Integrative Taxonomy of Nitraria (Nitrariaceae), Description of the New Enigmatic Species and Key to All Currently Known Species

A new species, Nitraria iliensis sp. nov., is described from the Ili basin, Almaty region, Kazakhstan. It belongs to section Nitraria ser. Sibiricae and is morphologically similar to N. sibirica Pall. An integrative taxonomic approach based on molecular, biochemical and morphological analyses, along with palynological data, was used to delimit this new species. The studied species of the genus are illustrated, and photographs of authentic specimens of the new species, as well as a distribution map of the new species and segregate taxa, are provided. Morphological characters were investigated, more important traits for identification were found, and a new key to distinguish between all species of the genus was prepared.

The first data on Nitraria were reported by G. Schober, a famous scientist who explored the Volga region and the Caucasus of the Russian state in 1717-1720 [5]. K. Linnaeus grew plants from seeds collected by G. Schober in the lower reaches of the Volga river. These plants were later identified as the N. schoberi lectotype [6].
Later, P.S. Pallas [7] identified specimens of Nitraria growing in the vicinity of Astrakhan (lower reaches of the Volga river) as N. schoberi var. caspica and specimens from Siberia (according to the collections of G.V. Steller) as N. sibirica.
By the end of the 19th century, three more species of Nitraria had been described: in 1828, N. billardierei DC. from Australia; in 1876, N. retusa from North Africa; and in 1883, N. sphaerocarpa from Central Asia (Khami Gobi).
R.E. Trautvetter made an important contribution to the future description of another species from the eastern coast of the Caspian Sea near the town of Krasnovodsk (now known as Turkmenbashi, Turkmenistan) [8]. He distinguished this plant from N. schoberi by its long and narrow leaves gradually tapering toward the base. At the time, he named the taxon N. schoberi var. polygama Trautv. For the first time, the same plant was mentioned under a different name (N. komarovii) in the journal Priroda by M.M. Il'in [9] but without a legitimate diagnosis: "N. komarovii Iljin et Lava., sp. nov. (typus Krasnovodsk, littora maris Caspii, 22 October 1900, leg, Freyn)". N.K. Kovtonyuk, M.A. Tomoshevich, and E.V. Banaev [10] conducted a detailed study to choose a lectotype, N. komarovii Iljin & Lava    Analysis of variance (ANOVA, LSD test) showed significant differences between N. iliensis and N. sibirica closest to it in leaf blade length, p = 0.0008; number of flowers per inflorescence, p = 0.0031; drupe parameters (length p = 0.0382; width p = 0.0262) and stone parameters (width p = 0.0001; area p = 0.0002; perimeter p = 0.0273).
At the same time, the LSD test did not reveal significant differences (p ≤ 0.05) between populations within N. sibirica (11 populations) and N. iliensis (3 populations), which confirms the integrity and independence of these taxa.
Numerous researchers indicate indistinct morphological isolation of species of the genus Nitraria [14][15][16]32]. Some researchers [36,37] report the difficulty in identifying Nitraria species among herbarium specimens from West Siberia, as well as the need for studies of Nitraria species in natural populations. We previously showed that morphological characters of the species N. sibirica were not uniform across its distribution area [21]. In this study, for the first time, we performed a comprehensive analysis of the morphological characters of five species of the genus Nitraria from 31 populations, including a new species N. iliensis, and identified the most relevant diagnostic characters in the genus.  Table 1 for character number, Table 2 for specimen number.   Table 1 for character number, Table 2 for specimen number.

