Molecular phylogenetic data and seed coat anatomy resolve the generic position of some critical Chenopodioideae (Chenopodiaceae – Amaranthaceae) with reduced perianth segments

Abstract The former Chenopodiumsubgen.Blitum and the genus Monolepis (Chenopodioideae) are characterised in part by a reduced (0–4) number of perianth segments. According to recent molecular phylogenetic studies, these groups belong to the reinstated genera Blitum incl. Monolepis (tribe Anserineae) and Oxybasis (tribe Chenopodieae). However, key taxa such as C.antarcticum, C.exsuccum, C.litwinowii, C.foliosumsubsp.montanum and Monolepisspathulata were not included and so their phylogenetic position within the Chenopodioideae remained equivocal. These species and additional samples of Blitumasiaticum and B.nuttallianum were incorporated into an expanded phylogenetic study based on nrDNA (ITS region) and cpDNA (trnL-trnF and atpB-rbcL intergenic spacers and rbcL gene). Our analyses confirm the placement of C.exsuccum, C.litwinowii and C.foliosumsubsp.montanum within Blitum (currently recognised as Blitumpetiolare, B.litwinowii and B.virgatumsubsp.montanum, respectively); additionally, C.antarcticum, currently known as Oxybasisantarctica, is also placed within Blitum (reinstated here as B.antarcticum). Congruent with previous studies, two of the three accepted species of Monolepis – the type species M.trifida (= M.nuttalliana) as well as M.asiatica – are included in Blitum. The monotypic genus Carocarpidium described recently with the type C.californicum is not accepted as it is placed within Blitum (reinstated here as B.californicum). To date, few reliable morphological characters have been proposed that consistently distinguish Blitum (incl. two Monolepis species) from morphologically similar Oxybasis; however, two key differences are evident: (1) the presence of long-petiolate rosulate leaves in Blitum vs. their absence in Oxybasis and (2) a seed coat structure with the outer wall of the testa cells lacking stalactites (‘non-stalactite seed coat’) but with an obvious protoplast in Blitum vs. seed coat with the outer walls of the testa cells having stalactites (‘stalactite seed coat’) and a reduced protoplast in Oxybasis. Surprisingly, the newly sequenced North American Monolepisspathulata nested within the tribe Dysphanieae (based on ITS and trnL-trnF + rbcL + atpB-rbcL analyses).The phylogenetic results, as well as presence of the stalactites in the outer cell walls of the testa and lack of the rosulate leaves, confirm the distinctive nature of Monolepisspathulata from all Blitum and, therefore, the recent combination Blitumspathulatum cannot be accepted. Indeed, the morphological and molecular distinctive nature of this species from all Dysphanieae supports its recognition as a new monotypic genus, named herein as Neomonolepis (type species: N.spathulata). The basionym name Monolepisspathulata is also lectotypified on a specimen currently lodged at GH. Finally, while Micromonolepispusilla is confirmed as belonging to the tribe Chenopodieae, its position is not fully resolved. As this monotypic genus is morphologically divergent from Chenopodium, it is retained as distinct but it is acknowledged that further work is required to confirm its status.

