The Genus Pinnularia Ehrenberg (Bacillariophyta) from the Transbaikal Area (Russia, Siberia): Description of Seven New Species on the Basis of Morphology and Molecular Data with Discussion of the Phylogenetic Position of Caloneis

Seven Pinnularia species from the Transbaikal area, Russia, are described as new for science. These are P. baicalgenkalii, P. baicalflexuosa, P. microfrauenbergiana, P. pergrunowii, P. siberiosinistra, P. baicalodivergens, and P. baicalislandica. All species are described by original LM and SEM microphotographs and molecular phylogeny. We provide comparisons between the taxa and document variability in the features found in the species. The number of Pinnularia species in the Transbaikal area is the largest number of species of the genus anywhere in the world.


Introduction
As is known, Lake Baikal is the world's largest freshwater lake, famous for its high biodiversity in many groups of organisms and abundance of endemic species [1,2].The research of microalgae was started in the 19th century, and the first works already demonstrated the high diversity and peculiarity of diatoms in the bottom sediments [3][4][5][6][7][8][9][10][11].According to Pomazkina et al. [12], already at the time about 800 diatom taxa had been found, 40% of which were endemic.In a review summarizing the results of long-standing research, Popovskaya et al. [13] indicate that diatoms comprise 49 taxa from 19 genera.Notably, the dominant taxa are exclusively endemic [13].
Pinnularia (1843) is one of the most diverse genera of diatoms and currently includes 861 accepted species names, 503 accepted varieties, and 126 accepted formae [14,15].However, in the publications dedicated to Lake Baikal, information on representatives of this genus is limited.Among the literature sources from the beginning of the 20th century that are available to us, the most comprehensive list of taxa is provided in a review of works by Boris Skvortzov (Skvortzow), featured in vol.23 of Iconographia Diatomologica [16].A significant part of Skvortzov's research was dedicated to the diatoms of Lake Baikal, including deepwater species (from the depth of 30-33 m).He described many new species and varieties.The author of the review Maria Gololobova studied about 60 publications and compiled a list of Skvortzov taxa that comprised 1562 diatom names [17].From those, 333 taxa are noted for Lake Baikal, including only 21 Pinnularia taxa.Meanwhile, Navicula (58 taxa), Cymbella (35), and Didymosphaenia (25) are represented more diversely.Other works of that period mention no more than two to three taxa from this genus.For example, in the list of new and interesting diatoms from Baikal compiled by Vladislav Jasnitsky [10], Plants 2023, 12 only two taxa Pinnularia are noted, previously described by Skvortzov: P. hemiptera (Kützing) Cleve var.baicalensis Skvortzov and P. passargei Reich var.baicalensis Skvortzov.In a work dedicated to diatoms from the periphyton of the northern part of Lake Baikal, Alexander Skabichevsky (Skabichevskij) describes new species, three of which belong to Pinnularia: P. braunii (Grunow) Cleve var.scabrosa Skab., P. polyonca (Brebisson) O. Müller var.scabrosa Skab., and P. timofeevii Skab.It is worth noting that all of them are considered rare species and were found at the depth of 26 m [11].The results of the processing of samples gathered by Niels Foged in 1975 were published posthumously [1], edited by Hannelore Håkansson.Out of 260 taxa, only 7 belong to Pinnularia (five species and two varieties).All were described as oligohalobous (indifferent), pH-circumneutral, and cosmopolitan.The taxa were found in samples from the Angara, which is the only river that drains out of the lake.Many taxa were observed from other genera: Navicula (37 taxa), Gomphonema (29), Nitzschia (20), and Cymbella (20).H. Håkansson notes that the samples were in general less diverse than those described earlier "by Skvortzow and Meyer (1928) and particularly by Skvortzow (1937)" [8,9].Currently, the unique algal flora of Lake Baikal is being intensively studied.Several monographs were published during the last two decades, dedicated to the results of long-term studies of diatoms from plankton [18], the littoral zone [19,20], and benthos [21].The results of the study of samples collected by Skabitschewsky in July 1965 and samples collected during a Darwin Initiative project in 1997 were presented in volumes 23 and 26 of Iconographia Diatomologica [22,23].Only in these works, 22 new genera and 554 new species were described.Publications dedicated to descriptions of new taxa from Baikal come out regularly.However, representatives of Pinnularia are not mentioned in these works.Brief reports are presented in several works by Pomazkina et al. on microphytobenthos [24][25][26].In south Baikal, P. microstauron (Ehrenberg) Cleve is mentioned among taxa that are dominant in winter, and P. brevicostata Cleve is often found [24].During a study in Olkhon Gate and Maloe More straits, representatives of Pinnularia were reported only as single finds.Only four species were found: P. major var.hyalina (Hust.)Skab.and P. pectinalis var.rostrata Skvortzow, as well as the endemic P. braunii var.scabrosa Skab.and P. timofeevii Skab.[25].In a publication on the microphytobenthos of Lake Baikal in areas close to rivers, the diversity of this genus is mentioned; however, there is no information on the number of species or list of taxa [26].
Research of diatoms from Baikal that includes the molecular genetic approach is still scarce.So far, only six new species from the genera Geissleria, Sellaphora, Placoneis, Cymbopleura, and Cymbella have been described with the use of molecular data [27][28][29][30][31].For five more previously known species from the genera Planothidium, Stauroneis, Craticula, and Stephanodiscus, there are mentions of genetic sequences of strains from Baikal [32][33][34].In the studies dedicated to the diversity of protist communities in Lake Baikal using metabarcoding, there are only brief reports on diatoms [35][36][37].
The aim of this publication is the molecular investigation and description of the morphology of seven new Pinnularia species from the Transbaikal area.