Palynological Analysis
The main features of the investigated pollen grains of Nitraria were summarized (Table 3) and presented with scanning electron microscopy (SEM) micrographs ( Figure 2). The Nitraria species analyzed here had medium pollen grains and varied from subprolate to prolate shape. P/E ratios of pollen grains showed that the highest P/E values belong to N. sibirica (1.70-1.97), which are of a prolate shape. The lowest was detected in N. pamirica (1.22), which are characterized by a subprolate pollen grains. Table 3. Sizes (µm) and shape of pollen grains of Nitraria. The data are presented as the mean (X), standard error (Sx), and the coefficient of variation (CV, %). (1.10-1.53). They are subprolate or prolate in equatorial view and triangular convex or pseudo-hexagonal, circular in polar view and monad, isopolar. The aperture is tricolporate. Ectoaperture-colpus, almost as long as the polar axis, open (4/5 of polar axis), straight, narrow, occasionally constricted at equator with ends acute; polar area asymmetric. Margin observed in polar view, costae colpi and fastigium conspicuous in equatorial view. Endoaperture-porus, conspicuous, lalongate, elliptic to rhombic in shape. Exine-tectate, exine slightly thicker in polar areas in relation to equatorial region; nexine is thicker than sexine. The surface ornamentation is striate and perforated. Striae are relatively loose, packed in the mesocolpia, and short ( Figure  2e1,e2).
The specimens of N. iliensis have a longer equatorial axis and a shorter polar axis compared with N. sibirica specimens; they exhibit a different striate-perforate ornamentation of pollen grains. The pollen morphology of Nitraria and Peganum is of a distinctly phylogenetic structure. Ancient species such as N. sphaerocarpa and N. retusa are considered to have pollen with a polar axis shorter than that in later divergent taxa, and a perforated exine [3,38]. Previously, we showed that striate-perforate ornamentation is typical only of the populations Basshi, Taskarasu, Karatal of N. sibirica (now N. iliensis) and it does not occur in the other four studied species of the genus Nitraria [4].  The aperture is tricolporate. Ectoaperture-colpus, almost as long as the polar axis, open (4/5 of polar axis), straight, narrow, occasionally constricted at equator with ends acute; polar area asymmetric. Margin observed in polar view, costae colpi and fastigium conspicuous in equatorial view. Endoaperture-porus, conspicuous, lalongate, elliptic to rhombic in shape. Exine-tectate, exine slightly thicker in polar areas in relation to equatorial region; nexine is thicker than sexine. The surface ornamentation is striate and perforated. Striae are relatively loose, packed in the mesocolpia, and short (Figure 2e1,e2).

Molecular Analysis
The specimens of N. iliensis have a longer equatorial axis and a shorter polar axis compared with N. sibirica specimens; they exhibit a different striate-perforate ornamentation of pollen grains. The pollen morphology of Nitraria and Peganum is of a distinctly phylogenetic structure. Ancient species such as N. sphaerocarpa and N. retusa are considered to have pollen with a polar axis shorter than that in later divergent taxa, and a perforated exine [3,38]. Previously, we showed that striate-perforate ornamentation is typical only of the populations Basshi, Taskarasu, Karatal of N. sibirica (now N. iliensis) and it does not occur in the other four studied species of the genus Nitraria [4].

Molecular Analysis
Phylogenetic trees constructed using UPGMA and ML were congruent. The phylogenetic tree (Figures 3 and 4) showed that the new species (specimens of Nitraria sp.) is sister to N. sibirica. The specimen of N. komarovii included in this clade, the sequences of which were taken from the Genbank (KP087774; KP087766), is subject to debate (2.5.8. Notes). The monophyly of the new species, N. sibirica and N. schoberi, was supported.
Phylogenetic trees constructed using UPGMA and ML were congruent. The phylogenetic tree (Figures 3 and 4) showed that the new species (specimens of Nitraria sp.) is sister to N. sibirica. The specimen of N. komarovii included in this clade, the sequences of which were taken from the Genbank (KP087774; KP087766), is subject to debate (2.5.8. Notes). The monophyly of the new species, N. sibirica and N. schoberi, was supported.
The molecular approach is now becoming a common aspect of plant research at various taxonomic levels. Non-encoded regions of internal transcribed spacers (ITS) of nuclear ribosomal DNA genes are the most promising molecular markers for plant taxa identification [39]. Analysis of the sequence polymorphism of the internal transcribed spacers (ITS1, ITS2) revealed the Siberian and the Kazakh N. sibirica ribotypes [32,33]. Complete sequences of the chloroplast genomes reported for N. sibirica [40], N. tangutorum and N. roborowskii [41] determine the phylogenetic position of the species and the entire family Nitrariaceae in the Sapindales clade.
ISSR analysis revealed high interpopulation differentiation in N. sibirica in 22 natural populations (Russia and Republic of Kazakhstan) [22]. The authors report that the maximum genetic differences were recorded in Kazakhstani Nitraria specimens from the Ili basin. ISSR analysis of two marginal populations of N. schoberi from Romania, in contrast, showed low interpopulation diversity (He ≈ 0.2), which may be due to founder effects since populations most likely originated from a limited number of ancestral individuals [42].

