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

Abstract

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.

1. Introduction

The genus Nitraria L. belongs to the monotypic family Nitrariaceae Bercht & J. Pres; this genus was previously assigned to the family Zygophyllaceae R. Br. [1,2]. It includes 9–11 species restricted to steppe, semi-desert and desert regions of Asia, North Africa, South-Eastern Europe (Romania), Papua New Guinea and Australia [3,4]. GBIF, Kew, Plant list, and IPNI databases list nine species: N. tangutorum Bobrov, N. sphaerocarpa Maxim., N. sibirica Pall., N. schoberi L., N. komarovii Iljin & Lava ex Bobrov, N. roborowskii Kom., N. pamirica L.I. Vassiljeva, N. billardierei DC., and N. retusa (Forsk.) Aschers.

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 ex Bobrov, which was identified as a specimen of this plant from the herbarium of V.L. Komarov Botanical Institute, RAS: LE00050757. This specimen had been identified in 1960 by V.P. Bochantsev as a lectotype of N. komarovii but was not published properly.

The first major analysis of the genus Nitraria was carried out by V.L. Komarov [5]. He noted substantial variation of the morphological characters of N. schoberi vegetative organs without a clear-cut hiatus across the geographic range. In addition, he described another new species: N. roborowskii. According to his data, the plant from the Cherchen oasis (Kashgar) differed sharply from known specimens of N. schoberi in larger leaves, inflorescences, and drupes.

It should be pointed out that the taxonomic position and distribution of N. roborowskii is the subject of debate among researchers [11,12,13]. In particular, E.G. Bobrov [14] believed that if only quantitative traits (gigantism) are taken into account, N. roborowskii should be regarded not as a species but as an ecological form: N. sibirica f. majus.

The most complete review of Nitraria systematics can be credited to E.G. Bobrov [14,15]. He divided the genus into two sections (sect. Tridentatae and sect. Nitraria) and described two new species: N. tangutorum (from Tsaidam, China) and N. praevisa (from the Alashan Gobi, China).

The tenth species of Nitraria described by L.I. Vasilieva [16] grows at an altitude of ~4000 m a.s.l. in the Eastern Pamirs: N. pamirica. According to that author’s description, the new species and closely related N. sibirica and N. schoberi differ mainly in an elongated conical stone with a reticulate-veined and irregularly lacunous surface, whereas in the two species, the stone surface is free of veins and contains only a few, rounded, deeper dimples. Furthermore, according to L.I. Vasilieva, this new species differs from N. schoberi in shape, leaf size, and bush habitus, and from N. sibirica in a larger cherry (not black) drupe, inflorescence axes pubescent with adpressed hairs, and in the pistil that is almost twofold shorter than the petals.

Recent studies have revealed the morphological and genetic diversity, phylogeny, and putative origin of some species of the genus Nitraria [17,18,19,20,21,22]. Along with species polymorphism, there are data on natural hybridization between species of the genus Nitraria [23].

For the studied region (Siberia, Crimea, Kazakhstan, and Tajikistan), only three Nitraria species (N. schoberi, N. sibirica, N. pamirica) are indicated in the literature [24,25,26,27,28,29,30]. Recently, another species, N. komarovii, has been recorded in the territory of Kazakhstan in the Balkhash-Alakol basin [31].

Recent studies of plants of the genus Nitraria in the territory of Kazakhstan have revealed a high genetic and morphological polymorphism of N. sibirica. Analysis of the sequence of the ITS of the nuclear ribosomal DNA identified two main N. sibirica ribotypes [32,33]. The heterogeneity of N. sibirica is also supported by data on composition and content of phenolic compounds in plants from Siberian and Kazakh populations [34] and the presence of morphological differences in plants from these populations [4,35].

The aim of the study was to comprehensively investigate the morphological (including palynological), molecular, genetic and biochemical traits of N. sibirica, N. schoberi, N. komarovii, and N. pamirica to confirm the possible taxonomic heterogeneity of the species. The relationship between these species and the new species described and referred to as Nitraria iliensis Banaev&Tomoshevich, sp. nov. was analyzed.

