The Charophytes (Charophyceae, Characeae) from Dagestan Aquatic Habitats, North Caucasus: Biogeographical and Barcoding Perspectives

: The charophytes in many regions of the world are still poorly understood. This hampers the synthesis of distributional and ecological datasets at worldwide and continental scales, as well as complicates the generalization of species concepts for widely distributed and local taxa. To fill in the blanks for charophytes in the Caucasus and to improve our knowledge of species distribution areas in Eurasia, a field survey and study of available specimens from Dagestan (North Caucasus, Russia) was conducted based on morphological observation using light and scanning electron microscopy and molecular genetic analyses allowing for the precise identification and testing of the presence of cryptic and undescribed taxa. Nineteen new localities for seven Chara species and one Tolypella species, seven new species, and one new genus were identified in the studied region, and one new species in the Caspian Sea region was found. Some species records changed the outline or filled in the gaps in species distribution data. The presence of species distributed mainly in central Eurasia ( C. globata , C. neglecta ) with mainly Mediterranean–Middle Eastern species ( C. gymnophylla ) is notable for this region, as well as for other studied regions of the Caucasus characterized by a mixture combination of species with different distribution patterns. Chara gymnophylla was frequent in Dagestan, similar to the Mediterranean and Middle Eastern regions. Small brackish waterbodies on the coast of the Caspian Sea, freshwater mountain rivers, small associated waterbodies, and water reservoirs are the main habitats of charophytes in the studied region. Based on habitat preference and distribution in the Caucasus, recommendations for the protection of some species were suggested. The lack of endemic species among charophytes from Dagestan and Caucasus contrasts with the flora of terrestrial magnoliophytes that is rich in species endemism.


Introduction
The charophytes comprise a distinct group of macroscopic algae widely known mainly as ecosystem engineers and pioneer species [1].Their life strategies and ecology are diverse [2][3][4] and still poorly studied for many species, especially outside Europe and Australia.Regional surveys of charophytes are an essential step toward better understanding their distribution and ecology.They allow further testing and improvement of species concepts and clarification of the bioindication potential of charophytes.The DNA barcoding of widely and narrowly distributed species fits the same targets, allowing testing of the presence of cryptic and undescribed taxa.Lesser known and remote areas are especially important from this perspective.Some species of charophytes are typical for the protected freshwater habitat types "Hard oligo-mesotrophic waters with benthic stoneworts Chara spp." of the Habitat Directive from the European Union and "C1.2a.Permanent oligotrophic to mesotrophic waterbody with Characeae", a vulnerable one according to the European Red List of Habitats [5].The charophytes are one of the most threatened groups of plants in Europe [6], easily illustrated by their proportionally high representation among endangered species on European and national Red Lists [7,8].The key areas important for the protection of vulnerable and endangered species were outlined in only a few European countries [9,10].
The current approach in taxonomical studies of charophytes is detailed morphological studies using light and scanning electron microscopy [11][12][13][14][15][16][17][18][19][20], usually in combination with phylogenetic analyses .Different regions of Eurasia were the focus of floristic research on charophytes in the last decade [10,, covering distribution, ecology, and protection, but only Europe has contemporary comprehensive flora mostly based on integrative taxonomy for its whole territory [77].
Knowledge about the charophytes of Dagestan, a region east of the North Caucasus, is limited and almost unknown worldwide.All available data are basically limited to three old specimens of Chara vulgaris L. from rivers Akusha and Yaloma and small water bodies near the village of Kajagent collected by Th.Alexeenko and reported in two articles covering large areas of Northern and Central Eurasia [78,79], later summarised by Hollerbach [80].A few recent records, mainly at the genus level (Chara L.), are available at iNaturalist.org[81].A similar situation could be noted for the entire North Caucasus, based on a longer list of references with only a few records in each of them (see note for Table 1

below).
As a first step towards better understanding this group of freshwater macrophytes, a keystone in some ecosystems and notable for their ecological role, we present here the main results of some recent field and herbarium investigations.In previous studies of Caucasian charophytes, species identification was carried out using only a morphological approach, which did not allow precise species identification in all cases.The aim of this study was to investigate the species composition of the charophytes of Dagestan using a polyphasic approach, which included morphological observations and molecular genetic analyses of the DNA of the studied species according to contemporary charophyte research standards.