HPLC-MS Analysis
Secondary metabolites of plants of the genus Nitraria are mainly represented by kaloids and flavonoids [43]. Saleh et al. [44] highlighted the relationship between t composition of these compounds and the phylogeny of taxa of the Zygophyllaceae fa ily. Literature analysis suggests the presence of specificity of individual components secondary metabolites in species of the genus Nitraria [45][46][47][48][49]. A number of papers rep on the specificity of phenolcarboxylic acids and flavonoids isolated from the leaves of tangutorum; therefore, they can be used in the taxonomy of the genus [50]. N. Barbhan al. [51] showed that the chemical composition of the above-ground part of N. retusa d fers from that of other species of the genus Nitraria.
Our phytochemical studies of N. sibirica, N. schoberi, N. komarovii, and N. pamir from 58 populations of Russia, Kazakhstan, and Tajikistan performed using HPL showed that the species differ in the composition and content of phenolic compoun [52,53]. A total of 27 phenolic compounds were identified. The maximum number (16compounds) was found in the leaves of N. sibirica. Nitraria specimens from the popu tions of the Almaty region, Kazakhstan, lack individual phenolic compounds found in populations of N. sibirica. The phenolic composition of N. schoberi is weaker compar with that of N. sibirica. Plants of this species mainly contain not more than 14 compoun In the leaves of N. pamirica, 12 compounds were found, and not less than 6-8 compoun were identified in the leaves of N. komarovii.
HPLC-MS analysis of N. sibirica and N. iliensis ( Figure 5) showed a significant d ference in the phenolic profiles of these species. The N. iliensis specimen contains smaller number of phenolic compounds compared with N. sibirica (11 and 13 co pounds, respectively). Only four compounds are common to both species (hyperosid compounds 5,16,17); hyperoside is inherent in all studied species. Compound 5, a The molecular approach is now becoming a common aspect of plant research at various taxonomic levels. Non-encoded regions of internal transcribed spacers (ITS) of nuclear ribosomal DNA genes are the most promising molecular markers for plant taxa identification [39]. Analysis of the sequence polymorphism of the internal transcribed spacers (ITS1, ITS2) revealed the Siberian and the Kazakh N. sibirica ribotypes [32,33]. Complete sequences of the chloroplast genomes reported for N. sibirica [40], N. tangutorum and N. roborowskii [41] determine the phylogenetic position of the species and the entire family Nitrariaceae in the Sapindales clade.
ISSR analysis revealed high interpopulation differentiation in N. sibirica in 22 natural populations (Russia and Republic of Kazakhstan) [22]. The authors report that the maximum genetic differences were recorded in Kazakhstani Nitraria specimens from the Ili basin. ISSR analysis of two marginal populations of N. schoberi from Romania, in contrast, showed low interpopulation diversity (He ≈ 0.2), which may be due to founder effects since populations most likely originated from a limited number of ancestral individuals [42].

