Molecular phylogenetic and biogeographical analysis of based on nuclear and chloroplast DNA sequences

Kamshat Temirbayeva & Ming-Li Zhang

Plant Systematics and Evolution

ISSN 0378-2697 Volume 301 Number 7

Plant Syst Evol (2015) 301:1897-1906 DOI 10.1007/s00606-015-1202-5

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1 23 Author's personal copy

Plant Syst Evol (2015) 301:1897–1906 DOI 10.1007/s00606-015-1202-5

ORIGINAL ARTICLE

Molecular phylogenetic and biogeographical analysis of Nitraria based on nuclear and chloroplast DNA sequences

Kamshat Temirbayeva • Ming-Li Zhang

Received: 3 January 2014 / Accepted: 1 February 2015 / Published online: 5 March 2015 Ó Springer-Verlag Wien 2015

Abstract Based upon DNA sequences from six plastid Introduction regions (rbcL, psbB-psbH, trnL-trnF, rpS16, psbA-trnH, rpS16-trnK) and the internal transcribed spacer (ITS) re- is a family of and annual and perennial gion of nuclear ribosomal DNA, the phylogenetic rela- herbs, mainly distributed in arid and semi-arid regions of tionships in the genus Nitraria and family Nitrariaceae are the Old World, as well as Australia’s deserts, and the New investigated by using methods of maximum parsimony, World from Texas to Mexico (Agnew 1980; Komarov maximum likelihood, and Bayesian inference. Our study 1908; Noble and Whalley 1978a; Ronse Decraene et al. strongly supports the monophyly of Nitraria. Nitraria can 1996; APG 1998, 2003; Sheahan 2011a, b). This family be divided into four parts, namely, the N. sphaerocarpa comprises four genera and around nineteen species group, N. retusa group, the N. roborowskii and N. worldwide (Pan et al. 1999; Stevens 2001 onwards; APG tangutorum group, and a group consisting of N. schoberi, 2009; Bachelier et al. 2011). The taxonomy of all genera N. komarovii, N. sibirica, and N. billardieri. Ancestral area has been the subject of disagreement for a lengthy period reconstruction using S-Diva shows that eastern Central (Cronquist 1981; Dahlgren 1980; El-Hadidi 1975; Gold- Asia is most likely the place of origin, and then dispersals berg 1986; Takhtajan 1969, 1980). In Engler’s work occurred to western Central Asia, Africa, and Australia. (1896a, b, 1931) the members of this family, based on seed and characters, were included in the family Zygo- Keywords Nitrariaceae Á Nitraria L. Á Peganum L. Á phyllaceae, that consisted of subfamilies Nitrarioideae, Molecular phylogeny Á Monophyly Á Place of origin Tetradiclidoideae, and Peganoideae (Peganum L. and Malacocarpus Fisch. & C.A.Mey.). Dahlgren (1980, 1983, 1989) suggested that Zygophyllaceae should be divided Handling editor: Pablo Vargas. into four families i.e., Zygophyllaceae, Nitrariaceae, Pe- Electronic supplementary material The online version of this ganaceae, and Balanitaceae. Molecular phylogenetic stud- article (doi:10.1007/s00606-015-1202-5) contains supplementary ies have shown that Nitrariaceae is closely related to material, which is available to authorized users. (Gadek et al. 1996; Sheahan and Chase 1996; Bakker et al. 1998; Savolianen et al. 2000), where Nitraria, K. Temirbayeva Á M.-L. Zhang Key Laboratory of Biogeography and Bioresource in Arid Land, Peganum, Malacocarpus, and Tetradiclis form a clade, Xinjiang Institute of Ecology and Geography, Chinese Academy with Nitraria sister to the other three genera, and Te- of Sciences, Urumqi 830011, China tradiclis close to Peganum ? Malacocarpus (Sheahan and Chase 1996; Savolianen et al. 2000; Muellner et al. 2007). K. Temirbayeva University of Chinese Academy of Sciences, Beijing 100049, Based on rbcL sequence data (Sheahan and Chase 1996) China and anatomical data (Sheahan and Cutler 1993), it was proposed that Nitraria and Peganum (and Malacocarpus) & M.-L. Zhang ( ) be separated from the family Zygophyllaeceae and defined Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China as distinct families, Nitrariaceae and Peganaceae. The e-mail: [email protected] floral development and vascular anatomy of Nitrariaceae 123 Author's personal copy