Further changes were subsequently proposed by Theodorova (2014), provided without a detailed explanation, suggesting that Blitum should be expanded to include Lipandra, Oxybasis and Chenopodiastrum, resulting  The recent split of Chenopodium sensu lato into genera belonging to different tribes as suggested by Fuentes-Bazan et al. (2012b) is supported in part by morphological characters. First, all species of Chenopodium with obvious glandular hairs, ovoid or roundish, yellow or orange subsessile glands and simple hairs now belong to the tribe Dysphanieae (placed in either Dysphania R.Br. or Teloxys Moq.), while the remaining former Chenopodium (now included in Chenopodieae and Anserineae) have an indumentum of white bladder ("mealy") hairs, sometimes with scattered simple hairs (Reimann and Breckle 1988;Simón 1997;Sukhorukov et al. 2015b). The number of perianth segments was also traditionally thought to be a good diagnostic character, which usually corresponds to the number of stamens. Chenopodium s.str., Lipandra and Chenopodiastrum are characterised by the presence of five perianth segments and five stamens, while various genera across the subfamily are characterised by a lower number (1-4) of perianth segments and stamens, as observed in some Oxybasis and Micromonolepis (Chenopodieae), Blitum incl. Monolepis (Anserineae) and many Dysphania (Dysphanieae), especially amongst Australian species (e.g. Ulbrich 1934;Wilson 1984;Judd and Ferguson 1999;Holmgren 2003). However, this character may not be consistently informative as species such as Oxybasis urbica usually has 5 perianth segments and 5 stamens.
It has become apparent in recent years that fruit and seed characters are also useful in distinguishing members of the former Chenopodium, particularly amongst groups that are quite morphologically similar (Sukhorukov 2006(Sukhorukov , 2014Sukhorukov et al. 2015a). A good example is Chenopodium gubanovii Sukhor. Originally this species was described as a member of the former Chenopodium subgen. Blitum sect. Pseudoblitum (Sukhorukov 1999). Its generic status was discussed by Fuentes-Bazan et al. (2012b) and finally resolved by  as being a part of Oxybasis [Oxybasis gubanovii (Sukhor.) Sukhor. et Uotila] based on molecular phylogenetic data supported by morphological and seed characters. Almost all Chenopodieae (Archiatriplex, Chenopodium, Chenopodiastrum, Exomis, Holmbergia, Lipandra, Manochlamys, Microgynoecium, Proatriplex and all Atriplex with red or black seeds) possess a seed-coat testa with thickened outer cell walls impregnated with vertical or oblique stalactites and a reduced protoplast (hereafter 'stalactite seed coat') (Sukhorukov 2006;Kadereit et al. 2010;Sukhorukov 2014). There are a few exceptions, however, for example the seed coat in Halimione and three Chenopodium species endemic to Juan Fernández Archipelago (Chile) (C. nesodendron Skottsb., C. sanctae-clarae Johow, C. sancti-ambrosii Skottsb.), does not contain the stalactites in the outer cell walls and possesses a visible protoplast (hereafter 'non-stalactite seed coat') (Sukhorukov 2014). These three geographically isolated Chilean species are closely allied and highly unusual, as they not only possess a non-stalactite seed coat but have a tree-like habit and fruits with an apically swollen pericarp. Of these, only C. sanctae-clarae has been included in molecular analyses (Kadereit et al. 2010), which confirmed its phylogenetic position within this genus.  (Sukhorukov 2014).
Amongst the species of the former Chenopodium or Monolepis investigated carpologically but not included in recent molecular phylogenetic studies, two taxa are of special interest. The first, Monolepis spathulata, is endemic to western states of USA and North Mexico and was transferred to Blitum (as B. spathulatum) due to morphological affinities with other species of the genus. The second taxon, Chenopodium antarcticum, is another poorly known taxon endemic to Tierra del Fuego (southernmost parts of Argentina and Chile) that still occupies a pending position within Chenopodioideae. Previously, it was described as Blitum antarcticum Hook.f. (Hooker 1847) and later transferred by the same author to Chenopodium as C. antarcticum (Hook.f.) Hook.f. (Bentham and Hooker 1880). The latter name was widely accepted in subsequent taxonomic treatments (Reiche 1911;Aellen 1929Aellen , 1931Aellen and Just 1943;Moore 1983;Giusti 1984;Zuloaga and Morrone 1999). Recently, Chenopodium antarcticum was transferred into Oxybasis by Mosyakin [2013, as O. antarctica (Hook.f.) Mosyakin] based on its morphological similarity to other Oxybasis. However, the stalactite seed coat morphology of Blitum spathulatum and non-stalactite seed coat of Oxybasis antarctica contrast with those of other members of Blitum and Oxybasis, respectively (Sukhorukov 2014), which raises the question of their true phylogenetic position.
To resolve this issue, we have included these two species, in addition to several accessions of taxa sampled for the first time [Chenopodium antarcticum, C. exsuccum (C.Loscos) Uotila, C. litwinowii (Paulsen) Uotila, C. foliosum (Moench) Asch. subsp. montanum Uotila and Monolepis spathulata], as well as an additional sample of Blitum asiaticum (Fisch. & C.A.Mey.) S.Fuentes, Uotila & Borsch. in expanded molecular analyses based on nrDNA (ITS region) and cpDNA (atpB-rbcL intergenic spacers + rbcL and trnL-trnF intergenic spacer + rbcL, hereafter as atpB-rbcL and trnL-trnF, respectively) to determine their phylogenetic position within the Chenopodioideae. Furthermore, we discuss the role of fruit and seed characters for delimitating morphologically similar but phylogenetically distant taxa and conclude with proposed taxonomic changes that reflect our findings.