Morphological Identification
The specimens were usually collected by hand during a careful survey of water bodies.They were dried as herbarium specimens and stored in LE (acronyms according to [82]).Some specimens collected in the 19th and 20th centuries were found in LE.Almost all of them had no identification before this study.Our efforts to search charophyte specimens from Dagestan stored elsewhere yielded no results.More than 30 pressed specimens were examined in the present study.A few records of charophytes were available at iNaturalist.org[81], but only two can be identified at the species level (Chara globata Migula).
The morphological features of the specimens were studied using an Olympus SZ61 stereomicroscope (Olympus Corporation, Shinjuku, Tokyo, Japan).Photographs of diagnostic traits were taken using a digital camera.Oospores taken from some recent specimens for scanning electron microscopy (SEM) were treated according to a previously described method [26].The cleaned oospores were coated with gold and studied using a Jeol JSM 6390LA scanning electron microscope (JEOL Ltd., Tokyo, Japan).Taxonomy followed the most recent reference [77].The Ecoregions' mapping program was used to map the individual charophyte species distribution in the studied territory [83].The BioDiversity Pro 2.0 program was used for the similarity calculation [84].

DNA Extraction, Amplification, and Sequencing
Total genomic DNA was extracted as described previously by Echt et al. [85] with some modifications [86].Part of the rbcL gene was amplified as described previously [37].The PCR products were purified with ExoSAP-IT PCR Product Cleanup Reagent (Affymetrix Inc., Santa Clara, CA, USA) and sequenced in both directions using an ABI 3500 genetic analyzer (Applied Biosystems, Waltham, MA, USA) with a BigDye terminator v. 3.1 sequencing kit (Applied Biosystems, Waltham, MA, USA).Sequences were assembled using the Staden Package v.1.4[87] and aligned manually in the SeaView program [88].Before the phylogenetic analyses, the sequences of the rbcL gene were compared with those available at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) using a BLAST search [89] to estimate their taxonomic position.

Phylogenetic Analyses
The rbcL dataset was used to access the affinity of our Chara species with the genus representatives retrieved from the NCBI.This dataset was assembled as described by Romanov et al. [37,38].Maximum likelihood (ML) analyses were carried out using PAUP 4.0b10 [90].Bayesian inference (BI) was performed using MrBayes 3.1.2[91].To determine the most appropriate DNA substitution models for our datasets, we used the Akaike information criterion (AIC; [92]), which was applied using jModelTest 2.1.1 [93].The GTR+I+G model was selected as the best fit for the rbcL dataset.ML analyses were carried out using heuristic searches with a branch-swapping algorithm (tree bisection-reconnection).Using BI, four parallel MCMC runs were carried out for 3 million generations.Sampling was carried out every 100 generations for a total of 30,000 samples.The convergence of these two chains was assessed, and stationarity was determined according to the 'sump' plot (the first 25% of samples were discarded as 'burn-in').The posterior probabilities were calculated from the trees sampled during the stationary phase.The robustness of the trees was estimated based on bootstrap percentages (BP; [94]) in ML and posterior probabilities (PP) in BI.A BP < 50% and PP < 0.95 were not considered.An ML-based bootstrap analysis was inferred using the web service RAxML version 7.7.1 (http://embnet.vitalit.ch/raxml-bb/;[95]; accessed on 1 March 2023).

Charophyte Diversity and Distribution
Eight species of charophytes, including seven species of Chara and one species of Tolypella (A.Braun) A.Braun, were found at 24 localities in Dagestan according to all available data (see below).The presence of a few taxa known before from this territory, i.e., C. vulgaris var.vulgaris and var.longibracteata (Kütz.)Kütz.[78,79] was confirmed with the studied specimens.Twenty two localities of charophytes were found for the region, and all of them were based on vouchers available for study.
Habitat: small lowland water bodies, bays, and shallows of mountain water reservoirs, the mouth of lowland rivers.
A few other records of Chara are known from Dagestan [81], but they cannot be identified at the species level according to available photos.

Species Identification and Phylogenetic Analysis
The identification of some Chara samples was based on a combination of morphological and molecular genetic approaches.DNA was isolated from most species, but our efforts were successful for only eight samples (five species: C. connivens, lobateata, C. gymnophylla, C. neglecta, C. vulgaris var.longibracteata).The topologies of the ML and BI trees based on the rbcL dataset were similar to that of the BI tree, except for some differences in clade support (Figure 6).Samples of C. connivens, C. vulgaris var.longibracteata, and lobateata were placed within species clades (60/0.96,-/-and 65/-, respectively), being part of the species haplotypes (Supplementary Materials).Samples of C. neglecta fell in shared haplotype with sequences of C. galioides DC. and C. aspera Willd.Three samples of C. gymnophylla represented a single haplotype that differed from the nearest (C.gymnophylla MN793052) by one substitution.