1898 K. Temirbayeva, M. Zhang was studied by Ronse Decraene and Smets (1991), Ronse separation of Nitraria retusa from the Zygophyllaceae. Su Decraene et al. (1996), and Bachelier et al. (2011), who and Zhang (2013) studied the chloroplast phylogeography confirmed the exclusion of these genera from Zygophyl- of Nitraria sphaerocarpa, and its evolutionary response to laceae and their integration with Sapindales. Quaternary climate aridification and oscillations in north- Nitraria consists of 10–12 species with a world-wide western China, and estimated divergence times between the distribution including Southeast Europe, Southwest and lineages within N. sphaerocarpa as early and early middle Central Asia (Usturt, Turan, Turgai, Kazakh uplands, and Pleistocene. partially the mountain ranges Kopetdag, Pamir-Alai, Tian- The genus Peganum L., including 5–6 species, is common Shan, Dzungar Alatau, Saur, and Tarbagatai), Mongolia in arid regions of Eurasia, North America, and North Africa. and China, North Africa, and Australia. The members of It consists of perennial herbs with twice or three times ir- this genus are salt desert shrubs of 0.5–2 m height, with regularly dissected leaves and herbaceous stipules (Ronse entire, alternate, fleshy leaves. They grow in great abun- Decraene et al. 1996). Peganum was divided into two genera, dance and produce salty-sweet edible (drupes) which Peganum and Malacocarpus (Hutchinson 1967; Sheahan are important as a food (Komarov 1908, 1947; Pan et al. and Cutler 1993); Malacocarpus had previously been a 1999; Noble and Whalley 1978a; Sheahan 2011a, b). These section of Peganum (Engler 1931). Malacocarpus is a liana shrubs are used in protective afforestation for enhancement with alternate leaves regularly dissected into linear of sand deposits and banks, and reduction of soil salinity, segments, and with stipules. This is a monotypic genus, in- and improvement of the concentration of organics sub- cluding only one species, M. crithmifolius (Retz.) C.A.M., stances (Noble and Whalley 1978b; Parida and Das 2005; distributed in the eastern Mediterranean basin (Sheahan Vysochina et al. 2011). There are several opinions re- 2011a, b). Tetradiclis Steven ex. M.Bieb is an annual herb garding the taxonomy of Nitraria. Bobrov (1965) separated with linear entire or irregularly dissected leaves with stip- the genus into two sections, Nitraria and Tridentatae Ilyin. ules, distributed in desert and semi-desert regions of the Section Nitraria was in turn divided into two series: Middle East, Asia Minor, southern and eastern Europe, the Sibiricae Bobr. (N. sibirica Poir., N. roborowskii Kom.,N. Caucasus, Central Asia, and Afghanistan (Namzaoglu et al. tangutorum Bobr., N. praevisa Bobr., and N. sphaerocarpa 2005; Sheahan 2011a, b). Initially, these were placed Maxim.) and Schoberianae Bobr. (N. schoberi L., N. ko- in the family Rutaceae, and later were included in Zygo- marovii Iljin & Lava, N. senegalensis Poir., and N. bil- phyllaceae (Engler 1896a, 1931). More recently, El-Hadidi lardieri DC.). Section Tridentatae included only N. retusa (1977) and Takhtajan (1980, 1983) excluded them from Asch. Khalkuziev (1990) divided the genus into two sec- Zygophyllaceae and awarded them familial status (Takhta- tions, Primaria Chalkuziev (based on characters of the jan 1986, 1987). stone, with anastomosing veins, a reticulate surface, and One interesting issue in Nitrariaceae concerns the place with sculpturing) (N. pamirica L. I. Vassilyeva, N. retusa, of origin of Nitraria. Distribution of the genus had attracted N. sphaerocarpa), and Nitraria (the stone solid or with the attention of researchers trying to understand the origin, deep rounded pits, with anastomosing veins, a smooth route and time of settlement of its representatives. Ko- surface, and without sculpturing) (N. schoberi, N. robor- marov (1908) suggested a tropical and western origin from owskii). According to pericarp structure, evolutionary Africa, from where it penetrated to Mongolia through the trends, and pollen morphology, Pan et al. (1999) divided Aral-Caspian basin during its desiccation; Ilyin (1944) and the genus into three groups: the honeycomb group, hard Vassilchenko (1982) supported this. According to Ko- shell-like group, and bubble-shaped fruit group. Based on marov (1908), the original location of N. schoberi was, in the chromosome number, as well as morphological and all probability, the sea shore. Pan et al. (1999) also thought geo-biological data of Nitraria species, another author by African and Australian species to be primitive, whereas the this surname, Pan et al. (2003), divided them into a Central Asian species were derived with respect to pollen diploid (2n = 2x = 24) and a tetraploid group characters. Popov (1927) suggested an opposite concept (2n = 4x = 48). Zhang (2006) studied molecular system- from Komarov’s, stressing the antiquity of the flora of the atics, genetic diversity, and drought resistant physiology of Central Asian deserts. He insisted that ‘‘Mongolia and the five species collected from the Hexi Corridor in Gansu desert country of Tien Shan had their own, older than the Province, China and used three DNA markers (ITS, rbcL, Miocene-Pliocene, sources of desert forms, some of which, trnL-F) for estimations using maximum parsimony, and after drying of the Aral-Caspian (Turan) plain began to produced a phylogenetic tree. A large distance was de- penetrate there from the northeast, moving to the south- tected between Nitraria L. and Zygophyllaceae s. s., but west’’. His opinion was fully accepted by Korovin (1935), the genus had a close affinity with Peganum. Based on and later almost completely agreed with by Bobrov (1946), numerical analysis of morphological, anatomical, and while Grubov (1963) thought the genus to have had a na- chemical characters, Hussein et al. (2009) suggested the tive origin, i.e., from Central Asia. Based on the ploidy 123 Author's personal copy

