Charophytes (Charophyceae, Charales) of South Kazakhstan: Diversity, Distribution, and Tentative Red List

The presented research was conducted during 2019–2022 in south and southeast Kazakhstan to document the species richness, distribution, and ecology of charophytes (Characeae) as a first step towards to estimate the need for species protection. Across the 54 sites, we found ten species and one variety. Chara vulgaris Linnaeus and C. contraria A.Braun ex Kützing were the most common species, followed by C. canescens Loiseleur, C. kirghisorum C. F. Lessing, C. tomentosa Linnaeus, C. dominii J. Vilhelm, C. globata W. Migula, Nitellopsis obtusa (Desvaux) J. Groves, and Nitella hyalina (De Candolle) C. Agardh. The list of localities for each species was compiled. The distribution of each taxon was mapped in relations to the ecoregions studied. The two most frequent species were found in a wide spectrum of ecoregions, whereas all other species occurred in only a few regions in Kazakhstan. The Kaskelen River Valley had the most sampled sites with the highest number of co-occurring species (up to five together). Statistical maps were plotted in attempt to outline key environmental variables explaining the distribution of each species. A comparison of species and environmental variables distribution maps lets us assume that C. vulgaris prefers low altitude habitats with higher water temperatures, organic enrichments, and color, but low oxygen and pH. Other species prefer clear, alkaline, organically unpolluted, and well-oxygenated waters in lowland habitats. The redundancy detrended analysis (RDA) defined pH and altitude as negative factors for Nitellopsis obtusa whereas an increase in water temperature was positive. Altitude and water temperatures affected Chara contraria positively while altitude negatively influenced the rare species: Chara tomentosa, C. kirghisorum, and C. dominii. The matK sequences were obtained for C. contraria and C. vulgaris to confirm their identity according to morphological traits and to compare populations of C. gymnophylla and C. vulgaris from an arid region in Israel. Our data allowed for the preparation of a tentative red list from the study region. One species was recognized as endangered, four species and one variety as vulnerable, and two species as least concern. There was insufficient data to determine the status of two species and one variety.


Introduction
Charophytes are members of the large phylum Charophyta [1] with six classes. Our study investigates the diversity from the family Characeae in the order Charales. This group is monophyletic [2] and consists of highly developed benthic macroalgae. Charales Lake Balkhash drainage basin below the Kapchagay Reservoir. This was observed during 1975-1978 because the Ili River discharge regulations changed, impacting the reservoir [53].
There are no species included in the national Red Data Book from Kazakhstan (https: //www.inaturalist.org/projects/red-book-of-kazakhstan-plants, accessed on 20 May 2022). Therefore, we investigated the main threatened species during our study to identify charophyte diversity and species regional distribution. Since the two species of Characeae, C. vulgaris and C. contraria, are widespread and extremely similar in morphology, it was necessary to compare DNA sequences to determine speciation between the two species and subsequent comparison in the NCBI data.
In this investigation, we aimed to describe species of charophytes from the south and southeast Kazakhstan. We use ecomorphological and polyphasic approaches (i.e., genotypic, chemotaxonomic, and phenotypic methods) to determine the taxonomic position of organisms [54,55] and present the results on their diversity, ecology, relationship with the main environmental factors, and distribution as a first step towards their protection.