Morphology and Ultrastructure
The studies performed with light and scanning electron microscopy showed that the isolates belong to the new species Pinnularia baicalgenkalii, P. baicalflexuosa, P. microfrauenbergiana, P. pergrunowii, P. siberiosinistra, P. baicalodivergens, and P. baicalislandica.
SEM, external view (Figure 2C).Proximal raphe ends are drop-like and deflected to the same side, but to the opposite direction than the terminal ends.Terminal raphe fissures are externally hooked and unilaterally deflected, reaching the valve mantle at the apex.Striae alveolate, multiseriate (about 7-9 areolae rows).SEM, internal view (Figure 2D).Central raphe ends are uninterrupted with knot and notch (shown by arrows), raphe branches end in polar simple helictoglossae, deflected to one side.Alveolar openings are covered 2/3 by an axial plate and 1/3 by a mantle plate, leaving an internal opening which is shorter than the entire alveolus.In LM, this covering gives the impression of two longitudinal lines.Striae are composed of areolae with irregularly rounded openings.Etymology.The species is named for the species locality, Lake Baikal, and the similarity with Pinnularia genkalii Krammer & Lange-Bertalot.
Distribution.As yet known only from the type locality.
Comments.P. baicalgenkalii sp.nov. is similar to P. reichardtii Krammer, differing from it by wider valves (19.5-20.0µm in new species vs. 14.7-18.8µm in P. reichardtii) and  Sequence data.Partial 18S rDNA gene sequence comprising V4 domain sequence (GenBank accession number KM350092) and partial rbcL sequence (GenBank accession number KM350002) for the strain B194.
Etymology.The species is named for the species locality, Lake Baikal, and the similarity with Pinnularia genkalii Krammer & Lange-Bertalot.
Distribution.As yet known only from the type locality.
Description.LM (Figure 3A-E, Figure 4A-E and Figure 5A,B).Frustule rectangular in girdle view (Figure 5B).Valve outline linear with parallel sides and broadly rounded ends.Length 109-116 µm, width 17.5-19.0µm.Axial area linear, narrow, tapering on the ends and widening towards the central area.Central area small, asymmetrically elliptic.Raphe semicomplex, undulate.Striae radiate at the center, becoming convergent at the ends, 7-8 in 10 µm.SEM, external view (Figure 5C).Proximal raphe ends are drop-like and deflected to the same side but in the opposite direction than the terminal ends.Terminal raphe fissures are externally hooked and unilaterally deflected, reaching the valve mantle at the apex.Striae alveolate, multiseriate (about 5-6 areolae rows).SEM, internal view (Figure 5D).Central raphe ends are uninterrupted with knot, raphe branches end in polar simple helictoglossae, deflected to one side.Alveolar openings are covered 2/3 by an axial plate and 1/3 by a mantle plate, leaving an internal opening which is shorter than the entire alveolus.In LM, this covering gives the impression of two longitudinal lines.Sequence data.Partial 18S rDNA gene sequence comprising V4 domain sequence (GenBank accession number KM350068) and partial rbcL sequence (GenBank accession number KM349984) for the strain B054-3.
Etymology.The species is named for the species locality, Lake Baikal, and the similarity with Pinnularia flexuosa P.T. Cleve.
Distribution.As yet known only from the type locality.Holotype here designated: Slide no.18959, Figure 3C, from oxidized culture strain no.B054-3, isolated from sample no.40, deposited in herbarium of MHA, Main Botanical Garden, Russian Academy of Science, Moscow, Russia.
Isotype.Slide no.18959a, collection of Maxim Kulikovskiy at the Herbarium of the Institute of Plant Physiology, Russian Academy of Science, Moscow, Russia.
SEM, external view (Figure 6V).Proximal raphe ends are drop-like and deflected to the same side but in the opposite direction than the terminal ends.Terminal raphe fissures are externally hooked and unilaterally deflected, reaching the valve mantle at the apex.Striae are alveolate, multiseriate; longitudinal lines are absent.SEM, internal view (Figure 6W).Central raphe ends are uninterrupted with knot and notch, internal raphe branches end in polar simple helictoglossae, deflected in the same direction.On each of the interstriae there is a single small outgrowth of a rounded or rectangular shape.Sequence data.Partial 18S rDNA gene sequence comprising V4 domain sequence (GenBank accession number KM350062 and partial rbcL sequence (GenBank accession number KM349979) for the strain B025.
Etymology.The species is named for the smaller size, and the similarity with Pinnularia frauenbergiana Reichardt.
Distribution.As yet known only from the type locality.
Comments.Pinnularia microfrauenbergiana sp.nov. is distinguished from P. frauenbergiana Reichardt by the outline, stria density, and axial area, differing in general by stria density (14-15 in 10 µm in P. microfrauenbergiana sp.nov. vs. 18-22 in 10 µm in P. frauenbergiana).P. microfrauenbergiana sp.nov. is also close to P. bullacostae Krammer et Lange-Bertalot in Lange-Bertalot & Genkal; however, new species differs by more radiate striae and less blunted valve ends, and its valve sides are not concave as in P. bullacostae (Table 3).Pinnularia microfrauenbergiana sp.nov. is close to P. pinseelliana Zidarova, Kopalová & Van de Vijver, described from the Antarctic [42].However, P. microfrauenbergiana sp.nov.does not exhibit protracted valve ends at all, while this feature is present in P. pinseelliana.The valves of P. pinseelliana are more widened in the center part relative to the ends than in P. microfrauenbergiana sp.nov.Other morphological features are similar in these two species.
An interesting feature of Pinnularia microfrauenbergiana sp.nov., P. bullacostae, and P. pinseelliana is the presence of a morphological structure located on the inner side of the raised interstriae (virgae) that is described by researchers in various ways: "knopfartige höcker" [43], "papillae-like structures" [38], "elevated siliceous outgrowth" [42].Pinnularia pergrunowii Kulikovskiy, Glushchenko, Kezlya and Maltsev sp.nov.(Figure 7).Description.LM (Figure 7A-E).Cells solitary, two parallel plastids on either side of the apical axis are present (Figure 7A-D).Frustule rectangular in girdle view (Figure 7D,M,N) with slightly undulating margins.Valve outline linear with capitate ends and shoulders that are broader than the central part.Length 49.5-51.0µm, width of central part 7 µm, width of shoulders 8.0-8.5 µm.Axial area linear, narrow and widening towards the central area.Central area is represented by a wide transverse fascia.Raphe straight, filiform, and well noticeable under LM.Striae strongly radiate to radiate at the center, becoming convergent to strongly convergent towards the ends, 10-11 in 10 µm.
SEM, external view (Figure 7F).Proximal raphe ends are drop-like and deflected to the same side but in the opposite direction than the terminal ends.Terminal raphe fissures are externally hooked and unilaterally deflected, reaching the valve mantle at the apex.Striae are alveolate, multiseriate, composed of areolae with irregularly rounded openings.SEM, internal view (Figure 7G).Central raphe ends are continuous with knot; raphe branches end in polar simple helictoglossae.There is no covering over the alveoli.Longitudinal lines are absent.
Etymology.The species is named for the similarity with Pinnularia grunowii Krammer.Distribution.As yet known only from the type locality.
Comments.Pinnularia pergrunowii sp.nov. is close to P. grunowii Krammer (Table 4); however, P. grunowii has distinctly triundulate sides of the valve, while new species has a small constriction at the center of the valve.The valve ends are less constricted in our species than in P. grunowii.The stria density in P. pergrunowii is generally lower than in P. grunowii (10-11 in 10 µm in P. pergrunowii vs. 11-14 in 10 µm in P. grunowii).Among similar species that are the same size, with capitate ends and a fascia, we should note P. rhombofasciata Krammer & Metzeltin and P. dicephala (Ehrenberg) W. Smith (Table 4).P. pergrunowii can be easily differentiated from these species by the concave sides.Biundulate valves of P. ferrophila Krammer are similarly shaped but can be differentiated by the valve width (8.0-8.5 µm in P. pergrunowii sp.nov. vs. 8.8-10 µm in P. ferrophila).Also, we should note the similarity of P. pergrunowii sp.nov.with illustrations of P. latarea Krammer provided by Siver et al. [45] (p.581, plate 168, Figures 1-14).These species are similar in size and valve shape (linear with concave sides and capitate ends) (Table 4).
A clear difference is in the structure of the axial and central area.In P. latarea, they form together a wide, lanceolate space with a very broad fascia, whereas in P. pergrunowii sp.nov. the axial area is distinctly separated, narrow, linear.The valve sides in P. pergrunowii are more concave than in P. latarea.We also studied vouchers of strains (images and metadate on the culture collection website or in publications associated with the nucleotide sequence) close in phylogenetic position: P. anglica AT100Gel01, P. mesolepta AT_160Gel30, P. grunowii Pin 889 MG, P. termitina UTEX FD484.All of them are clearly different from the new species (Table 4).Pinnularia siberiosinistra Kulikovskiy, Glushchenko, Kezlya and Maltsev sp.nov.(Figure 8).Description.LM (Figure 8A-V).Cells solitary, two parallel plastids on either side of the apical axis are present (Figure 8A-F).Frustule rectangular in girdle view (Figure 8E,F,V).Valve outline narrowly elliptical with subcapitate ends.Length 25-29 µm, width 5 µm.Axial area narrowly lanceolate and widening towards the central area.Central area is represented by a wide transverse fascia.Raphe straight, filiform, and weakly noticeable under LM.Striae strongly radiate to radiate at the center, becoming convergent to strongly convergent towards the ends, 12-14 in 10 µm.
SEM, external view (Figure 8W).Proximal raphe ends are drop-like and deflected to the same side but in the opposite direction than the terminal ends.Terminal raphe fissures are externally hooked and unilaterally deflected, reaching the valve mantle at the apex.SEM, internal view (Figure 8X).Central raphe ends are continuous with knot; internal raphe branches end in polar simple helictoglossae.There is no internal covering over the alveoli.Longitudinal lines are absent.Striae alveolate, multiseriate (about 5-6 areolae rows), composed of areolae with irregularly rounded openings.
Etymology.The species is named for the species locality-the extensive geographical region Siberia-and the similarity with Pinnularia sinistra Krasske.T Distribution.As yet known only from the type locality.
Comments.Pinnularia siberiosinistra sp.nov. is morphologically similar to P. sinistra Krammer (Table 5).It differs from the type population of the Krammer species by a stronger tapering of the valve ends and a more widened axial area than in P. sinistra (Table 5).New species could also be compared with the material illustrated by Souffreau et al. [39] (p.867, Figure 1o) marked as "P.sp.(Tor4)r".P. siberiosinistra sp.nov.differs from P. sp. (Tor4)r by a narrow elliptical valve shape, while P. sp. (Tor4)r has linear valves with slightly undulate ends.Other features are quite similar in these two species (Table 5).Sequence data.Partial 18S rDNA gene sequence comprising V4 domain sequence (GenBank accession number KM350075) and partial rbcL sequence (GenBank accession number KM349990) for the strain B112.
Etymology.The species is named for the species locality, Lake Baikal, and the similarity with Pinnularia divergens W. Smith.
Distribution.As yet known only from the type locality.
Description.LM (Figure 12A-O).Cells solitary, two parallel plastids on either side of the apical axis are present (Figure 12A-D).Frustule rectangular in girdle view (Figure 12D,O).Valve outline linear with parallel sides and cuneiform rounded ends.Length 54-60 µm, width 10-11 µm.Axial area linear, narrow, tapering on the ends and widening towards the central area.Central area asymmetric, rhomboid.Raphe straight, filiform, and well noticeable under LM.Striae strongly radiate to radiate at the center, becoming parallel to convergent at the ends, 10 in 10 µm.Comments.This species is distinguished from P. islandica Østrup by the valve outline and the shape of axial and central areas (Table 7).New species has a lower valve width (10-11 µm vs. 12-14 µm in P. islandica).The axial area in P. baicalislandica sp.nov. is linear, distinctly separated from the central area, while in the valves of P. islandica this separation is not clear, and the axial area is wedge-shaped.New species is distinguished from P. subcommutata Krammer and P. subcommutata var.nonfasciata Krammer by the valve outline (in P. subcommutata the sides are slightly convex whereas in P. baicalislandica sp.nov.they are parallel).P. rupestris Hantzsch differs from P. baicalislandica sp.nov.by stria density (12-13 in 10 µm in P. rupestris vs. 10 in 10 µm in the new species).P. perspicua Krammer can be differentiated by the presence of crescent-like markings on the central area; in P.  13E-G).Central raphe ends are continuous with knot; internal raphe branches end in polar simple helictoglossae.There is no internal covering over the alveoli.Longitudinal lines are absent.