2. Results and Discussion

2.1. Morphological Analysis

We compared 26 characters to distinguish between N. sibirica, N. schoberi, N. komarovii, N. pamirica, and N. iliensis (

Table 1. Morphological characters of Nitraria species.

Number of Character	Character	N. schoberi	N. sibirica	N. komarovii	N. pamirica	N. iliensis
1	Height of bush (m)	0.7–1.5	0.2–0.8	0.5–1	0.1–0.3	0.6–1.8
2	Habit	Spreading-branching	Spreading-branching, dense	Branching, graceful	Spreading, dense	Spreading-branching, dense
3	Leaf length (mm)	20–26	10–13	25–28	13–17	14–16
4	Leaf width (mm)	3–6	2–4	2–3,5	2–3	2–3
5	Distance from the base to the widest point of the leaf blade (mm)	15–16	8–9	22	10	10–12
6	Leaf color	Dark-green, shiny	Glaucescent-green	Pale green	Green, matte	Green
7	Leaf shape	Oblanceolate	Oblanceolate	Narrow, linear-spathulate	Oblanceolate, with a pointed apex	Oblanceolate
8	Flowers	Yellowish-white	White (buds pale-violet)	Yellowish-white	White	White
9	Number of flowers per inflorescence	11–28	20–48	17–30	9–15	40–80
10	Flower spacing (mm)	0.4–0.8	0.2–0.3	0.3–0.7	0.2–0.3	0.3–0.5
11	Petal shape	Ovate or rhombic; claws short	Acuminate-elliptica; claws narrow	Ovate; claws short	Ovate; claws short	Ovate or rhombic; claws short
12	Petal length (mm)	3–4.7	2.6–4	1.7–3.9	2.1–2.3	3.5
13	Petal width (mm)	2–3.6	1–2.8	1–2.9	0.7–0.9	2.6
14	Anther length (mm)	1.2–1.4	0.6–0.9	1.3	-	0.8
15	Anther width (mm)	0.5–0.7	0.3–0.5	0.7	-	0.5–0.6
16	Pestle length (mm)	2.02–4.5	1.35–3.19	2.01–3.59	-	2.05
17	Pestle width (mm)	1.38–2.23	0.97–1.41	1.09–1.82	-	1.22
18	Fruit color	Dark-red to black	Black	Yellow, orange, or pale to bright red	Cherry red	Black
19	Fruit length (mm)	7–10	4–9	8–12	7–8	4–6
20	Fruit width (mm)	6–11	4–8	7–11	4–5	4–4.5
21	Weight of 100 fruits (g)	270–600	100–270	380–490	80–90	80–130
22	Fruit shape	Oval	Globate or oval	Oval	Oval	Globate or oval
23	Fruit sap	Pale reddish	Dark-blue	Pale pink	Pale pink	Black–green
24	Stone length (mm)	7–10	3,6–6	8,6–11	5–6	3.5–5
25	Stone width (mm)	4.5–6.5	2.5–3.5	4.7–6	2.0–2.5	2–2.6
26	Stone shape	Ovate, obtuse	Ovate, pointed	Conic-ovate with a pointed apex	Oblong-conical,	Narrow ovoid with a narrow pointed apex

). Principal component analysis showed that the first two principal components accounted for 65.1% of the variance of the entire array of parameter data and exhibited the best species discrimination (Figure 1a). It was found that in terms of the first factor, which brought about 48% of the variance, all the studied populations (species) were divided into two groups; the most relevant characters were leaf length, leaf width, distance from the base to the widest point of the leaf blade, fruit length, fruit width, and stone length (Table 1, Figure 1b). The populations of the new species were clearly separated in the plane of the second factor from the populations of N. sibirica and N. pamirica; the most significant characters were height of bush and number of flowers per inflorescence (Table 1, Figure 1b).

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.

2.2. Palynological Analysis

The main features of the investigated pollen grains of Nitraria were summarized (

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, %).