Charophytes Diversity and Distribution
Altogether, there were 10 species and one variety of charophytes identified from 54 investigated regional sites (Appendix B). Taxon distributions were mapped in accordance to the ecoregion they were found (Figure 1a,b). From Figure A1, charophyte thickets usually formed far from the shoreline, apparently as a result of a significant fluctuation of the water levels in arid climates. Some sites were polluted which were confirmed by high BOD (Appendix A Table A1) which inhibits the formation of large mats of charophytes.
Chara vulgaris (26 sites) and C. contraria (20 sites) were widely distributed in the regions (Figure 1e,g). These two species were found in two regions: b, Central Asian riparian woodlands, and f, Tian Shan montane steppe and meadows. At the same time, C. canescens, C. kirghisorum, C. tomentosa, C. dominii, C. globata, and Nitellopsis obtusa were concentrated in the central Asian riparian woodland ecoregion (Figure 1d,f,h). Nitella hyalina was found in two closely related ecoregions in southeast Kazakhstan: central Asian riparian woodlands and Emin Valley Steppe (Figure 1d). Only one species Chara aspera and its variety subinermis is widely distributed across the studied territory ( Figure 1c). Moreover, this variety was found separately from the variety only in the central Asian riparian woodland ecoregion. The site with the largest species richness was Kaskelen River pond 1, in which there were five species: Chara aspera, C. contraria, C. kirghisorum, C. vulgaris, and C. tomentosa. The Kaskelen River is not large and located on the flat landscape where it is dammed and divided into three water bodies in the river delta close to the Kapchagai Reservoir. These ponded areas of the river delta were rich in species, such as C. aspera, C. contraria, C. kirghisorum, C. tomentosa, C. dominii, C. globata, and C. vulgaris, with addition of C. vulgaris and C. contraria in the upper reaches of the Kaskelen River. Therefore, the Ili River basin was the richest area and had the largest diversity of charophytes from the studied regions.
Specimens collected, housed in the Institute of Botany and Phytointroduction in Almaty, are presented in Appendix B, along with morphological descriptions and distribution data regarding the studied areas and herbarium numbers.
Statistical maps were constructed for each identified species with known environmental variables (Appendix A Table A1 and Appendix A Table A2).   Species distribution maps were used to visualize and demonstrate the tendencies of each species in the study. Chara aspera and C. tomentosa preferred north and east regional environments (Figure 3a,b), whereas C. dominii and C. globata were found across the Ili River basin (Figure 3c,d). The tendency of two similar species was very interesting. C. vulgaris and C. contraria tended to differ in dissimilar environmental conditions and geographic areas. Species distribution maps were used to visualize and demonstrate the tendencies of each species in the study. Chara aspera and C. tomentosa preferred north and east regional environments (Figure 3a,b), whereas C. dominii and C. globata were found across the Ili River basin (Figure 3c,d). The tendency of two similar species was very interesting. C. vulgaris and C. contraria tended to differ in dissimilar environmental conditions and geographic areas. C. vulgaris occupied southern water habitats (Figure 3e) while C. contraria preferred more northern habitats (Figure 3f). Their distributions seemed not to be linked, as if mutually exclusive, even though both species sometimes occupy some habitats together. Statistical maps of other Chara species demonstrates a limited distribution for C. canescens and C. kirghisorum (Figure 4a,b). Nitella hyalina and Nitellopsis obtusa (Figure 4c,d) were found in a few occupied areas. Nitella hyalina has a unique distribution, whereas Nitellopsis obtusa's distribution was similar to that of C. dominii. To clarify the factors influencing the distribution of species in Kazakhstan, RDA analysis was carried out, as detailed in Appendix Table A2. Average species richness was 11 species in 34 sites that are dependent on biological variables, whereas environmental variables of water and air temperature, pH, index of organic pollution S, and site altitude were independent variables.
There are species that are highly influenced by pH, temperature, and altitude, while organic pollution (Index S) is not an important factor for regulating species distributions ( Figure 5). RDA shows that pH and altitude are negatively correlated to Nitellopsis obtusa's distribution, but higher water temperatures are positively correlated. Increasing site altitude and water temperatures were positive influences for Chara contraria. At the same time, the habitat altitude is a negative factor for the rare species Chara tomentosa, C. kirghisorum, and C. dominii's distributions. For the other species, the RDA does not indicate specific influencing factors.
Therefore, RDA helped to identify major environmental variables affecting each species, groups of species, and rare species' preferences. The RDA results confirm the visualized distribution of species and environmental variable map comparisons, as well as provide some characteristics concerning the preferred environments of the rare species.
From the available data on the ecology of 38 species [57], it is known that they prefer waters slightly saturated with organic matter. They are more represented by indicators To clarify the factors influencing the distribution of species in Kazakhstan, RDA analysis was carried out, as detailed in Appendix A Table A2. Average species richness was 11 species in 34 sites that are dependent on biological variables, whereas environmental variables of water and air temperature, pH, index of organic pollution S, and site altitude were independent variables.
There are species that are highly influenced by pH, temperature, and altitude, while organic pollution (Index S) is not an important factor for regulating species distributions ( Figure 5). RDA shows that pH and altitude are negatively correlated to Nitellopsis obtusa's distribution, but higher water temperatures are positively correlated. Increasing site altitude and water temperatures were positive influences for Chara contraria. At the same time, the habitat altitude is a negative factor for the rare species Chara tomentosa, C. kirghisorum, and C. dominii's distributions. For the other species, the RDA does not indicate specific influencing factors.
Therefore, RDA helped to identify major environmental variables affecting each species, groups of species, and rare species' preferences. The RDA results confirm the visualized distribution of species and environmental variable map comparisons, as well as provide some characteristics concerning the preferred environments of the rare species.
From the available data on the ecology of 38 species [57], it is known that they prefer waters slightly saturated with organic matter. They are more represented by indicators of oligosaprobic waters, where the saprobity index ranges from 0.8 to 1.3, and the trophic status has a wide amplitude from oligo-to eutrophic. Among the 11 species identified in Kazakhstan, only seven species are known to prefer organic pollution and trophic status. All preferred to exist in oligosaprobic communities with an index amplitude of 1.1-1.2 and a trophic status from oligo-to eutrophic waters. In our case, for the populations of the studied charophytes, the saprobity index varied in amplitude of 1.26-2.11, which shows adaptability to the more organically saturated waters in our study but from the same wide range of trophic status.

