Kazakhstan Has an Unexpected Diversity of Medicinal Plants of the Genus Acorus (Acoraceae) and Could Be a Cradle of the Triploid Species A. calamus

The Acorus calamus group, or sweet flag, includes important medicinal plants and is classified into three species: A. americanus (diploid), A. verus (tetraploid), and A. calamus (sterile triploid of hybrid origin). Members of the group are famous as components of traditional Indian medicine, and early researchers suggested the origin of the sweet flag in tropical Asia. Subsequent research led to an idea of the origin of the triploid A. calamus in the Amur River basin in temperate Asia, because this was the only region where both diploids and tetraploids were known to co-occur and be capable of sexual reproduction. Contrary to this hypothesis, triploids are currently very rare in the Amur basin. Here, we provide the first evidence that all three species occur in Kazakhstan. The new records extend earlier data on the range of A. verus for c. 1800 km. Along the valley of the Irtysh River in Kazakhstan and the adjacent Omsk Oblast of Russia, A. verus is recorded in the south, A. americanus in the north, and A. calamus is common in between. We propose the Irtysh River valley as another candidate for a cradle of the triploid species A. calamus. It is possible that the range of at least one parent species (A. americanus) has contracted through competition with its triploid derivative species, for which the Irtysh River floods provide a tool for downstream range expansion. We refine our earlier data and show that the two parent species have non-overlapping ranges of variation in a quantitative metric of leaf aerenchyma structure.


Introduction
Acorus is the only genus of the family Acoraceae and the order Acorales, holding a phylogenetic position sister to the rest of the monocotyledons [1][2][3][4][5][6].The genus comprises importance of this river system, we extended our interest from Kazakhstan to the adjacent Russian part of the Irtysh basin in Omsk Oblast.
Our earlier study, using extensive data from across Asia, Europe, and North America, revealed that A. calamus, A. americanus, and A. verus can be precisely distinguished using sequences of the nuclear ribosomal internal transcribed spacer [9].Acorus verus differs from A. americanus in as many as 12 single nucleotide substitutions plus 10 indels in nuclear ribosomal ITS sequences (nrITS).We called the sequence found in A. americanus ribotype I and that of A. verus ribotype II.We found no infraspecific variation in our extensive material from Russia, Mongolia, India, and the USA.Acorus calamus has both ribotypes with no traces of contamination, apparently because of its exclusively clonal reproduction.Therefore, nrITS is an efficient tool for molecular barcoding Acorus species [9].DNA content as inferred from flow cytometry provides another direct tool to distinguish between the triploid A. calamus (2C = 1.23-1.31pg), the diploid A. americanus (2C = 0.88-0.95pg), and the tetraploid A. verus (2C = 1.47-1.61pg) [9,33,34].Seeds of A. americanus (morphotype I) and A. verus (morphotype II) differ in the absence vs. presence of long, branched hairs in the micropylar region [9].Acorus calamus never produces any seeds, and its pollen shows a high percentage of sterility, as measured through its stainability with acetocarmine or cotton blue in lactophenol [9,11,26,35].The pollen of two other species is largely fertile (the stainability is, in most cases, over 90%).Our earlier study covered leaf anatomy in 449 individual plants of Acorus from 253 localities in Austria, Belarus, Bhutan, Canada, China, Germany, India, Japan, Kazakhstan, Mongolia, Nepal, Russia, Ukraine, the USA, and Uzbekistan [9].We found that the mean cell number in the septa of leaf lamina aerenchyma as seen in cross-section (N cell ) ranges from 0.5 to 3.2 (1.02-2.85 in 95% of samples studied) in A. verus and from 2.95 to 7.8 (3.40-7.15 in 95% of samples studied) in A. americanus.The range of the leaf aerenchyma metric in A. calamus (N cell ) overlapped with those of the two parent species.To summarise, various tools for identification showed excellent correlations with each other.Each of the two tools-flow cytometry and nrITS barcoding-alone is sufficient for precise identification.Seed micromorphology, leaf anatomy, and pollen stainability, taken together, also allow for precise identification.When seeds are available or when the pollen is sterile, seed and pollen characters alone provide for precise identification.On the other hand, features of external morphology (e.g., those available in scanned herbarium specimens) are insufficient for the precise recognition of the three species, at least in Asia.They can therefore be considered cryptic species in the wide sense of the term [36], or pseudocryptic, because of the existence of micromorphological differences [37].