Taxa	Polar Axis (P)			Equatorial Axis (E)			P/E			Shape
	Range	X ± Sx	CV, %	Range	X ± Sx	CV, %	Range	X ± Sx	CV, %
N. sibirica	36.11–43.33	40.24 ± 0.34	4.3	17.07–23.50	20.48 ± 0.30	7.5	1.73–2.28	1.97 ± 0.02	5.8	prolate
N. komarovii	23.38–29.50	27.15 ± 0.30	5.6	14.23–18.91	16.42 ± 0.30	9.1	1.44–1.89	1.66 ± 0.02	8.0	prolate
N. schoberi	38.19–49.90	42.84 ± 0.62	7.2	22.28–32.59	27.84 ± 0.65	11.0	1.24–2.05	1.57 ± 0.05	16.5	prolate
N. pamirica	30.13–36.07	32.29 ± 0.32	4.9	24.87–30.62	26.50 ± 0.28	5.3	1.18–1.27	1.22 ± 0.005	2.0	subprolate
N. iliensis	31.41–36.00	33.82 ± 0.32	4.8	21.10–27.08	25.81 ± 0.50	9.7	1.10–1.51	1.32 ± 0.02	6.7	subprolate or prolate

) 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.

Pollen grains of N. iliensis are medium in size and are radially symmetrical. The mean length of the polar axes of them is 33.82 (28.71–37.29) μm, and the mean length of the equatorial axes is 25.81 (21.10–27.08) μm. The mean P/E ratio of the pollen is 1.32 (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].

2.3. Molecular Analysis

Phylogenetic trees constructed using UPGMA and ML were congruent. The phylogenetic tree (Figure 3 and Figure 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].

2.4. 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.

2.5. 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. schoberi.

Nitraria iliensis Banaev&Tomoshevich, sp.nov.

Type. Republic of Kazakhstan, Almaty region, vicinity of Basshi village, 44°10′51′′ N, 78°44′31′′ E, 1021 m a.s.l., 25 May 2016, E. V. Banaev & M. A. Tomoshevich (holotype, NSK3001499) (Figure 6).

Paratype Republic of Kazakhstan, Almaty region, vicinity of Basshi village, 44°10′51′′ N, 78°44′31′′ E, 1021 m a.s.l., 30 July 2013, E. V. Banaev & M. A. Tomoshevich (NSK3001277) (Figure 7).

2.5.1. 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.

2.5.2. 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, Figure 8 and Figure 9).

2.5.3. 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.

2.5.4. Distribution

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

2.5.5. Habitat and Ecology

Nitraria iliensis grows at an altitude of 400–1000 m a.s.l., in halophytic cenoses including Tamarix, Halimodendron, Nitraria schoberi, Artemisia, Achnatherum, Halocnemum, and Swaeda.

2.5.6. Etymology

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

2.5.7. Additional Specimens Examined

Republic of Kazakhstan, Almaty region, Karatalskii district, vicinity of Ushtobe city, on the terrace of the Karatal river, 45°21.995′ N, 77°55.048′ E, 29 May 2016, E.V. Banaev & M.A. Tomoshevich, 3000922 (NSK); Republic of Kazakhstan, Almaty region, Karatalskii district, vicinity of Ushtobe city, on the terrace of the Karatal river, 45°21.995′ N, 77°55.048′ E, 15 August 2017, E.V. Banaev & M.A. Tomoshevich, 3001272 (NSK); Republic of Kazakhstan, Almaty region, vicinity of Taskarasu village, 43°46′54.60′′ N, 79°27′16.56′′ E, 26 May 2016, E.V. Banaev & M.A. Tomoshevich, 3001244 (NSK).

2.5.8. 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.

A similar situation can be observed for N. komarovii recorded from the flora of Iran. H. Akhani [54] reports the absence of N. komarovii in the flora of Iran, and Iranian records of N. komarovii Iljin & Lava [55] are referred to Atraphaxis suaedifolia Jaub. & Spach (Polygonaceae).