SEM, internal views (Figure
Holotype here designated: Slide no.18997, Figure 12E, from oxidized culture strain B238, isolated from sample no.Sequence data.Partial 18S rDNA gene sequence comprising V4 domain sequence (GenBank accession number KM350101) and partial rbcL sequence (GenBank accession number KM350009) for the strain B024-1.
Etymology.The species is named for the species locality, Lake Baikal, and the similarity with Pinnularia islandica Østrup.
Distribution.As yet known only from the type locality.
Comments.This species is distinguished from P. islandica Østrup by the valve outline and the shape of axial and central areas (Table 7).New species has a lower valve width (10-11 µm vs. 12-14 µm in P. islandica).The axial area in P. baicalislandica sp.nov. is linear, distinctly separated from the central area, while in the valves of P. islandica this separation is not clear, and the axial area is wedge-shaped.New species is distinguished from P. subcommutata Krammer and P. subcommutata var.nonfasciata Krammer by the valve outline (in P. subcommutata the sides are slightly convex whereas in P. baicalislandica sp.nov.they are parallel).P. rupestris Hantzsch differs from P. baicalislandica sp.nov.by stria density (12-13 in 10 µm in P. rupestris vs. 10 in 10 µm in the new species).P. perspicua Krammer can be differentiated by the presence of crescent-like markings on the central area; in P. baicalislandica sp.nov.such markings are not found.It may be complicated to differentiate the new species from P. levkovii Metzeltin, Lange-Bertalot & Soninkhishig (Table 7); the structure of the axial area should be considered (it is expanded towards the central area in P. levkovii, while in P. baicalislandica sp.nov. it is linear, narrow).The central area is distinctly separated, asymmetric, rhomboid in P. baicalislandica sp.nov., and in P. levkovii it is variably shaped and is not clearly differentiated.The stria density is lower in P. levkovii than in P. baicalislandica sp.nov.(8-10 in 10 µm in P. levkovii vs. 10 in 10 µm in P. baicalislandica sp.nov.).