HPLC-MS Analysis
Secondary metabolites of plants of the genus Nitraria are mainly represented by alkaloids and flavonoids [43]. Saleh et al. [44] highlighted the relationship between the composition of these compounds and the phylogeny of taxa of the Zygophyllaceae family. Literature analysis suggests the presence of specificity of individual components of secondary metabolites in species of the genus Nitraria [45][46][47][48][49]. A number of papers report on the specificity of phenolcarboxylic acids and flavonoids isolated from the leaves of N. tangutorum; therefore, they can be used in the taxonomy of the genus [50]. N. Barbhan et al. [51] showed that the chemical composition of the above-ground part of N. retusa differs from that of other species of the genus Nitraria.
Our phytochemical studies of N. sibirica, N. schoberi, N. komarovii, and N. pamirica from 58 populations of Russia, Kazakhstan, and Tajikistan performed using HPLC showed that the species differ in the composition and content of phenolic compounds [52,53]. A total of 27 phenolic compounds were identified. The maximum number (16-18 compounds) was found in the leaves of N. sibirica. Nitraria specimens from the populations of the Almaty region, Kazakhstan, lack individual phenolic compounds found in all populations of N. sibirica. The phenolic composition of N. schoberi is weaker compared with that of N. sibirica. Plants of this species mainly contain not more than 14 compounds. In the leaves of N. pamirica, 12 compounds were found, and not less than 6-8 compounds were identified in the leaves of N. komarovii.
HPLC-MS analysis of N. sibirica and N. iliensis ( Figure 5) showed a significant difference in the phenolic profiles of these species. The N. iliensis specimen contains a smaller number of phenolic compounds compared with N. sibirica (11 and 13 compounds, respectively). Only four compounds are common to both species (hyperoside, compounds 5,16,17); hyperoside is inherent in all studied species. Compound 5, and minor compounds 16 and 17 were not detected in N. schoberi, N. komarovii, and N. pamirica.

Taxonomy
A comprehensive analysis of the obtained data distinguished a new species and showed its uniqueness in comparison with four related species growing in the study area : N. sibirica, N. komarovii, N. pamirica, and N. (Figure 6).

Description
Bushes tend to be 0.6-1.8 m high, densely branched from the base, with slightly arched shoots sticking out in the center, and multispinous. The branches are bare with ash-gray, cracked bark; one-year-old shoots are yellowish, shiny, pubescent. The leaves (12) measure 14-17 (20) × 2-3 mm, are oblanceolate, gradually tapering towards the base, acute or obtuse at the apex, entire, green, fleshy. All leaves are pubescent on both sides. The ultimate inflorescence scorpioid cyme, with peduncle 5.5-15 cm; flowers 40-80. Flowers hermaphrodite, typically pentamerous. Peduncles and inflorescence axes slightly hairy. Resistant calyx up to 2.5 mm, fleshy, pubescent. Petals white, oblong-ovate, 2.8-3.9 × 1.5-2.6 mm, concave with incurved margins, claws short. The fruit is a fleshy black drupe, with black-green sap; oval or spherical, 4-6 mm long, 4-4.5 mm in diameter; finely pubescent; edible and salty-bitter. The sap of ripe berries stains white paper black-green. The stone is dark, reddish brown, narrow ovoid with a narrow pointed apex, 3.5-5 mm long, 2-2.6 mm in diameter.

Affinity
The new species N. iliensis belongs to the sect. Nitraria ser. Sibiricae Bobrov [14], which is evidenced by the results of molecular phylogenetic analysis. Differences between the five species studied in Russia, Tajikistan, and Kazakhstan are summarized in Table 1.
Nitraria iliensis is morphologically similar to N. sibirica ( Figure 8) in spreading-branching, dense bush habit, oblanceolate leaf shape, flower shape and size, globate or oval black drupe.
The new species differs from other related species in the height of the bush (0.6-1.8 m), size and color of the leaf blade, showing a smaller size of the fruit and stone, and a larger number of flowers per inflorescence ( Table 1, Figures 8 and 9).

Phenology
Flowering: late May-early June, 5-7 days later than that in N. schoberi, but 2-3 days earlier compared with N. sibirica. Fruiting: end of July-beginning of August.

Distribution
Nitraria iliensis is confined to the Almaty region, Republic of Kazakhstan, the Ili basin.

Etymology
The specific epithet of the new species comes from the type locality, Ili basin, Almaty region, Republic of Kazakhstan.

Notes
The genebank contains sequences of nuclear and plastid DNA fragments isolated from voucher P. Farse, 13 May 1964, Afghanistan. This voucher specimen is stored in the herbarium of the Institute of Botany, Chinese Academy of Sciences (PE) and referred to as N. komarovii. However, N. komarovii is exclusively littoral and grows on the sands along the coasts of large lakes. E.G. Bobrov [14] considered it the youngest of all known species associated with the history of formation of the Caspian basin. Previously, it was only known from three habitats: the Krasnovodsk Peninsula, in vicinity of Turkmenbashi city (Turkmenistan), the Apsheron peninsula (Azerbaijan), and the mouth of the Volga river. We have identified one more locality for N. komarovii [31] on the coast of the lake Balkhash. Therefore, the presence of N. komarovii in the flora of Afghanistan is debatable.