Materials and Methods
We followed all protocols used in our earlier study [9], including those of DNA extraction, amplification, sequencing, and alignment, flow cytometry, scanning electron microscopy (SEM), anatomy with light microscopy (LM), pollen stainability, and creating a distribution map.Herbarium collections of AA, ALTB, LE, MW, and TK were used (Table 1).Identification tools applied to each examined specimen varied depending on the age of the specimen, its developmental stage, and the parts kindly allocated for destructive sampling by the curators.GenBank accessions for sequences of nrITS are provided in Table 2.
Table 1.Examined specimens of Acorus from Kazakhstan and Omsk Oblast of Russia and their taxonomic identities as inferred from our data.

RUSSIA. Omsk Oblast
Table 2. Data on leaf anatomy, pollen stainability, seeds, nuclear DNA content and nrITS ribotypes of examined specimens.Ncell is the mean cell number per septum in leaf lamina aerenchyma as seen in cross-section (corner cells not counted), based on counts of at least 20 septa per specimen.Legend to the field "Seeds": A, inflorescence at anthesis; PANS, no seeds in postanthetic inflorescence; SM = I, seed morphotype I; SM = II, seed morphotype II; v, vegetative sample.

Taxonomic Identification of Herbarium Collections
Our data document the occurrence of all three species of the A. calamus group in Kazakhstan and show that both A. calamus s.str.and A. americanus occur in Omsk Oblast of Russia.Full lists of species records in each region based on the present study are provided in Table 1.Table 2 summarises diagnostic characters used to identify each specimen.Figure 1 provides a distribution map of Acorus species in Kazakhstan and adjacent parts of septum in leaf lamina aerenchyma, as seen in a cross-section.Like in the previous study, the mean was calculated using cell counts in at least 20 septa in each specimen.As such, Ncell does not provide a useful tool to identify triploids, but the range of variation of Ncell found here in examined samples of A. calamus (2.15-3.60)fits very well with the range observed for this species in other regions of Eurasia [9].[38] and from the Ishim River in Soltüstık Qazaqstan oblysy [39] for which we had no access to voucher specimens.The only specimen of A. americanus from Pavlodar oblysy has no precise locality information and is therefore omitted from the map.
Seed micromorphology provided unequivocal evidence for the identification of collections of A. americanus and A. verus from Kazakhstan.The specimen of A. americanus (Figure 2A,B) has well-developed seeds bearing only rare, very short (<0.05 mm), unbranched hairs (seed morphotype I [9]).Specimens of A. verus (Figure 3) also have fruits with well-developed seeds, but these bear numerous long (>0.2 mm), branching hairs (seed morphotype II [9]).Figures of Ncell inferred from the specimens of A. verus and A. americanus examined here (Table 2) fit very well with the ranges of variation inferred for each of these species in our earlier study (Figure 4); also, these figures do not fall into the interval of Ncell = 1.95-4.15that includes 95% of samples of A. calamus included in the earlier study [9].[38] and from the Ishim River in Soltüstık Qazaqstan oblysy [39] for which we had no access to voucher specimens.The only specimen of A. americanus from Pavlodar oblysy has no precise locality information and is therefore omitted from the map.
The present work revealed the same two haplotypes of nrITS as the earlier study, and the occurrence of haplotype I, II or their combination allowed identification of A. americanus, A. verus, and A. calamus, respectively (Table 2).Figures of the nuclear DNA content of 2C = 1.24-1.26pg and/or very low percentages of stainable pollen grains allowed for the precise recognition of the sterile triploid A. calamus, while the absence of any seed set in well-postanthetic inflorescences was considered indirect evidence of sterility (Table 2).The main metric of leaf anatomy used here was N cell , which is the mean cell number per septum in leaf lamina aerenchyma, as seen in a cross-section.Like in the previous study, the mean was calculated using cell counts in at least 20 septa in each specimen.As such, N cell does not provide a useful tool to identify triploids, but the range of variation of N cell found here in examined samples of A. calamus (2.15-3.60)fits very well with the range observed for this species in other regions of Eurasia [9].
Seed micromorphology provided unequivocal evidence for the identification of collections of A. americanus and A. verus from Kazakhstan.The specimen of A. americanus (Figure 2A,B) has well-developed seeds bearing only rare, very short (<0.05 mm), unbranched hairs (seed morphotype I [9]).Specimens of A. verus (Figure 3) also have fruits with well-developed seeds, but these bear numerous long (>0.2 mm), branching hairs (seed morphotype II [9]).Figures of N cell inferred from the specimens of A. verus and A. americanus examined here (Table 2) fit very well with the ranges of variation inferred for each of these species in our earlier study (Figure 4); also, these figures do not fall into the interval of N cell = 1.95-4.15that includes 95% of samples of A. calamus included in the earlier study [9].
Plants 2024, 13, x FOR PEER REVIEW 8 of 20 We recognised two subspecies of A. americanus based on characters of leaf anatomy and morphology [9].The American taxon, subsp.americanus, has leaves typically with several equally developed large vascular bundles on either side.Leaves of the Asian subsp.triqueter in most instances have a conspicuous major bundle.In addition, specimens from Asia and North America tend to differ in the shape of the leaf lamina, as seen in a cross-section.The Kazakhstan specimen of A. americanus studied here has the quantitative metric of the shape of the lamina cross-section that does not allow its unequivocal placement into either subspecies (Figure 5).Leaf vasculature is also variable in this specimen, with the occurrence of one, two, or three larger veins, sometimes in various parts of the same leaf.
The material of A. verus from Kazakhstan is similar to collections of this species from other countries regarding the quantitative metric of the shape of the lamina cross-section.Acorus americanus, Kazakhstan Acorus americanus, other countries, samples identified using DNA and/or seeds Acorus americanus, samples identified using leaf aerenchyma only

