Musca domestica and Calliphoravicina), butterflies (Plebejusargus) and others in Formicidae.

Breeding systems

The results of the pollination experiment suggested that species in the complex had analogous mating systems (Tables 3 & 4), as both geitonogamy and cross-pollination conducted by hand yielded better fruit sets compared with natural pollination. They also had similar pollen characters and indices (P & E) (Table 5 & Figure 2). They interbred amongst each other (OCI = 4). The spontaneous self-pollination did not occur because when pollinators were excluded in the bagging treatment, no fruits were produced. It resulted in a very low (if any) fruit set in the self-pollination treatment. The fruit set using geitonogamy treatment shows self-compatibility within each species. The apomixis did not occur in these species as exclusion of both pollinators and emasculation did not result in any fruit set.

Table 3.

Comparison of actual fruit set of species in the Calligonum mongolicum complex under each pollination treatment in 2011 to 2013 (n = the total number of flowers manipulated in each treatment, data shown are mean ± SE).

Treatment Species
Calligonum mongolicum Calligonum gobicum Calligonum chinense Calligonum pumilum Calligonum zaidamense
No emasculation, bagged, self-pollination 0 0 0 0 0
Emasculation, bagged, hand geitonogamy 2.00±1.00 1.67±0.58 1.00±1.00 1.00±1.00 1.00±1.00
Emasculation, bagged, hand cross pollination in same individual 15.12±1.00 16.58±1.22 17.24±1.31 17.32±1.23 14.42±1.25
Emasculation, bagged, no pollination 0.00 0.00 0.00 0.00 0.00
Emasculation, unbagged, natural pollination 11.21±2.13 9.15±2.54 12.48±2.41 12.47±1.21 13.56±2.15
Unemasculation, unbagged, natural pollination 11.23±1.23 15.45±1.58 8.35±3.35 14.28±3.69 10.25±2.36
Open in a new tab

Table 4.

Fruit set (%) for the five Calligonum species under different cross-pollination treatments (n = the total number of flowers manipulated in each treatment, mean ± SE).

Species cross Calligonum mongolicum ♂ Calligonum gobicum ♂ Calligonum chinense ♂ Calligonum pumilum ♂ Calligonum zaidamense ♂
Calligonum mongolicum ♀ 65±1.25 54±3.21 41±1.15 47±1.68 45±1.25
Calligonum gobicum ♀ 47±2.34 44±2.47 59±4.21 57±1.51 47±2.36
Calligonum chinense ♀ 58±1.21 46±2.11 59±4.18 66±2.12 48±3.25
Calligonum pumilum ♀ 48±2.24 59±4.56 54±3.06 65±2.14 52±2.48
Calligonum zaidamense ♀ 44±2.14 58±1.63 47±1.85 60±1.23 51±4.21
Open in a new tab

Table 5.

The characteristics of the pollen grains of five species of the Calligonum mongolicum complex.

Species Shape Length (μm) width(μm) P/E Aperture ornamentation
Calligonum mongolicum Prolate 38.90 23.20 1.68 tricolporate reticulate
Calligonum gobicum Prolate 38.35 19.51 1.97 tricolporate reticulate
Calligonum chinense Prolate 33.45 21.15 1.58 tricolporate reticulate
Calligonum pumilum Prolate 31.52 22.40 1.41 tricolporate reticulate
Calligonum zaidamense Prolate 37.79 20.04 1.89 tricolporate reticulate
Open in a new tab

Figure 2.

Figure 2.

Open in a new tab

Equatorial view of pollen grains of the Calligonum mongolicum complex under SEM micrographs.1 Calligonum mongolicum 2 Calligonum chinense 3 Calligonum gobicum 4 Calligonum pumilum and 5. Calligonum zaidamense.