Phenology
Flowering: late May-early June, 5-7 days later than that in N. schoberi, but 2-3 days earlier compared with N. sibirica. Fruiting: end of July-beginning of August.

Distribution
Nitraria iliensis is confined to the Almaty region, Republic of Kazakhstan, the Ili basin.

Plant Material
The specimens of N. sibirica (11 habitats), N. schoberi (14 habitats), N. komarovii, N. pamirica and N. iliensis were collected in expeditions in Siberia (Novosibirsk region, Altai Territory, Republic of Tuva), Crimea, the Republics of Kazakhstan and Tajikistan in 2011-2017 ( Figure 10). Field work was carried out in different seasons to observe species both in the flowering stage and in the fruiting stage. In each population, 25-30 herbarium leaves were collected (more than 800 herbarium specimens in total), and specimens of flowers, fruits, and seeds, which were packed in paper bags, marked and delivered to the laboratory of dendrology of the CSBG SB RAS (Novosibirsk, Russia) for morphometric analysis.
The specimens collected during the expeditions were deposited in the collection of the NSC CSSB SB RAS (Novosibirsk, Russia) and are available in the digital herbarium of the CSBG SB RAS "http://herb.csbg.nsc.ru:8081 (accessed on 22 December 2022)". Sample voucher data are shown in Table 2.
Revision of herbarium materials was undertaken in the herbaria at LE, MW, NS, NSK, PE.

Morphological Analysis
The vegetative and reproductive morphology was studied on well-developed specimens of the generative age state. For numerical analysis, not less than 25 specimens were studied in each population of each species. Table 1 presents 26 morphological characters studied.
Morphological analysis was carried out using a Carl Zeiss Stereo Discovery V12 stereo microscope equipped with a high-resolution color digital camera AxioCam HRc and Revision of herbarium materials was undertaken in the herbaria at LE, MW, NS, NSK, PE.

Morphological Analysis
The vegetative and reproductive morphology was studied on well-developed specimens of the generative age state. For numerical analysis, not less than 25 specimens were studied in each population of each species. Table 1 presents 26 morphological  characters studied. Morphological analysis was carried out using a Carl Zeiss Stereo Discovery V12 stereo microscope equipped with a high-resolution color digital camera AxioCam HRc and AxioVision 4.8 software for image acquisition, processing and analysis (Carl Zeiss Ltd., Göttingen, Germany), and the instrumental platform of the SIAMS Photolab image analysis system (SPF AVEK, 2013-2020) with the module morphometric analysis of plants.
Morphometric data were subjected to ANOVA using the STATISTICA 6.0 software (StatSoft Inc., Tulsa, OK, USA). The differences between means were tested for significance using the LSD test at p ≤ 0.05. In addition, clustering was performed with PCA. For PCA, relative metric parameters were additionally included: 4/3 is the ratio of leaf width to leaf length, 5/3 is the ratio of distance from the base to the widest point of the leaf blade to leaf length, and 24/25 is the ratio of stone length to stone width.

Palynological Analysis
For SEM examination, the air-dried pollen grains were dispersed evenly and put in double-sided adhesive-tape-covered aluminum stubs. The studs were coated with gold in a Mini SC 7620 sputter coater (Quorum Technologies, Laughton, Great Britain) and photographed under 20.0 kV voltage using the EVO MA10 (Carl Zeiss, Göttingen, Germany) scanning electron microscope.
About 25 pollen grains of each species were selected randomly, and the polar axis length (P) and equatorial axis length (E) of them were measured. All data were analyzed to calculate the mean (X), standard error (Sx), and the coefficient of variation (CV, %). Morphometric data were subjected to ANOVA using the STATISTICA 6.0 software (StatSoft Inc., Tulsa, OK, USA). The differences between means were tested for significance using the LSD test at p ≤ 0.05. In addition, clustering was performed with PCA. For PCA, relative metric parameters were additionally included: 4/3 is the ratio of leaf width to leaf length, 5/3 is the ratio of distance from the base to the widest point of the leaf blade to leaf length, and 24/25 is the ratio of stone length to stone width.