Discussion
Eight species of charophytes from two genera, Chara and Tolypella, are reliably known from Dagestan.Seven species are reported here for the first time in the area studied.The investigation of the biodiversity of charophytes of Dagestan using a polyphasic approach allowed us to conduct precise taxa identification of Chara species.Morphological traits of oospores studied with SEM were considered.They are in good agreement with species variability [40,52,77,96].The images of gyrogonites and the surface of the oospore of C. neglecta were taken for the first time.
The overall topology of our rbcL tree (Figure 6) was similar to that presented in the previous studies of the genus Chara [37,38,41,97,98].We somewhat extended taxon sampling in the genus by adding sequences for C. neglecta.Neither cryptic nor new species were found in Dagestan, and all Chara accessions were resolved within already-known haplotypes.
Figure 6.Maximum likelihood phylogenetic tree inferred in PAUP with the GTR+I+G nucleotide substitution model from 92 rbcL sequences of Chara.ML BP (>50%) and BI PP (>0.95).Branches received 100% BP and 1.00 PP support, and the newly obtained sequences are shown in bold.Sequences carrying one genotype are marked with grey.Chara sections and subsections are based on [99] with changes from [15,38].
The subsection Hartmania R.D. Wood of the genus Chara is widely known as "crux et scandalum botanicorum."In other words, it represents a group of species with still debatable and uncertain boundaries [52,77].The molecular markers applied so far failed to be helpful in this case [27,31,97,98,100] except for C. globata, a species delineable with rbcL sequences [26,35], in this study.
Some further perspectives could be outlined based on the phylogenetic data presented here.It was found that C. neglecta is undistinguishable from C. galioides and C. aspera despite well-pronounced differences between these species in stem cortex arrangement (e.g., weakly tylacanthous to isostichous triplostichous in C. galioides and weakly aulacanthous diplo-triplostichous to isostichous triplostichous in C. neglecta [47,77]).A detailed study on C. neglecta phenotypic plasticity is required and will be conducted by the authors in the future with more populations for evaluation of its delineation or merging with C. galioides.
There are 25 species of charophytes known in the Caucasus (Table 1), but some species records require confirmation.Georgia has the richest charophyte flora among the compared regions.The species lists of Caucasian regions largely overlap but are not identical to each other.Seven species known from Dagestan represent less than a third of the species richness of the Caucasus.This number seems low, and new species records are expected according to the species distribution in the Caucasus and neighboring regions (Table 1).The species composition of charophytes from Dagestan is similar to those from Caucasian regions, whereas Azerbaijan and Georgia, as well as Armenia and Dagestan, are closer to each other (Figure 7).16) 1 A comprehensive bibliography is impossible to report within the scope of this article, so the cited references are those that include all known species from the territory. 1this study, 2   A few old species records [80] and specimens could not be georeferenced, even up to administrative regions or the whole territory of the Caucasus, but they could be evidence of the possible occurrence of some species in the studied area.The collection by F.A. Marschall von Bieberstein (1768-1826) stored in LE (LE 01157109-01157113) and checked by the first author contained Chara cf.globularis Thuill., C. vulgaris, Nitella opaca (C.Agardh ex Bruzelius) C.Agardh and Tolypella glomerata (Desv. in Loisel.)Leonh.Some of them were collected in "deserto Cumanum" [118], i.e., in an arid region of the drainage basin of the Kuma River that belongs to the Dagestan, Kalmykia, and Stavropol Territory of Russia.The exact georeferencing of these specimens is not possible.Old records of C. aspera and C. tomentosa L. from the Lake Atu-Kol of the Terek Oblast of the Russian Empire [107] could belong to the current territory of the Republic of Kalmykia.The occurrence of some species of Nitella C.Agardh, Sphaerochara Mädler, Nitellopsis obtusa (Desvaux) J.Groves and Lamprothamnium papulosum (Wallr.)J.Groves seems to be possible in Dagestan, although they are rarely found in West Asia [101,119,120].
Despite a long interval of collecting, 1861-2020, only 22 localities are still known from this region, which could be explained by the fact that the charophytes were a neglected group in this region for a long time.The new species records from Dagestan improve species distribution ranges, filling the gaps and changing their outlines in several cases.New localities of C. connivens are situated between a few localities in the South Caucasus and the Lower Volga region [45,77,78,101,107].
New localities of C. contraria, C. globularis,C.vulgaris, and, especially, C. gymnophylla add essential details to their distribution in the Caucasus.Chara contraria, C. globularis, and C. vulgaris are widely distributed species.They are generalists in many temperate regions [121].Chara gymnophylla is the second most frequently found species in the area studied.It has a wide distribution area in Eurasia, but most records are concentrated in the Mediterranean region and the Middle East [49,77,101,122,123].It is the second most common species in Iran [49] and northern Israel [123].Its records are unknown north of the area studied [77,101], i.e., in the southeastern margin of Eastern Europe, and its northern distribution range can be tentatively outlined with the Piedmont area of the North Caucasus.