DNA Sequencing and Species Definition
DNA extractions taken from samples from the studied populations in Kazakhstan were compared to populations from Israel that had similar climates. DNA was isolated from two species (Chara vulgaris and C. contraria) from Kazakhstan and Israel. Thus, sequencing was carried out for the two most common species from climatically similar regions. In Table 1, environmental data for the DNA sequenced populations are presented for Chara species from two sites in south and southeast Kazakhstan and ten sites in Israel. For comparison with charophytes from Israel, we selected the C. vulgaris from seven habitats and the closely morphologically related C. gymnophylla A. Braun from three habitats, but C. contraria from this region has not yet been sequenced.

DNA Sequencing and Species Definition
DNA extractions taken from samples from the studied populations in Kazakhstan were compared to populations from Israel that had similar climates. DNA was isolated from two species (Chara vulgaris and C. contraria) from Kazakhstan and Israel. Thus, sequencing was carried out for the two most common species from climatically similar regions. In Table 1, environmental data for the DNA sequenced populations are presented for Chara species from two sites in south and southeast Kazakhstan and ten sites in Israel. For comparison with charophytes from Israel, we selected the C. vulgaris from seven habitats and the closely morphologically related C. gymnophylla A. Braun from three habitats, but C. contraria from this region has not yet been sequenced. Figure 6 demonstrates high similarity of matK sequences of Chara vulgaris samples from the Kakpatas River (yellow) with other populations of C. vulgaris from NCBI. It confirms the identity of C. vulgaris according to morphological traits only from different habitats, which includes NCBI data in the tree that is very closely related to our data.  Figure 6 demonstrates high similarity of matK sequences of Chara vulgaris samples from the Kakpatas River (yellow) with other populations of C. vulgaris from NCBI. It confirms the identity of C. vulgaris according to morphological traits only from different habitats, which includes NCBI data in the tree that is very closely related to our data.    Figure 7 reflects a high similarity of matK sequences for Chara contraria samples from the Anniversary Lake (yellow) and populations from other regions according to NCBI data. These data confirm our identification of C. contraria based on morphological traits and sequencing data.
The phylogenetic tree presents a high similarity of matK sequences for Chara contraria, C. vulgaris, and C. gymnophylla samples from the south and southeast Kazakhstan and Israel sites in Figure 8. There are a few clusters with high similarity of sequences shown in Table 1. Most similar were populations of C. vulgaris from Mediterranean coastal zone with high salinity and pH (Table 1). C. vulgaris populations from Oren can be included in cluster 1 too. C. gymnophylla was a slightly different form from northern Israel (Dafna) that is in cluster 2. The phylogenetic tree presents a high similarity of matK sequences for Chara contraria, C. vulgaris, and C. gymnophylla samples from the south and southeast Kazakhstan and Israel sites in Figure 8. There are a few clusters with high similarity of sequences shown in Table 1. Most similar were populations of C. vulgaris from Mediterranean coastal zone with high salinity and pH (Table 1). C. vulgaris populations from Oren can be included in cluster 1 too. C. gymnophylla was a slightly different form from northern Israel (Dafna) that is in cluster 2. Cluster 3 combined C. vulgaris populations from Carmel Mountain Biosphere Reserve and Ein Afeq Natural Reserve, Israel and C. gymnophylla from the Upper Jordan River valley habitat Ein Tao. High mountain habitat Nevoria in northern Israel included C. gymnophylla in cluster 4. C. vulgaris from Kakpatas River in Kazakhstan and Ein El Verde from the mountainous region of northern Israel was grouped into cluster 5. We analyzed the environmental variables and their importance to regulate the species into clusters but only found similar parameters for cluster 1. C. vulgaris species was found from coastal zone sites of Israel where salinity was highest ( Table 1). All other sites differed in salinity, pH, nitrates, and altitude and did not correlate with the distribution of specific species of Chara. Previously, we analyzed the molecular differentiation between the C. vulgaris-C. contraria complex and C. gymnophylla using the AFLP method in the habitats of Israel and found not only distinct species differentiation [58], but also that C. gymnophylla is more sensitive to arid environments, since it only occurs in northern Israel [40]. This allows us to assume that C. vulgaris and C. contraria demonstrated a tolerance for arid habitat conditions.