Taxon sampling
Several new taxa were included in the phylogenetic analysis for the first time:  Table 1.

DNA extraction
Total genomic DNA was extracted from herbarium samples according to Krinitsina et al. (2015). Following the homogenisation of plant fragments (MiniLys, Bertin Technologies, France), total DNA was extracted using the CTAB-method (Doyle and Doyle 1987) and further purified using AMPure Beads (Beckman Coulter, USA).
PCRs for two chloroplast markers (atpB-rbcL and trnL-trnF) and nrDNA (ITS region) were carried out in a Thermal Cycler T100 (Bio-Rad, USA) using primers and cycler programmes listed in Table 2. A 10 ng aliquot of DNA was used to make a 25 μl total volume reaction, containing 1 μM of each primer, 200 μM of each dNTP and 0.5 U Encyclo polymerases (Evrogen, Russia). PCR products were checked on 1.2% agarose gels and purified using AMPure Beads (Beckman Coulter, USA) according to the owner's manual. AMPure Beads suspension was mixed with a solution containing PCR-product ratio 1 vol. PCR-mix: 1.2 vol. AMPure Beads for atpB-rbcL and ITS primer pairs and 1 vol. PCR-mix: 1.4 vol. AMPure Beads for rbcL, Tab C/Tab D and Tab E/Tab F primer pairs. Table 1. Voucher information and GenBank accession numbers for the species of Chenopodioideae and outgroups included in the phylogenetic analysis (arranged in alphabetical order). The newly sequenced samples are highlighted in bold. Some vouchers in GenBank may be stored under old names.

Species
Old names (if applicable) GenBank accession number

Sequencing and alignment
Sequencing was performed following Sanger methods on an Applied Biosystems 3730 DNA Analyser using ABI PRISM BigDye Terminator v. 3.1 (Center of Collective Use "Genome", Institute of Molecular Biology, Moscow, Russia). The sequencing primers were the same as the amplification primers. The raw forward and reverse sequences were checked and combined in BioEdit sequence alignment editor v. 7.0.5.3 (Hall 1999). Sequences were edited and aligned using Muscle 3.6 (Edgar 2004). The obtained alignments were manually edited using PhyDe (version 0.9971: Müller et al. 2010) following the rules outlined in Löhne and Borsch (2005). Mutational hotspots (regions of uncertain homology) were excluded from the analysis . Gaps were treated as missing data during the phylogenetic inference.

Phylogenetic inference
To show the relationships between taxa, we reconstructed various phylogenies using Bayesian analysis, maximum likelihood (ML) and maximum parsimony (MP) methods for the ITS and combined trnL-trnF + rbcL + atpB-rbcL datasets. Models of nucleotide substitution were selected using the MrModeltest 2.1.7 (Nylander 2004) via the Akaike information criterion (AIC: Akaike 1974). The substitution model was set to GTR + G + I. For the ML analyses, we employed RAxML Version 8 (Stamatakis 2014). Bootstrap analyses were conducted with 2500 replicates for ML. Parsimony analyses were conducted in PAUP* 4.0a162 (Swofford 2002) with the following settings: all characters have equal weight, MaxTrees set to 1000 (auto increased by 1000), TBR branch swapping and with 20000 jackknife (JK) replicates to calculate node support. Bayesian analyses were conducted in BEAST 2.5.0 (Bouckaert et al. 2014). Four Markov Chain Monte Carlo analyses with four chains were run for 20 million generations for every dataset, sampling every 1000 generations. Burn-in was set to remove 5% of the total trees sampled after assessing likelihood convergence by inspection of the trace plots in the programme Tracer v.1.6 (Rambaut et al. 2014). A birth and death prior was chosen for branch lengths (Gernhard 2008). The maximum clade credibility tree was calculated in the programme TreeAnnotator v1.4.8 (Drummond and Rambaut 2007) with a posterior probability limit of 0.7. Final trees were edited in the programme TreeGraph ver. 2.14.0 (Stöver and Müller 2010).