Chara globata is mainly a Central Asian species with several localities in Southeast Europe, North Africa, the Middle East, and China [26,35,49,75,101,124].Its distribution area extends north of the Caucasus to the arid regions of Volga and Don Interfluve [75,107].It is known from a few localities in the Caucasus, although its remarkable habit and size nearly exclude its undersampling during targeted searches for charophytes and allow it to be used as a flagship species for biogeographical studies [26].The records from small water bodies on the coast of the Caspian Sea add a new type of habitat known for this species, mostly associated with much bigger permanent lakes [77,124].
Chara neglecta is a species mostly known from the south of Eastern Europe and Central Asia [77].In the Caucasus, it was formerly known only in Azerbaijan [96,115].New records in Dagestan point towards its wider distribution in coastal regions of the Caspian Sea.
Tolypella nidifica is a species occurring mostly in coastal regions of Europe, where it is mostly known from North and West Europe as well as the West Mediterranean [77].Only two reliable small areas are known from the North Black Sea region [77,101,104,125,126], and one recent record from inland Georgia by V.S. Vishnyakov [81,112, https://www.inaturalist.org/observations/156074494,accessed on 13 July 2023].The record from the inland of Crimea [53,126] belongs to Sphaerochara prolifera, according to published images.Lake Issyk Kul is the sole locality for this species in Central Asia [96,101].The unexpected new record from Dagestan is the first for the Caspian Sea region and fills the gap in the eastern part of the species distribution area in Eurasia.
The combination of species with distribution areas mainly in the center of Eurasia, such as C. globata and C. neglecta with mainly Mediterranean-Middle East C. gymnophylla, is a notable trait of charophyte flora of the area studied.It seems to be a trait common to other Caucasian regions.
The habitat preference of species recorded from the area studied allows tentative suggestion of two main groups of habitats having dissimilar species compositions.Chara contraria, C. gymnophylla, and C. vulgaris have been found mostly in freshwater small water bodies usually associated with rivers.Chara globata, C. neglecta, and Tolypella nidifica are known only from small brackish water bodies at the coast of the Caspian Sea.They grew together here with Ruppia maritima L. during our survey, which evidently indicates a brackish environment.Chara connivens and C. globularis fall outside this scheme because they are known only from two large artificial mountain and coastal water reservoirs.Surveys of the region, especially of the oligotrophic mountain, brackish coastal, and lowland temporal spring water bodies, seem to be the most fruitful in further clarification of the biogeography of charophytes in the North Caucasus.
Based on habitat preference and distribution in the Caucasus, recommendations can be given to protect some of the species.Coastal water bodies threatened from both land and sea sides are some of the most endangered and shrinking habitats worldwide [127,128].There is no assessment of charophyte habitat loss at the coast of the Caspian Sea available, but a long-term negative trend is notable for the Black Sea region, where it has already resulted in a significant decrease in charophyte biomass and their community areas [47,[129][130][131].Some lagoons in this region harbor gyrogonites and oospores in bottom sediments, whereas charophyte stands seem to be lacking [132,133].The evident loss of coastal habitat can be recognized as too late for the restoration of the initial state [134] and may result in the irreversible disappearance of brackish water charophytes [135].Charophyte communities of Albufera de València lagoon in the West Mediterranean vanished due to long-lasting and still ongoing eutrophication, although viable oospores and gyrogonites were stored in bottom sediments, leaving a small hope for future restoration of aquatic vegetation [136].The hydrological change, habitat destruction, and expansion of reed stands because of a decline in livestock grazing can lead to similar results in brackish environments [137,138].Therefore, Chara globata, C. neglecta, Tolypella nidifica, and their habitats are suggested here as primary targets charophyte protection in Dagestan.The same need to protect coastal brackish habitats of charophytes was highlighted for Scotland [137], Great Britain [139], the Baltic Sea [138], France [140], and Sardinia [10].
The territory of Dagestan was recognized as a floristic province at the beginning of the study of its flora.It is one of the key areas for the speciation of xerophytic flora in the Caucasus, which can be proven by the high number of local endemics of magnoliophytes [141].The absence of endemic species among charophytes from Dagestan and the Caucasus disagrees with the flora of terrestrial magnoliophytes, which have a large number of endemic species [142][143][144].In contrast, the flora of the submersed aquatic magnoliophytes of this area consists of mostly widely distributed species [111,[143][144][145][146], although recent speciation of hygrophyte species of Cardamine L. (Brassicaceae) probably driven by both geographic separation and ecological divergence was revealed [147].Therefore, the aquatic ecosystems of this region do not seem to be an area with ongoing diversification and speciation of submersed aquatic macroscopic plants.This paradox looks similar to Tajikistan, which has no unique species of charophytes in contrast with its original flora of terrestrial magnoliophytes [55].