Molecular phylogeny and biogeography of Nitraria 1899 variation, Pan et al. (2003) speculated the location with genomic DNA at 5 ng/ll. The protocol for amplification primitive diploid species should be the place of origin, consisted of an initial hotstart at 95 °C for 4 min, followed namely, the Tethys seacoast, presently North Africa and by 30 cycles of denaturation at 94 °C for 30 s, annealing at the Mediterranean basin. We aim to examine these hy- 52 °C for 30 s, extension at 72 °C for 90 s, and a final potheses on the basis of molecular phylogeny. extension at 72 °C for 10 min; 4 °C hold. PCR products Overall, disagreements on the taxonomy of Nitrariaceae were electrophoresed using a 0.8 % agarose gel in a and Nitraria have continued, due to the lack of a good 0.5 9 TAE (pH 8.3) buffer, then stained with ethidium molecular phylogeny. Previously, only a few species within bromide to confirm a single product. The PCR products the family were investigated phylogenetically, and only a were purified using purification kits (Qiagen) following the few DNA regions used. In this paper, we aim to determine recommended protocol, and sequenced using an ABI Prism the relationships of the Nitrariaceae, especially Nitraria, 3770 Genetic Analyzer (Shanghai Sanggon Biological based on a larger number of species samples, and seven Engineering Technology and Service, Shanghai, China). DNA regions—the nuclear ribosomal DNA (nrDNA) in- We selected seven DNA fragments to examine phylo- ternal transcribed spacer (ITS) and six chloroplast DNA genetics of the Nitrariaceae, including nrDNA ITS, cpDNA (cpDNA) regions (rbcL, psbB-psbH, trnL-trnF, rpS16, rbcL, psbB-psbH, trnL-trnF, rpS16, psbA-trnH, rpS16- psbA-trnH, and rpS16-trnK). We then examine hypotheses trnK. The ITS gene was amplified with primers ITSl: 50- for the place of origin using the biogeographical approach AGA AGT CGT AAC AAG GTT TCC GTA G-30 (Kang of ancestral area reconstruction. et al. 2003); ITS4: 50TCC TCC GCT TAT TGA TAT GC- 30 (White et al. 1990). The psbB-psbH region was ampli- fied using the primers psbB-psbH-f (50-AGA TGT TTT Materials and methods TGC TGG TAT TGA-30) and psbB-psbH-r (50-TTC AAC AGT TTG TGT AGC CA-30) (Xu et al. 2000). For rbcL Taxon sampling sequences, we used four standard primers 1F (50-ATG TCA CCA CAA ACA GAA ACT AAA GC-30), 875F (50-GCA We sampled five species of Nitraria obtained from the GTT ATT GAT AGA CAG A-30), 972 R (50-TCT CCA Turfan Botanical Garden, Xinjiang Province, and one CCA GAC ATA CG -30) and 1460R (50-TTT AGT AAA species of Peganum from Gansu Province, in China. Other AGA TTG GGC CGA G-30) (Zhang 2006). For trnL-trnF Nitraria species were sampled from the Dead Sea area, region trnL-trnF-f (50-CGA AAT CGG TAG ACG CTA Israel, and Kangaroo Island, Australia. Three additional CG-30), trnL-trnF-r (50-ATT TGA ACT GGT GAC ACG samples of Nitraria came from herbaria: PE (Herbarium, AG-30) (Taberlet et al. 1991) were used. The psbA-trnH Institute of Botany, Chinese Academy of Sciences, Bei- region was amplified using the primers psbA-trnH-f (50- jing), and MHA (Main Botanical Garden, Russia, Mos- GTT ATG CAT GAA CGT AAT GCT C-30) (Sang et al. cow). Previously reported sequences were also downloaded 1997) and psbA-trnH-r (50-CGC GCA TGG TGG ATT from Genbank (Zhang et al. 2005). Two species, Tribulus CAC AAT CC-30) (Tate and Simpson 2003). For the rpS16 terrestris L. and Zygophyllum xanthoxylum Maxim. (Zy- region rpS16-f (50-GTG GTA GAA AGC AAC GTG CGA gophyllaceae) were chosen as outgroups for the phyloge- CTT-30), rpS16-r (50-TCG GGA TCG AAC ATC AAT netic analysis of Nitrariaceae. We considered that all these TGC AAC-30) (Oxelman et al. 1997) and for rpS16-trnK species have been viewed as closely related within fragment rpS16-trnK-f (50- AAA GTG GGT TTT TAT Zygophyllaceae in the past, and it seems that Zygophyl- GAT CC -30), rpS16-trnK-r (50-TTA AAA GCC GAG laceae is an old family, because of its cosmopolitan dis- TAC TCT ACC-3) (Shaw et al. 2007) primers were used. tribution (Sheahan and Chase 1996; Zhang et al. 2005) The DNA sequences were assembled by SeqMan (Table 1). (Lazergene, DNASTAR Inc., Madison, Wisconsin, USA), and visually aligned with Clustal X v.1.81 (Thompson et al. DNA sequencing 1997), followed by manual correction in BioEdit (Hall 2001). Alignment is available in Online Resource 1. Total genomic DNA was extracted using the CTAB (cetyltrimethylammonium bromide) method of Doyle and Phylogenetic analyses Doyle (1987). PCR amplifications were carried out in a total volume of 30 ll, containing 1.5 llof109 PCR reaction To find incongruence between ITS and six cpDNA se- buffer (Takara, Japan), 1.5 ll of 25 mM MgCl2, 1.2 llof quences (rbcL, psbB-psbH, trnL-trnF, rpS16, psbA-trnH, each primer (Shanghai Sangon, China) at 50 ng/ll, 2.4 llof and rpS16-trnK), we used MrBayes (version 3.0b4, 2.5 mM dNTP solution in an equimolar ratio, 0.6 llofTaq Huelsenbeck and Ronquist 2001) to estimate the difference DNA-polymerase (5 U/ll, Takara, Japan), and 2 llof of phylogenetic tree topologies. The option was set up 123 1900 123