The Tentative Red List for Charophytes of South and Southeastern Kazakhstan
The thorough application of all IUCN Red List criteria is impossible due to a la essential data. From the available records, some species distributional, biological, and logical traits were used to suggestion a tentative Red List for the species observed below).
Estimation of trends in abundance and occurrence: not possible due to an abs of details from previous records and a recent survey of formerly known sites. A dr decline of species occurrence in Lake Balkhash drainage basin below the Kapchagay ervoir was found during 1975-1978 because of Ili River discharge regulation into this ervoir [53].
Other evidence for evaluation of Red List Category: shallow water species ab withstand moderate eutrophication in brackish waters of subarid regions (R. Roma pers. observation).

The Tentative Red List for Charophytes of South and Southeastern Kazakhstan
The thorough application of all IUCN Red List criteria is impossible due to a lack of essential data. From the available records, some species distributional, biological, and ecological traits were used to suggestion a tentative Red List for the species observed (see below).
Estimation of trends in abundance and occurrence: not possible due to an absence of details from previous records and a recent survey of formerly known sites. A drastic decline of species occurrence in Lake Balkhash drainage basin below the Kapchagay Reservoir was found during 1975-1978 because of Ili River discharge regulation into this reservoir [53].
Other evidence for evaluation of Red List Category: shallow water species able to withstand moderate eutrophication in brackish waters of subarid regions (R. Romanov, pers. observation).
Estimation of trends in abundance and occurrence: not possible due to an absence of details from previous records and recent survey of formerly known sites.
Other evidence for evaluation of Red List Category: shallow water species able to withstand moderate eutrophication in brackish waters of subarid regions (R. Romanov, pers. observation). C. aspera var. subinermis could be a common species in bays of the Lake Balkhash and neighboring water bodies. Actual scale of its distribution and threats in the regions studied cannot be estimated due to a lack of data.
Estimation of trends in abundance and occurrence: not possible because an absence of details from previous records and recent survey of formerly known sites. C. canescens could be a common species in bays of the Lake Balkhash and neighboring water bodies with higher salinity. Actual scale of its distribution and threats in the regions studied cannot be estimated because of small amount of data.
Other evidence for evaluation of Red List Category: unknown. General distribution: Holarctic, non-native in Australia. IUCN Red List Category: DD.
Estimation of trends in abundance and occurrence: not possible due to an absence of details from previous records and recent survey of formerly known sites.
Other evidence for evaluation of Red List Category: This is one of the most common, generalist species in many temperate regions [42].

Chara dominii
Estimation of trends in abundance and occurrence: not possible due to an absence of details from previous records and recent survey of formerly known sites except the Kaskelen River where its populations are stable. C. dominii could be a common species in the bays of Lake Balkshash, Kapchagay Reservoir, and neighboring water bodies.
Other evidence for evaluation of Red List Category: The species had a scattered distribution in arid and semiarid regions of Eurasia [63] with most localities known from stable lakes. C. dominii seems to be able to form perennial stable stands in stable environments. This species is unable to grow in shallow waters (less than 0.5 m) and coarse substrates, which could explain its susceptibility to decrease water transparence as a consequence of eutrophication.
IUCN Red List Category: VU.
Estimation of trends in abundance and occurrence: not possible due to an absence of details from previous records and recent survey of formerly known sites except the Kapchagay Reservoir where recent populations were found. C. globata could be a common species in bays of the Lake Balkhash, the Kapchagay Reservoir, and neighboring water bodies.
Other evidence for evaluation of Red List Category: The species has a scattered distribution in arid and semiarid regions of Eurasia and North Africa with most localities known from stable lakes [20,64]. C. globata seems to be able to form perennial stable stands in stable environment. Some localities in Middle East are lost [64]. This species is unable to grow in shallow waters (less than 0.5 m) and coarse substrates, which could explain its susceptibility to decrease water transparence as a consequence of eutrophication.
IUCN Red List Category: VU.
Estimation of trends in abundance and occurrence: not possible due to an absence of details from previous records and recent survey of formerly known sites.
Other evidence for evaluation of Red List Category: Globally, a rare species with limited distribution and few localities known.
General distribution: Central Eurasia: Russia, Kazakhstan, Uzbekistan, Iran, few localities in each region.
IUCN Red List Category: EN.
Estimation of trends in abundance and occurrence: not possible due to an absence of details from previous records and recent survey of formerly known sites.
Other evidence for evaluation of Red List Category: This species seems to be able to form perennial stable stands in stable environment and in arid regions. C. tomentosa could be a common species in bays from Lake Balkshash, Kapchagay Reservoir, and neighboring water bodies. Actual scale of its distribution and threats in the regions cannot be estimated.
General distribution: Palearctic. IUCN Red List Category: DD.
Estimation of trends in abundance and occurrence: not possible due to an absence of details from previous records and recent survey of formerly known sites.
Other evidence for evaluation of Red List Category: The most common species in Central Asia is able to grow in a wide spectrum of habitats including newly created and maintained for millennia for irrigation in the region studied [63]. This is one of the common, generalist species from many temperate regions [42].
Estimation of trends in abundance and occurrence: not possible due to an absence of details from previous records and recent survey of formerly known sites. Stable presence seems to be confirmed for Ili River delta and the Alakol Lake.
Other evidence for evaluation of Red List Category: All species of Nitella are really rare in Central Asia [63].
General distribution: cosmopolite, but really rare in many regions. IUCN Red List Category: VU.
Estimation of trends in abundance and occurrence: not possible due to an absence of details from previous records and recent survey of formerly known sites. It was found from 1975-1978, but drastically declined in occurrence in the Lake Balkhash drainage basin below the Kapchagay Reservoir when the Ili River discharge regulation into this reservoir changed [53]. N. obtusa could be a common species in the Ili River delta lakes, neighboring bays of Lake Balkshash and the Kapchagay Reservoir.
Other evidence for evaluation of Red List Category: This species seems to be able to form perennial stable stands in stable environments and in arid regions. N. obtusa is unable to grow in shallow waters (less than 0.5 m) and coarse substrates, which could explain its susceptibility to decrease water transparence as a consequence of eutrophication [31,32].
General distribution: Palearctic, non-native in North America IUCN Red List Category: VU.