It should be noted that data on the genus Nitraria in various databases are currently controversial. For example, in the Kew, Plant list, and IPNI databases “https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:873338-1 (accessed on 22 December 2022)”, N. praevisa is a synonym for N. roborowskii according to E.G. Bobrov [14]. However, E.G. Bobrov is the author of the species N. praevisa, which he described in this work in 1965. In the key, he included N. praevisa along with N. roborowskii.

2.5.9. Key to Nitraria Species

All characters in the key of species of the genus Nitraria are described from the studied material from natural populations and authentic specimens stored in the LE herbarium.

1. Drupe juicy, oval or spherical; stone ovoid or conical.............................................................2

- Drupe dry, swollen, spherical up to 1 cm in diameter, with a membranous swollen pubescent shell; narrow stone, almost fusiform, 0.8 × 0.2–0.3 cm. Thorny bush, prostrate, up to 20–40 cm high, with curved branches, pillow-shaped. Branches are grayish-white in color. Leaves narrow, linear-lanceolate, 20–40 × 1–3 mm. Inflorescence shoot short, up to 5–10 cm. Flowers white, small, up to 0.5 cm....................................................N. sphaerocarpa Maxim

2. Leaves obovate, oblanceolate or linear, entire above, very rarely 1–2-toothed; stipules membranous, deciduous....3

- Leaves spatulate or cuneiform, entire above or 3–5 crenate-toothed; stipules present........N. retusa (Forsk.) Aschers

3. Leaves entire, blunt at the apex, rarely pointed...................................................................4

- Leaves are the largest of all species of the genus, 25–46 × 6–9 mm, either entire or 1–2-toothed at the apex (on vegetative shoots); drupe large, round, juicy, pale pink to maroon in color, stone 10–12 mm long, 3.5–4.5 mm wide, ovoid pointed to apex, barely pointed or almost blunt; bush 1–1.5 m high................................N. roborowskii Kom.

4. Bush above 0.5 m, spreading-branching..........................................................................5

- Bush 0.1–0.3 m high, spreading, dense; leaves green, matte, oblanceolate with a pointed apex; number of flowers in the inflorescence 9–15; petals white, oblong-oval with short claws; drupe cherry, 7–8 × 4–5 mm, stone oblong-conical, 5–6 × 2–2.5 mm.............................................................................N. pamirica L.I. Vassiljeva

5. Drupe maroon to black in color, oval or spherical.................................................................7

- Drupe yellow, orange, pale to bright red, oval, confined to Central Asia............................................6

- Bush grows in Australia, leaves narrow at the apex, slightly pointed, stone conically ovoid.........N. billardierei DC.

6. Stems ascending, leaves greenish-yellow, narrow, long, linear spatulate, 25–28 mm long, 2–3.5 mm wide; petals yellowish-white, ovate with short claws; number of flowers per inflorescence 17–30; drupe oval, 8–12 × 7–11 mm; stone conically ovoid with a recurved apex, 8.5–11 mm long, 4.5–6 mm wide; drupe sap light pink.......................................................................................................N. komarovii Iljin & Lava ex Bobrov

7. Drupe black, oval or spherical; stone smaller than 7 mm..........................................................8

- Drupe oval, maroon to black in color, 7–10 × 6–9 mm; stone ovoid, obtuse, 7–10 mm long, 4.5–6.5 mm wide; bush 0.7–1.5 m high, spreading-branching; stems arcuate, with large spines; leaves dark green, shiny, oblong-spatulate, 20–26 mm long, 3–6 mm wide; flowers yellowish-white in color; petals ovoid or diamond-shaped with short claws; number of flowers per inflorescence 11–28; fruit sap pale reddish................................................N. schoberi L.