Table 1 List of sampled taxa, vouchers and Genbank accession numbers Species Voucher Sourse GenBank accession nos ITS trnL-trnF rbcL rpS16 psbA- psbB- rpS16- trnH psbH trnK

Nitrariaceae Author's Nitraria schoberi L. M.L. Zhang (2008.5.6) Turfan, Xinjiang, China KP087771 KP087727 KP087789 KP087747 KP087759 KP087735 KP087779 Nitraria sibirica Pall. M.L. Zhang (2008.5.6) Turfan, Xinjiang; China DQ267178 DQ267168 DQ267160 KP087748 KP087760 KP087736 KP087780 Nitraria sphaerocarpa Maxim. M.L. Zhang (2008.5.6) Turfan, Xinjiang; China DQ267177 DQ267167 DQ267159 KP087749 KP087761 KP087737 KP087781 Nitraria roborowskii Kom. M.L. Zhang (2008.5.6) Turfan, Xinjiang; China DQ309042 DQ267169 DQ267161 KP087750 KP087762 KP087738 KP087782 Nitraria tangutorum Bobr. M.L. Zhang (2008.5.6) Turfan, Xinjiang; China DQ267176 DQ267166 DQ267158 KP087751 KP087763 KP087739 KP087783 personal Nitraria retusa Aschers. T. Konovalova (1996.3.3) 28°N33°E, Egypt, Africa KP087772 KP087728 KP087790 KP087752 KP087764 KP087740 KP087784 (MHA) Nitraria retusa Aschers. M. Blecher (2012.7.10) Ein Gedi,Dead Sea area, KP087773 KP087729 KP087791 KP087753 KP087765 KP087741 KP087785 Israel

Nitraria komarovii Ili & Lav. P. Farse (1964.5.13) (PE) Afganistan KP087774 KP087730 KP087792 KP087754 KP087766 KP087742 KP087786 copy Nitraria billardieri D. C. M&P Rismiller (2013.3) Kangaroo Ireland; S KP087775 KP087731 – KP087755 KP087767 KP087743 – Australia Peganum harmala L. Z.H. Su (2011) Jin Ta, Gansu, China KP087776 KP087732 KP087793 KP087756 KP087768 KP087744 KP087787 Outgroup Tribulus terrestris L. Z.H. Su (2011) Hami, Xinjiang, China KP087777 KP087733 KP087794 KP087757 KP087769 KP087745 – Zygophyllum xanthoxylum X.J. Shi (2010) Zhongwei, Ninxia, China KP087778 KP087734 KP087795 KP087758 KP087770 KP087746 KP087788 Maxim. .Tmraea .Zhang M. Temirbayeva, K. Author's personal copy