Description of Study Site
Charophyte algae samples were collected during June-October of 2019-2022 from rivers, canals, ponds, and lakes from 3 regions (Turkestan I, Zhambyl II, and Almaty III administrative regions) (Figure 9). The surveyed localities are situated between 41.00 to 46.40 N and 68.12 to 81.45 E, at an elevation of 245-3629 m above sea level (a.s.l.) (Appendix A Table A1). The climate varied across the study areas [67]. Going from the southwest to the northeast direction, the average annual temperature decreased (13.2 °C in the Turkestan Region to 11.2 °C in the Zhambyl Region [68] and then to 8.6 °C in the Almaty Region) [69], while annual precipitation increased from 502.4 mm to 511.83 mm.
The regions studied include the two largest basins: the Aral-Syrdarya Basin and the Balkhash-Alakol Basin [70]. The Syrdarya and Chu rivers flow into the Aral-Syrdarya Basin. The Syrdarya River belongs in the Turkestan Region, sites 1-4 (Appendix Table A1). The Chu River is part of the Zhambyl Region, sites 5, 6, 8-12. Lake Mynaral, site 7, is in the south part of Zhambyl Region. The Balkhash-Alakol FEOW (Freshwater Ecoregions of the World) Basin includes the whole Ili River catchment basin. It belongs to the Almaty Region III. The studied sites are divided into three different parts of the Ili River catchment basin: 1. The first region before the Kapchagai Reservoir includes sites 42-51 and belongs to the mountainous area of the territory. 2. The second region contains sites studied from Ili River basin, including sites from the rivers Talgar (sites [35][36][37][38][39][40] and Kaskelen (sites [25][26][27][28]30) as well as the Kapchagai Reservoir (sites 31-34). 3. The third region contains sites from the Ili River that start at site 29 after Kapchagai Reservoir dam and continue in the Lake Balkhash direction with sites in Arystan (sites 13-18), Zhidely (sites [19][20][21][22], and Bakanas (site 23) canals. There are two endorheic Lake Sorbulak (site 24) and Kurti River (site 41) belonging to the Almaty Region III. Lake Alakol (sites 52, 53, and 54) is in the eastern Almaty Region III and within a paleo-basin of Lake Balkhash, but currently there is not a connection between them.
Thus, we found that the studied habitats in the southern part of Kazakhstan are climatically like the semi-arid area of the eastern Mediterranean, and therefore our data on the environment and diversity of charophytes can be used to compare key species using methods applied for both regions. The climate varied across the study areas [67]. Going from the southwest to the northeast direction, the average annual temperature decreased (13.2 • C in the Turkestan Region to 11.2 • C in the Zhambyl Region [68] and then to 8.6 • C in the Almaty Region) [69], while annual precipitation increased from 502.4 mm to 511.83 mm.
The regions studied include the two largest basins: the Aral-Syrdarya Basin and the Balkhash-Alakol Basin [70]. The Syrdarya and Chu rivers flow into the Aral-Syrdarya Basin. The Syrdarya River belongs in the Turkestan Region, sites 1-4 (Appendix A Table A1). The Chu River is part of the Zhambyl Region, sites 5, 6, 8-12. Lake Mynaral, site 7, is in the south part of Zhambyl Region. The Balkhash-Alakol FEOW (Freshwater Ecoregions of the World) Basin includes the whole Ili River catchment basin. It belongs to the Almaty Region III. The studied sites are divided into three different parts of the Ili River catchment basin: 1. The first region before the Kapchagai Reservoir includes sites 42-51 and belongs to the mountainous area of the territory. 2. The second region contains sites studied from Ili River basin, including sites from the rivers Talgar (sites [35][36][37][38][39][40] and Kaskelen (sites 25-28, 30) as well as the Kapchagai Reservoir (sites 31-34). 3. The third region contains sites from the Ili River that start at site 29 after Kapchagai Reservoir dam and continue in the Lake Balkhash direction with sites in Arystan (sites 13-18), Zhidely (sites [19][20][21][22], and Bakanas (site 23) canals. There are two endorheic Lake Sorbulak (site 24) and Kurti River (site 41) belonging to the Almaty Region III. Lake Alakol (sites 52, 53, and 54) is in the eastern Almaty Region III and within a paleo-basin of Lake Balkhash, but currently there is not a connection between them.
Thus, we found that the studied habitats in the southern part of Kazakhstan are climatically like the semi-arid area of the eastern Mediterranean, and therefore our data on the environment and diversity of charophytes can be used to compare key species using methods applied for both regions.