Hybridisation experiments in the complex resulted in a fruit set and the results (in percentage terms) are shown in Table 4. The flowering of the complex was synchronised. The pollen morphology of the five species showed similarities in major pollen characteristics such as shape, size and exine characters (Figure 2, Table 5). The hybridisation experiments and interspecific hand pollination yielded some viable seeds (Table 5). The maximum of the fruit set is amongst the Calligonum mongolicum (65±1.25) and the Calligonum pumilum (65±2.14) themselves; the minimum is that between Calligonum chinense and Calligonum mongolicum (41±1.15). In general, the fruit set amongst the five species is similar (p>0.05).

Phylogenetic analysis

The aligned matrix with 45 accessions of nrITS and ETS is 807bp long. The Phi test did not find statistically significant (p= 0.0323) evidence for the presence of chimeric sequences in the nrITS and ETS data matrix. The nrITS and ETS sequence alignment used for phylogenetic tree reconstruction included 44 sequences: 43 from the in-group and one of Calligonum caput-medusae as the out-group. The data sets included 20 newly generated nrITS, 23 ITS sequences from GenBank and 20 new ETS sequences (Table 2).

The model test suggested F81 for ETS (nucleotide frequencies A = 0.2023, C = 0.3494, G = 0.2778, T = 0.1706) and TPM2uf for ITS (nucleotide frequencies A = 0.1873, C = 0.3265, G = 0.3277, T = 0.1586; substitution rates: RAC = 0.3484, RAG = 3.4478, RAT = 0.3484, RCG = 1.0000, RCT = 3.4478, RGT = 1.0000). The Bayesian inference used the partition of ITS and ETS based on the respective models. The ML analyses used GTR+G as the model. Topologies inferred by the two phylogenetic tree reconstruction methods were congruent (Figure 3). The most morphologically distinctive Calligonum caput-medusae from Central Asia was used as the out-group, the first diverged clade in the analyses being Calligonum arich (six accessions included, PP 1.00, BS 92) from western Asia, the remaining species forming a large clade A. Of interest, all species from the Calligonum mongolicum complex formed a clade. The five species of the Calligonum mongolicum complex, Calligonum ebinuricum and two other species Calligonum roborowskii and Calligonum taklamakan were distributed within the broad geographic region of the Calligonum mongolicum complex, but Calligonum roborowskii and Calligonum taklamakan were of a more restricted distribution in the Taklamakan Basin of Xinjiang province, China. The three individuals of Calligonum ebinuricum which form an independent clade, have specific fruit characters different from the complex. The individuals of Calligonum mongolicum and Calligonum pumilum each did not form a clade, but they were intermixed with Calligonum alashanicum, Calligonum zaidamense and Calligonum chinense, Calligonum roborowskii and Calligonum taklamakan, forming a large clade C (Figure 3). It is of interest to note that the p-distance amongst taxa of Calligonum for the ITS and ETS region is as high as 11.364% between the out-group species Calligonum caput-medusae and Calligonum mongolicum JX259384. Within the clade C, the p-distance was as high as 0.564% between Calligonum ebinuricum and Calligonum mongolicum JX259384. A neighbour-net was constructed for the Calligonum mongolicum complex using ITS and ETS sequences which also supported the complex in one branch (Figure 4).

Figure 3.

Figure 3.

Open in a new tab

Maximum likelihood tree for 43 (in-group) Calligonum nrITS and ETS sequences produced with RAxML. Numbers adjacent to (relevant) nodes represent maximum likelihood value and Bayesian posterior probabilities. Branches marked with an asterisk collapse on the maximum likelihood strict consensus tree of the same dataset. The branch marked with a number sign collapses on the Bayesian majority rule consensus tree of the same dataset.

Figure 4.

Figure 4.

Open in a new tab

Neighbour-net analyses of the Calligonum mongolicum complex, Calligonum ebinuricum, Calligonum calliphysa and closely related taxa based on uncorrected p-distances. Numbers indicate bootstrap values over 1000 replicates.

Discussion

Evidence for interbreeding of species in the Calligonum mongolicum complex

Species isolation is frequently caused by the temporal heterogeneity of blooming amongst sympatric species (Levin 1971; Adams 1983; Grant 1992, 1994). The flowering periods of five species in the complex showed a high degree of overlapping, with some differences in peak blooming periods (also see cases in Wilson 1983; Burd 1995).