Supplementary Materials:
The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/environments10090153/s1,Supplementary Materials: Species name, GenBank accession number, and the haplotypes for the taxa used in our analyses.The sequences obtained in this study are in bold.Shared haplotypes are highlighted in yellow.

Figure 3 .
Figure 3. Chara globata and Tolypella nidifica from water bodies on the coast of the Caspian Sea (LE): (a,c)-upper parts of first morphotype of C. globata lacking evident lime incrustation and having long abaxial bract cells (arrowhead); (b)-upper part of the second morphotype of C. globata showing evident lime incrustation and abaxial bract cells of moderate length; it is really similar to C.
Russian regions of the Caucasus excl.The Republic of Dagestan: Chechen Republic, Kabardino-Balkarian Republic, Karachayevo-Circassian Republic, Krasnodar Territory, Republic of Adygeya, Republic of Ingushetia, Republic of North Ossetia-Alania, Stavropol Territory [63,80,81,99,101-106], 3 [80,106-109], 4 [77,80,81,107,110-113], 5 [41,79-81,101,107,114-117].(+) -: Other Russian regions of the Caucasusrecords need confirmation with specimen study; existence of vouchers is unknown; Armenia-a record of C. papillosa from Lake Sevan needs confirmation because it could actually be based on misidentification of C. globata; a record of C. connivens from Lake Sevan needs checking because of similarity of its images to C. aspera [cf.81]; Azerbaijan-the recent record of C. baltica[81,117] needs to be confirmed with an independent study of the specimen; the old record of C. hispida[7] needs confirmation because it could be based on another species.

Figure 7 .
Figure 7.The similarity of charophytes species composition of different regions of the Caucasus according to Bray-Curtis cluster analysis (single link) based on reliable records; others: Russian regions of the Caucasus excl.The Republic of Dagestan: Chechen Republic, Kabardino-Balkarian Republic, Karachayevo-Circassian Republic, Krasnodar Territory, Republic of Adygeya, Republic of Ingushetia, Republic of North Ossetia-Alania, Stavropol Territory.

Author
Contributions: Conceptualization, R.E.R.; methodology, R.E.R., M.M.M. and S.B.; software, R.E.R., S.B., V.Y.N. and A.A.G.; validation R.E.R., M.M.M., S.B., V.Y.N. and A.A.G.; formal analysis, R.E.R., M.M.M., S.B., V.Y.N. and A.A.G.; investigation, R.E.R., M.M.M., S.B., V.Y.N. and A.A.G.; data curation, R.E.R., V.Y.N. and A.A.G.; writing-original draft preparation, R.E.R., M.M.M., S.B., V.Y.N. and A.A.G.; writing-review and editing, R.E.R., M.M.M., S.B., V.Y.N. and A.A.G.; visualization, R.E.R., M.M.M., S.B., V.Y.N. and A.A.G.; funding acquisition, R.E.R., S.B., V.Y.N. and A.A.G.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the project "Flora and taxonomy of algae, lichens and bryophytes in Russia and phytogeographically important regions of the world" (no.121021600184-6) of the Komarov Botanical Institute of the Russian Academy of Sciences, within the state assignment of Ministry of Science and Higher Education of the Russian Federation (theme No. 121031000117-9), with the financial support the Ministry of Science and Higher Education of the Russian Federation under the agreement dated 28 September 2021, no.075-15-2021-1056 (placement of the algal specimens in the LE collection), and the Israel Ministry of Aliyah and Integration.

Table 1 .
Species of charophytes from different regions of the Caucasus.