Sampling and Laboratory Study
Temperature and pH were measured at the same time of sampling with a Waterproof Portable pH/Temperature meter HI991001 (HANNA instruments, USA) at Kazakhstan sites, and while in Israel conductivity and total dissolved solids (TDS) were measured with a HANNA HI 9813-0, and N-NO 3 with a HANNA HI 93728 (HANNA Instruments, USA) with three repetitions. GPS coordinates for the sampling sites were obtained with a GARNMIN GISMAP 64. The air temperature was measured with a standard thermometer.
Dissolved oxygen, biological oxygen demand (BOD), and water color (Pt/Co scale) data were taken from the reference [8], and the monthly data from the Ministry of Ecology, Geology, and Natural Resources website of the Republic of Kazakhstan Department of Environmental Monitoring RSE "Kazhydromet". The environmental data were defined according to [71]. The data from the documents cited in [8] were taken during the same month and year that charophytes were sampled.
Charophytes were collected in situ. Usually, charophyte mats were visible at a depth of 0-0.5 m. Each point where visible aggregations of charophytes were found was designated as a sampling point and GPS coordinates were recorded. Thus, several sampling points were established in some water bodies. Samples of charophytes were collected in a tenmeter radius from each sampling point by scrapping with anchor tugging and pulling by hands at a depth of 0-0.5 m in 5-10 samples. Samples were dried, transported to the laboratory, and labelled for permanent deposition in the Herbarium at the Institute of Botany and Phytointroduction (Kazakhstan) (label # AA 1-1 to AA 54-1). Samples were studied at the Institute of Botany and Phytointroduction, the Institute of Evolution, University of Haifa (Israel); and the Arkansas State University Beebe, Beebe in Arkansas (USA). The MBS-9 stereomicroscope (SCOPICA, Russian Federation), MicroOptix light microscope), and Leica DM2500 light microscope were used for species identification. The dimensions were taken with a microscopic eyepiece micrometer at 400-1000× magnification. The specimens were photographed with a modern Motic BA-400 microscope (Motic Asia, Hong Kong, China) and OMAX 9.0 MP USB Digital Camera. A thick layer of calcium carbonate did hamper a few of the morphological investigations by covering the plant. These specimens were treated with 4% acetic acid to dissolve the CaCO 3 .
The most relevant taxonomic reference books were used for identification of taxa [72][73][74][75]. Taxon names were checked according to the Algaebase.org website [1] for synonyms and updated.
The Ecoregions mapping program was used to create individual species distributions [56]. Statistica 12.0 was used to create maps that reflect the probability of mapped variable distribution over the lake surface according to parameter values, geospatial coordinates, and the environmental variables, which were partly measured by us and partly from reference [8] for each site [57].
Additionally, the saprobic index S was used, which describes the organic matter pollution and ecosystem state. Saprobity indices were obtained for each algal community as a function of the number of saprobic species and their relative abundances as described earlier [8]: where S is index of saprobity for algal community (unitless) according to Sládeček [76], s is species-specific saprobity index, and n is the cell density of each species (Appendix A Table A2).
The linear ordination method redundancy detrended analysis (RDA) was processed in CANOCO 4.5 program to determine the importance of main environmental factors to the species [77]. The analysis of environmental data was performed only for those 34 sites where complete environmental data were available. Species ecological preferences of studied charophytes were taken from reference [78].
The conservation status and rarity of species was assessed according to the IUCN (The International Union for Conservation of Nature) criteria [79] using the following scheme ( Figure 10 Species were assessed using five criteria [79] according to species distr population size, and population change, in combination with extinction sessment. These criteria determined which category was most significant fo