Molecular phylogeny and biogeography of Nitraria 1901 using 20,000,000 generations of Markov Chain Monte Operational areas Carlo (MCMC) and a sample frequency of 1,000. Satura- tion was reached after a burn-in of 10 % generations. Two Central Asia is the distribution center of Nitraria. Based on trees have no incongruence among the main nodes. the floristic divisions of Grubov (1999), we can divide it Therefore, seven sequence combined dataset of ITS and six into two parts, eastern, consisting of Mongolia, and west- cpDNA sequences was used for phylogenetic analyses ern, including the Turan Basin. Other areas clearly in- which employed approaches of maximum parsimony (MP), volved include Africa and Australia, as well as East Asia. maximum likelihood (ML) and Bayesian inference (BI). Thus, five areas were distinguished for the present bio- MP and ML were executed in PAUP* 4.0b1.0 (Swofford geographical study, namely, A: eastern Central Asia, 2002). The MP analyses used heuristic searches (1,000 Mongolia; B: western Central Asia, Turan; C: Africa, in- random addition cycles, with tree bisection-reconnection cluding locations around the Mediterranean; D: Australia; and branch-swapping), and swapping was run to comple- E: East Asia, north of A and B, but mainly the northern tion for all random addition replicates. Clade support was Chinese mountains near the Mongolian deserts. estimated with 1,000 heuristic bootstrap replicates (100 random addition cycles per replicate, with tree bisection- Ancestral area reconstruction reconnection and branch-swapping) (Felsenstein 1985; Hillis and Bull 1993). We used S-Diva (or Bayes-DIVA, Nylander et al. 2008;Yu For ML analysis, the index of appropriate model of et al. 2010) to reconstruct the ancestral area. S-Diva can DNA substitution estimated from Modeltest 3.06 (Posada assess the relevant explanatory processes of vicariance, and Crandall 1998) was used. The model selected by the dispersal, and extinction in biogeography. Akaike information criterion (AIC) was GTR ? I ? G. S-Diva determines statistical support for ancestral range BI analysis was conducted using MrBayes, version reconstructions using multiple trees from Bayesian analysis 3.0b4 (Huelsenbeck and Ronquist 2001; Huelsenbeck and (Nylander et al. 2008). This has the advantage that uncer- Rannala 2004). The option was set up using 20,000,000 tainties in phylogenetic inference can be taken into ac- generations of Markov Chain Monte Carlo (MCMC) and a count. In general, 100 Bayesian MCMC trees with stable sample frequency of 1,000. Saturation was reached after a topologies were input into the S-Diva program RASP (Yu burn-in of 10 % generations. et al. 2010). Here for Nitrariaceae, the 711 ultimate stable Bayesian inference trees were input into the program RASP. Setting Maxarea = 2. Biogeographical events such Zygophyllum xanthoxylum as dispersal, vicariance, and extinction were calculated under Tree View Form in RASP. Tribulus terrestris

Peganum harmala Results Nitraria sphaerocarpa I 100 2n=24 1.00 Phylogenetic analysis Nitraria retusaAfr 100 2n=24 100 1.00 II 1.00 Nitraria retusaLsr Analyses of the three phylogenetic approaches MP, ML, 2n=24 and BI yielded identical tree topologies. The ML tree of the Nitraria roborowskii 84 combined matrix is presented in Fig. 1. Zygophyllum 68 2n=48 1.00 III 0.70 xanthoxylum and Tribulus terrestris were defined as out- Nitraria tangutorum 2n=48 groups. The family Nitrariaceae formed a monophyly (Fig. 1), and Peganum harmala was close to it 100 Nitraria sibirica 1.00 2n=24 (bt = 100 %; pP = 1.00). Four groups formed within Ni- 69 1.00 Nitraria komarovii trariaceae. Nitraria sphaerocarpa was resolved as sister to 93 2n=24 IV 97 1.00 the rest of Nitraria and formed group I. Two N. retusa 1.00 Nitraria billardieri 2n=48 samples from North Africa and Israel constituted a clade, i.e., group II. Nitraria roborowskii and N. tangutorum in Nitraria schoberi 2n=24 eastern Central Asia, i.e., northwestern China and Mon- golia, formed a clade, indicated as group III (bt = 68 %; Fig. 1 Maximum likelihood tree conducted from the combined pP = 0.70). Nitraria schoberi, N. sibirica, and N. ko- seven-gene analysis (ITS, rbcL, psbB-psbH, trnL-trnF, rpS16, psbA-trnH, rpS16-trnK). Maximum parsimony bootstrap values are marovii, as well as Australian N. billardieri were included shown above branches, and Bayesian posterior probabilities below in group IV (bt = 97 %; pP = 1.00). 123 Author's personal copy