Acorus verus, Kazakhstan
Acorus verus, other countries, samples identified using DNA and/or seeds Acorus verus, samples identified using leaf aerenchyma only We recognised two subspecies of A. americanus based on characters of leaf anatomy and morphology [9].The American taxon, subsp.americanus, has leaves typically with several equally developed large vascular bundles on either side.Leaves of the Asian subsp.triqueter in most instances have a conspicuous major bundle.In addition, specimens from Asia and North America tend to differ in the shape of the leaf lamina, as seen in a crosssection.The Kazakhstan specimen of A. americanus studied here has the quantitative metric of the shape of the lamina cross-section that does not allow its unequivocal placement into either subspecies (Figure 5).Leaf vasculature is also variable in this specimen, with the occurrence of one, two, or three larger veins, sometimes in various parts of the same leaf.

Notes on Critical Historical Specimens
Medvedev & Melvil s.n.(AA002771) is the only specimen of A. americanus known so far from Kazakhstan.It was collected in Pavlodar Krai (now Pavlodar oblysy) in 1930 but has no precise locality information.We believe that it indeed comes from Kazakhstan, because the collectors were employed by the Kazakhstan Institute for Soil Studies (operated in Kyzyl-Orda Town during 1929-1933) for surveying pastures and hayfields [40] (Bykov & Kubanskaya, 1960), and, according to further herbarium specimens kept at TK, it is known that they conducted a vegetation survey of Pavlodar Krai during this year.The material of A. verus from Kazakhstan is similar to collections of this species from other countries regarding the quantitative metric of the shape of the lamina cross-section.