DNA Barcoding
The purpose of the polyphasic approach [54,55] was to determine th complete genomic DNA was extracted from dry material taken from her mens using the standard DNeasy Plant Mini Kit (Geneaid Biotech Ltd., Ne wan) according to the manufacturer's instructions. Species sequences from stan populations were compared to populations in Israel that are from sim This specimen choice was determined by the availability of samples and in which the study was conducted.
Sequencing was performed using a 3730 DNA Analyzer (Applied Bios quarters, Thermo Fisher Scientific, Waltham, MA USA) with identical seque to those used for PCR reactions.

Phylogenetic Analysis
Sequences were analyzed and aligned using the BioEdit sequence ali (version 7.2). The resulting sequences were corrected manually. For phyloge we used the matK kit containing 8 Chara vulgaris specimens (7 specimens f 1 specimen from Kazakhstan); 4 specimens of C. gymnophylla (A.Braun) A. rael and 1 specimen of C. contraria from Kazakhstan. The datasets were a maximum likelihood (ML), maximum parsimony (MP), and distance (nei tion (NJ)) in MAFFT (version 7). Using the BLAST program, areas of simi the obtained nucleotide sequences were found and compared with the N Figure 10. Scheme of conservation status of species according to the IUCN criteria [79].
Species were assessed using five criteria [79] according to species distribution range, population size, and population change, in combination with extinction probability assessment. These criteria determined which category was most significant for each species.

DNA Barcoding
The purpose of the polyphasic approach [54,55] was to determine the species. The complete genomic DNA was extracted from dry material taken from herbarium specimens using the standard DNeasy Plant Mini Kit (Geneaid Biotech Ltd., New Taipei, Taiwan) according to the manufacturer's instructions. Species sequences from the Kazakhstan populations were compared to populations in Israel that are from similar climates. This specimen choice was determined by the availability of samples and the laboratory, in which the study was conducted.
Amplification of the matK gene region was performed using 2× Taq Mix Red PCR MasterMix with advanced hot-start technology (PCR Biosystems Ltd., London, UK) using F-Chara (AGAATGAGCTTAAACAAGGAT) and R-Chara (ACGATTTGAACATCCAC-TATAATA) primers. For each PCR product, both strands were sequenced on an Applied Biosystems VeritiTM Thermal Cycler Genetic Analyzer (Applied Biosystems, CA, USA). PCR was performed with an initial two-minute denaturation step at 95 • C and one minute each for denaturation (95 • C), annealing (56 • C), and polymerization (72 • C) for 10 cycles, followed by one-minute denaturation (95 • C), annealing (52 • C), and polymerization (72 • C) for 25 cycles before the last elongation step (10 min). PCR products were visualized by 1.5% agarose gel electrophoresis with GelRed staining (GelRed ® Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA)) and UV illumination. PCR products were purified using the Wizard ®SV Gel and PCR Clean-up Systems (Promega, Promega Corporation, Madison, WA USA) kit.
Sequencing was performed using a 3730 DNA Analyzer (Applied Biosystems, Headquarters, Thermo Fisher Scientific, Waltham, MA, USA) with identical sequencing primers to those used for PCR reactions.

Phylogenetic Analysis
Sequences were analyzed and aligned using the BioEdit sequence alignment editor (version 7.2). The resulting sequences were corrected manually. For phylogenetic analysis, we used the matK kit containing 8 Chara vulgaris specimens (7 specimens from Israel and 1 specimen from Kazakhstan); 4 specimens of C. gymnophylla (A.Braun) A.Braun from Israel and 1 specimen of C. contraria from Kazakhstan. The datasets were analyzed using maximum likelihood (ML), maximum parsimony (MP), and distance (neighbor connection (NJ)) in MAFFT (version 7). Using the BLAST program, areas of similarity between the obtained nucleotide sequences were found and compared with the NCBI (National Center for Biological Information) sequence databases, and statistical significance was calculated. The UPGMA (unweighted pair group method with arithmetic) clustering method was used for phylogenetic tree construction.
Data concerning morphology, matK sequences, and environmental variables from a climatically similar region of Israel were used to confirm the identity Chara contraria, C. vulgaris, and C. gymnophylla.