1902 K. Temirbayeva, M. Zhang

Fig. 2 Ancestral area optimal distributions at each node: reconstruction by S-Diva run by RASP. Pie charts at the internal (A) Zygophyllum xanthoxylum nodes represent the calculated probabilities (relative (ABE) Tribulus terrestris frequencies) of alternative 1.00 ancestral area reconstructions. (ABCE) Peganum harmala The most likely areas and 1.00 frequencies calculated by RASP (A) are illustrated on the node at A Nitraria sphaerocarpa right. The arrowheads indicate 1.00 dispersals. The five areas are A: (C) Nitraria retusaAfr eastern Central Asia, Mongolia; A 1.00 1.00 B: western Ancestral Asia, (C) Nitraria retusaLsr Turan; C: Africa, including location around Mediterranean; AC (A) Nitraria roborowskii D: Australia; E: East Asia, 1.00 mainly northern China 1.00 mountains near the Mongolian (A) Nitraria tangutorum deserts AB 1.00 (B) Nitraria schoberi

B 1.00 (AB) Nitraria sibirica B 1.00 (B) Nitraria komarovii BD 1.00 (D) Nitraria billardieri

Ancestral area reconstruction Malacocarpus) and Nitraria were transferred from Zygo- phyllaceae into two respective families of Sapindales based The S-Diva result is shown in Fig. 2. Eastern Central Asian on rbcL sequence analysis (Sheahan and Chase 1996). In Mongolia (A) appeared at nodes of the genus Nitraria and their results, Peganum and Malacocarpus were in a clade, the family Nitrariaceae. Therefore, we recognized eastern but the appearance of Nitraria as a sister to it was unex- Central Asia to be the place of origin of Nitraria and pected. According to several morphological and anatomical Nitrariaceae. dissimilarities between Peganum and Nitraria, such as Several dispersals are indicated in Fig. 2. Distributions leaves (pinnatifid in Peganum and simple, clustered from in western Central Asian Turan (B), Africa (C), and Aus- branchlets in Nitraria), fruits (capsular in Peganum and tralia (D), are marked as dispersals, while the distribution drupaceous in Nitraria), it was proposed they should be- of N. sibirica in Mongolia (A) was a dispersal from western long to separate families (Sheahan and Cutler 1993; Central Asian Turan (B). The distributions represented by Sheahan and Chase 1996). Peganum, Malacocarpus, and group I with N. sphaerocarpa, and group III with N. Nitraria formed a distinct and robustly supported clade roborowskii and N. tangutorum, were geographically in- (decay ?7) within Sapindales, where Peganum and herited from eastern Central Asian Mongolia (A), whereas Malacocarpus were strongly clustered (decay ?8) pro- group II with N. retusa, and group IV distributions in viding good support for their inclusion in the single family western Central Asian Turan (B) and Australia (D) were Peganaceae (Gadek et al. 1996). The study of floral de- shown to be dispersals. velopment and morphology of Peganum as well as the flower structure of Nitraria and Tetradiclis, showed that the flower features of these three taxa shared are not Discussion common in the remainder of Zygophyllaceae, but wide- spread in sapindalean families (Bachelier et al. 2011). Ni- The relationships between Peganum and Nitraria traria and Peganum have more than ten (fifteen) stamens, and the unusual androecium is most likely derived from a The present phylogenetic analysis shows the monophyly of haplostemonous condition, similar to that of Tetradiclis. Nitrariaceae. A close relationships between Peganum and The syncarpous structure of the gynoecium gives good Nitraria is indicated with high support (bt = 100 %; support for a close relationship between the three genera, pP = 1.00). In the previous studies, Peganum (with and Peganum and Tetradiclis are even closer (Ronse