Morphology and anatomy
The carpology of the tribe Chenopodioideae was described in detail in a previous study by Sukhorukov (2014). In this study, we pay particular attention to the fruit and seed of Chenopodium antarcticum and to the general structure of the reproduc-tive shoot of Monolepis spathulata that were not illustrated in Sukhorukov (2014). The samples were observed using a scanning electron microscope (SEM) JSM-6380 (JEOL Ltd., Japan) at 15 kV after sputter coating with gold-palladium in the laboratory of Electron Microscopy at Lomonosov Moscow State University. Prior to SEM, the fruits were dehydrated in aqueous ethyl alcohol solutions of increasing concentration, followed by alcohol-acetone solutions and pure acetone. No dehydration of the seeds is required prior to SEM observation due to the absence of soft tissues (e.g. papillae or trichomes) on their surface.
The cross-sections of the seeds were prepared using a rotary microtome Microm HM 355S (Thermo Fisher Scientific, USA) and then examined using a Nikon Eclipse Ci (Nikon Corporation, Japan) light microscope and photographed using a Nikon DS-Vi1 camera (Nikon Corporation, Japan) at the Department of Higher Plants, Lomonosov Moscow State University. Before sectioning, the seeds were soaked in water:alcohol:glycerine (1:1:1) solution, dehydrated in ethanol dilution series and embedded in the Technovit 7100 resin (Heraeus Kulzer, Germany).
In the ITS analysis (Figure 1), the tribe Axyrideae is placed sister to the remaining Chenopodioideae. The next diverging lineage is a well-supported Dysphanieae, with Monolepis spathulata + Teloxys forming a sister lineage to the remaining representatives of the tribe. Chenopodium antarcticum, C. litwinowii, C. exsuccum and C. foliosum subsp. montanum fall well within Blitum, which is sister to a well-supported Chenopodieae. Blitum californicum and B. bonus-henricus (L.) C.A.Mey. form part of the polytomy with the rest of the genus.
Like the ITS phylogenetic analysis, the combined trnL-trnF + rbcL + atpB-rbcL tree (Figure 2) shows the Axyrideae as an early branching lineage in Chenopodioideae, sister to a polytomy of Dysphanieae, Anserineae and Chenopodieae. Within the Dysphanieae, Monolepis spathulata and Teloxys form a polytomy with the remaining representatives of the tribe, which includes Cycloloma nested within Dysphania. Chenopodium antarcticum, C. litwinowii and C. exsuccum are nested within Blitum (C. foliosum subsp. montanum is not included in the combined tree). Chenopodium antarcticum is sister to Chenopodium exsuccum + C. litwinowii -Blitum virgatum.

Carpological studies
This study highlighted the fact that these species, with the exception of Monolepis spathulata, possess the same fruit and seed anatomy as other Blitum species such as a mamillate pericarp (Figure 3) and non-stalactite seed-coat with obvious (visible) protoplast (Table 3; Figure 4). In contrast, the carpology of Monolepis spathulata somewhat resembles the morphology observed in species of Oxybasis and many other Chenopodieae in having a papillate pericarp and a stalactite seed coat with a highly reduced protoplast ( Figure 5). Other important characters such as life history, the degree of fusion of reduced perianth segments, pericarp structure and adherence, the colour, shape and morphology of seeds and an embryo position, are recorded for representative species of each genus, as summarised in Table 3.

Discussion
The     this study. Indeed, the results were predictable due to the shared morphological and carpological affinities of these species to B. virgatum, such as the presence of a leaf rosette, tight adherence of the pericarp to the seed coat and the ovoid and keeled seeds having the same anatomical structure (e.g. Uotila 1993Uotila , 1997Sukhorukov 2014). For this reason, while Chenopodium korshinskyi (Litv.) Minkw. has not been included in any molecular phylogenies to date, it should be treated as Blitum korshinskyi Litv. (Fuentes-Bazan et al. 2012b) due to the shared presence of these diagnostic traits. It is also evident, based on phylogenetic and carpological data from this study, that Oxybasis antarctica (formerly Chenopodium antarcticum) must be treated as Blitum antarcticum as proposed by Hooker (1847)