Phylogenetic Analysis
Our phylogenetic analysis was carried out using the genetic markers rbcL and 18S rDNA and included 76 strains of Pinnularia and 17 strains of Caloneis.It is these genes that are most often used in work with this group [39,52,53].In this case, the largest number of nucleotide sequences is available for the rbcL gene, somewhat less for 18S rRNA.Significantly fewer sequences are available for 28S rRNA and cox1 genes.Therefore, we have chosen a strategy for using 18S rRNA and rbcL genes to minimize the loss of Pinnularia strains and species for which other genes are not available.Previous studies already showed that Pinnularia and Caloneis form a monophyletic group and there are three well supported clades within this group, designated as A, B, C by Souffreau et al. [39,52,53].Our phylogeny confirms these findings and new strains are added to the clades and subclades.Strains of Caloneis amphisbaena, C. cf.westii SZCZCH1002 and C. sp.21IV14_6A formed an additional clade.All new species described in this study occupy separate positions in the corresponding subclades (Figure 14).
Subclade "divergens" includes species that are morphologically close to P. divergens, with linear or linear-elliptic valves, capitate, subcapitate or rostrate valve ends, a fascia on the central area, and internally open alveoli.However, a specific feature of the P. divergens type is the presence of rounded thickenings at the margin of the fascia.These structures can only be supposedly confirmed for P. sp.B027-1 (this study, Figure 15A-C).On the LM image of the P. sp. 7 Tor1b voucher thickenings at the margin of the fascia cannot be seen; the LM and SEM images of P. sp. 1 Tor7c also do not show any structures on the central area [39] (p.867, Figure 1a,b,e).On the contrary, in the new species P. baicalodivergens we have found crescent-shaped hollow markings on the central area (Figure 10A,B), which brings this species close to representatives of the "stomatophora" subclade.
The "stomatophora" subclade species are united by the presence of well discernible crescent-shaped or irregular hollow markings on the external surface of the central area (P.stomatophora, P. ministomatophora, P. valida).
Most taxa belong to the "subgibba" subclade.A characteristic feature of this subclade for almost all of the taxa is the presence of ghost striae on the inner surface of the central area and fascia.
Taxa from the "grunowii" subclade do not have any differentiating features in their valve structure, they do not have any markings on the inner or outer surface of the central area.A common feature is an H-shaped chloroplast [39].Two new species described in the current study are included in this subclade.P. pergrunowii occupies a stable position next to P. grunowii (Figure 14), and morphologically these species differ clearly (Table 4).
P. nodosa and P. acrosphaeria form separate subclades.Subclade "nodosa" now includes only P. nodosa Pin855 TM with characteristic markings on the valve sufrace-heavily structured on the outer surface and smooth on the inside [38].
Subclade "acrosphaeria" includes only two strains of P. acrosphaeria and is defined by a mottled, distinct structured area on the outer surfaces and slightly structured on the inner surfaces.
Subclade "borealis" is formed by taxa with a characteristic morphotype similar to representatives of the P. borealis species complex.Currently the subclade includes taxa with relatively small linear or linear-elliptic valves, widely spaced coarse striae, and a lateral or fusiform raphe.
A new clade "caloneis 3"has been defined with high support, formed by five strains of Caloneis.Three strains are represented by the type species of the genus C. amphisbaena, they form a subclade with maximum support, and separate lines are formed by C. cf.westii SZCZCH1002 and C. sp.21IV14-6A.