Discussion
Ten charophyte species and one variety were confirmed from the regions studied. Some other species, namely Chara aculeolata Kütz. in Rchb. ( [7,10,[13][14][15][16][17][18][19][20]23,53] but not identified during our study. Most of their records are known from the delta of the Ili River without any environmental or location details in many cases [17,53]. The charophyte abundance across the study region was not totally confirmed during our survey because previous known sites were not rechecked. At least four of them, C. globularis, C. gymnophylla, C. papillosa (as C. aculeolata sensu Hollerbach et Krassavina), and N. tenuissima, were found in previous studies by the authors [8,61,65]. Lychnothamnus barbatus was confirmed with the specimen studied [20]. In addition, C. globata was found in the Kapchagay Reservoir [19]. Some published species records can be misidentified [8,23] (cf. Jumakhanova et al., 2021 [23] and this work). The presence of C. aculeolata, C. baltica, C. fragifera, C. galioides, C. hispida, C. schaffneri, C. strigosa, and N. confervacea is questionable from the perspective of species distribution and ecology [7,[80][81][82], and Romanov (personal communication) needs confirmation. Therefore, actual species richness before our studies could be greatly overestimated because of some misidentifications. Nevertheless, the negative trends in species richness and distribution of charophytes were noted before our studies [8,53].
A pattern was observed in that surveyed sites were inhabited by more than one species of Chara and only a few sites in Israel had two and more species in the same site. In our previous study, Chara vulgaris and C. contraria were shown to be distinguishable by their genome structure, whereas their morphological identification is hindered by the similarity and uncertainty of most morphological features [83]. The revealed relationship between the genome sequence and the habitat characteristics in semi-arid regions in the Israel is an example which suggests that adaptive genetic divergence in charophytes is associated with the intensity of sunlight, water level, and pH, and consequently with climatic differentiation and local environmental stresses. This plays a critical role in shaping modern charophyte diversity. Maps in Figure 3e,f demonstrate strictly the differences of both species' distribution where Chara vulgaris preferred southern localities but C. contraria is distributed in northern sites, confirmed by RDA. As known, charophytes are usually closed to new migration, especially when an existing population becomes established in one habitat and invasive oospores compete with natives [84]. Previously, we hypothesized that site ecology is the main limiting factor for oospore establishment in a new site, meaning that gene flow is more likely to occur in sites with similar environments [83]. The discussion about the ecological preferences of these two morphologically similar species usually did not include the impact of UV radiation as a regulatory factor, but this was suggested with our observations in Israel [85]. This gives more evidence and emphasizes the importance of studying the distribution of charophytes in Kazakhstan, to have a reliable identification of both species, which is possible only with the involvement of molecular methods. In Figure 3e,f, C. vulgaris prefers southern sites while C. contraria inhabited a more northernly, less insolated sites. Therefore, the differences in distribution for these critical species are defined and confirmed by its genome sequencing and confirms the importance of species ecology. This lets us assume that the conditions for the growth of charophytes in Kazakhstan might be more favorable than for charophytes in Israel, where insolation and hydrology could regulate diversity in water bodies more strictly [83]. The cohabitation of several species of charophytes indicates a long-term non-disturbance of habitat and the fact that at least two species find it suitable, i.e., they can be excluded from the IUCN Threatened List [79]. In those cases, where only one species was found, its habitat could be severely impacted. Regarding the management of water bodies to prevent eutrophication to maintain stable charophyte stands, excessive water abstraction, salinization, and alteration of the hydrological regime as a consequence of river discharge regulation seem to be the most important threats to charophytes in the region studied. Further studies need to be conducted to evaluate the present and future stability of the ecoregions.
As a result of this study, it was possible to identify 10 species and one variety of charophyte in the studied region. As 54 sites were examined, 30 of these charophytes were found for the first time. The study of their ecology and distribution made it possible to characterize the identified species according to IUCN categories as one endangered species (C. kirghisorum) from a pond on the Kaskelen River, five species of the vulnerable category, and for the rest there were not enough data to determine the category.

Conflicts of Interest:
The authors declare no conflict of interest.
Appendix A Table A1. Investigated sites coordinates and known environmental variables [8] of aquatic habitats in south and southeast Kazakhstan, 2019-2022. "*", first investigated sites; "-", no data. The environmental data is presented only for those 34 sites where complete environmental data were available.

Site
Name

Appendix B
Descriptions of the species based on their morphology, with distribution data in the studied areas and herbarium numbers of the Institute of Botany and Phytointroduction in Almaty.

Appendix B
Descriptions of the species based on their morphology, with distribution data in the studied areas and herbarium numbers of the Institute of Botany and Phytointroduction in Almaty.
Habitat: both fresh and brackish waters; 4 sites; lakes, large ponds, canals, and rivers.

Chara canescens Loiseleur
Description: The plants are dioecious (only female ones) with haplostichous stem cortex, appearing spiny because of clustered well developed acute spine cells, acuminate stipulodes in 2 well developed rows, verticillate bract cells (Figure A2c,d).