Diagnostic characters for Blitum and Oxybasis
The importance of morphological characters used to delineate species within the genus Chenopodium that are now considered to belong to either Blitum or Oxybasis have been discussed by various authors (e.g. Moquin-Tandon 1840, 1849; Aellen and Just 1943;Scott 1978;Fuentes-Bazan et al. 2012b). However, the morphological similarity of some species has led to taxonomic confusion. For example, many macromorphological characters overlap in Blitum and Oxybasis, including previous diagnostic traits such as: reduced (1-4) number of perianth segments, presence of the vertical seed embryo position and emergence of spatial heterospermy. Such characters are clearly homoplastic in Chenopodieae, Anserineae and some other groups of the Chenopodioideae (Sukhorukov and Zhang 2013). Only one trait visible to the naked eye, the presence of leaf rosette in Blitum ( Figure 6) and its absence in Oxybasis, can be used for the delimitation of both genera (see diagnostic key and generic descriptions in Fuentes-Bazan et al. 2012b). However, it should be noted that the leaf rosette in some Blitum, especially in species previously included in Monolepis (B. asiaticum, B. nuttallianum), is reduced to 1-2 leaves that may wither away completely by anthesis. From this study and from previous work Sukhorukov 2014), it is evident that another character, the structure of the testa cells of the seed coat, is also diagnostic. In Oxybasis, as well as almost all other Chenopodieae, the seed testa cells have a reduced protoplast and "stalactites" hanging vertically in the outer wall (stalactite seed coat). In contrast, the presence of non-stalactite seed coat with a highly visible protoplast, unambiguously distinguishes Blitum. Other characters, such as reduced perianth segments, mamillate pericarp, red seeds, seed keel, vertical embryo position of note for representative species of each genus, are summarised in Table 3 and they play a role for the diagnostics at the species level or species group (see Sukhorukov 2014 for further detail).
In the absence of molecular phylogenetic data, it is clear that carpological characters must be taken into consideration when determining the generic placement of taxa in either Blitum or Oxybasis. Molecular data from this study and previous investigations (Kadereit et al. 2010;Fuentes-Bazan et al. 2012a, 2012b, when examined in conjunction with carpological evidence (Sukhorukov 2014), show that two taxonomic changes recently proposed: (1) the merger of Oxybasis, Lipandra and Chenopodiastrum (Chenopodieae) into an extended Blitum (Anserineae) as suggested by Theodorova (2014) and (2) the description of a new monotypic genus Carocarpidium S.C.Sanderson et G.L.Chu with the type C. californicum (≡Blitum californicum) by Zhu and Sanderson (2017), cannot be accepted. Additionally, it should be noted that the pericarp of B. californicum is not fleshy as previously described (Zhu and Sanderson 2017), but its outer layer consists of spongy (mamillate) cells that imitate a "fleshy" pericarp. This type of mamillate pericarp is present in some Blitum and Oxybasis (Figure 3, see also Table 3) and so this character is clearly not unique to Carocarpidium.

Micromonolepis pusilla
This species was initially described as Monolepis pusilla Torr. ex Watson (Watson 1871) and it is noteworthy to consider its morphology and phylogenetic position in context with other species previously known as Monolepis. It is a small annual herb covered with bladder hairs that has fleshy leaves (Figure 7), unisexual flowers with reduced (1-3) perianth segments and tiny papillate fruits. Due to its unusual habit, M. pusilla was transferred into a new monotypic genus Micromonolepis (Ulbrich 1934). The species was included in a atpB-rbcL molecular analysis, where it was unexpectedly placed within the "Chenopodieae I" clade comprising Rhagodia, Einadia and a part of Chenopodium s.l. (Kadereit et al. 2010). The papillate pericarp and the stalactite seed coat provide a good support for its placement into Chenopodieae, based on cpDNA being a part of Chenopodium s.str. (Kadereit et al. 2010, as Chenopodieae I; Figure 2). However, the limited number of taxa used in the atpB-rbcL analysis, the lack of additional molecular data and the significant morphological differences evident between Micromonolepis and the remaining Chenopodium species in this clade, such as the presence of fleshy leaves and reduced perianth segments, precludes the formal transfer of M. pusilla to Chenopodium. Further work is needed to evaluate the exact position of Micromonolepis pusilla within Chenopodieae.