Palynological Analysis
For SEM examination, the air-dried pollen grains were dispersed evenly and put in double-sided adhesive-tape-covered aluminum stubs. The studs were coated with gold in a Mini SC 7620 sputter coater (Quorum Technologies, Laughton, Great Britain) and photographed under 20.0 kV voltage using the EVO MA10 (Carl Zeiss, Göttingen, Germany) scanning electron microscope.
About 25 pollen grains of each species were selected randomly, and the polar axis length (P) and equatorial axis length (E) of them were measured. All data were analyzed to calculate the mean (X), standard error (Sx), and the coefficient of variation (CV, %).

Molecular Analysis
Two spacer regions, one in the nuclear genome (ITS) and one in the plastid genome (trnH-psbA), were subjected to the molecular analysis. DNA was extracted from dried leaves using the conventional CTAB-based method [61]. The concentration and amount of the extracted DNA were evaluated in 0.8% agarose gel and using a spectrophotometer (NanoPhotometer P-Class, P-360, Implen, Munich, Germany). For amplification of different DNA sequences, a ready-made kit of GenePak ® PCR Core reagents (Laboratory Izogen, Moscow, Russia) was used. For amplification of the ITS operon, which includes intergenic spacers ITS1, ITS2, and the 5.8 s gene, primers ITS6 and ITS9 developed for East Asian species of the tribe Spiraeeae [62] were used. The amplification cycle included denaturation at 94 • C for 1 m, primer annealing at 58 • C for 50 s, and elongation at 72 • C for 1 m within 30 cycles.
For amplification of the trnH-psbA chloroplast locus, universal primers were used [63]. The PCR cycle included denaturation at 95 • C for 40 s, primer annealing at 59 • C for 50 s, and elongation at 72 • C for 90 s within 32 cycles. The quality of the obtained PCR fragments was verified in 1.5% agarose gel and purified with a kit for rapid DNA elution from agarose gels Diatom DNA Elution (Laboratory Izogen, Moscow, Russia).
Sequencing was performed in both directions for ITS and trnH-psbA at ZAO Evrogen, using an automatic analyzer model ABI PRISM 3500. Sequencing was performed using the BigDye Terminator v. 1.1 Cycle Sequencing Kit. The subsequent purification of products was performed using the BigDye XTerminator Purification Kit.

HPLC-MS Analysis
Samples of air-dried and ground plant material (0.5 g leaves) were extracted with ethanol-water (50:50, v/v) in a water bath at 60-70 • C. The extract was purified using a C16 Diapack cartridge and dissolved in 70% ethanol. Mass spectrometric analysis was carried out at the Novosibirsk Institute of Organic Chemistry SB RAS (Novosibirsk, Russia). HPLC-MS analysis was performed using an Agilent 1200 liquid chromatograph (Agilent Technologies, USA) and a micrOTOF-Q hybrid quadrupole-time-of-flight mass spectrometer (Bruker, Germany) with API-ES. Positive ions were identified in the range of 100-3000 m/z. Chromatographic separation was carried out at 30 • C using a Zorbax SB-C18 column (2.1 mm × 150 mm, inner diameter 3.5 µm) with a ZorbaxSB-C8 guard column (2.1 mm × 12.5 mm, inner diameter 5 µm). The composition of the mobile phase changed in a linear gradient from 15:85 (v/v) methanol (phase A) and 2% formic acid in water (phase B) to 100:0 (v/v) in 30 min, then in isocratic mode from 30 to 45 min. The volume of the injected sample was 10 µL. UV detection was carried out at four wavelengths/bandwidths: 255/16, 270/16, 320/16, 340/32 nm. Mass detection operating parameters were as follows: dryer gas flow (nitrogen) 8 L/min, nitrogen temperature 230 • C, nebulizer pressure 1.6 bar.