123 Author's personal copy

Molecular phylogeny and biogeography of Nitraria 1903

Decraene and Smets 1991; Ronse Decraene et al. 1996; The N. retusa clade forms our group II. Khalkuziev Bachelier et al. 2011). Peganum was recently indicated to (1990) treated N. retusa as the primitive taxon relative to N. be monophyletic based on the trnL-F and psbA-trnH se- pamirica and N. sphaerocarpa, and this species has a high quences (Zhao et al. 2011). They sampled five Nitraria variability, showing subspecific descendants, such as N. species in China and sequenced the rbcL and trnL-F loci, retusa (Forsk.) Aschers. ssp. retusa, ssp. senegalensis which in combination with morphology, biochemistry, and (Lam.) Chalk., and ssp. sericea (Jaub. et Spach. A dis- embryology indicates a close relationship between Nitraria tinctive feature of N. retusa is a triangular stone with very and Peganum. Our results are consistent with these previ- precise sculpturing of the outer layer, with a system of ous conclusions. anastomosing veins. The mysterious holes in the bottom part of the stones in other species are explained as remnants Monophyly of Nitraria and relationships of the large gaps between these original lignified, heavily within the genus overgrown vascular bundles (veins) on their surface. Fur- ther, N. retusa has stipules remaining at the base of the Our phylogenetic analyses suggests that Nitraria sphaero- leaves and even becoming lignified, with an elongated carpa is sister to the rest of the members of Nitraria, as it is peduncle (Komarov 1947). Pan et al. (1999) allocated it to shown to be at a position of early divergence in the den- the section Retusae Pan together with N. pamirica. drogram (bt = 84 %; pP = 1.00). Przewalskii (1883) Group III consists of N. roborowskii and N. tangutorum considered this shrub to be characteristic of the Hami desert (bt = 68 % and pP = 0.7); N. roborowskii was collected (eastern Tianshan Mountains, China), where it is found in by Grubov (1963) in Mongolia and by Petrov (1964) in the great abundance. Bobrov (1965) did not include this species Alashan desert (China), where they indicated its habitat as in either of his sections. It is the only species with a dry solonchaks (saline soils), dry arenaceous localities, and dry spherical drupe, while all the others in the genus have sites with cane along riverbeds, i.e., areas with temporary succulent drupes (Komarov 1908, 1947; Bobrov 1965; watercourses. It was noted that all the habitats of this Khalkuziev 1990; Liu and Zhou 1998; Liu and Huang 2000; species are relatively favorable and moist compared to Su and Zhang 2013). Like other dune plants, this species is most desert areas. Bobrov (1965) noted that this species adapted to an existence on relatively shifting sands (Bobrov differs from the Siberian species only in quantitative at- 1965). Khalkuziev (1990) included this species in section tributes, such as gigantism. Nitraria roborowskii and N. Primaria Chalkuziev (N. pamirica, N. retusa, and N. tangutorum are included in the bubble-shaped fruit group sphaerocarpa), where characters such as primitive structure (section Nitraria) (Pan et al. 1999), and also have the same of stones, placentation, and reduction of ovules indicated chromosome number (2n = 4x = 48) (Pan et al. 2003). the antiquity of the taxa. He defined N. sphaerocarpa and N. Our results maintain this division. pamirica as relicts. Pan et al. (1999) put this species in his The last clade consists of four species. It matches the section Sphaerocarpae Pan (hard shelled group), indicating previous section in a Nitraria treatment (Pan et al. 1999), its difference from the other species. Based on chromosome where N. schoberi, N. komarovii, N. sibirica together with numbers, the other Pan et al. (2003) divided Nitraria in- N. roborowskii and N. tangutorum (but without N. bil- to diploid and tetraploid groups. To the diploid he referred lardieri) were included in the bubble-shaped fruit group. species distributed in North Africa and adjacent Arabia, the Nitraria komarovii, related to the recent history of the Mediterranean (N. retusa, N. senegalensis, N. tridentate), Caspian Basin, is thought to be the youngest species. Caspian (N. komarovii), and Aral Sea areas and northwest N. billardieri is distributed in the dry savanna and saline China (N. schoberi, N. sibirica, N. sphaerocarpa), and subdeserts of southwestern Australia. In our studies, two indicated them as primitive. In the tetraploid group he in- desert species of the northern and southern hemispheres, cluded species distributed in Central Asia, northwest China N. komarovii and N. billardieri, are nested together (N. pamirica, N. tangutorum, N. roborowskii = N. prae- (bt = 93 %; pP = 1.00), sharing close affinities, and sister visa,and N. sinensis), and Australia (N. billardieri) and to N. sibirica. The distribution of N. sibirica is primarily in indicated them as advanced. In this division, N. sphaero- the mountainous countries of Central Asia, stretching from carpa was again assigned to the primitive group. Based on the western Tianshan to the Liaodong Peninsula in China, ITS sequence data, Nitraria in Gansu Province in China and from the Trans-Baikal in Siberia to the northern edge was divided into three groups by Zhang (2006). The first of of Tibet (Bobrov 1946). Nitraria schoberi occurs in the their three groups consisted of N. sibirica; the second Aral Sea–Caspian Sea region, i.e., the Caspian and Turan consisted of N. sphaerocarpa, and the third included N. lowlands, in sand and solonchak (saline) deserts covering praevisa, N. roborowskii, and N. tangutorum. Our N. the Cretaceous–Tertiary plateau. It expands eastward to sphaerocarpa clade thus endorses this aspect of the previ- Kashgar and Dzungaria, northward to the Kazakh ous studies. uplands, southward to Afghanistan and northern Iran, 123 Author's personal copy

1904 K. Temirbayeva, M. Zhang southwestward to Transcaucasia and westward to Syria As seen in Fig. 2, it is apparent that several dispersals (Bobrov 1946). Individuals of N. schoberi are distinctive in were from Mongolia, eastern Central Asia (A) to Turan, their fluffy appearance, leaf size, and flower cluster type western Central Asia (B), and to Africa (C). Nitraria si- (Komarov 1908, 1947; Khalkuziev 1990); N. schoberi is birica dispersed from western Central Asia (B) to Mon- probably a complex containing many intraspecific taxa golia (A). It is peculiar that the Australian species N. within group IV. billardieri is derived from western Central Asian taxa On the whole, our four groups are equal to Zhang’s (B) and related to N. schoberi, N. sibirica and N. ko- (2006) groups, and roughly correspond to the sections of marovii, rather than N. retusa. Komarov (1908, 1947) hy- Pan et al.’s (1999) classification system, but there are pothesized that N. billardieri is derived from Africa, based several differences, such as the placement of N. billardieri. upon paleogeographical evidence of former contact be- She put it in section Retusae Pan, whereas we place it in tween the Australian and African plates. It currently re- group IV with the species N. schoberi, N. komarovii, and N. mains unclear how N. billardieri came to originate from sibirica. Meanwhile, our groups III ? IV are equal to Turan, western Central Asia (B), and further work is section Nitraria of Pan et al. (1999). needed to establish the nature of the paleogeologic and paleoclimatic linkages between Central Asia and Australia. Ancestral area reconstruction

According to our ancestral area reconstruction from the Conclusions S-Diva analyses (Fig. 2), eastern Central Asia, mainly Mongolia (A), is confirmed to be the place of origin of the We documented seven sequences and obtained the results genus Nitraria and family Nitrariaceae. This is different that the family Nitrariaceae (Nitraria, Peganum) and the from the previous hypotheses, such as an African origin genus Nitraria are monophyletic. Our phylogenetic tree (Komarov 1908, 1947; Vassilchenko 1982; Pan et al. shows that Nitraria can be divided into four major clades: 1999), or Tethys origin (Pan et al. 2003). group I includes N. sphaerocarpa, group II N. retusa, group There are four endemic species and two groups in III N. roborowskii and N. tangutorum, while group IV eastern Central Asia (Fig. 1) in terms of the present paper. consists of N. schoberi, N. komarovii, N. sibirica, and N. Importantly, group I, with N. sphaerocarpa, is located at a billardieri. Nitraria sphaerocarpa is found as the most basal position of the phylogenetic tree. These most likely basal among the species. These results are concordant with strongly influenced the ancestral area reconstruction, re- previous studies, such as Pan et al.’s (1999) classification sulting in eastern Central Asia being regarded as the system. Our group I corresponds to section Sphaerocarpae ancestral area or place of origin. In contrast with the Pan (N. sphaerocarpa), group II to section Retusae Pan (N. Central Asian flora cases, which is hypothesized to have retusa), and groups III ? IV to section Nitraria (N. originated from the Mediterranean area and/or North roborowskii, N. tangutorum, N. schoberi, N. komarovii, N. Africa, then migrating eastward to Central Asia, Mongolia, sibirica, N. billardieri). Eastern Central Asia is most likely or the Tibetan Plateau, Nitraria originated from eastern the place of origin, with subsequent dispersals to western Central Asia and dispersed westward to western Central Central Asia, Africa, and Australia. Asia and Africa, as well as Australia. From this point, Nitraria resembles Biebersteinia, which expanded in its Acknowledgments We thank Dr. Stewart C. Sanderson (Shrub eastern distribution range (i.e. Tibet and Inner Mongolia), Sciences Laboratory, USDA, Utah, USA) for his careful English improvement to the manuscript. This research was financially sup- then westward to the Mediterranean and North Africa ported by the National Key Basic Research Program of China (Muellner et al. 2007). (2012FY111500, 2014CB954201), and Xinjiang Institute of Ecology The Australian species N. billardieri was previously and Geography, Chinese Academy of Sciences. regarded to be related to and/or derived from the African species N. retusa (Komarov 1908, 1947; Vassilchenko 1982; Pan et al. 1999, 2003). However, our result (Fig. 2) References indicates that it is derived from western Central Asian Agnew ADQ (1980) Zygophyllaceae. In: Townsend CC, Guest E species (B) rather than N. retusa. In group IV (Fig. 2), N. (eds) Flora of Iraq, vol 1. Ministry of Agriculture, Baghdad billardieri is tetraploid (2n = 48), but its three allied spe- APG (Angiosperm Phylogeny Group) (1998) An ordinal classification cies N. schoberi, N. sibirica, and N. komarovii, are all for the families of flowering plants. Ann Missouri Bot Gard diploid (Pan et al. 2003). Thus, our results show that N. 85:531–553 APG (Angiosperm Phylogeny Group) (2003) An update of the billardieri has a relationship to these species but also has Angiosperm Phylogeny Group classification for the orders and the difference of being a derived species, since it is families of flowering plants: APG II. Bot J Linn Soc tetraploid. 141:399–436 123 Author's personal copy

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