Phylogeny of the Genera Pinnularia and Caloneis
The issue of separating Pinnularia and Caloneis was repeatedly raised by algologiststaxonomists [54][55][56].Pinnularia was described in 1843 by Ehrenberg with the type species P. viridis [57].Caloneis was described later by Cleve in 1894 on the basis of longitudinal lines on the valve surface.Even then the unclear differentiation of small-celled forms and a close connection with "some marine, panduriform Pinnulariae" were noted [58].Nevertheless, to this day, Caloneis remains a valid genus and diatomists identify its representatives in flora and describe new species [22,[59][60][61].Traditionally, the genera are delineated by stria density, species with finer and denser spaced striae being attributed to Caloneis.According to the opinion of Mann [56] (p.33), ". ..there is no adequate basis for the traditional Pinnularia-Caloneis distinction. ..", and more than 100 years ". ..people learn to recognize not the genus itself, but individual species and species complexes, which they then learn to associate with a particular genus name".In his discussion of the closeness of Pinnularia and Caloneis and the great diversity of both genera, Mann from the first page quotes E. Cox, F. Round, and K. Krammer, who speak of the heterogeneity of these genera and of suggestions to divide them into many smaller units.
It would seem that the application of molecular methods would give a clearer picture of the relationship between Pinnularia and Caloneis.In the first work of Bruder et al., published in 2008 [52], it was shown that the assumptions summarized by David Mann [56] had been confirmed.Phylogenetic analysis does not support the traditional division Pinnularia-Caloneis; these genera form a single monophyletic clade.In turn, Caloneis turned out to be non-monophyletic, its representatives (C.lauta, C. budensis, C. amphisbaena) forming separate lines among Pinnularia.For a morphological feature that corresponded with the division of phylogenetic groups, the suggestion made by Krammer and Lange-Bertalot [54] of using the degree of alveoli closure ("nearly open alveoli", "partially closed" and "nearly closed") was confirmed at that moment.
In the study of Souffreau et al. [39], dedicated to a time-calibrated multi-gene phylogeny of the Pinnularia, representatives of Caloneis are positioned in different clades (A and C), which confirms the heterogeneity of this genus.A more comprehensive phylogenetic analysis has been performed, and a third clade of Pinnularia-Caloneis has been added to the two defined by Bruder et al. [52].The analysis of the morphological features of the obtained subclades discusses shapes of the valves and apices, external central raphe endings (linear or rounded), raphe fissures (straight or undulate), chloroplasts (H-shaped or elongated, with pyrenoids or not, etc.), and specific markings on the central area (ghost striae, fascia, wart-like bodies, etc.).
In our phylogenetic analysis, the range of Caloneis strains has been significantly expanded compared to previous studies and includes 17 strains.Their positions in the clades remain the same: in clade A C. lauta forms separate line; the suclades "caloneis1" and "caloneis2" are separated, but they have low external support (Figure 14); C. budensis forms lineage with P. brebissonii UTEX FD274 and P. cf. microstauron (B2)c in clade C; strains of C. amphisbaena together with two other representatives of the genus form a separate clade "caloneis 3" (Figure 14).