Monolepis spathulata is neither Monolepis nor Blitum
Recently, Monolepis spatulata was transferred to Blitum (as B. spathulatum) based on its resemblance to other species of the genus due to the presence of a reduced number of perianth segments (Fuentes-Bazan et al. 2012b). It is evident, however, that the reduced number of perianth segments independently evolved in Chenopodieae (e.g. in Micromonolepis and some Oxybasis), Anserineae and many Dysphanieae . In light of carpological evidence (Sukhorukov 2014), it seemed doubtful that M. spathulata should be included in Blitum, as this species possesses a stalactite seed coat with a reduced protoplast. Our phylogenetic results show that Monolepis spathulata is not closely related to the other species in Monolepis (M. asiatica, and M. nuttalliana) that are now included in Blitum (Anserineae) as B. asiaticum and B. nuttallianum, respectively. This species falls within Dysphanieae forming a polytomy with Teloxys and Dysphania + Cycloloma. M. spathulata is a glabrous annual and differs from all Dysphanieae by the absence of simple hairs and subsessile glands that are diagnostic characters of this tribe. Additionally, M. spathulata is found to have the stalactite seed coat, a character missing in all Dysphanieae (Sukhorukov 2014). The close relationship between M. spathulata and the Dysphanieae, evidenced by molecular data, is unexpected given the obvious morphological and carpological differences. Indeed, M. spathulata is considered so distinct that it warrants recognition at the generic level. Description. Annual, glabrous, branched or not; lateral branches if present ascending; leaves cauline (rosulate leaves absent), densely located, spatulate-oblong, with a short petiole up to 1 cm or sessile, entire; inflorescence leafy (bracts similar to stem leaves); flowers sessile or shortly pedicellate, unisexual intermixed in small glomerules ( Figure 8); male flowers with 2-lobed hyaline perianth, stamens 1-2, anthers 0.10-0.15 mm long; female flowers without perianth, fruits 0.55-0.65 mm in diameter, almost round, with blackish papillate pericarp (when dry) that is easily raptured, styles 2(3); seeds 0.4 × 0.3 mm, reddish, with smooth surface, with small irregular pits (seen at a higher magnification), seed-coat testa with stalactites in the outer cell walls and reduced protoplast; embryo vertical.

Morphological notes.
As Neomonolepis is a monotypic genus, the description of N. spathulata corresponds to the generic description above. Neomonolepis spathulata is morphologically distant from all Dysphanieae (Teloxys, Suckleya A.Gray, Dysphania R.Br. and Cycloloma Moq.) in being glabrous in all parts (vs. glandular and/or simple hairs), having unisexual flowers (vs. bisexual or polygamous) and 'stalactite' seed-coat testa (vs. 'non-stalactite'). For this reason, we prefer to refer to the clade with the abovementioned genera as the 'Dysphanieae + Neomonolepis' clade.
Typification. The type specimen lodged at GH contains several plants collected from different areas in California and almost all of them were collected after the description of Monolepis spathulata (Gray 1868). The lectotype selected here (lower righthand specimen on the GH00037208 sheet) is a part of original material cited in the protologue as "Sierra Nevada, at Mono Pass, in loose soil, Bolander" (Gray 1868) and it is chosen in accordance with Art. 9 of ICN (Turland et al. 2018). The description of the species is consistent with the image of the lectotype. Gray (1868) also noted that the seeds of Monolepis spathulata are notably smaller than those of M. chenopodioides [= Blitum nuttallianum]. The small seed dimensions of Neomonolepis spathulata (0.4 × 0.3 mm) are similar to those observed in many Australian Dysphania (Wilson 1984 sub Chenopodium;Sukhorukov 2014).
Distribution. South-western North America (USA, North Mexico). Etymology. The new generic name is composed by the prefix "neo" (new) and the core name Monolepis.

Conclusion
In the Chenopodioideae, some phylogenetically distant taxa often look similar due to convergence of various morphological characters, some of which were previously thought to be diagnostic such as the number of perianth segments. A remarkable example is highlighted by the different phylogenetic positions occupied by members of the former genus Monolepis, which are currently included in Anserineae (M. nuttalliana ≡ Blitum nuttallianum; M. asiatica ≡ B. asiaticum), Dysphanieae (Neomonolepis spathulata ≡ Monolepis spathulata) and Chenopodieae (Monolepis pusilla ≡ Micromonolepis pusilla). This study shows that fruit and seed characters such as seed-coat structure are valuable traits for taxonomic study. These features are particularly useful in distinguishing the morphologically similar but phylogenetically distinct genera Blitum and Oxybasis.
of the Department of Higher Plants, Lomonosov Moscow State University (revision of the herbaria in Moscow and St.-Petersburg) and Russian Foundation for Basic Research (project 18-04-00029: revision of the herbarium collection in UK) supported the study of AS, MN and AK. The study of AE was financially supported by the Scientific programme АААА-А17-117012610055-3 of the Central Siberian Botanical Garden, SB RAS (sampling herbarium specimens from NS) and Tomsk State University competitiveness improvement programme (sampling herbarium specimens from TK).