Notes on Critical Historical Specimens
Medvedev & Melvil s.n.(AA002771) is the only specimen of A. americanus known so far from Kazakhstan.It was collected in Pavlodar Krai (now Pavlodar oblysy) in 1930 but has no precise locality information.We believe that it indeed comes from Kazakhstan, because the collectors were employed by the Kazakhstan Institute for Soil Studies (operated in Kyzyl-Orda Town during 1929-1933) for surveying pastures and hayfields [40] (Bykov & Kubanskaya, 1960), and, according to further herbarium specimens kept at TK, it is known that they conducted a vegetation survey of Pavlodar Krai during this year.Furthermore, the occurrence of A. americanus in areas of Kazakhstan adjacent to the border with Altai Krai of Russia looks more than plausible.In Altai Krai, the species was found in wetlands associated with so-called ribbon pine forests [9].The ribbon forests are long and narrow belts that extend for hundreds of kilometres from the Ob River valley in Russia in the east towards the Irtysh River valley in Kazakhstan in the west.
Karelin & Kiriloff 1066 (MW0812267, TK) has no label information on the collection locality; only the year is indicated (1840).G.S. Karelin and I.P. Kirilov were famous and pioneering plant collectors in Central Asia, and the history of their collections is known in detail [41].A full catalogue of their collections made in 1840 is published [42].While more than one locality is indicated for many other species in the catalogue, only one is provided for Acorus ("In paludosis prope Buchtarminsk.Fl.Julio."), which indicates the precise geographical origin of the collection (Bukhtarma, Bukhtarminsk or Ust-Bukhtarminskaya, formerly populated place at the confluence of the Bukhtarma and Irtysh rivers in Kazakhstan).
Golde s.n.(LE01249648) and Golde s.n.(LE01249647) are specimens with similar but not completely identical labels.Karl L. Golde made extensive plant collections from the present-day Omsk Oblast of Russia during his service as pharmacy administrator in Omsk for the Ministry of War of the Russian Empire in 1884-1886 [43].During that period, his collecting activities and botanical works were facilitated and financed by the West-Siberian Department of the Imperial Russian Geographical Society [44].Golde accurately labelled his herbarium specimens with locality information and collection dates according to the Julian calendar (as evidenced by explanations of his collection activities [45].The Siberian collections of Golde have been acquired by predecessors of the present-day Komarov Botanical Institute (LE) in three ways.One part was sent by the collector for identification and revision to K.F.Meinshausen, who was a conservator at the Botanical Museum of the Imperial Saint Petersburg Academy of Sciences [46].Such specimens (LE01249647) were curatorially labelled by Meinshausen.A smaller portion of this collection was deposited by Golde in 1894 to the Imperial Botanical Garden [47].Finally, the main set of Golde's collections was purchased by the Botanical Museum after his death [48].The latter specimens (LE01249648) bear original collector's labels, and all specimens (acquired in 1886 and 1907) were curatorially labelled by Litvinov with printed forms of the Botanical Museum (Sennikov, pers.obs. in herbarium collections).The Museum's collections were kept unmounted at that time, and specimens of the same species may have been accidentally mixed during that period, so the geographical origin of some particular specimens may become unreliable (Sennikov, pers.obs. in herbarium collections).
We confidently identify LE01249648 as A. calamus.This specimen has an original handwritten label of Golde, but the plant material includes only a spadix with its peduncle.Neither vegetative leaves nor a spatha are available.LE01249647 has two plant fragments that are not connected to each other.The left fragment is an inflorescence that we with certainty identify as A. americanus.The right fragment is vegetative.In our previous study [9], we used LE01249647 as a representative specimen of A. americanus from Omsk Oblast, and its species identification was based on the left fragment bearing seeds.We inferred data on leaf anatomy from its vegetative fragment and interpreted the results as belonging to A. americanus.The value of N cell = 2.95 that we found in Golde s.n.(LE01249647) was the lowest among the total of 173 samples of A. americanus collected from all across the species range, including Western and Eastern Siberia, Mongolia, the Far East of Russia, and North America [9].Now, we can take into consideration the specimen Golde s.n.(LE01249648) that has a similar label and belongs to A. calamus.Since the specimens had been originally kept loose together with other collections, we see no proof that the right fragment of LE01249647 belongs to A. americanus.It could well belong to A. calamus and be part of the collection mounted in LE01249648.The figure of N cell = 2.95 falls among typical values of A. calamus.
It is a good question whether Golde's collections indicate that A. calamus and A. americanus were growing side-by-side near Omsk in the 1880s.Based on our extensive fieldwork experience, we are not aware of other localities where these two clonal species grow in mixed populations.A report of diploids and triploids growing together in Iran [49] requires further study; in the absence of precise taxonomic identification, we consider the chromosome counts in that work unreliable because the characters other than ploidy level were not investigated.LE01249648 has a handwritten collector's label with a more precise locality, 'behind the camp', which should be read as behind the military camp.Where the military camp was located at that time near Omsk is well documented.One of us (I.V.K.) visited the locality in 2023 and found that Acorus no longer occurs there because an embankment of the Irtysh River has been constructed since Golde's time.Both LE01249648 and LE01249647 indicate the collection date as 28 July 1886 according to the Julian calendar, which corresponds to 9 August in the Gregorian calendar.The inflorescence of A. calamus in LE01249648 is at the male stage of anthesis, which would be not realistic for a plant collected in August.This stage of Acorus anthesis normally takes place in June and early July in the southern part of Siberia.For example, specimens of A. calamus collected 20 km northeast of Omsk on 31 July 1900 (Julian calendar) are postanthetic (Skalozubov 1384, LE01249642, LE01249645).The inflorescence of Golde s.n.(LE01249647) has fruits with well-developed seeds.This stage perfectly fits the first decade of August of the modern calendar.We conclude that the specimen of A. americanus was collected by Golde in Omsk, whereas the material of A. calamus in LE01249648 and LE01249647 comes from another collection of unknown origin.Thus, the collections of Golde should not be taken as proof that diploid and triploid plants of Acorus occurred in a mixed population.