Morphological Features of Some Phylogenetical Groups
Currently, it is generally quite hard to connect division into clades with any kind of morphological patterns, since species in every clade are very variable in valve size and shape.For example, clade A contains both the large-celled, morphologically close to the viridis group P. valida and the small-celled Caloneis silicula, C. fontinalis.For the most part clade B includes taxa with linear valves and capitate or subcapitate ends, but there are exceptions, like the small-celled species with an elliptic valve outline P. microfraubergiana sp.nov.The presence of a fascia on the central area is characteristic for all species of clade B except P. acrosphaeria.Clade C also includes very different forms: large, elongated elliptical viridis-like valves in the species from "viridiformis" and "subcommutata" subclades and small-celled P. altiplanensis, Caloneis budensis, etc.
The connection of phylogenetic groups and morphological features is more defined at the subclade level.Our further discussion is based on a study of voucher images that can be openly accessed (Table S1).For the species that do not have accessible voucher images or the images do not contain the necessary morphological features (for example, there are only live cells pictured, there are no SEM images on which the ultrastructure of the central area or the degree of closure of the alveoli could be studied, etc.), we considered taxa descriptions and features given in the relevant literature and descriptions from [39,52].In the end, we could not find images only for 8 unidentified taxa (less than 10%) (see Table S1).
To determine the significance of a specific morphological feature as a phylogenetic signal, we compiled a comparison table for the main morphological features mentioned in previous works [39,52] and compared them with the phylogenetic groups.Unique features, i.e., those that appear in one or two clade and can be used as differentiating, are highlighted in bold (Table 8).We do not, however, speak of a 100% conclusion, since our phylogeny only includes a small part of the whole Pinnularia-Caloneis diversity, we are only making a suggestion based on an analysis of a concrete set of taxa.So, the structure of internal alveoli aperture can be used to define subclade "caloneis1" and a monospecies subclade "acrosphaeria", which are characterized by nearly closed alveoli.A preliminary analysis indicates that nearly closed alveoli are a rare and specific feature, the importance of which is confirmed by our phylogeny.In the future it can be used as differentiating.Generally, we can conclude that for each subclade the structure of internal alveoli aperture is a unifying feature (Table 8).Markings on the valves surface are present in representatives of five subclades, however, the ultrastructure of these markings is distinctive in each subclade.
In the subclades "divergens" and "stomatophora", the markings are crescent-shaped or irregular hollow on the external surface of the central area.However, among the representatives of "divergens", the presence of such markings is confirmed only for P. baicalodivergens sp.nov.(this study, Figure 10A,B).After studying LM images of the P. divergens D31_023 voucher, we can also assume the presence of these structures.Note that on the voucher images of Caloneis representatives from clade A we did not find such structures on the central area (Table S1).However, according to literary data, crescentshaped hollows are shown for Caloneis lewisii and C. silicula [62,63].In any case these crescent-shaped or irregular hollows on the external surface of the central area have been found only in representatives of clade A.
Most of the taxa from subclade "subgibba" have the so-called ghost striae (slight thinnings of the valve that correspond in size and spacing to the normal striae [64] on the internal surface of the central area.The exception are the strains of Pinnularia sp.6 Tor4r, on the voucher image ghost striae are not distinct [39], p. 867, Figure 1o), and in P. siberiosinistra (this study, Figure 8 W,X) ghost striae are absent.
The monospecies lines "nodosa" and "acrosphaeria" have a heavily structured central area; "nodosa" has a relief-like structure on the outer surface and "acrosphaeria" has mottled, wart-like structures on the outer surface and is slightly structured on the inner surface.
Subclade "viridiformis" clearly stands out by the raphe structure, because only this clade unites taxa with a complex or semicomplex raphe.Other subclades contain species with a lateral and/or fusiform raphe.The border lineage between subclades "viridiformis" and "subcommutata" includes P. substreptoraphe AT 70.09 with a complex raphe and P. acuminata Pin876 TM with a lateral raphe, but this lineage is poorly supported (Figure 14).
Concerning the structure of the chloroplasts, most species in our analysis have two plate-like chloroplasts.The two plate-like chloroplasts are widest in girdle view, with the small parts of each edge extending to valve view.Some species with one H-shaped chloroplast are united in the subclade "grunowii".Also, H-shaped chloroplasts are present in P. sp. (Wie)a, P. cf. altiplanensis, P. cf. isselana ("subcommutata" subclade), P. microstauron ("microstauron" subclade), and one species in subclade "caloneis1"-Caloneis silicula (according to Bruder et al. [52] and Souffreau et al. [39]).Thus, the significance of chloroplast structure for phylogenetic differentiation is not yet clear.