These five diploid species of Calligonum have similar pollen characters in both with spheroidal shape and tricolporate apertures with each other (Figure 2). The other species in Medusae also have the similar pollen characters but without specific pollen indexes (P&E) analysis (Qiu 1988; Gulinuer 2008). The hand-pollination tests suggested the five species are self-compatible (geitonogamous, not autophilous). Furthermore, pollinators were necessary for the sexual reproduction in the complex, although some fruit sets were resulted with exclusion of pollinators. The results of test crosses suggest the existence of a strong internal hybridisation potential in each of these species.

Crossing compatibility between the species of the Calligonum mongolicum complex is largely the same as that between individuals within the same species (Table 4). The crossing behaviour amongst them is consistent with the view from Soskov (1975a, 1975b) by treating these various segregate species as one variable biological species of Calligonum mongolicum.

Lack of phylogenetic structure and nrDNA sequence variation as indirect evidence for interbreeding in the Calligonum mongolicum complex

Although phylogenetic inference based on nrITS needs to be considered carefully (Alvarez and Wendel 2003, Feliner and Rossello 2007), some conclusions may be drawn based on the ITS and ETS analyses of the target species. As shown by the ML and Bayesian trees of nrITS and ETS sequences (Figure 3), a striking divergence exists between Calligonum arich (clade A) and other species. Yet species of the Calligonum mongolicum complex had very similar or identical sequences (Clade C in Figure 3). The nrITS and ETS tree together with the network of ribotypes (Figure 4) suggest the lack of phylogenetic structure within the complex. Excluding Calligonum arich (5 individuals), Calligonum ebinuricum (3 individuals) can be easily differentiated from the Calligonum mongolicum complex (13 individuals) (Figures 3 and 4). The intermixed patterns of sequences from different “species” of the Calligonum mongolicum complex may indicate past or present introgressive potential of the Calligonum mongolicum complex and argues for the existence of hybridisation or interbreeding (if these “species” represent the same taxon).

Implications on taxonomy and conservation of Calligonum

Calligonum is one of the medium-sized genera of Polygonaceae with approximately 60–80 species and represents a rapid diversification in the hot and arid deserts of Central Asia to western China (Mabberley 1990). Molecular analyses of both nrDNA ITS and some cpDNA sequences (trnL-F, matK, atpB-rbcL, psbA-trnH, psbK-psbL and rbcL) have not resolved relationships amongst species of Calligonum (Sanchez et al. 2011, Sun and Zhang 2012, Li et al. 2014). Our study showed that Calligonum ebinuricum possesses highly distinct nrITS sequences (Figures 3 & 4); yet the ITS and ETS sequences of the Calligonum mongolicum complex generated a topology with the species of the complex highly intermixed with each other in the tree. The authors’ results both in this paper and in their previous studies (Shi et al. 2011, 2012, 2013, 2016, Shi and Pan 2015) argue for the merging of Calligonum chinense, Calligonum gobicum, Calligonum pumilum and Calligonum zaidamense with Calligonum mongolicum as proposed by Soskov (1975a, 1975b). Detailed evidence was also recently presented on merging Calligonum pumilum with the more widespread Calligonum mongolicum (Shi et al. 2016). Detailed morphological comparisons of the other species in the complex will be pursued by the authors as was done for Calligonum pumilum and Calligonum mongolicum (Shi et al. 2016) and the phylogeographic structure of the complex will be further explored with phylogenomic methods (Wen et al. 2015, Zimmer and Wen 2015).