Acorus verus and A. americanus Have Non-Overlapping Ranges of Variation in Quantitative Leaf Anatomy
Röst [25] compared leaf aerenchyma structure in Acorus to ploidy level in a common garden experiment.It was logical to expect that with the use of herbarium collections covering entire geographical ranges of the diploid A. americanus and the tetraploid A. verus, the picture of quantitative differences between diploids and tetraploids would become less sharp.Climatic conditions vary considerably across species ranges in Acorus (for example, between middle Yakutia, Altai Krai, and Minnesota in A. americanus).Moreover, when public herbarium collections are used, one should consider that they were made in different seasons and that no more than one leaf per specimen can be normally investigated.Some herbarium specimens allowed for sampling vegetative leaves, while for others, only the leaf was associated with inflorescence (the spatha).Nevertheless, using a refined metric of leaf aerenchyma (N cell ), our earlier study found only a narrow zone of overlapping in variation ranges between 173 wild-source samples of A. americanus and 132 samples of A. verus [9].In the present study, we were able to reconsider the interpretation of leaf anatomy in Golde s.n.(LE01249647) that had the lowest figure of N cell (2.95) in our data set of A. americanus.Our new data show that this count cannot be safely attributed to A. americanus and most likely belongs to A. calamus.With the count of 2.95 omitted, the ranges of variation of N cell no longer overlap between the diploid A. americanus and the tetraploid A. verus.Having this conclusion, we decided to re-investigate our earlier data on the highest figure of N cell in A. verus, namely N cell = 3.18 for Bobrov & Mochalova 694 (MW0163317, Khabarovsk Krai, Russia).This specimen has only one vegetative leaf.We re-sampled and re-investigated anatomically the Bobrov & Mochalova 694 specimen.For a better confidence, we studied two different sections of this leaf and in each instance counted cells in 50 aerenchyma septa.These two sections provided N cell values of 2.22 and 2.18, which are rather typical of A. verus.We therefore conclude that a lab mistake likely took place in our earlier study of Bobrov & Mochalova 694.
With the refined dataset, A. americanus and A. verus have completely non-overlapping ranges of the variation of leaf aerenchyma.The difference can be easily described in an identification key: A. verus has N cell < 3 while A. americanus has N cell > 3 (Figure 4).