Conclusions
Based on our analysis presented herein, with a greater degree of taxon sampling than past studies, we can say there is better resolution of the genera Caloneis and Pinnularia and a reason to continue to recognize them as distinct.The genera Caloneis and Pinnularia are each monophyletic but not with the composition of species traditionally assigned to them.For Caloneis, the generitype (C.amphisbaena) and other species form a strongly supported monophyletic group that is distinct from Pinnularia sensu lato.Small species traditionally assigned to Caloneis, such as C. silicula, C. lewisii, C. lauta, etc, however, are not part of this lineage; they fall out within a broader concept of Pinnularia.The genus Pinnularia s. l. is also a monophyletic group, but it is not strongly supported.Within Pinnularia, the three subgroups (A, B, and C) are monophyletic, though only one in our analysis (Clade B) has strong support--however, this group does not contain the generitype (P.viridis).The large number of taxa described in Pinnularia (with over 4200 named taxa; [15]) makes it tempting to begin recognizing subgroups within Pinnularia s.l. as distinct genera, and assigning morphological characters to these groups supports that approach.But, further analyses and better support for the groups may be a crucial next step in the direction of creating a refined, natural classification of the Pinnlariaceae.

Sampling
The samples used in the present report were collected from Eastern Siberia, Russia, by Maxim Kulikovskiy.The samples were collected in deltas of rivers that drain into Lake Baikal (the rivers Kapustinskaja, Selenga, Zagza, Vydrinnaja, Bolshaya Suhaya) as well as in Lake Baikal itself (see Figure 16, Table 9).Water mineralization and temperature measurements were performed using the Hanna Combo (HI 98129) multiparameter probe (Hanna Instruments, Inc., Woonsocket, RI, USA).A list of all strains examined in this study with their GenBank accession numbers and geographic location of sampling sites with measured ecological parameters is presented in Table 9.

Culturing
A subsample of each collection was added to WC liquid medium [65].Monoclonal strains were established by micropipetting a single cell under an inverted microscope Axio Vert.A1 (Zeiss, Oberkochen, Germany).Non-axenic unialgal cultures were maintained in WC liquid medium at 22-25 • C in a growth chamber with a 12:12 h light:dark photoperiod.Strains were analyzed after one month of culturing.
by Maxim Kulikovskiy.The samples were collected in deltas of rivers that drain into Lake Baikal (the rivers Kapustinskaja, Selenga, Zagza, Vydrinnaja, Bolshaya Suhaya) as well as in Lake Baikal itself (see Figure 16, Table 9).Water mineralization and temperature measurements were performed using the Hanna Combo (HI 98129) multiparameter probe (Hanna Instruments, Inc., Woonsocket, RI, USA).A list of all strains examined in this study with their GenBank accession numbers and geographic location of sampling sites with measured ecological parameters is presented in Table 9.

Preparation of Slides and Microscope Investigation
Strains for LM and SEM investigations were processed by means of a standard procedure involving treatment with concentrated hydrogen peroxide.The material was washed with distilled water.Permanent diatom preparations were mounted in Naphrax ® (Brunel Microscopes Ltd., Chippenham, UK; refractive index = 1.73).Light microscopic (LM) observations were performed using the microscope AxioScope A1 (Zeiss, Germany) equipped with an oil immersion objective (×100/n.a.1.4,DIC).Ultrastructure of the valves was examined with the scanning electron microscope JSM-6510LV (Jeol, Tokyo, Japan).
Amplifications were carried out using premade polymerase chain reaction (PCR) mastermixes (ScreenMix by Evrogen, Moscow, Russia).Amplification conditions for the 18S rRNA gene were as follows: initial denaturation for 5 min at PCR products were visualized by horizontal electrophoresis in 1.0% agarose gel stained with SYBRTM Safe (Life Technologies, Carlsbad, CA, USA).The products were purified with a mixture of FastAP, 10× FastAP Buffer, Exonuclease I (Thermo Fisher Scientific, Waltham, MA, USA), and water.The sequencing was performed using a Genetic Analyzer 3500 instrument (Applied Biosystems, Waltham, MA, USA).
Plants 2023, 12, x FOR PEER REVIEW 7 of 41 Reference strain.B054-3, isolated from the sample no.40, deposited in the collection of Maxim Kulikovskiy at the Herbarium of the Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia.

Plants 2023 ,
12, 3552   Isotype.Slide no.18931a, collection of Maxim Kulikovskiy at the Herbarium of the Institute of Plant Physiology, Russian Academy of Science, Moscow, Russia.Reference strain.B025, isolated from the sample no.51.1, deposited in the collection of Maxim Kulikovskiy at the Herbarium of the Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia.