- Drupe oval, burgundy to dark purple in color, 8 × 6 mm; stone ovoid, obtuse, 6 × 3 mm; leaves oblong-lanceolate, 24–25 x 4.5–6 mm; bush 1–2 m high (up to 3–4)...................................................N. tangutorum Bobrov

8. Leaves pubescent or slightly pubescent...................................................9

- Leaves silvery from dense pubescence, calyx densely hairy, petals and ovaries hairy on the outside; leaf 10–15 × 4–6 mm..........................................................................N. praevisa Bobrov

9. Bush 0.6–1.8 m high, dense, with small spines; leaves oblanceolate, elongated, green, 14–16 × 2–3 mm; number of flowers per inflorescence 40–80 (90); petals white, ovate or rhombic with short claws; drupe black, oval or spherical, 4–6 × 4–4.5 mm; stone narrowly ovoid with a narrow pointed apex; 3.5–5 mm long, 2–2.5 mm wide; fruit sap green-black.....................................................................................N. iliensis Banaev&Tomoshevich

- Bush 0.2–0.8 m high, dense, with tightly arranged small spines and oblanceolate bluish-green leaves, 10–13 mm long, 2–3 mm wide; number of flowers per inflorescence 20–48; flowers white, pale-purple in buds; petals white, pointed-elliptical with narrow claws; drupe black, 4–9 × 4–8 mm; stone ovoid-acuminate, 3.6–6 mm long and 2.5–3.5 mm wide; fruit sap dark blue..............................................................................N. sibirica Pall.

3. Materials and Methods

3.1. 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. Vaucher specimens of Nitraria (Nitrariaceae).