The Importance of the Discovery of A. verus in the Lake Zaisan Area
This is the first report of A. verus in Kazakhstan and Central Asia.The importance of our new records from the Lake Zaisan area is not limited by the fact that they are at least 1800 km away from any previously documented locality of the species.All earlier records of A. verus were located within the Asian monsoon precipitation domain (as defined by Wang and Ding [50]) or close to its borders.The conditions in eastern Kazakhstan are far from the monsoon climate.Earlier records of A. verus lie within the area with August mean precipitation above 2.88 mm/day (and mostly with even much higher precipitation in August).In contrast, August mean precipitation is below 0.93 mm/day in the Lake Zaisan area [51].Therefore, our new data considerably broaden our understanding of the climatic requirements of A. verus.They further support the idea that temperature regime plays a more important role than precipitation in shaping distribution ranges of aquatic species [9,52].With respect to temperature regime, our new records fit well the earlier idea that A. verus requires a warmer climate than A. americanus, and therefore the two species tend to separate geographically along a latitudinal gradient (Figures 1 and 4).
All three samples of A. verus from Kazakhstan have fruits with well-developed seeds.This is in contrast with the material of this species from tropical Asia, including India, where A. verus never sets seeds despite high pollen fertility.We speculated that the absence of the seed set in tropics is because of self-incompatibility within large clones derived from ancient introduction and/or because of the absence of pollinators.Both A. verus and A. americanus are specialised for pollination by two beetles, Platamartus jakowlewi and Sibirhelus corpulentus (Kateretidae), that lay eggs into the inflorescences [9,53,54].All known records of these beetles do not extend beyond the area where Acorus amerianus and/or A. verus set fruits.Sibirhelus corpulentus is known from Russia (Altai Krai, Irkutsk Oblast, Primorsky Krai) and Japan; P. jakowlewi is recorded from Altai Krai, Republic of Tuva, Yakutia, Irkutsk Oblast, and Krasnoyarsk Krai in Russia as well as from Japan [9,53,55].Now, we should expect findings of Platamartus jakowlewi and Sibirhelus corpulentus in Kazakhstan.These records will indicate a re-consideration of the climatic requirements of the beetles, because no species of Acorus was previously known to set fruits in a climate with as dry summer weather as in the Lake Zaisan area.
It is interesting that one of the Kazakhstan specimens of A. verus (Samusev s.n., AA002770) has been examined by K.V. Dobrokhotova, which is evidenced by her identification as A. calamus s.l.The whole label, including the name of the expert, is written by the same hand, most likely soon after the sample was collected in 1950.The fact that Dobrokhotova [32] indicated the absence of seed ripening in Acorus in Kazakhstan shows that she did not pay attention to the occurrence of fully ripening and abscising fruits with mature seeds in this specimen.It is most likely that the information on non-ripening seeds was taken from the general account of Acorus for Flora of the USSR [56], where this feature is mentioned in the context of the introduction of A. calamus in Europe, but the text can be read as if the author intended to extend the absence of seed ripening into the Asiatic part of the range, too.
The fact that A. verus sets seeds in Kazakhstan suggests that it is native rather than introduced here.The habitats of A. verus in the Lake Zaisan area, as indicated in herbarium labels, suggest quite natural wetlands.All three collections of A. verus were made before the construction of the Bukhtarma hydroelectric power station that started operating in 1960s and changed the ecosystem of Lake Zaisan by elevating its water level by several meters.Therefore, the localities of A. verus discussed here are currently underwater.New fieldwork is needed to figure out whether the species still occurs in Kazakhstan.We believe that it does, because large wetlands still occur in the delta of the Black Irtysh [57][58][59], apparently close to the locality of Samusev s.n.(AA002770).The collector indicated the locality as 'Old Irtysh', which is one of the channels of the delta of the Black Irtysh [58].Dimeyeva et al. [59] mentioned 'Acorus palustris' while characterising the vegetation of the wetlands of the Black Irtysh delta.

Taxonomy Is a Key Tool in All Studies Related to Medicinal Plants
Our new data show that Kazakhstan has important genetic resources of Acorus, and these resources can be of practical use.Acorus is well-known as a medicinal plant.Studies based on Acorus collections from other countries revealed important between the ploidy level and the composition of essential oils, especially the content of β-asarone, which is highest in tetraploids [29,[59][60][61][62]. Unfortunately, published studies of chemical composition, medicinal properties of Acorus and stocks of these medical plants in Kazakhstan [39,[63][64][65][66][67][68] did not consider the ploidy level of the material.At the current level of knowledge, as outlined above, plants with different ploidy levels can be easily distinguished using micromorphology because they belong to different taxonomic species.A phytochemical investigation of A. verus from the Lake Zaisan area and an analysis of its resources will be important.
We highlight the critical importance of botanical taxonomy in all studies related to medicinal plants.Not only is it important with respect to plant resources existing today, but also with respect to the history of the uses of spices and medicinal plants.It is still unclear whether the calamus of the Bible should be identified as Acorus [10].It is equally unclear whether Acorus or Cymbopogon was used as an important aromatic plant in ancient Mesopotamia [69].Reports that certain Ancient Greek and Ancient Egyptian perfume or medicinal recipes included exactly Acorus calamus var.angustatus [70] seem not to be supported by convincing evidence.Acorus calamus var.angustatus was suggested by Röst [25] as a name for tetraploid plants, but this name is illegitimate and cannot be used for any taxon [34].More importantly, with only verbal descriptions in the historical literature, there is no chance of distinguishing the tetraploid Acorus (A. verus) from diploid (A.americanus) and triploid (A.calamus) plants.However, if the archaeological record has preserved rhizomes, we believe that paleogenetics may help in disentangling their taxonomic identity.Such research will be of mutual interest to botany and history.