Figure 9 .
Figure 9. Pinnularia baicalodivergens Kulikovskiy, Glushchenko, Kezlya and Maltsev sp.nov.Strain B 112. Slide no.19170.Light microscopy, differential interference contrast.(A,B).Live cells with plastids structure.(C-N).Oxidized material, size diminution series.(N).Frustule in girdle view.(C).Holotype.Scale bar = 10 µm.Description.LM (Figure 9A-N).Cells solitary, two parallel plastids on either side of the apical axis are present (Figure 9A,B and Figure 11A).Frustules rectangular in girdle view (Figures 9N and 11H).Valve outline linear with parallel sides and subcapitate rounded ends.Length 46-50 µm, width 8-9 µm.Axial area linear, narrow, tapering at the ends and widening towards the central area.Central area is represented by an asymmetric transverse fascia.On the central area are often present irregular structures as diverse flecks, chaotic or united in a line.Raphe straight, filiform, and well noticeable under LM.Striae strongly radiate to radiate at the center, becoming convergent to strongly convergent at the ends; longitudinal lines are absent, 11-12 in 10 µm.

Figure 14 .
Figure 14.Phylogenetic position of the new Pinnularia species (indicated in bold) based on Bayesian inference for the partial rbcL and 18S rRNA genes.The total length of the alignment is 1797 characters.Bootstrap supports (BS) from ML (constructed by RA × ML) and posterior probabilities (PP) from BI (constructed by Beast) are presented on the nodes in order.Only BS and PP above 50 and 0.9 are shown.Strain numbers (if available) and GenBank numbers are indicated for all sequences.

Figure 14 .
Figure 14.Phylogenetic position of the new Pinnularia species (indicated in bold) based on Bayesian inference for the partial rbcL and 18S rRNA genes.The total length of the alignment is 1797 characters.Bootstrap supports (BS) from ML (constructed by RA × ML) and posterior probabilities (PP) from BI (constructed by Beast) are presented on the nodes in order.Only BS and PP above 50 and 0.9 are shown.Strain numbers (if available) and GenBank numbers are indicated for all sequences.

Figure 16 .
Figure 16.Map showing the sampling locations.Figure 16.Map showing the sampling locations.

Figure 16 .
Figure 16.Map showing the sampling locations.Figure 16.Map showing the sampling locations.
95 • C followed by 35 cycles of 30 s denaturation at 94 • C, 30 s annealing at 52 • C, and 50 s extension at 72 • C, with the final extension for 10 min at 72 • C. Amplification conditions for the rbcL gene were as follows: initial denaturation for 4 min at 94 • C followed by 40 cycles of 50 s denaturation at 94 • C, 50 s annealing at 53 • C, and 80 s extension at 72 • C, with the final extension for 7 min at 72 • C.

Table 1 .
Comparison of morphological features of P. baicalgenkalii sp.nov.and related species.

Table 2 .
Comparison of morphological features of P. baicalflexuosa sp.nov.and related species.

Table 3 .
Comparison of morphological features of P. microfrauenbergiana sp.nov.and related species.
.2, deposited in herbarium of MHA, Main Botanical Garden, Russian Academy of Science, Moscow, Russia.Isotype.Slide no.18989a, collection of Maxim Kulikovskiy at the Herbarium of the Institute of Plant Physiology, Russian Academy of Science, Moscow, Russia.Reference strain.B162-3, isolated from the sample no.28.2, deposited in the collection of Maxim Kulikovskiy at the Herbarium of the Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia.Type locality.Russia, Bolshaya Suhaya River near the Zarech'e Settlement, sample no.28.2, benthos (52 • 33.418 N 107 • 08.564 E), collected by M. Kulikovskiy, 17.07.2011.

Table 4 .
Comparison of morphological features of P. pergrunowii sp.nov.and related species.

Table 5 .
Comparison of morphological features of P. siberiosinistra sp.nov.and related species.
P. siberiosinistra sp.nov.P. sinistra P. sp. (Tor4)r .1 deposited in herbarium of MHA, Main Botanical Garden, Russian Academy of Science, Moscow, Russia.Isotype.Slide no.18930a, Collection of Maxim Kulikovskiy at the Herbarium of the Institute of Plant Physiology, Russian Academy of Science, Moscow, Russia.Reference strain.B024-1, isolated from the sample no.51.1, deposited in the collection of Maxim Kulikovskiy at the Herbarium of the Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia.Type locality.Russia, Vydrinnaja River, sample no.51.1, periphyton (51 • 29.383 N 104 • 50.986E), collected by M. Kulikovskiy, 20.07.2011.

Table 5 .
Comparison of morphological features of P. siberiosinistra sp.nov.and related species.
delineated from the new species by the shape of the valve ends: subcapitate in P. baicalodivergens sp.nov. vs. rostrate in P. submicrostauron.

Table 6 .
Comparison of morphological features of P. baicalodivergens sp.nov.and related species. P.

Table 6 .
Comparison of morphological features of P. baicalodivergens sp.nov.and related species.
11.2, deposited in herbarium of MHA, Main Botanical Garden, Russian Academy of Science, Moscow, Russia.Isotype.Slide no.18997a, Collection of Maxim Kulikovskiy at the Herbarium of the Institute of Plant Physiology, Russian Academy of Science, Moscow, Russia.

Table 7 .
Comparison of morphological features of P. baicalislandica sp.nov.and related species.

Table 8 .
Correlation of morphological features in phylogenetic clades and subclades of Pinnularia-Caloneis.
* Unique features highlighted in bold.** There are exceptions.

Table 9 .
List of strains examined in this study, with their GenBank accession numbers.Geographic locality of samples and measured ecological parameters are indicated.