No.	Taxon	Specimen	Locality	Date	Herbarium, Specimen Number
1	N. sibirica	Noven’koe	Russia, Altai Krai, Loktevsky District, vicinity of Noven’koe village	31 May 2011	NSK3001284
2	N. sibirica	Veseloyarsk	Russia, Altai Krai, Rubtsovsky District, vicinity of Veseloyarsk village	31 May 2011	NSK3001286
3	N. sibirica	Kulunda	Russia, Altai Krai, Slavgorodskiy District, on the shore of Lake Kulundinskoe	2 June 2011	NSK3001276
4	N. sibirica	Bele	Russia, Republic of Tyva, Tandinsky kozhuun, northern shore of Lake Bele	22 July 2011	NSK3001016
5	N. sibirica	Balansor	Russia, Altai Krai, Uglovskiy District, on the shore of Lake Balansor	1 June 2011	NSK3001280
6	N. sibirica	Dzhira	Russia, Altai Krai, Kulundinsky District, eastern shore of Lake Dzhira	2 June 2011	NSK3001283
7	N. sibirica	Gornyak	Russia, Altai Krai, Loktevsky District, vicinity of Gornyak village	31 May 2011	NSK3001289
8	N. sibirica	Kuchuk	Russia, Altai Krai, Blagoveshchensky District, vicinity of Nizhny Kuchuk village	2 June 2011	NSK3001274
9	N. sibirica	Yarovoe	Russia, Altai Krai, Tabunsky District, southern shore of Bolshoye Yarovoe Lake	3 June 2011	NSK3001282
10	N. sibirica	Shara-Nur	Russia, Tyva Republic, Tes-Khemsky kozhuun, the shore of Lake Shara-Nur	28 July 2011	NSK3000991
11	N. sibirica	Rubtsovsk	Russia, Altai Krai, vicinity of Rubtsovsk city	15 June 2013	NSK3001785
12	N. schoberi	Balhash	Republic of Kazakhstan, Almaty Region, on the shore of Lake Balkhash, sandy desert	31 May 2016	NSK3000948
13	N. schoberi	Sariozek	Republic of Kazakhstan, Almaty Region, 30 km north of Sariozek village	25 May 2016	NSK3000947
14	N. schoberi	Basshi	Republic of Kazakhstan, Almaty Region, vicinity of Basshi village	25 May 2016	NSK3000982
15	N. schoberi	Aidarli	Republic of Kazakhstan, Almaty Region, Zhambylskii District, 17 km south of Aidarli village	21 August 2017	NSK3000958
16	N. schoberi	Koktal	Republic of Kazakhstan, Almaty Region, vicinity of Koktal village	30 July 2013	NSK3000999
17	N. schoberi	Lepsi	Republic of Kazakhstan, Almaty region, on the bank of the Lepsi River in outskirts of Lepsi village	30 May 2016	NSK3000981
18	N. schoberi	Raz’ezd_47	Republic of Kazakhstan, Almaty region, vicinity of the village of Molaly (railroad junction No. 47)	15 August 2017	NSK3000944
19	N. schoberi	Bagan	Russia, Novosibirskaya Oblast’, southwest of the village Grushevka, on the terrace of Lake Bol’shoy Bagan	4 June 2011	NSK3000973
20	N. schoberi	Kaspii	Republic of Kazakhstan, Mangistauskaya Oblast’, vicinity of Aktau city, on sandy mound	12 June 2012	NSK3000979
21	N. schoberi	Actau	Republic of Kazakhstan, Mangistauskaya Oblast’, vicinity of Aktau city	12 June 2012	NSK3000978
22	N. schoberi	Krim	Crimea, on the sandy coast of the Black Sea in Dvuyakornaya Bay	16 September 2013	NSK3000960
23	N. schoberi	Pyandzh	Republic of Tajikistan, Gorno-Badakhshan Autonomous Region, on the sandy bank of the Pyandzh River	8 August 2014	NSK3000994
24	N. schoberi	Kulunda	Russia, Altai Krai, Slavgorodskiy District, on the shore of Lake Kulundinskoe	2 June 2011	NSK3000975
25	N. schoberi	Malinovoe	Russia, Altai Krai, Mikhailovskiy District, on the shore of Lake Malinovoe	1 June 2011	NSK3000971
26	N. komarovii	Balhash	Republic of Kazakhstan, Almaty Region, on the shore of Lake Balhash, sandy desert	31 May 2016	NSK3000920
27	N. iliensis	Basshi 1	Republic of Kazakhstan, Almaty Region, vicinity of Basshi village	25 May 2016	NSK3001499
28	N. iliensis	Basshi 2	Republic of Kazakhstan, Almaty Region, vicinity of Basshi village	30 July 2013	NSK3001277
29	N. iliensis	Taskarasu	Republic of Kazakhstan, Almaty Region, vicinity of Taskarasu village	26 May 2016	NSK3001244
30	N. iliensis	Karatal	Republic of Kazakhstan, Almaty Region, Karatalskii District, vicinity of Ushtobe city, on the terrace of the Karatal River	29 May 2016	NSK3000922
31	N. pamirica	Shaimak	Republic of Tajikistan, Gorno-Badakhshan Autonomous Region, Eastern Pamir, on the cliff of the Djilga River	10 August 2014	NSK3001238

.

Revision of herbarium materials was undertaken in the herbaria at LE, MW, NS, NSK, PE.

3.2. 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.

3.3. 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, %).

Palynological terminology was referenced from former works [56,57,58,59]. The pollen shape class, based on the P/E ratio, was identified using Erdtman’s system [60].

3.4. 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.

The sequences were viewed using Data Collection v. 3.1 and were read using Sequencing Analysis Software v.6. The sequences were pairwise-aligned using the BioEdit v.7.1.9 program [64], multiple alignment was performed using the ClustalW2 program, followed by verification of ambiguous positions in the chromatograms and manual editing. Aligned sequences were analyzed using the MEGA X program [65]. The phylogenetic tree was constructed using the UPGMA method and the maximum likelihood method based on the Tamura–Ney model [66]. To construct the phylogenetic tree, we employed fragments deposited in GenBankNCBI (N. sphaerocarpa: DQ267177; KP087761; N. retusa: KP087772; KP087764; N. roborowskii: DQ309042; KP087762; N. tangutorum: DQ267176, KP087763; N. billardieri: KP087775; KP087767; N. komarovii: KP087774; KP087766). Peganum harmala L. (KP087776; KP087768) was used as the outgroup closely related to the genus Nitraria.

3.5. 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.