Kazakhstan Could Be a Cradle of the Triploid Species A. calamus
Some early researchers suggested the origin of A. calamus in tropical Asia (e.g., [71,72]).They accepted the wide circumscription of the species to include both fertile and sterile plants.A more specific idea of the origin of the triploid A. calamus in the Himalayan region of India has been proposed [29][30][31].The absence of the fruit set of both sterile and fertile plants in tropical Asia makes this hypothesis problematic [9].Subsequent research led to an idea of the origin of the triploid A. calamus in the Amur River basin in temperate Asia because this was the only region where both diploids and tetraploids were known to co-occur and be capable of sexual reproduction [9,25].However, triploids are currently very rare in the Amur basin.For example, even though rare diploids are recorded along with the common tetraploids near Lake Khanka in Primorsky Krai, our study of voucher specimens allowed for rejecting an earlier report of triploids from this area [73], and no triploid plant was found during our extensive fieldwork there [9].
Here, we provide the first evidence that all three species of the group occur in Kazakhstan.Along the valley of the major River Irtysh in Kazakhstan and the adjacent Omsk Oblast of Russia, A. verus is recorded in the south, A. americanus in the north, and A. calamus is common in between.We propose the Irtysh River valley as another candidate for a cradle of the triploid species A. calamus.It is possible that the range of at least one parent species (A.americanus) has contracted through competition with the triploid species, for which the Irtysh River floods provide a tool for downstream range expansion.The Irtysh River valley has been at an intersection of trade routes and human migrations since pre-historic times.One can easily imagine human-mediated dispersals of triploid plants from the Irtysh River valley to the Ob River valley in Altai Krai, where the species is currently common, southwards to other regions of Middle Asia and eastwards to China.Molecular phylogeographic analysis of the entire group will help in testing the Amur basin and the Irtysh valley hypotheses of the origin of A. calamus.The distance between the two potential regions of the origin of A. calamus exceeds 3000 km.We hope that the two parent species exhibit a certain geographical differentiation at the molecular level over such distances and that this differentiation will be sufficient for the evaluation of the cradle of A. calamus.
The Irtysh and Ob valleys provide model systems to investigate the relative impacts of ice-mediated downstream dispersal, differential climatic requirements and potential competition between different species of Acorus.The Irtysh River valley has three species of Acorus, whereas the Ob River valley has only two species (A.calamus and A. americanus).In both instances, species differ in their distribution along the valley, with at least A. calamus being common in the areas where it occurs.Acorus plants (at least those of A. calamus) are perfectly adapted for vegetative dispersal along large river valleys where their rhizomes can be transported during spring floods as frozen into ice [39,74].In both Irtysh and Ob valleys, the direction of the downstream transport is towards the north, and the plants thus potentially migrate into areas with a less optimal temperature regime.It has been hypothesised that the extensive vegetative propagation of the triploid A. calamus provides certain adaptive advantages over the diploid A. americanus in floodplains of a large river [33].This idea can be tested by a targeted study of all populations of Acorus at the zone of overlap between two species.Potential zones of overlap between A. calamus and A. americanus in Novosibirsk Oblast of Russia and between A. verus and A. calamus in Şy gys Qazaqstan oblysy of Kazakhstan are affected by the construction of hydroelectric power stations.On the other hand, the contact zone between diploids and triploids in Omsk Oblast is not affected and would serve an excellent area for ecological research.

1.
All three species of the Acorus calamus group occur in Kazakhstan.

2.
Along the valley of the Irtysh River in Kazakhstan and the adjacent Omsk Oblast of Russia, the tetraploid A. verus is recorded in the south, the diploid A. americanus in the north, and the sterile triploid A. calamus is common in between.

3.
The Irtysh River valley could be a more plausible area of the origin of the triploid species A. calamus than the Amur River basin.The dilemma will be disentangled using molecular phylogeography.4.
The Irtysh valley provides a model system to investigate relative impacts of icemediated downstream dispersal, differential climatic requirements and potential competition between different species of Acorus.

5.
Micromorphology allows for precise diagnostics of all three species of the A. calamus group, except for vegetative specimens.The two fertile species have non-overlapping ranges of variation of leaf aerenchyma.6.
The discovery of A. verus in the Lake Zaisan area suggests the overlooked occurrence of its specialised beetle pollinators Platamartus and Sibirhelus in the region and facilitates further work on the genetic resources of the important medicinal plant in Kazakhstan.