Distributional ranges of some species in clade C (Figure 3) do not overlap but are geographically close or adjacent to each other. Calligonum roborowskii (2n=36) grows at the edge of Taklamakan basin; Calligonum taklamakan occurs in the central part of the basin; and the other species in the complex except Calligonum mongolicum are confined to the south-eastern edge of the basin and Calligonum ebinuricum is in North Xinjiang and also in Mongolia but never in South Xinjiang. According to their morphological comparisons (Gulinuer 2008, Kang et al. 2008), the taxonomic relationship of Calligonum ebinuricum and Calligonum taklamakan with other species needs further analyses. The fact that most of the collected seeds can germinate without any pre-treatment suggests that the five Calligonum species produce enough seeds to renew the populations. On the other hand, the ex-situ conservation of genetic diversity for the long-term survival of species of Calligonum needs a new management strategy due to their reproductive biology and the potential for hybridisation/interbreeding (Kramer and Havens 2009, Swarts and Dixon 2009). Special efforts are needed to ensure isolation of genetic sources in ex situ conditions.

Acknowledgments

This work was supported by a grant from the National Natural Science Foundation of China (Project No 31100150), the West Light Talents Cultivation Program of Chinese Academy of Sciences (XBBS201202) and the Laboratories of Analytical Biology of the National Museum of Natural History, the Smithsonian Institution.

The authors declare that they have no conflict of interest.

Citation

Shi W, Wen J, Zhao Y, Johnson G, Pan B (2017) Reproductive biology and variation of nuclear ribosomal ITS and ETS sequences in the Calligonum mongolicum complex (Polygonaceae). PhytoKeys 76: 71–88. https://doi.org/10.3897/phytokeys.76.10428

References

  1. Abdurahman M, Sabirhazi G, Liu B, Yin L, Pan BR. (2012) Comparison of five Calligonum species in Tarim basin based on morphological and molecular data. EXCLI Journal 11: 776–782. [PMC free article] [PubMed] [Google Scholar]
  2. Adams VD. (1983) Temporal patterning of blooming phenology in Pedicularis on Mount Rainier. Canadian Journal Botany 61: 786–791. https://doi.org/10.1139/b83-088 [Google Scholar]
  3. Alvarez I, Wendel JF. (2003) Ribosomal ITS sequences and plant phylogenetic inference. Molecular Phylogenetics and Evolution 29: 417–434. https://doi.org/10.1016/S1055-7903(03)00208-2 [DOI] [PubMed] [Google Scholar]
  4. Baitenov MB, Pavlov NV. (1960) Calligonum L – Flora of Kazakhstan. Science Press, Astana, 246–382. [Google Scholar]
  5. Bao BJ, Grabovskaya-Borodina AE. (2003) Calligonum L. In: Wu CY, Raven PH. (Eds) Flora of China (Vol. 5). Science Press; (Beijing: ) and Missouri Botanical Garden Press; (St. Louis: ), 324–328. [Google Scholar]
  6. Burd M. (1995) Ovule packaging in stochastic pollination and fertilization environments. Evolution 49: 100–109. https://doi.org/10.2307/2410296 [DOI] [PubMed] [Google Scholar]
  7. Darriba D, Taboada GL, Doallo R, Posada D. (2012) jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9(8): 772 https://doi.org/10.1038/nmeth.2109 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doyle JJ, Doyle JL. (1990) Isolation of plant DNA from fresh tissue. Focus 12: 13–15. [Google Scholar]
  9. Drobov VP. (1953) Calligonum L. In: Kudrjaschev SN. (Ed.) Flora of Uzbekistan (Vol. 2). Editor Academic Science Press, Tashikanit, 54–230. [Google Scholar]
  10. Edgar RC. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32: 1792–1797. https://doi.org/10.1093/nar/gkh340 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feliner GN, Rossello JA. (2007) Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants. Molecular Phylogenetics and Evolution 44: 911–919. https://doi.org/10.1016/j.ympev.2007.01.013 [DOI] [PubMed] [Google Scholar]
  12. Felsenstein J. (1985) Phylogenies and the comparative method. American Naturalist 125: 1–15. https://doi.org/10.1086/284325 [DOI] [PubMed] [Google Scholar]
  13. Grant V. (1992) The Evolutionary Process: A Critical Study of Evolutionary Theory. Columbia University Press, New York. [Google Scholar]
  14. Grant V. (1994) Mechanical and ethological isolation between Pedicularis groenlandica and P. attllens (Scrophulariaceae). Biologisches Zentralblatt 113: 43–51. [Google Scholar]
  15. Gulinuer S. (2008) Study on endemic species of Calligonum in Tarim basin. PhD Thesis, Chinese University Academic Science, Beijing, 154 pp. [Google Scholar]
  16. Huson DH, Bryant D. (2006) Application of phylogenetic networks in evolutionary studies. Molecular Biology and Evolution 23: 254–267. https://doi.org/10.1093/molbev/msj030 [DOI] [PubMed] [Google Scholar]
  17. Kang XS, Zhang Y, Pan BR, Duan SM, Tan Y. (2008) Research on fruits of morphological characteristics of Calligonum ebinuricum population. Bulletin of Botanical Research 28: 1213–1221. [Google Scholar]
  18. Kang XS, Pan BR, Duan SM, Shi W, Zhang YZ. (2011) Is ex-situ conservation suitable for Calligonum L.? A research program in Turpan Eremophyte Botanical Garden. Natureza & Conservação 9(1): 47–53. https://doi.org/10.4322/natcon.2011.005 [Google Scholar]
  19. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A. (2012) Geneious basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28: 1647–1649. https://doi.org/10.1093/bioinformatics/bts199 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kovalevskaja SS. (1971) Calligonum L. In: Kovalevskaja SS. (Ed.) Conspectus Florae Asise Mediae (Vol. 2). Editio Academiae Science Press, 82–200. [Google Scholar]
  21. Kramer AT, Havens K. (2009) Plant conservation genetics in a changing world. Trends in Plant Science 14: 599–607. https://doi.org/10.1016/j.tplants.2009.08.005 [DOI] [PubMed] [Google Scholar]
  22. Levin DA. (1971) The origin of reproductive isolating mechanism in flowering plants. Taxon 20: 91–113. https://doi.org/10.2307/1218538 [Google Scholar]
  23. Li Y, Feng Y, Wang XY, Liu B, Lv GH. (2014) Failure of DNA barcoding in discriminating Calligonum species. Nordical Journal Botany 32: 511–517. https://doi.org/10.1111/njb.00423 [Google Scholar]
  24. Losinskaja AS. (1927) Perennial Calligonum L. Izv Glavn. Botany Sada USSR 26(1): 596–609. [Google Scholar]
  25. Mabberley DJ. (2008) Mabberley’s Plant-Book: a Portable Dictionary of Plants, Their Classifications, and Uses. Cambridge University Press, Cambridge, 1021 pp. [Google Scholar]
  26. Mao ZM, Pan BR. (1986) The classification and distribution of the genus Calligonum L. in China. Acta Phytotaxonomica Sinica 24(2): 98–107. [Google Scholar]
  27. Mao ZM, Yang G, Wang CG. (1983) Studies on chromosome numbers and anatomy of young branches of Calligonum of Xinjiang in relation to the evolution of some species of the genus. Acta Phytotaxonomica Sinica 21(2): 44–49. [Google Scholar]
  28. Oldfield SF. (2009) Botanic gardens and the conservation of tree species. Trends in Plant Science 14: 581–583. https://doi.org/10.1016/j.tplants.2009.08.013 [DOI] [PubMed] [Google Scholar]
  29. Pavlov NV. (1936) Calligonum L. In: Komarov VL. (Ed.) Flora of USSR (Vol. 5). Academiae Scientiarum Press, USSR: 5: 527–594. [Google Scholar]
  30. Posada D. (2008) jModelTest: Phylogenetic model averaging. Molecular Biology and Evolution 25: 1253–1256. https://doi.org/10.1093/molbev/msn083 [DOI] [PubMed] [Google Scholar]
  31. Pyankov VI, Gunin PD, Tsoog S, Black CC. (2000) C-4 plants in the vegetation of Mongolia: their natural occurrence and geographical distribution in relation to climate. Oecologia 123: 15–31. https://doi.org/10.1007/s004420050985 [DOI] [PubMed] [Google Scholar]
  32. Qi Y, Pan BR. (2010) Anatomical structure of assimilating shoots and cotyledons of Calligonum L. Acta Botanica Boreali-Occidentalia Sinica 30(3): 512–518. [Google Scholar]
  33. Qiu GY. (1988) The pollen characters and the evolutional relationships of Calligonum L. Journal of Inner Mongolia Forest (Science) 2(1): 73–81. [Google Scholar]
  34. Ren J, Tao L, Liu XM. (2002) RAPD study on inter-species relationships in Calligonum. Acta Botanica Boreali-Occidentalia Sinica 22(2): 338–343. [Google Scholar]
  35. Rieseberg LH, Brunsfeld SJ. (1992) Molecular evidence and plant introgression. Chapman and Hall press, 151–176. https://doi.org/10.1007/978-1-4615-3276-7_7 [Google Scholar]
  36. Ronquist F, Huelsenbeck JP. (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19(12): 1572–1574. https://doi.org/10.1093/bioinformatics/btg180 [DOI] [PubMed] [Google Scholar]
  37. Sanchez A, Schuster TM, Burke JM, Kron KA. (2011) Taxonomy of Polygonoideae (Polygonaceae): A new tribal classification. Taxon 60: 151–160. [Google Scholar]
  38. Sang T. (2002) Utility of low-copy nuclear gene sequences in plant phylogenetics. Critical reviews in Biochemistry and Molecular Biology 37(3): 121–147. https://doi.org/10.1080/10409230290771474 [DOI] [PubMed] [Google Scholar]
  39. Sergievskaya LP. (1961) Calligonum L. In: Krasnoborov IM. (Ed.) Flora of Siberia (Vol. 3). Tomsk University Press, 3237–3250. [Google Scholar]
  40. Shi W, Pan BR. (2015) Karyotype analysis of the Calligonum mongolicum complex (Polygonaceae) from Northwest China. Caryologia 68: 125–131. https://doi.org/10.1080/00087114.2015.1032572 [Google Scholar]
  41. Shi W, Pan BR, Kang XS, Duan SM. (2011) Difference analysis on the fruit morphological characteristics of Calligonum mongolicum and its related species. Journal of Desert Research 31: 121–128. [Google Scholar]
  42. Shi W, Pan BR, Shomurodov H. (2012) Correlation of soil properties and fruit size of Calligonum mongolicum and related species. Journal Arid Land 4: 63–70. https://doi.org/10.3724/SP.J.1227.2012.00063 [Google Scholar]
  43. Shi W, Wen J, Pan BR. (2016) A comparison of ITS sequence data and morphology for Calligonum pumilum and C. mongolicum (Polygonaceae) and its taxonomic implications. Phytotaxa 261: 157–167. https://doi.org/10.11646/phytotaxa.261.2.5 [Google Scholar]
  44. Shi W, Zhao YF, Kong FK, Pan BR. (2013) Species redress in the Calligonum mongolicum complex (Polygonaceae) - A multidisciplinary approach. Vegetos 26: 249–261. https://doi.org/10.5958/j.2229-4473.26.1.036 [Google Scholar]
  45. Soskov YD. (1975a) The distribution of Calligonum L. The New Higher Plants 12(1): 147–159. [Google Scholar]
  46. Soskov YD. (1975b) The new series of subspecies and hybrids of Calligonum L. Botanicheskii Zhurnal 60(1): 1–6. [Google Scholar]
  47. Soskov YD. (2011) The genus Calligonum L.: Taxonomy, distribution, evolution, introduction. Russian Academy of Agricultural Sciences (Siberian Branch), Novosibirsk, 361 pp. [Google Scholar]
  48. Stamatakis A. (2006) RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22(21): 2688–2690. https://doi.org/10.1093/bioinformatics/btl446 [DOI] [PubMed] [Google Scholar]
  49. Stanford AM, Harden R, Parks CR. (2000) Phylogeny and biogeography of Juglans (Juglandaceae) based on matK and ITS sequence data. American Journal of Botany 87: 872–882. https://doi.org/10.2307/2656895 [PubMed] [Google Scholar]
  50. Stebbins GL. (1950) Variation and Evolution in Plants. Columbia Univ Press, New York. [Google Scholar]
  51. Sun Y, Zhang M. (2012) Molecular phylogeny of tribe Atraphaxideae (Polygonaceae) evidenced from five cpDNA genes. Journal of Arid Land 4: 180–190. https://doi.org/10.3724/SP.J.1227.2012.00180 [Google Scholar]
  52. Swarts ND, Dixon KW. (2009) Perspectives on orchid conservation in botanic gardens. Trends in Plant Science 14: 590–598. https://doi.org/10.1016/j.tplants.2009.07.008 [DOI] [PubMed] [Google Scholar]
  53. Tavakkoli S, Osaloo SK, Maassoumi AA. (2010) The phylogeny of Calligonum and Pteropyrum (Polygonaceae) based on nuclear ribosomal DNA ITS and chloroplast trnL-F sequences. Iranian Journal of Biotechnology 8: 7–15. [Google Scholar]
  54. Wang CG, Guan SC. (1986) The geographical distributions of chromosomes of Calligonum in Xinjiang. Arid Zone Research 2: 28–31. [Google Scholar]
  55. Wang CG, Yang G. (1985) Investigation of chromosome number and chromosomal ploidy of Calligonum in Xinjiang. Arid Zone Research 1: 62–64. [Google Scholar]
  56. Wen J, Liu JQ, Ge S, Xiang QY(J), Zimmer EA. (2015) Phylogenomic approaches to deciphering the tree of life. Journal of Systematics and Evolution 53: 369–370. https://doi.org/10.1111/jse.12175 [Google Scholar]
  57. Wen J, Nowicke JW. (1999) Pollen ultrastructure of Panax (the ginseng genus, Araliaceae), an eastern Asian and eastern North American disjunct genus. American Journal of Botany 86: 1624–1636. [PubMed] [Google Scholar]
  58. Wen J, Zimmer EA. (1996) Phylogeny and biogeography of Panax L. (the ginseng genus, Araliaceae): inferences from ITS sequences of nuclear ribosomal DNA. Molecular Phylogenetics and Evolution 6: 167–177. https://doi.org/10.1006/mpev.1996.0069 [DOI] [PubMed] [Google Scholar]
  59. Wen Z-B, Li Y, Zhang H-X, Meng H-H, Feng Y, Wei S. (2016) Species-level phylogeographical history of the endemic species Calligonum roborovskii and its close relatives in Calligonum section Medusa (Polygonaceae) in arid north-western China. Botanical Journal of the Linnean Society 180: 542–553. https://doi.org/10.1111/boj.12381 [Google Scholar]
  60. Wilson MF. (1983) Plant Reproductive Ecology. Wiley-Interscience, New York. [Google Scholar]
  61. Xu BS. (1998) The species problem in plant taxonomy in China. Acta Phytotaxonomica Sinica 36(5): 470–480. [Google Scholar]
  62. Zhu KZ, Wan MW. (1999) Phenology. Science Press, Beijing. [Google Scholar]
  63. Zimmer EA, Wen J. (2012) Using nuclear gene data for plant phylogenetics: Progress and prospects. Molecular Phylogenetics and Evolution 65: 774–785. https://doi.org/10.1016/j.ympev.2012.07.015 [DOI] [PubMed] [Google Scholar]
  64. Zimmer EA, Wen J. (2015) Using nuclear gene data for plant phylogenetics: Progress and prospects II. Next-gen approaches. Journal of Systematics and Evolution 53: 371–379. https://doi.org/10.1111/jse.12174 [Google Scholar]

Articles from PhytoKeys are provided here courtesy of Pensoft Publishers

RESOURCES