Figure 1 .
Figure 1.Distribution of Acorus species in Kazakhstan and adjacent parts of Russia.Inset: Kazakhstan in the map of Asia and the position of the main map.Red stars indicate records of Acorus from Zhideli channel in the valley of the Ili River[38] and from the Ishim River in Soltüstık Qazaqstan oblysy[39] for which we had no access to voucher specimens.The only specimen of A. americanus from Pavlodar oblysy has no precise locality information and is therefore omitted from the map.

Figure 1 .
Figure 1.Distribution of Acorus species in Kazakhstan and adjacent parts of Russia.Inset: Kazakhstan in the map of Asia and the position of the main map.Red stars indicate records of Acorus from Zhideli channel in the valley of the Ili River[38] and from the Ishim River in Soltüstık Qazaqstan oblysy[39] for which we had no access to voucher specimens.The only specimen of A. americanus from Pavlodar oblysy has no precise locality information and is therefore omitted from the map.

Figure 4 .
Figure 4. Quantitative data on the variation of leaf aerenchyma in the fertile species Acorus americanus and A. verus as related to the geographical latitude of collection localities.Ncell is the mean number of cells in the septa between the air canals visible in the cross-section of the leaf blade.The mean was calculated using cell counts in at least 20 septa in each specimen.The latitude of the Kazakhstan specimen of A. americanus is arbitrary indicated as that of Pavlodar city.Data on countries other than Kazakhstan are taken from[9].See text for discussion on samples Golde s.n.(LE01249647) and Bobrov & Mochalova 694 (MW0163317).The dashed line shows that Ncell = 3 serves as a clear boundary between the ranges of variation recorded in the two fertile species.

Figure 4 .
Figure 4. Quantitative data on the variation of leaf aerenchyma in the fertile species Acorus americanus and A. verus as related to the geographical latitude of collection localities.N cell is the mean number of cells in the septa between the air canals visible in the cross-section of the leaf blade.The mean was calculated using cell counts in at least 20 septa in each specimen.The latitude of the Kazakhstan specimen of A. americanus is arbitrary indicated as that of Pavlodar city.Data on countries other than Kazakhstan are taken from[9].See text for discussion on samples Golde s.n.(LE01249647) and Bobrov & Mochalova 694 (MW0163317).The dashed line shows that N cell = 3 serves as a clear boundary between the ranges of variation recorded in the two fertile species.

Plants 2024 , 20 Figure 5 .
Figure 5. Quantitative analysis of the shape of lamina cross-section in the fertile species Acorus americanus and A. verus.Inset shows a diagram of leaf lamina in cross-section with a legend to the metrics A, B, and C. Data from localities other than in Kazakhstan are taken from[9].

Figure 5 .
Figure 5. Quantitative analysis of the shape of lamina cross-section in the fertile species Acorus americanus and A. verus.Inset shows a diagram of leaf lamina in cross-section with a legend to the metrics A, B, and C. Data from localities other than in Kazakhstan are taken from[9].

Longitude Specimen Herbarium Acronym and Barcode N cell Pollen: % Stained (Total Counted) Seeds 2C, pg nrITS Ribotype and GenBank Accession No. Acorus calamus s.str.
* All these URLs were accessed on 23 May 2024.
The work of GVD, EES, and MVR on micromorphology, pollen fertility and molecular barcoding was supported by the Ministry of Science and Higher Education of Russia (project 075-15-2021-1064).Flow cytometry and fieldwork in Kazakhstan were supported by the State Assignment of the Altai State University (project FZMW-2023-0008).The research of SSB was funded by the Ministry of Science and Higher Education of the Republic of Kazakhstan, the Scientific Program «Assessment of biological resources of the Kazakh part of the transboundary Irtysh basin in the context of climate change (BR18574062)».The research of IVK was supported by the State Order of the Tyumen State University (project FEWZ-2024-0007).The work of ANS received no external funding.High-quality images of the original material of Acorus are available through the data portal of the Herbarium of the Komarov Botanical Institute (https://en.herbariumle.ru/?t=occ, accessed on 17 June 2024) and the Herbarium of Moscow State Univeristy (https://plant.depo.msu.ru/open/public/search?collection=MW, accessed on 17 June 2024). Funding: