Description of Encyonemataceae fam. nov. and Witkowskiaceae fam. nov. (Bacillariophyceae, Cymbellales) based on molecular and morphological analyses

Description of Encyonemataceae fam. nov. and Witkowskiaceae fam. nov. 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(Bacillariophyceae, Cymbellales) based on molecular and morphological analyses Andrei Mironov, Yevhen Maltsev, John Patrick Kociolek, Maxim Kulikovskiy This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4764030/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted 16 You are reading this latest preprint version Abstract In this study, based on the results of molecular analysis of SSU rRNA and rbc L sequences, we propose the descriptions of two new families in the order Cymbellales. Molecular data demonstrates that diatoms of the genus Encyonema constitute an independent monophyletic clade, which represents a new family described herein – Encyonemataceae fam. nov. Another family introduced in this article, with regards to molecular data, is Witkowskiaceae fam. nov. In our research, it is comprised of three closely related genera – Geissleria , Paraplaconeis and Witkowskia . The results of performed molecular investigation are supported by morphological analysis. Morphological diagnoses of the new families are based on a combination of features: type of valve symmetry, raphe structure, presence and position of stigmata or stigmoids, number and location of apical pore fields and morphology of chloroplast. In addition, we discuss the phylogeny of selected genera of the order Cymbellales. In addition, based on their morphology, 3 and 5 genera are assigned with provisionary position in Encyonemataceae fam. nov. and Witkowskiaceae fam. nov., respectively. Biological sciences/Evolution Biological sciences/Genetics Biological sciences/Microbiology Biological sciences/Molecular biology Biological sciences/Plant sciences Diatoms new families Encyonemataceae Witkowskiaceae molecular phylogenetics morphology Figures Figure 1 Figure 2 Introduction Cymbellales D.G. Mann is a widely represented taxon of pennate biraphid diatoms. The order was originally described by Round et al. [ 1 ] to include diatoms with different kinds of valves – cymbelloid (apically asymmetric), gomphonemoid (transpically asymmetric), naviculoid (apically and transapically symmetric) and rhoicosphenioid (flexed in girdle view). In the proposed system, Cymbellales was represented by four families – Anomoeoneidaceae D.G. Mann, Rhoicospheniaceae J.Y. Chen and H.Z. Zhu, Cymbellaceae Kützing and Gomphonemataceae Kützing. Two latter families, which have been described nearly two centuries ago [ 2 ], nowadays subsume the majority of species and genera of the order – around two thousand taxa [ 3 ]. However, taxonomic competence of Cymbellaceae and Gomphonemataceae were not widely supported throughout the XIX and XX centuries. In particular, many diatomists questioned the concept of valve symmetry being a sufficient taxonomic feature for family-level identification. Hence, diatoms with cymbelloid or gomphonemoid types of valve symmetry were consistently included in the catch-all genus Navicula Bory s.l. [ 4 ] or in the family Naviculaceae Kützing [ 5 – 7 ]. Others, e.g., Patrick and Reimer [ 8 ], distinguished Cymbellaceae and Gomphonemataceae (under the incorrect name “Gomphonemaceae”) from Naviculaceae. The perception changed when Krammer [ 9 ] revised the morphology of Cymbella Agardh s.l. In his study, Krammer listed such features as raphe organization, fissure orientation, areolae structure and presence or position of apical pore fields (APFs), stigmata and stigmoids as essential for identification of cymbelloid and gomphonemoid diatoms. In the years to come, a lot of new genera have been described in both groups, and the original list of taxa, belonging to Cymbellaceae and Gomphonemataceae (sensu Round et al. [ 1 ]), has significantly expanded. In particular, in Round et al. [ 1 ] Cymbellaceae was represented by 5 genera: Placoneis Mereschkowsky, Cymbella, Brebissonia Grunow, Encyonema Kützing and Gomphocymbella O. Müller, while Gomphonemataceae was comprised by 4 genera: Didymosphenia Schmidt, Gomphoneis Cleve, Gomphonema Ehrenberg, Gomphopleura Reichelt ex Tempère and Reimeria J.P. Kociolek and Stoermer [ 1 ]. To date, Cymbellaceae constitutes a total of 29 genera and Gomphonemataceae includes 9 genera. Several genera, for example Crucicostulifera Taylor and Lange-Bertalot, Gomphonella Rabenhorst, Rexlowea Kociolek and Thomas, Khursevichia Kulikovskiy, Lange-Bertalot and Metzeltin, Skvortzowia Kulikovskiy, Lange-Bertalot and Metzeltin, Ochigma Kulikovskiy, Lange-Bertalot and Metzeltin, were described with postulating of close relationships with cymbelloid or gomphonemoid taxa but were not included in either of the families [ 10 – 12 ]. It is important to mention that most of the taxa in Cymbellales were described based solely on morphology. Moreover, diatomists made attempts to propose new systems of the order relying on an insufficient set of selected morphological features of the valve and protoplast, e.g., type of valve symmetry and morphology of chloroplast [ 13 , 14 ]. However, classic system of Round et al. [ 1 ], as well as “alternative” systems, proposed by Cox [ 13 ] and Kulikovskiy et al. [ 14 ] were poorly supported by phylogeny. For example, close relationships between species and genera were often refuted with the help of cladistics and molecular analysis. For example, Mann [ 15 ], raised the idea of Cymbella s.l. being polyphyletic after revealing the crucial role of Voigt discontinuities in phylogeny. Later, cladistic analysis was applied by Kociolek and Stoermer [ 16 ] to suggest close relationships between Cymbella and Didymosphenia , which was formerly considered to be a member of Gomphonemataceae [ 1 ]. Didymosphenia , was once again subsumed in Cymbellaceae by Kermarrec et al. [ 17 ] based on the results of SSU rDNA sequencing. Furthermore, multigene molecular studies [ 18 , 19 ] demonstrated that Placoneis and Geissleria Lange-Bertalot and Metzeltin both belong to Cymbellales despite their naviculoid symmetry. Thereby, family- and level classification of Cymbellales, has become obsolete due to largest genera (e.g., Cymbella , Cymbopleura (Krammer) Krammer, Gomphonema , and Witkowskia Kulikovskiy, Mironov, Glushchenko and Kociolek) being indeed para- or polyphyletic [ 19 – 26 ]. Thus, since the introduction of the order Cymbellales [ 1 ], taxonomists have been using morphological and molecular data to describe dozens of new genera and hundreds of new species within this order, but no improvements have been made to the family-level classification of Cymbellales. Two most species-rich genera Cymbella and Gomphonema [ 3 ], for instance, are included in Cymbellaceae and Gomphonemataceae, respectively [ 1 , 13 ], while other large genera Encyonema and Witkowskia are repeatedly treated as the members of Cymbellaceae [ 1 , 14 ] or Gomphonemataceae [ 13 ] in different systems. Because of that, currently accepted descriptions of Cymbellaceae and Gomphonemataceae are not precise and contain multiple exceptions, e.g. type of valve symmetry, structure of the raphe and APFs, morphology of pore occlusions. To solve this problem, we propose the descriptions of two new families of the order Cymbellales – Encyonemataceae Kulikovskiy, Mironov, Maltsev and Kociolek fam. nov. and Witkowskiaceae Kulikovskiy, Mironov, Maltsev and Kociolek fam. nov. in accordance with previously acquired molecular data and results of the new two-gene (SSU rRNA and rbc L) molecular analysis. In order to prevent further taxonomic intrications, we complement the molecular data with morphological diagnoses of both families and discuss their characteristic features. Representatives of Encyonemataceae fam. nov. and Witkowskiaceae fam. nov. can be distinguished from other diatoms of the order by a combination of morphological features: type of valve symmetry, structure of the raphe, raphe fissures orientation, presence or absence of stigmata and stigmoids, position and number of APFs, morphology of chloroplast. An important role of these characters in diatom systematics has been previously highlighted in several studies [ 1 , 13 , 16 , 27 , 28 ] and is upheld in this paper. Results Molecular analysis The phylogeny of Cymbellales is presented in Figure 1. As demonstrated, Cymbellales is a monophyletic order consisting of three monophyletic clades, each supported by maximum likelihood analysis. The three clades correspond to three families of the order: Gomphonemataceae, Encyonemataceae fam. nov. and Witkowskiaceae fam. nov. Gomphonemataceae is constituted by a monophyletic clade with Gomphonema and 4 closely related genera – Gomphonella , Reimeria , Gomphoneis and Gomphadelpha R.Jahn and N.Abarca, each, apart from Gomphoneis , being monophyletic with high statistical support (likelihood bootstrap, LB>71). Based on our data, Gomphonemataceae is most closely related to a clade constituted by Witkowskia , Paraplaconeis Kulikovskiy, Lange-Bertalot and Metzeltin and Geissleria . This relationship however, is unsupported. On the contrary, the “ Witkowskia + Paraplaconeis + Geissleria ” clade is strongly supported (LB=94), which is therefore is described as an independent family, Witkowskiaceae fam. nov., herein. The next clade, comprised of Encyonema species is also strongly supported (LB=70), but its relationship to other clades of the order Cymbellales is uncertain (LB<50). On the basis of molecular data, we suggest erecting Encyonemataceae fam. nov. from this clade. The phylogeny of remaining taxa of the order Cymbellales is confusing. The fourth, paraphyletic clade of Cymbella , Cymbopleura , Didymosphenia , Encyonopsis Krammer and Karthickia Kociolek, Glushchenko and Kulikovskiy (“CCDEK”) is unsupported, and Cymbella and Cymbopleura are polyphyletic within this clade. Descriptions of new families Given the strong support for the lineage containing Enyconema species as a distinct group, and for those genera with naviculoid symmetry, we propose two new families for each of these lineages within the Cymbellales: Encyonemataceae fam. nov. and Witkowskiaceae fam. nov., subsuming 4 genera in them based on the results of molecular analysis. 8 genera are allocated among the newly described families as provisional members, as their taxonomic positions have not been confirmed by molecular data, yet. These allocations are supported by the results of morphological analysis, discussed below. Witkowskiaceae Kulikovskiy, Mironov, Maltsev and Kociolek fam. nov. Subordinate taxa: Geissleria , Paraplaconeis , Witkowskia . Provisional taxa: Chudaevia Kulikovskiy, Mironov, Glushchenko and Kociolek, Khursevichia , Ochigma , Placoneis , Rexlowea . Description: Cells solitary. Valves isopolar, apically symmetrical or rarely moderately dorsiventral. A single chloroplast is H-shaped, formed by two X-shaped plates connected by an isthmus. Central portion of the chloroplast aligned with the apical axis, two pairs of lobes extend under the valve surface, appressed against the valve mantle. Pyrenoid located in the central region of the valve. Two libroplasts lie near the valve ends. Raphe usually filiform, sometimes lateral. Proximal raphe ends generally expanded and slightly deflected to the primary valve side. Distal ends dislocated to the same side or to the opposite sides of the valve. In the latter case, one of the fissures can recurve to form a hook-like ending, while the other fissure is simply curved (typical for some Witkowskia species). Striae uniseriate, sometimes biseriate. Areolae externally round or oval, internally round to square. Areolae openings occluded by tectula s.l. (i.e. tectula, paratectula, pseudotectula, etc.). One or more stigmoids may be located at the central area, often very close to striae. Stigmoids absent, single or numerous, located on the one or both valve sides. External and internal openings of stigmoids round or oval, generally without projections. APFs absent. Synapomorphic features for this family within the Cymbellales is the secondarily-derived symmetrical valves. Encyonemataceae Kulikovskiy, Mironov, Maltsev and Kociolek fam. nov. Subordinate taxa: Encyonema . Provisional taxa: Cymbellopsis Krammer, Kurtkrammeria Bahls, Pseudencyonema Krammer. Description: Cells solitary or colonial, growing in mucilage tubes. Valves isopolar, more or less dorsiventral. A single chloroplast consists of two H-shaped plates, similar to chloroplast of Cymbellaceae, but with a broad isthmus dislocated towards the ventral side of the valve (in case of conspicuous dorsiventrality). Pyrenoid is round, large, lies in the isthmus against the girdle. Raphe lies along or near the middle part of the valve. Raphe filiform to lateral. Proximal raphe endings expanded, dorsally bent in species with apical asymmetry, or, otherwise, practically straight. Distal raphe ends ventrally curved (“ Encyonema ”-type raphe sensu Krammer, 1982). Striae uniseriate. Areolae openings of different shape, from round to slit-like. Internally, areolae are occluded by volae or foricula. One or more stigmoids may be located dorsally, beside the central area. Externally, openings of stigmoids are round or oval, internally – slit-like, inner surface not convoluted or with minute projections. In dorsiventral species Voigt discontinuities are located on the ventral side of the valve. APFs absent in all genera except Kurtkrammeria , a provisional member of the family (several species possess groups of smaller areolae at the apices which may be treated as porelli of the APFs). Synapomorphic features for this lineage are the features associated with the position of the stigma, deflection of the external distal raphe ends, and the arrangement of the nucleus and pyrenoid in relationship to the symmetry of the valves. Discussion The modern concept of species and genera in diatom systematics implies that infra-familiar taxa should be distinguishe­­d from each other by a combination of morphological features or by unique characters of the valve. It is traditionally considered that ultrastructural characters of the diatom frustule, e.g., the structure of pore occlusions, should be stable in monophyletic groups [27, 28]. This approach is adopted in our study, as well. Moreover, morphological data serves as supportive evidence for phylogenetic reconstruction in case of genera with no molecular data available. Additionaly, morphological analysis is applied to highlight the differences between the new families, as well as Cymbellaceae and Gomphonemataceae. The results of morphological comparison are presented in Table 1. Valve symmetry and raphe structure The newly proposed families can be distinguished by a combination of essential characters of the frustule. However, as the taxonomic concept of diatoms transformes, phylogenetic importance of some features reduces. For example, valve symmetry (cymbelloid, gomphonemoid or naviculoid) originally had an important role in the systematics of Cymbellales [8]. Nowadays, the significance of this feature has decreased, and in the current study the type of valve symmetry is not specific among the families of Cymbellales. In particular, Cymbellaceae and Gomphonemataceae, according to the traditional taxonomic beliefs [1, 16] are both comprised of taxa with cymbelloid and gomphonemoid valves. Nevertheless, as suggested in the current study, valve outlines among the diatoms of Witkowskiaceae fam. nov. are symmetric about both apical and transapical axes, e.g. Geissleria ([29]: fig. 31), Chudaevia ([30]: figs 6, 9), Placoneis ([30]: fig. 3) Paraplaconeis ([11]: figs 133–135), Ochigma ([11]: figs 139–140), Khursevichia ([11]: fig. 63) and Witkowskia ([22]: fig. 8), while Encyonemataceae fam. nov. includes genera specifically with apical asymmetry expressed to a greater or lesser. However, valve symmetry can serve as a distinctive feature for high-level taxonomy, if analyzed in conjunction with other essential and robust morphological features of the valve, e.g. raphe organization. Thus, for morphological analysis of Cymbellales, we bound the type of valve symmetry with location of raphe fissures and, consequently, Voigt discontinuities (VDs). In this case, our suggestions correspond to Mann’s [15] ideas on the homology of valve sides of the diatoms of Cymbellales. As he stated, in Cymbella sensu stricto. VDs are situated dorsally, while in Encyonema these structures are always located ventrally [15]. Hence, species with different location of Voigt discontinuities should belong to separate taxonomic groups. This principle is obeyed herein: if dorsiventrality is evident, the discontinuities are dorsal in Cymbellaceae and ventral in Encyonemataceae fam. nov. Distal raphe fissures are orientated analogously, which corresponds to Krammer’s classification [9], differentiating “ Cymbella ”-type (Figure 2A) and “ Encyonema ”-type (Figure 2B) raphes. As a result, in Encyonemataceae fam. nov. proximal raphe fissures are dorsal, distal – ventral; ventral side with VDs. In Cymbellaceae, on the contrary, proximal fissures are ventral, distal – dorsal; VDs dorsal (Figure 2A, B). Diatoms of Witkowskiaceae fam. nov. possess symmetric valves, but, nevertheless, are equipped with raphes, typical for the order Cymbellales, i.e. distal fissures are unilaterally deflected in relation to each other, but oppositely to the proximal fissures. Additionally, some species of Witkowskia and Paraplaconeis have heteromorphic distal fissures (one is simply curved, another – recurved in the same direction at the valve margin) (Figure 2C; [11, 30, 31]). Pore occlusions Another feature, hereby demonstrated as non-family-specific is the structure of pore occlusions. Mann [27] suggested that pore occlusions should be morphologically similar in closely related taxa that comprise monophyletic groups. Mann’s principle was later adopted by Cox [28], who made an attempt to improve the classification of pore occlusions solving the confusion with the use of the term “vola”. Additionally, she described tectulum as typical for Placoneis and foricula as a type of areolae, common for several species-rich genera – Cymbella , Cymbopleura , Encyonema and Gomphonema . Cox [28] stated that foricula are variable among cymbelloid taxa – unilateral in Gomphonema , symmetrical in Cymbella sensu stricto, uneven in Cymbella delicatula Kützing (= Delicatophycus delicatulus (Kützing) M.J. Wynne), round in Reimeria and dendritic in Didymosphenia . Latest investigations, involving SEM studies, demonstrated that the diversity of pore occlusions among diatoms of Cymbellales is greater than estimated before [30]. In this article, the structure of pore occlusions is variable among the genera, belonging to the same family. The structure of pore occlusions in Encyonemataceae fam. nov. seems to be unstable: the provisionary members of this family – Cymbellopsis and Pseudencyonema – have polymorphic areolar openings with volate occlusions [32, 33], while areolae of Kurtkrammeria , another provisionary member of Encyonemataceae fam. nov., might be occluded by simple hymens [34]. Morphologically, pore occlusions in Witkowskiaceae fam. nov. are more conservative and, in general, can be described as tectula sensu lato [30]. Hence, pore occlusions of the diatoms, belonging to Cymbellales demonstrate a high level of polymorphism. Because of that, monophyletic groups in this order might not be inevitably characterized by a similar type of pore occlusions. In the same way, similarities in the structure of areolar occlusions do not always indicate close phylogenetic relationships among separate taxa. It is possible that evolutionary course of pore occlusions in the order Cymbellales is complicated, i.e. various types of occlusions erected independently in different groups on several occasions. Consequently, the morphology of pore occlusions, as a distinctive feature, should be applied for species- and genus-level taxonomy, rather than identification of families. Stigmata, stigmoids and apical pore fields Another important feature of the frustule that should be taken into account during separation of the families in the order Cymbellales, is position of stigmata and stigmoids. With this feature, the terminology is confusing. Krammer [9], understood stigmata as isolated pores, morphologically distinguishable from areolae in striae, with slit-like, convoluted internal openings. Stigmoids, on the other hand, are barely isolated from areolae and similar to them in external morphology. Internally, the openings of stigmoids are slit-like, with unconvoluted surface. However, diatomists tend to confuse these features and some authors use an unclear term “pseudostigmoid” [35, 36]. Bahls [34], described Kurtkrammeria as a genus with stigmata, but his SEM microphotographs show typical stigmoids – with inner openings, not equipped with any projections (34]: figs 46, 86, 104). In addition, new types of stigmoids and stigmata were designated, e.g., stigmata with two slit-like internal openings were discovered in Oricymba and Karthickia [37, 38]. To make this feature suitable for taxonomic use, an improved classification of stigmata and stigmoids is desperately required. As no kind of comprehensive classification has been adopted since Krammer’s [9] revision of Cymbella , the understanding of stigmata and stigmoids in this paper matches his definitions. In our system, both the structure and position of stigmata and stigmoids are treated as valuable taxonomic features. Thus, according to our description, diatoms of Encyonemataceae fam. nov., e.g., Kurtkrammeria , sometimes possess stigmoids, that are invariably situated dorsally [34]. In Cymbellopsis and Pseudencyonema stigmoids are absent [32, 33, 39]. In Witkowskiaceae fam. nov. (and genera with provisionary position in this family) stigmoids are present in only four genera, closely related to each other – Chudaevia , Geissleria , Placoneis and Witkowskia . In some species with multiple stigmoids they are positioned to the both sides from the central nodule [20, 30, 40]. In addition, the newly erected families are similar to each other by the absence of the APFs but, at the same time, can be distinguished from Cymbellaceae and Gomphonemataceae based on this feature. An exception, which seems to be of minor importance, is the genus Kurtkrammeria with APFs at both poles [34]. Some species of this taxon possess weakly developed APFs. Porelli are very similar to the areolae of striae, which indicates the primitivity of the APFs. Chloroplasts The structure of chloroplast in cymbelloid diatoms was originally described by Mereschkowsky [41]. He proposed an idea of using the morphology of chloroplast to distinguish high-level taxa from each other. In his system, diatoms, currently regarded as the members of the order Cymbellales, were placed in “Monoplacatae” due to the presence of a single chloroplast. Both Geitler [42] and Cox [43] stated that position of chloroplast is stable at the generic level in Cymbella and Encyonema . In both taxa, the chloroplast is H-shaped, valve-appressed and connected by a broad bridge (isthmus). The bridge is dorsal in Cymbella and ventral in Encyonema [1, 43], which is why diatoms Encyonemataceae fam. nov. can be distinguished from Cymbellaceae by the ventrally located isthmuses. Diatoms of Witkowskiaceae fam. nov. are mostly apically-symmetric and have H-shaped chloroplasts with isthmuses offset to secondary side [30]. As only a few genera in Cymbellales have their chloroplasts studied, it is impractical to use the morphology and position of chloroplasts to distinguish the genera of this order from each other. But, because the chloroplasts are well studied in the most species-rich genera of Cymbellales – Cymbella , Encyonema , Gomphonema [16, 42] and Witkowskia [21, 22, 40] in order to separate the families of the order Cymbellales, chloroplast morphology should be examined along with features of the valve. Table 1. Comparison of Encyonemataceae fam. nov. and Witkowskiaceae fam. nov. to Cymbellaceae and Gomphonemataceae. Feature of morphology Encyonemataceae fam. nov. Witkowskiaceae fam. nov. Cymbellaceae Gomphonemataceae Valve symmetry Apically asymmetric Apically and transapically symmetric Apically asymmetric, rarely transapically asymmetric ( Didymoshenia ) Transapically asymmetric, rarely apically asymmetric ( Afrocymbella , Reimeria ) Raphe organization “ Encyonema ”-type (proximal fissures dorsal, distal fissures ventral) Proximal fissures straight or unilaterally deflected. Distal fissures unilaterally deflected (and opposite in relation to proximal fissures), sometimes heteromorphic (one of them with a recurvature) “ Cymbella ”-type (proximal fissures ventral, distal fissures dorsal) Proximal and distal fissures straight or unilaterally deflected Location of VDs Ventral valve side Secondary valve side Dorsal valve side Secondary valve side (=ventral in dorsiventral taxa) Pore occlusions Irregular, volae-like or hymens Tectula sensu lato (tectula, paratectula, pseudotectula, etc.) Symmetrical or asymmetrical foricula, volae Unilateral or asymmetric foricula Stigmata and stigmoids Dorsal stigmoid(-s) Stigmoid(-s). If multiple, at one or both sides of the central area Ventral stigma(-ta) Stigma(-ta) or stigmoid(-s), dorsal in dorsiventral species APFs Absent Absent Present, typically at both poles Present, typically at one, narrower pole (=footpole) Chloroplasts With ventrally offset isthmus With isthmus offset to the secondary valve side With dorsally offset isthmus With isthmus offset to the secondary valve side Reference [32–34, 43] [11, 30, 31, 40, 43] [9, 43, 44] [43, 45] Conclusions Our molecular and morphological investigations highlight the differences between high-level taxonomic groups within the order Cymbellales. Based on the results of rbc L and SSU rRNA analysis, we provide the descriptions of two new families. Taxonomic innovations are congruent with the results of the comprehensive morphological analysis. According to our study, the new families are distinguished from each other (and from other closely related taxa) based on the combination of features of the valves and living cells, e.g. type of valve symmetry, raphe organization, presence and position of the apical pore fields and chloroplast morphology. Features analyzed above are robust among the genera of the order Cymbellales and thus are suitable for separation of the families within the studied group. At the same time, we demonstrate that the results of morphological analysis should serve as supplementary evidence for molecular data, primarily because even the essential features may differ significantly in closely related groups. Materials and methods The technique of molecular analysis follows Mironov et al. [ 30 ] and Glushchenko et al. [ 46 ], as the same sequences were used in both studies. The dataset was comprised of 82 SSU rDNA, and 85 rbc L concatenated sequences, selected for available Cymbellales lineages and five diatom species from the family Rhopalodiaceae Topachevskyj and Oksiyuk chosen as the outgroup (taxa names and Accession Numbers are provided in Fig. 1 ). The obtained sequences of SSU rDNA and rbc L genes were aligned independently with the help of G-INS-I algorithm in the Mafft ver. 7 software (RIMD, Osaka, Japan) [ 47 ]. The consequent dataset included 1,731, and 1,401 nucleotide sites for nuclear SSU rDNA, and plastid rbc L regions, respectively. Unpaired regions were subsequently removed, the aligned SSU rRNA gene sequences were combined with the rbc L gene sequences into a single matrix. The Bayesian inference (BI) method was performed in Beast ver. 1.10.1 software (BEAST Developers, Auckland, New Zealand) [ 48 ]. Best matching partition-specific substitution models, shape parameter α and a proportion of invariable sites (pinvar) were established using the Bayesian information criterion (BIC) in jModelTest ver. 2.1.10 software (Vigo, Spain) [ 49 ]. Thus, the following criteria were selected: GTR + G + I, α = 0,4710 and pinvar = 0,5970 for SSU rDNA; TPM1uf + G + I, α = 0,3960, and pinvar = 0,7310 for the first codon position of the rbc L gene; JC + I, pinvar = 0,8690 for the second codon position of the rbc L gene; GTR + G + I, α = 1,1260, and pinvar = 0,2320 for the third codon position of the rbc L gene. Herewith, TPM1uf model was substituted with HKY, and JC – with F81, as the most similar applicable options for BI. Yule process tree prior was applied to perform a speciation model. Five MCMC analyses were run for 5 million generations (burn-in 1,000 million generations). Tracer ver. 1.7.1 software (MCMC Trace Analysis Tool, Edinburgh, United Kingdom) [ 48 ] was implemented for convergence diagnostics. The initial 15% trees were subsequently removed, the rest retained to construct a final chronogram with 90% posterior probabilities (PP). Tree topologies-robustness was evaluated by boot-strapping the dataset with Maximum Likelihood (ML) analysis (with 1,000 replicas) in RaxML software [ 50 ]. FigTree ver. 1.4.4 (University of Edinburgh, Edinburgh, United Kingdom) and Adobe Photoshop CC ver. 19.0 software were used for editing and viewing of acquired trees at the final stage. Terminology of the valve, used in morphological analysis, follows Krammer [ 9 , 44 ]. Terminology of pore occlusions corresponds to Cox [ 28 ] and Mironov et al. [ 30 ]. Declarations Conflict of interest The authors declare that they have no conflict of interest. Funding This publication is based on research carried out with financial support by the Russian Science Foundation (24-14-00165) for LM and SEM and by the framework of state assignment of the Ministry of Science and Higher Education of the Russian Federation (theme 122042700045-3) for finishing manuscript. Author Contribution Conceptualization, A.M. and M.K.; methodology, Y.M. and M.K.; validation, J.P.K and M.K.; formal analysis, A.M., J.P.K and M.K.; investigation, A.M. and Y.M.; resources, Y.M. and M.K.; data curation, M.K.; writing—original draft preparation, A.M.; writing—review and editing, J.P.K. and M.K.; visualization, Y.M.; supervision, M.K.; project administration, M.K.; funding acquisition, Y.M. and M.K. All authors have read and agreed to the published version of the manuscript. Data Availability Correspondence and requests for materials should be addressed to A. Mironov References Round, F.E., Crawford, R.M. & Mann, D.G. The Diatoms. 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Identification book of diatoms from Russia (Filigran, Yaroslavl, Russia, 2016). Mann, D.G. An ontogenetic approach to diatom systematics in Proceedings of the 7th Symposium on Recent and Fossil Diatoms (ed. Mann, D.G.) 113–144 (O. Koeltz, Koenigstein, Germany, 1984). Kociolek, J.P. & Stoermer, E.F. A preliminary investigation of the phylogenetic relationships among the freshwater, apical pore field-bearing cymbelloid and gomphonemoid diatoms (Bacillariophyceae). Journal of Phycology 24 , 377–385 (1988). Kermarrec, L., Ector, L., Bouchez, A., Rimet, F. & Hoffmann, L. A preliminary phylogenetic analysis of the Cymbellales based on 18S rDNA gene sequencing. Diatom Research 26(3) , 305–315 (2011). http://dx.doi.org/10.1080/0269249x.2011.633255 Bruder, K. & Medlin, L.K. Molecular assessment of phylogenetic relationships in selected species/genera in the naviculoid diatoms (Bacillariophyta). I. The genus Placoneis . Nova Hedwigia 85 , 331–352 (2007). http://dx.doi.org/10.1127/0029-5035/2007/0085-0331. Nakov, T., Ruck, E.C., Galachyants, Y., Spaulding, S.A. & Theriot, E.C. Molecular phylogeny of the Cymbellales (Bacillariophyceae, Heterokontophyta) with a comparison of models for accommodating rate variation across sites. Phycologia 53 , 359–373 (2014). Kulikovskiy, M., Gusev, E., Andreeva, S. & Annenkova, N. Phylogenetic position of the diatom genus Geissleria Lange-Bertalot and Metzeltin and description of two new species from Siberian mountain lakes. Phytotaxa 177(5) , 249–260 (2014). http://dx.doi.org/10.11646/phytotaxa.177.5.1. Kezlya, E., Glushchenko, A., Maltsev, Y., Gusev, E., Genkal, S., Kuznetsov, A., Kociolek, J.P. & Kulikovskiy, M. Placoneis cattiensis sp. nov.– a new diatom (Bacillariophyceae: Cymbellales) soil species from Cát Tiên National Park (Vietnam). Phytotaxa 460(4) , 237–248 (2020). http://dx.doi.org/10.11646/phytotaxa.460.4.1. Kezlya, E.; Glushchenko, A.; Maltsev, Y.; Gusev, E.; Genkal, S.; Kociolek, J.P.; Kulikovskiy, M. Three New Species of Placoneis Mereschkowsky (Bacillariophyceae: Cymbellales) with Comments on Cryptic Diversity in the P. elginensis —Group. Water 13 , 1–21 (2021). http://dx.doi.org/10.3390/w13223276. Kezlya, E., Glushchenko, A., Kociolek, J.P., Maltsev, Y., Genkal, S. & Kulikovskiy, M. A new species of Placoneis Mereschkowsky (Bacillariophyceae: Cymbellales) from wet soils in southern Vietnam. Cryptogamie, Algologie 43(11) , 177–188 (2022). http://dx.doi.org/10.5252/cryptogamie-algologie2022v43a11. Glushchenko, A.M., Maltsev, Y.I., Kociolek, J.P., Kuznetsova, I.V. & Kulikovskiy, M.S Molecular and morphological investigations of two giant diatom Cymbella species from the Transbaikal Area (Russia, Siberia) with comments on their distributions. Plants 11(2445) , 1–17 (2022). Yana, E., Nakkaew, S., Pekkoh, J., Peerapornpisal, Y., Tuji, A., Davis, M., Julius, M. & Mayama, S. Valve and ‘stigma’ structure and phylogeny of an enigmatic cymbelloid diatom Karthickia verestigmata Glushchenko, Kulikovskiy and Kociolek. Diatom Research 37 , 1–13 (2022). https://doi.org/10.1080/0269249X.2022.2101545 Abarca, N., Stancheva, R., Skibbe, O., Schimani, K., Kusber, W.H., Zimmermann, J. & Jahn, R. Gomphadelpha (Bacillariophyceae) - a new genus name for taxa formerly subsumed in the Gomphoneis heculeana -group. Nova Hedwigia, Beihefte 117(1–4) , 213–254 (2023). Mann, D.G. Sieves and flaps: siliceous minutiae in the pores of raphid diatoms. Proceedings of the 6th Symposium on recent and fossil diatoms, Budapest, Hungary, 1–5 September 1980 279–300 (Koeltz Sci. Books, Königstein, Germany, 1981). Cox, E.J. Pore occlusions in raphid diatoms — a reassessment of their structure and terminology, with particular reference to members of the Cymbellales. Diatom 20 , 33–46 (2004). https://doi.org/10.11464/diatom1985.20.0_33. Lange-Bertalot, H. &Metzeltin, D. Indicators of oligotrophy. 800 taxa representative of three ecologically distinct lake types, carbonate buffered-Oligodystrophic-weakly buffered soft water with 2428 figures on 125 plates. Oligotrophie-Indikatoren. Iconographia Diatomologica 2 , 1–390 (1996). Mironov, A., Glushchenko, A., Maltsev, Ye., Genkal, S., Kuznetsova, I., Kociolek, J.P., Liu, Y. & Kulikovskiy, M. Reassessment of pore occlusion in some diatom taxa with re-evaluation of Placoneis Mereschkowsky (Bacillariophyceae: Cymbellales) and description of two new genera. PeerJ 12 , e17278 (2024). http://dx.doi.org/10.7717/peerj.17278. Cox, E.J. Placoneis Mereschkowsky (Bacillariophyta) revisited: resolution of several typification and nomenclatural problems, including the generitype. Botanical Journal of the Linnean Society 141 , 53–83 (2003). Krammer, K. Die cymbelloiden Diatomeen. Eine Monographie der weltweit bekannten Taxa. Teil 1. Allgemeines und Encyonema Part. Bibliotheca Diatomologica 36 , 1–382 (1997). Krammer, K. Die cymbelloiden Diatomeen. Ein Monographie der weltweit bekannten Taxa. Teil 2. Encyonema , Encyonopsis and Cymbellopsis . Bibliotheca Diatomologica 37 , 1–463 (1997). Bahls, L.L. Kurtkrammeria , a new genus of freshwater diatoms (Bacillariophyta, Cymbellaceae) separated from Encyonopsis . Nova Hedwigia 101(1/2) , 165–190 (2015). Le Cohu, R., Marquié, J. & Tudesque, L. Three new species of Delicatophycus M.J. Wynne (Gomphonemataceae, Bacillariophyta) from New Caledonia. Notulae algarum 98 , 1–2 (2018). Liu, B., Zhou, Y.-Y., Blanco, S. & Williams, D.M. Three new species of Delicatophycus M.J. Wynne (Bacillariophyta) from China, all possessing apical pore fields. Fottea 22 , 137–151 (2022). Jüttner, I., Krammer, K., Van de Vijver, B., Tuji, A., Simkhada, B., Gurung, S., Sharma, S., Sharma, C. & Cox, E.J. Oricymba (Cymbellales, Bacillariophyceae), a new cymbelloid genus and three new species from the Nepalese Himalaya. Phycologia 49 , 407–423 (2010). Glushchenko, A., Kuznetsova, I., Kociolek, J.P. & Kulikoviskiy, M. Karthickia verestigmata gen. et sp. nov. – an interesting diatom with frustular morphology similar to several different cymbelloid diatom genera. Phycologia 58 , 605–613 (2019). Pomazkina, G.V. & Rodionova, E.V. Diatoms of the family Cymbellaceae of Lake Baikal. Atlas and key (Nauka: Novosibirsk, Russia, 2014). Cox, E.J. Placoneis Mereschkowsky: the re-evaluation of a diatom genus originally characterized by its chloroplast type. Diatom Research 2(2) , 145–157 (1987). http://dx.doi.org/10.1080/0269249x.1987.9704994. Mereschkowsky, C. Uber Placoneis , ein neues Diatomeen-Genus. Beihefte zum Botanischen Centralblatt 15 , 1–30 (1903). Geitler, L. Die Lage des Chromatophors in Beziehung zur Systematik von Cymbella -Arten ( Bacillariophyceae ). Plant Systematics and Evolution 138 , 153–156 (1981). Cox E.J. Identification of freshwater diatoms from live material (Chapman and Hall, London, UK, 1996). Krammer, K. Cymbella in Diatoms of Europe (ed. Lange-Bertalot, H.) 1–584 (A.R.G. Gantner Verlag K.G, Ruggell, Germany, 2002). Krammer, K. Cymbopleura , Delicata , Navicymbula , Gomphocymbellopsis , Afrocymbella in Diatoms of Europe (ed. Lange-Bertalot, H.) 1–530 (A.R.G Gantner Verlag K.G., Ruggell, Germany, 2003). Glushchenko, A., Mironov, A., Kuznetsova, I. & Kulikovskiy, M. Cymbella alexandrovichii sp. nov. (Cymbellaceae, Bacillariophyceae), a new cymbelloid diatom species with predominantly biseriate striae from South-East Asia. Phytotaxa 630(2), 112–122 (2023). Katoh, K. & Toh, H. Parallelization of the MAFFT multiple sequence alignment program. Bioinformatics 26 , 1899–1900 (2010). Drummond, A.J. & Rambaut, A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7 , 214 (2007). http://dx.doi.org/10.1186/1471-2148-7-214. Darriba, D., Taboada, G.L., Doallo, R. & Posada, D. ModelTest 2: More models, new heuristics and parallel computing. Nat. Methods 9 , 772 (2012). http://dx.doi.org/10.1038/nmeth.2109. Stamatakis, A., Hoover, P. & Rougemont, J. A Rapid Bootstrap Algorithm for the rAxML Web Servers. Syst. Biol. 57 , 758–771 (2008). http://dx.doi.org/10.1080/10635150802429642. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 28 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 28 Oct, 2024 Reviews received at journal 17 Oct, 2024 Reviews received at journal 15 Oct, 2024 Reviews received at journal 13 Oct, 2024 Reviews received at journal 10 Oct, 2024 Reviewers agreed at journal 06 Oct, 2024 Reviewers agreed at journal 04 Oct, 2024 Reviewers agreed at journal 04 Oct, 2024 Reviewers agreed at journal 04 Oct, 2024 Reviews received at journal 09 Sep, 2024 Reviewers agreed at journal 01 Sep, 2024 Reviewers invited by journal 01 Sep, 2024 Editor assigned by journal 01 Sep, 2024 Editor invited by journal 07 Aug, 2024 Submission checks completed at journal 05 Aug, 2024 First submitted to journal 18 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4764030","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":346724311,"identity":"2af3e2fc-54ac-40fe-9e14-7d15f9da7320","order_by":0,"name":"Andrei Mironov","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYDCCw2BSAsL5ACIOENbC2ADTwjiDKC0HwFoggJmHGC18x5mfP/iYY5HPL918TNq2zS6P73iPAXNlG24tkofZDBtnbpOwnDnnWJp0bltyseSZYwmMZ/FoMTjMYNjMu03CwOBGjrFxbhtz4oYbyQcYG/FqYf+I0GLZVp+44f7DBgJaeOC2GD5mbDsMtIUZvy2Sh3kKZwL9YiA5Iy3xYc+544kzz6QlHGw4h1sL3/njGz583FZnwC+RfODAj7LqxL7jZwwfNpTh1oIKGNkg9AFiNQDBHxLUjoJRMApGwYgBAL0bV5fcMz15AAAAAElFTkSuQmCC","orcid":"","institution":"К.А. Timiryazev Institute of Plant Physiology RAS, IPP RAS","correspondingAuthor":true,"prefix":"","firstName":"Andrei","middleName":"","lastName":"Mironov","suffix":""},{"id":346724312,"identity":"011a6bcd-528a-4d45-b4db-37dce92a3644","order_by":1,"name":"Yevhen Maltsev","email":"","orcid":"","institution":"К.А. Timiryazev Institute of Plant Physiology RAS, IPP RAS","correspondingAuthor":false,"prefix":"","firstName":"Yevhen","middleName":"","lastName":"Maltsev","suffix":""},{"id":346724313,"identity":"cd0f1422-96de-4afa-98e0-55a3a76c7774","order_by":2,"name":"John Patrick Kociolek","email":"","orcid":"","institution":"Museum of National History, Boulder","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"Patrick","lastName":"Kociolek","suffix":""},{"id":346724314,"identity":"16864920-16ff-4b08-afb1-221bb0bfdce0","order_by":3,"name":"Maxim Kulikovskiy","email":"","orcid":"","institution":"К.А. Timiryazev Institute of Plant Physiology RAS, IPP RAS","correspondingAuthor":false,"prefix":"","firstName":"Maxim","middleName":"","lastName":"Kulikovskiy","suffix":""}],"badges":[],"createdAt":"2024-07-18 16:39:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4764030/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4764030/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-82213-0","type":"published","date":"2024-12-28T15:57:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63596543,"identity":"bbb31e05-69cd-40c0-83f5-aa089910a967","added_by":"auto","created_at":"2024-08-30 04:13:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2357737,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogeny of the order Cymbellales based on BI from an alignment of 90 sequences and 3132 characters (\u003cem\u003erbc\u003c/em\u003eL and SSU rRNA genes). Values of likelihood bootstrap (LB) from ML analyses below 50 and values of Bayesian Post Priori (PP) below 0.90 are hidden. Available strain numbers and GenBank numbers are indicated for all sequences. The designated clades are colored in red (Gomphonemataceae clade), blue (Witkowskiaceae fam. nov. clade), green (Encyonemataceae fam. nov. clade) and yellow (“CCDEK” clade).\u003c/p\u003e","description":"","filename":"Fig1mod.png","url":"https://assets-eu.researchsquare.com/files/rs-4764030/v1/a5c7c4ccb38528b0f8574aa6.png"},{"id":63596542,"identity":"6bda96db-33ba-42bd-a94c-cce11e089dda","added_by":"auto","created_at":"2024-08-30 04:13:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":883358,"visible":true,"origin":"","legend":"\u003cp\u003eThree types of raphe organization in the order Cymbellales. (\u003cstrong\u003eA)\u003c/strong\u003e “\u003cem\u003eCymbella\u003c/em\u003e”-type raphe. Proximal fissures ventrally deflected (blue arrows), distal – dorsally (red arrows). Voigt discontinuities are dorsal (yellow arrows). Note the ventral stigmata (green arrows). \u003cstrong\u003e(B)\u003c/strong\u003e “\u003cem\u003eEncyonema\u003c/em\u003e”-type raphe. Proximal fissures dorsally deflected (blue arrows), distal – ventrally (red arrows). Voigt discontinuities are ventral (yellow arrows). Note the dorsal stigmoid (green arrow). \u003cstrong\u003e(C)\u003c/strong\u003e “\u003cem\u003eWitkowskia\u003c/em\u003e”-type raphe. Proximal fissures predominantly straight (blue arrows), distal – heteromorphic (red arrows), but unilaterally deflected to the secondary side (with Vogt discontinuities (yellow arrows)). Note the stigmoids (green arrows).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4764030/v1/513559fbf8946ece9e4c5156.png"},{"id":72640618,"identity":"42bfad3b-6fad-4440-83fc-657c05b49a5a","added_by":"auto","created_at":"2024-12-30 16:07:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3191591,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4764030/v1/1046b23c-8fbf-44b5-ad53-c76826ede8a2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Description of Encyonemataceae fam. nov. and Witkowskiaceae fam. nov. (Bacillariophyceae, Cymbellales) based on molecular and morphological analyses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCymbellales D.G. Mann is a widely represented taxon of pennate biraphid diatoms. The order was originally described by Round et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] to include diatoms with different kinds of valves \u0026ndash; cymbelloid (apically asymmetric), gomphonemoid (transpically asymmetric), naviculoid (apically and transapically symmetric) and rhoicosphenioid (flexed in girdle view). In the proposed system, Cymbellales was represented by four families \u0026ndash; Anomoeoneidaceae D.G. Mann, Rhoicospheniaceae J.Y. Chen and H.Z. Zhu, Cymbellaceae K\u0026uuml;tzing and Gomphonemataceae K\u0026uuml;tzing. Two latter families, which have been described nearly two centuries ago [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], nowadays subsume the majority of species and genera of the order \u0026ndash; around two thousand taxa [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, taxonomic competence of Cymbellaceae and Gomphonemataceae were not widely supported throughout the XIX and XX centuries. In particular, many diatomists questioned the concept of valve symmetry being a sufficient taxonomic feature for family-level identification. Hence, diatoms with cymbelloid or gomphonemoid types of valve symmetry were consistently included in the catch-all genus \u003cem\u003eNavicula\u003c/em\u003e Bory s.l. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] or in the family Naviculaceae K\u0026uuml;tzing [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Others, e.g., Patrick and Reimer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], distinguished Cymbellaceae and Gomphonemataceae (under the incorrect name \u0026ldquo;Gomphonemaceae\u0026rdquo;) from Naviculaceae.\u003c/p\u003e \u003cp\u003eThe perception changed when Krammer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] revised the morphology of \u003cem\u003eCymbella\u003c/em\u003e Agardh s.l. In his study, Krammer listed such features as raphe organization, fissure orientation, areolae structure and presence or position of apical pore fields (APFs), stigmata and stigmoids as essential for identification of cymbelloid and gomphonemoid diatoms. In the years to come, a lot of new genera have been described in both groups, and the original list of taxa, belonging to Cymbellaceae and Gomphonemataceae (sensu Round et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]), has significantly expanded. In particular, in Round et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Cymbellaceae was represented by 5 genera: \u003cem\u003ePlaconeis\u003c/em\u003e Mereschkowsky, \u003cem\u003eCymbella, Brebissonia\u003c/em\u003e Grunow, \u003cem\u003eEncyonema\u003c/em\u003e K\u0026uuml;tzing and \u003cem\u003eGomphocymbella\u003c/em\u003e O. M\u0026uuml;ller, while Gomphonemataceae was comprised by 4 genera: \u003cem\u003eDidymosphenia\u003c/em\u003e Schmidt, \u003cem\u003eGomphoneis\u003c/em\u003e Cleve, \u003cem\u003eGomphonema\u003c/em\u003e Ehrenberg, \u003cem\u003eGomphopleura\u003c/em\u003e Reichelt ex Temp\u0026egrave;re and \u003cem\u003eReimeria\u003c/em\u003e J.P. Kociolek and Stoermer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. To date, Cymbellaceae constitutes a total of 29 genera and Gomphonemataceae includes 9 genera. Several genera, for example \u003cem\u003eCrucicostulifera\u003c/em\u003e Taylor and Lange-Bertalot, \u003cem\u003eGomphonella\u003c/em\u003e Rabenhorst, \u003cem\u003eRexlowea\u003c/em\u003e Kociolek and Thomas, \u003cem\u003eKhursevichia\u003c/em\u003e Kulikovskiy, Lange-Bertalot and Metzeltin, \u003cem\u003eSkvortzowia\u003c/em\u003e Kulikovskiy, Lange-Bertalot and Metzeltin, \u003cem\u003eOchigma\u003c/em\u003e Kulikovskiy, Lange-Bertalot and Metzeltin, were described with postulating of close relationships with cymbelloid or gomphonemoid taxa but were not included in either of the families [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is important to mention that most of the taxa in Cymbellales were described based solely on morphology. Moreover, diatomists made attempts to propose new systems of the order relying on an insufficient set of selected morphological features of the valve and protoplast, e.g., type of valve symmetry and morphology of chloroplast [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, classic system of Round et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], as well as \u0026ldquo;alternative\u0026rdquo; systems, proposed by Cox [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and Kulikovskiy et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] were poorly supported by phylogeny.\u003c/p\u003e \u003cp\u003eFor example, close relationships between species and genera were often refuted with the help of cladistics and molecular analysis. For example, Mann [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], raised the idea of \u003cem\u003eCymbella\u003c/em\u003e s.l. being polyphyletic after revealing the crucial role of Voigt discontinuities in phylogeny. Later, cladistic analysis was applied by Kociolek and Stoermer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] to suggest close relationships between \u003cem\u003eCymbella\u003c/em\u003e and \u003cem\u003eDidymosphenia\u003c/em\u003e, which was formerly considered to be a member of Gomphonemataceae [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eDidymosphenia\u003c/em\u003e, was once again subsumed in Cymbellaceae by Kermarrec et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] based on the results of SSU rDNA sequencing. Furthermore, multigene molecular studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] demonstrated that \u003cem\u003ePlaconeis\u003c/em\u003e and \u003cem\u003eGeissleria\u003c/em\u003e Lange-Bertalot and Metzeltin both belong to Cymbellales despite their naviculoid symmetry. Thereby, family- and level classification of Cymbellales, has become obsolete due to largest genera (e.g., \u003cem\u003eCymbella\u003c/em\u003e, \u003cem\u003eCymbopleura\u003c/em\u003e (Krammer) Krammer, \u003cem\u003eGomphonema\u003c/em\u003e, and \u003cem\u003eWitkowskia\u003c/em\u003e Kulikovskiy, Mironov, Glushchenko and Kociolek) being indeed para- or polyphyletic [\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23 CR24 CR25\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThus, since the introduction of the order Cymbellales [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], taxonomists have been using morphological and molecular data to describe dozens of new genera and hundreds of new species within this order, but no improvements have been made to the family-level classification of Cymbellales. Two most species-rich genera \u003cem\u003eCymbella\u003c/em\u003e and \u003cem\u003eGomphonema\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], for instance, are included in Cymbellaceae and Gomphonemataceae, respectively [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], while other large genera \u003cem\u003eEncyonema\u003c/em\u003e and \u003cem\u003eWitkowskia\u003c/em\u003e are repeatedly treated as the members of Cymbellaceae [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] or Gomphonemataceae [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] in different systems. Because of that, currently accepted descriptions of Cymbellaceae and Gomphonemataceae are not precise and contain multiple exceptions, e.g. type of valve symmetry, structure of the raphe and APFs, morphology of pore occlusions.\u003c/p\u003e \u003cp\u003eTo solve this problem, we propose the descriptions of two new families of the order Cymbellales \u0026ndash; Encyonemataceae Kulikovskiy, Mironov, Maltsev and Kociolek fam. nov. and Witkowskiaceae Kulikovskiy, Mironov, Maltsev and Kociolek fam. nov. in accordance with previously acquired molecular data and results of the new two-gene (SSU rRNA and \u003cem\u003erbc\u003c/em\u003eL) molecular analysis. In order to prevent further taxonomic intrications, we complement the molecular data with morphological diagnoses of both families and discuss their characteristic features. Representatives of Encyonemataceae fam. nov. and Witkowskiaceae fam. nov. can be distinguished from other diatoms of the order by a combination of morphological features: type of valve symmetry, structure of the raphe, raphe fissures orientation, presence or absence of stigmata and stigmoids, position and number of APFs, morphology of chloroplast. An important role of these characters in diatom systematics has been previously highlighted in several studies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and is upheld in this paper.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMolecular analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phylogeny of Cymbellales is presented in Figure 1. As demonstrated, Cymbellales is a monophyletic order consisting of three monophyletic clades, each supported by maximum likelihood analysis. The three clades correspond to three families of the order: Gomphonemataceae, Encyonemataceae fam. nov. and Witkowskiaceae fam. nov. Gomphonemataceae is constituted by a monophyletic clade with \u003cem\u003eGomphonema\u003c/em\u003e and 4 closely related genera \u0026ndash; \u003cem\u003eGomphonella\u003c/em\u003e, \u003cem\u003eReimeria\u003c/em\u003e, \u003cem\u003eGomphoneis\u003c/em\u003e and \u003cem\u003eGomphadelpha\u003c/em\u003e R.Jahn and N.Abarca, each, apart from \u003cem\u003eGomphoneis\u003c/em\u003e, being monophyletic with high statistical support (likelihood bootstrap, LB\u0026gt;71). Based on our data, Gomphonemataceae is most closely related to a clade constituted by \u003cem\u003eWitkowskia\u003c/em\u003e, \u003cem\u003eParaplaconeis\u003c/em\u003e Kulikovskiy, Lange-Bertalot and Metzeltin and \u003cem\u003eGeissleria\u003c/em\u003e. This relationship however, is unsupported. On the contrary, the \u0026ldquo;\u003cem\u003eWitkowskia\u003c/em\u003e+\u003cem\u003eParaplaconeis\u003c/em\u003e+\u003cem\u003eGeissleria\u003c/em\u003e\u0026rdquo; clade is strongly supported (LB=94), which is therefore is described as an independent family, Witkowskiaceae fam. nov., herein. The next clade, comprised of \u003cem\u003eEncyonema\u003c/em\u003e species is also strongly supported (LB=70), but its relationship to other clades of the order Cymbellales is uncertain (LB\u0026lt;50). On the basis of molecular data, we suggest erecting Encyonemataceae fam. nov. from this clade. The phylogeny of remaining taxa of the order Cymbellales is confusing. The fourth, paraphyletic clade of \u003cem\u003eCymbella\u003c/em\u003e, \u003cem\u003eCymbopleura\u003c/em\u003e, \u003cem\u003eDidymosphenia\u003c/em\u003e, \u003cem\u003eEncyonopsis\u003c/em\u003e Krammer and \u003cem\u003eKarthickia\u003c/em\u003e Kociolek, Glushchenko and Kulikovskiy (\u0026ldquo;CCDEK\u0026rdquo;) is unsupported, and \u003cem\u003eCymbella\u003c/em\u003e and \u003cem\u003eCymbopleura\u003c/em\u003e are polyphyletic within this clade.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescriptions of new families\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the strong support for the lineage containing \u003cem\u003eEnyconema\u003c/em\u003e species as a distinct group, and for those genera with naviculoid symmetry, we propose two new families for each of these lineages within the Cymbellales: Encyonemataceae fam. nov. and Witkowskiaceae fam. nov., subsuming 4 genera in them based on the results of molecular analysis. 8 genera are allocated among the newly described families as provisional members, as their taxonomic positions have not been confirmed by molecular data, yet. These allocations are supported by the results of morphological analysis, discussed below.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWitkowskiaceae\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eKulikovskiy, Mironov, Maltsev and Kociolek\u0026nbsp;fam. nov.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubordinate taxa:\u0026nbsp;\u003c/strong\u003e\u003cem\u003eGeissleria\u003c/em\u003e, \u003cem\u003eParaplaconeis\u003c/em\u003e, \u003cem\u003eWitkowskia\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProvisional taxa:\u003c/strong\u003e \u003cem\u003eChudaevia\u003c/em\u003e Kulikovskiy, Mironov, Glushchenko and Kociolek,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003eKhursevichia\u003c/em\u003e, \u003cem\u003eOchigma\u003c/em\u003e, \u003cem\u003ePlaconeis\u003c/em\u003e, \u003cem\u003eRexlowea\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription:\u003c/strong\u003e Cells solitary. Valves isopolar, apically symmetrical or rarely moderately dorsiventral. A single chloroplast is H-shaped, formed by two X-shaped plates connected by an isthmus. Central portion of the chloroplast aligned with the apical axis, two pairs of lobes extend under the valve surface, appressed against the valve mantle. Pyrenoid located in the central region of the valve. Two libroplasts lie near the valve ends. Raphe usually filiform, sometimes lateral. Proximal raphe ends generally expanded and slightly deflected to the primary valve side. Distal ends dislocated to the same side or to the opposite sides of the valve. In the latter case, one of the fissures can recurve to form a hook-like ending, while the other fissure is simply curved (typical for some \u003cem\u003eWitkowskia\u003c/em\u003e species). Striae uniseriate, sometimes biseriate. Areolae externally round or oval, internally round to square. Areolae openings occluded by tectula s.l. (i.e. tectula, paratectula, pseudotectula, etc.). One or more stigmoids may be located at the central area, often very close to striae. Stigmoids absent, single or numerous, located on the one or both valve sides. External and internal openings of stigmoids round or oval, generally without projections. APFs absent. \u0026nbsp;Synapomorphic features for this family within the Cymbellales is the secondarily-derived symmetrical valves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEncyonemataceae\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eKulikovskiy, Mironov, Maltsev and Kociolek\u0026nbsp;fam. nov.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubordinate taxa:\u0026nbsp;\u003c/strong\u003e\u003cem\u003eEncyonema\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProvisional taxa:\u003c/strong\u003e \u003cem\u003eCymbellopsis\u003c/em\u003e Krammer, \u003cem\u003eKurtkrammeria\u003c/em\u003e Bahls, \u003cem\u003ePseudencyonema\u003c/em\u003e Krammer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription:\u003c/strong\u003e Cells solitary or colonial, growing in mucilage tubes. Valves isopolar, more or less dorsiventral. A single chloroplast consists of two H-shaped plates, similar to chloroplast of Cymbellaceae, but with a broad isthmus dislocated towards the ventral side of the valve (in case of conspicuous dorsiventrality). Pyrenoid is round, large, lies in the isthmus against the girdle. Raphe lies along or near the middle part of the valve. Raphe filiform to lateral. Proximal raphe endings expanded, dorsally bent in species with apical asymmetry, or, otherwise, practically straight. Distal raphe ends ventrally curved (\u0026ldquo;\u003cem\u003eEncyonema\u003c/em\u003e\u0026rdquo;-type raphe sensu Krammer, 1982). Striae uniseriate. Areolae openings of different shape, from round to slit-like. Internally, areolae are occluded by volae or foricula. One or more stigmoids may be located dorsally, beside the central area. Externally, openings of stigmoids are round or oval, internally \u0026ndash; slit-like, inner surface not convoluted or with minute projections. In dorsiventral species Voigt discontinuities are located on the ventral side of the valve. APFs absent in all genera except \u003cem\u003eKurtkrammeria\u003c/em\u003e, a provisional member of the family (several species possess groups of smaller areolae at the apices which may be treated as porelli of the APFs). \u0026nbsp; Synapomorphic features for this lineage are the features associated with the position of the stigma, deflection of the external distal raphe ends, and the arrangement of the nucleus and pyrenoid in relationship to the symmetry of the valves.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe modern concept of species and genera in diatom systematics implies that infra-familiar taxa should be distinguishe\u0026shy;\u0026shy;d from each other by a combination of morphological features or by unique characters of the valve. It is traditionally considered that ultrastructural characters of the diatom frustule, e.g., the structure of pore occlusions, should be stable in monophyletic groups [27, 28]. This approach is adopted in our study, as well. Moreover, morphological data serves as supportive evidence for phylogenetic reconstruction in case of genera with no molecular data available. Additionaly, morphological analysis is applied to highlight the differences between the new families, as well as Cymbellaceae and Gomphonemataceae. The results of morphological comparison are presented in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValve symmetry and raphe structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe newly proposed families can be distinguished by a combination of essential characters of the frustule. However, as the taxonomic concept of diatoms transformes, phylogenetic importance of some features reduces. For example, valve symmetry (cymbelloid, gomphonemoid or naviculoid) originally had an important role in the systematics of Cymbellales [8]. Nowadays, the significance of this feature has decreased, and in the current study the type of valve symmetry is not specific among the families of Cymbellales. In particular, Cymbellaceae and Gomphonemataceae, according to the traditional taxonomic beliefs [1, 16] are both comprised of taxa with cymbelloid and gomphonemoid valves. Nevertheless, as suggested in the current study, valve outlines among the diatoms of Witkowskiaceae fam. nov. are symmetric about both apical and transapical axes, e.g. \u003cem\u003eGeissleria\u003c/em\u003e ([29]: fig. 31), \u003cem\u003eChudaevia\u003c/em\u003e ([30]: figs 6, 9), \u003cem\u003ePlaconeis\u003c/em\u003e ([30]: fig. 3) \u003cem\u003eParaplaconeis\u003c/em\u003e ([11]: figs 133\u0026ndash;135), \u003cem\u003eOchigma\u003c/em\u003e ([11]: figs 139\u0026ndash;140), \u003cem\u003eKhursevichia\u003c/em\u003e ([11]: fig. 63) and \u003cem\u003eWitkowskia\u003c/em\u003e ([22]: fig. 8), while Encyonemataceae fam. nov. includes genera specifically with apical asymmetry expressed to a greater or lesser.\u003c/p\u003e\n\u003cp\u003eHowever, valve symmetry can serve as a distinctive feature for high-level taxonomy, if analyzed in conjunction with other essential and robust morphological features of the valve, e.g. raphe organization. Thus, for morphological analysis of Cymbellales, we bound the type of valve symmetry with location of raphe fissures and, consequently, Voigt discontinuities (VDs). In this case, our suggestions correspond to Mann\u0026rsquo;s [15] ideas on the homology of valve sides of the diatoms of Cymbellales. As he stated, in \u003cem\u003eCymbella\u003c/em\u003e sensu stricto. VDs are situated dorsally, while in \u003cem\u003eEncyonema\u003c/em\u003e these structures are always located ventrally [15]. Hence, species with different location of Voigt discontinuities should belong to separate taxonomic groups. This principle is obeyed herein: if dorsiventrality is evident, the discontinuities are dorsal in Cymbellaceae and ventral in Encyonemataceae fam. nov. Distal raphe fissures are orientated analogously, which corresponds to Krammer\u0026rsquo;s classification [9], differentiating \u0026ldquo;\u003cem\u003eCymbella\u003c/em\u003e\u0026rdquo;-type (Figure 2A) and \u0026ldquo;\u003cem\u003eEncyonema\u003c/em\u003e\u0026rdquo;-type (Figure 2B) raphes. As a result, in Encyonemataceae fam. nov. proximal raphe fissures are dorsal, distal \u0026ndash; ventral; ventral side with VDs. In Cymbellaceae, on the contrary, proximal fissures are ventral, distal \u0026ndash; dorsal; VDs dorsal (Figure 2A, B).\u003c/p\u003e\n\u003cp\u003eDiatoms of Witkowskiaceae fam. nov. possess symmetric valves, but, nevertheless, are equipped with raphes, typical for the order Cymbellales, i.e. distal fissures are unilaterally deflected in relation to each other, but oppositely to the proximal fissures. Additionally, some species of \u003cem\u003eWitkowskia\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Paraplaconeis\u003c/em\u003e have heteromorphic distal fissures (one is simply curved, another \u0026ndash; recurved in the same direction at the valve margin) (Figure 2C; [11, 30, 31]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePore occlusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnother feature, hereby demonstrated as non-family-specific is the structure of pore occlusions. Mann [27] suggested that pore occlusions should be morphologically similar in closely related taxa that comprise monophyletic groups. Mann\u0026rsquo;s principle was later adopted by Cox [28], who made an attempt to improve the classification of pore occlusions solving the confusion with the use of the term \u0026ldquo;vola\u0026rdquo;. Additionally, she described tectulum as typical for \u003cem\u003ePlaconeis\u003c/em\u003e and foricula as a type of areolae, common for several species-rich genera \u0026ndash; \u003cem\u003eCymbella\u003c/em\u003e, \u003cem\u003eCymbopleura\u003c/em\u003e, \u003cem\u003eEncyonema\u003c/em\u003e and \u003cem\u003eGomphonema\u003c/em\u003e. Cox [28] stated that foricula are variable among cymbelloid taxa \u0026ndash; unilateral in \u003cem\u003eGomphonema\u003c/em\u003e, symmetrical in \u003cem\u003eCymbella\u003c/em\u003e sensu stricto, uneven in \u003cem\u003eCymbella delicatula\u003c/em\u003e K\u0026uuml;tzing (= \u003cem\u003eDelicatophycus delicatulus\u003c/em\u003e (K\u0026uuml;tzing) M.J. Wynne), round in \u003cem\u003eReimeria\u003c/em\u003e and dendritic in \u003cem\u003eDidymosphenia\u003c/em\u003e. Latest investigations, involving SEM studies, demonstrated that the diversity of pore occlusions among diatoms of Cymbellales is greater than estimated before [30].\u003c/p\u003e\n\u003cp\u003eIn this article, the structure of pore occlusions is variable among the genera, belonging to the same family. The structure of pore occlusions in Encyonemataceae fam. nov. seems to be unstable: the provisionary members of this family \u0026ndash; \u003cem\u003eCymbellopsis\u003c/em\u003e and \u003cem\u003ePseudencyonema\u003c/em\u003e \u0026ndash; have polymorphic areolar openings with volate occlusions [32, 33], while areolae of \u003cem\u003eKurtkrammeria\u003c/em\u003e, another provisionary member of Encyonemataceae fam. nov., might be occluded by simple hymens [34]. Morphologically, pore occlusions in Witkowskiaceae fam. nov. are more conservative and, in general, can be described as tectula sensu lato [30].\u003c/p\u003e\n\u003cp\u003eHence, pore occlusions of the diatoms, belonging to Cymbellales demonstrate a high level of polymorphism. Because of that, monophyletic groups in this order might not be inevitably characterized by a similar type of pore occlusions. In the same way, similarities in the structure of areolar occlusions do not always indicate close phylogenetic relationships among separate taxa. It is possible that evolutionary course of pore occlusions in the order Cymbellales is complicated, i.e. various types of occlusions erected independently in different groups on several occasions. Consequently, the morphology of pore occlusions, as a distinctive feature, should be applied for species- and genus-level taxonomy, rather than identification of families.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStigmata, stigmoids and apical pore fields\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnother important feature of the frustule that should be taken into account during separation of the families in the order Cymbellales, is position of stigmata and stigmoids. With this feature, the terminology is confusing. Krammer [9], understood stigmata as isolated pores, morphologically distinguishable from areolae in striae, with slit-like, convoluted internal openings. Stigmoids, on the other hand, are barely isolated from areolae and similar to them in external morphology. Internally, the openings of stigmoids are slit-like, with unconvoluted surface. However, diatomists tend to confuse these features and some authors use an unclear term \u0026ldquo;pseudostigmoid\u0026rdquo; [35, 36]. Bahls [34], described \u003cem\u003eKurtkrammeria\u003c/em\u003e as a genus with stigmata, but his SEM microphotographs show typical stigmoids \u0026ndash; with inner openings, not equipped with any projections (34]: figs 46, 86, 104). In addition, new types of stigmoids and stigmata were designated, e.g., stigmata with two slit-like internal openings were discovered in \u003cem\u003eOricymba\u003c/em\u003e and \u003cem\u003eKarthickia\u003c/em\u003e [37, 38]. To make this feature suitable for taxonomic use, an improved classification of stigmata and stigmoids is desperately required. As no kind of comprehensive classification has been adopted since Krammer\u0026rsquo;s [9] revision of \u003cem\u003eCymbella\u003c/em\u003e, the understanding of stigmata and stigmoids in this paper matches his definitions. In our system, both the structure and position of stigmata and stigmoids are treated as valuable taxonomic features.\u003c/p\u003e\n\u003cp\u003eThus, according to our description, diatoms of Encyonemataceae fam. nov., e.g., \u003cem\u003eKurtkrammeria\u003c/em\u003e, sometimes possess stigmoids, that are invariably situated dorsally [34]. In \u003cem\u003eCymbellopsis\u003c/em\u003e and \u003cem\u003ePseudencyonema\u003c/em\u003e stigmoids are absent [32, 33, 39]. In Witkowskiaceae fam. nov. (and genera with provisionary position in this family) stigmoids are present in only four genera, closely related to each other \u0026ndash; \u003cem\u003eChudaevia\u003c/em\u003e, \u003cem\u003eGeissleria\u003c/em\u003e, \u003cem\u003ePlaconeis\u003c/em\u003e and \u003cem\u003eWitkowskia\u003c/em\u003e. In some species with multiple stigmoids they are positioned to the both sides from the central nodule [20, 30, 40].\u003c/p\u003e\n\u003cp\u003eIn addition, the newly erected families are similar to each other by the absence of the APFs but, at the same time, can be distinguished from Cymbellaceae and Gomphonemataceae based on this feature. An exception, which seems to be of minor importance, is the genus \u003cem\u003eKurtkrammeria\u0026nbsp;\u003c/em\u003ewith APFs at both poles [34]. Some species of this taxon possess weakly developed APFs. Porelli are very similar to the areolae of striae, which indicates the primitivity of the APFs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChloroplasts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe structure of chloroplast in cymbelloid diatoms was originally described by Mereschkowsky [41]. He proposed an idea of using the morphology of chloroplast to distinguish high-level taxa from each other. In his system, diatoms, currently regarded as the members of the order Cymbellales, were placed in \u0026ldquo;Monoplacatae\u0026rdquo; due to the presence of a single chloroplast. Both Geitler [42] and Cox [43] stated that position of chloroplast is stable at the generic level in \u003cem\u003eCymbella\u003c/em\u003e and \u003cem\u003eEncyonema\u003c/em\u003e. In both taxa, the chloroplast is H-shaped, valve-appressed and connected by a broad bridge (isthmus). The bridge is dorsal in \u003cem\u003eCymbella\u003c/em\u003e and ventral in \u003cem\u003eEncyonema\u0026nbsp;\u003c/em\u003e[1, 43], which is why diatoms Encyonemataceae fam. nov. can be distinguished from Cymbellaceae by the ventrally located isthmuses. Diatoms of Witkowskiaceae fam. nov. are mostly apically-symmetric and have H-shaped chloroplasts with isthmuses offset to secondary side [30].\u003c/p\u003e\n\u003cp\u003eAs only a few genera in Cymbellales have their chloroplasts studied, it is impractical to use the morphology and position of chloroplasts to distinguish the genera of this order from each other. But, because the chloroplasts are well studied in the most species-rich genera of Cymbellales \u0026ndash; \u003cem\u003eCymbella\u003c/em\u003e, \u003cem\u003eEncyonema\u003c/em\u003e, \u003cem\u003eGomphonema\u0026nbsp;\u003c/em\u003e[16, 42] and \u003cem\u003eWitkowskia\u003c/em\u003e [21, 22, 40] in order to separate the families of the order Cymbellales, chloroplast morphology should be examined along with features of the valve.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Comparison of Encyonemataceae fam. nov. and Witkowskiaceae fam. nov. to Cymbellaceae and Gomphonemataceae.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"648\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.465224111282843%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFeature of morphology\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.792890262751158%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEncyonemataceae fam. nov.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eWitkowskiaceae fam. nov.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCymbellaceae\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGomphonemataceae\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.465224111282843%\" valign=\"top\"\u003e\n \u003cp\u003eValve symmetry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.792890262751158%\" valign=\"top\"\u003e\n \u003cp\u003eApically asymmetric\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eApically and transapically symmetric\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eApically asymmetric, rarely transapically asymmetric (\u003cem\u003eDidymoshenia\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eTransapically asymmetric, rarely apically asymmetric (\u003cem\u003eAfrocymbella\u003c/em\u003e, \u003cem\u003eReimeria\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.465224111282843%\" valign=\"top\"\u003e\n \u003cp\u003eRaphe organization\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.792890262751158%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ldquo;\u003cem\u003eEncyonema\u003c/em\u003e\u0026rdquo;-type (proximal fissures dorsal, distal fissures ventral)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eProximal fissures straight or unilaterally deflected. Distal fissures unilaterally deflected (and opposite in relation to proximal fissures), sometimes heteromorphic (one of them with a recurvature)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ldquo;\u003cem\u003eCymbella\u003c/em\u003e\u0026rdquo;-type (proximal fissures ventral, distal fissures dorsal)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eProximal and distal fissures straight or unilaterally deflected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.465224111282843%\" valign=\"top\"\u003e\n \u003cp\u003eLocation of VDs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.792890262751158%\" valign=\"top\"\u003e\n \u003cp\u003eVentral valve side\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eSecondary valve side\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eDorsal valve side\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eSecondary valve side (=ventral in dorsiventral taxa)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.465224111282843%\" valign=\"top\"\u003e\n \u003cp\u003ePore occlusions\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.792890262751158%\" valign=\"top\"\u003e\n \u003cp\u003eIrregular, volae-like or hymens\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eTectula sensu lato (tectula, paratectula, pseudotectula, etc.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eSymmetrical or asymmetrical foricula, volae\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eUnilateral or asymmetric foricula\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.465224111282843%\" valign=\"top\"\u003e\n \u003cp\u003eStigmata and stigmoids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.792890262751158%\" valign=\"top\"\u003e\n \u003cp\u003eDorsal stigmoid(-s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eStigmoid(-s). If multiple, at one or both sides of the central area\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eVentral stigma(-ta)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eStigma(-ta) or stigmoid(-s), dorsal in dorsiventral species\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.465224111282843%\" valign=\"top\"\u003e\n \u003cp\u003eAPFs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.792890262751158%\" valign=\"top\"\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003ePresent, typically at both poles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003ePresent, typically at one, narrower pole (=footpole)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.465224111282843%\" valign=\"top\"\u003e\n \u003cp\u003eChloroplasts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.792890262751158%\" valign=\"top\"\u003e\n \u003cp\u003eWith ventrally offset isthmus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eWith isthmus offset to the secondary valve side\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eWith dorsally offset isthmus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003eWith isthmus offset to the secondary valve side\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.465224111282843%\" valign=\"top\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.792890262751158%\" valign=\"top\"\u003e\n \u003cp\u003e[32\u0026ndash;34, 43]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003e[11, 30, 31, 40, 43]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003e[9, 43, 44]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.24729520865533%\" valign=\"top\"\u003e\n \u003cp\u003e[43, 45]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur molecular and morphological investigations highlight the differences between high-level taxonomic groups within the order Cymbellales. Based on the results of \u003cem\u003erbc\u003c/em\u003eL and SSU rRNA analysis, we provide the descriptions of two new families. Taxonomic innovations are congruent with the results of the comprehensive morphological analysis. According to our study, the new families are distinguished from each other (and from other closely related taxa) based on the combination of features of the valves and living cells, e.g. type of valve symmetry, raphe organization, presence and position of the apical pore fields and chloroplast morphology. Features analyzed above are robust among the genera of the order Cymbellales and thus are suitable for separation of the families within the studied group. At the same time, we demonstrate that the results of morphological analysis should serve as supplementary evidence for molecular data, primarily because even the essential features may differ significantly in closely related groups.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThe technique of molecular analysis follows Mironov et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and Glushchenko et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], as the same sequences were used in both studies. The dataset was comprised of 82 SSU rDNA, and 85 \u003cem\u003erbc\u003c/em\u003eL concatenated sequences, selected for available Cymbellales lineages and five diatom species from the family Rhopalodiaceae Topachevskyj and Oksiyuk chosen as the outgroup (taxa names and Accession Numbers are provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The obtained sequences of SSU rDNA and \u003cem\u003erbc\u003c/em\u003eL genes were aligned independently with the help of G-INS-I algorithm in the Mafft ver. 7 software (RIMD, Osaka, Japan) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The consequent dataset included 1,731, and 1,401 nucleotide sites for nuclear SSU rDNA, and plastid \u003cem\u003erbc\u003c/em\u003eL regions, respectively. Unpaired regions were subsequently removed, the aligned SSU rRNA gene sequences were combined with the \u003cem\u003erbc\u003c/em\u003eL gene sequences into a single matrix.\u003c/p\u003e \u003cp\u003eThe Bayesian inference (BI) method was performed in Beast ver. 1.10.1 software (BEAST Developers, Auckland, New Zealand) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Best matching partition-specific substitution models, shape parameter α and a proportion of invariable sites (pinvar) were established using the Bayesian information criterion (BIC) in jModelTest ver. 2.1.10 software (Vigo, Spain) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Thus, the following criteria were selected: GTR\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;I, α\u0026thinsp;=\u0026thinsp;0,4710 and pinvar\u0026thinsp;=\u0026thinsp;0,5970 for SSU rDNA; TPM1uf\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;I, α\u0026thinsp;=\u0026thinsp;0,3960, and pinvar\u0026thinsp;=\u0026thinsp;0,7310 for the first codon position of the \u003cem\u003erbc\u003c/em\u003eL gene; JC\u0026thinsp;+\u0026thinsp;I, pinvar\u0026thinsp;=\u0026thinsp;0,8690 for the second codon position of the \u003cem\u003erbc\u003c/em\u003eL gene; GTR\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;I, α\u0026thinsp;=\u0026thinsp;1,1260, and pinvar\u0026thinsp;=\u0026thinsp;0,2320 for the third codon position of the \u003cem\u003erbc\u003c/em\u003eL gene.\u003c/p\u003e \u003cp\u003eHerewith, TPM1uf model was substituted with HKY, and JC \u0026ndash; with F81, as the most similar applicable options for BI. Yule process tree prior was applied to perform a speciation model. Five MCMC analyses were run for 5\u0026nbsp;million generations (burn-in 1,000\u0026nbsp;million generations). Tracer ver. 1.7.1 software (MCMC Trace Analysis Tool, Edinburgh, United Kingdom) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] was implemented for convergence diagnostics. The initial 15% trees were subsequently removed, the rest retained to construct a final chronogram with 90% posterior probabilities (PP). Tree topologies-robustness was evaluated by boot-strapping the dataset with Maximum Likelihood (ML) analysis (with 1,000 replicas) in RaxML software [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. FigTree ver. 1.4.4 (University of Edinburgh, Edinburgh, United Kingdom) and Adobe Photoshop CC ver. 19.0 software were used for editing and viewing of acquired trees at the final stage.\u003c/p\u003e \u003cp\u003eTerminology of the valve, used in morphological analysis, follows Krammer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Terminology of pore occlusions corresponds to Cox [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and Mironov et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis publication is based on research carried out with financial support by the Russian Science Foundation (24-14-00165) for LM and SEM and by the framework of state assignment of the Ministry of Science and Higher Education of the Russian Federation (theme 122042700045-3) for finishing manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, A.M. and M.K.; methodology, Y.M. and M.K.; validation, J.P.K and M.K.; formal analysis, A.M., J.P.K and M.K.; investigation, A.M. and Y.M.; resources, Y.M. and M.K.; data curation, M.K.; writing\u0026mdash;original draft preparation, A.M.; writing\u0026mdash;review and editing, J.P.K. and M.K.; visualization, Y.M.; supervision, M.K.; project administration, M.K.; funding acquisition, Y.M. and M.K. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eCorrespondence and requests for materials should be addressed to A. Mironov\u003c/p\u003e"},{"header":"References","content":"\u003col start=\"1\" type=\"1\"\u003e\n\u003cli\u003eRound, F.E., Crawford, R.M. \u0026amp; Mann, D.G. \u003cem\u003eThe Diatoms. Biology and Morphology of the Genera\u003c/em\u003e (Cambridge University Press, Cambridge, UK, 1990).\u003c/li\u003e\n\u003cli\u003eK\u0026uuml;tzing, F.T. \u003cem\u003eDie Kieselschaligen Bacillarien oder Diatomeen \u003c/em\u003e(Zu finden bei W. K\u0026ouml;hne, Nordhausen, Germany, 1844).\u003c/li\u003e\n\u003cli\u003eGuiry, M.D. \u0026amp; Guiry, G.M. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. https://www.algaebase.org/ (2024).\u003c/li\u003e\n\u003cli\u003eCleve, P.T. Synopsis of the naviculoid diatoms. 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Biol.\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 758\u0026ndash;771 (2008). http://dx.doi.org/10.1080/10635150802429642.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Diatoms, new families, Encyonemataceae, Witkowskiaceae, molecular phylogenetics, morphology","lastPublishedDoi":"10.21203/rs.3.rs-4764030/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4764030/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, based on the results of molecular analysis of SSU rRNA and \u003cem\u003erbc\u003c/em\u003eL sequences, we propose the descriptions of two new families in the order Cymbellales. Molecular data demonstrates that diatoms of the genus \u003cem\u003eEncyonema\u003c/em\u003e constitute an independent monophyletic clade, which represents a new family described herein \u0026ndash; Encyonemataceae fam. nov. Another family introduced in this article, with regards to molecular data, is Witkowskiaceae fam. nov. In our research, it is comprised of three closely related genera \u0026ndash; \u003cem\u003eGeissleria\u003c/em\u003e, \u003cem\u003eParaplaconeis\u003c/em\u003e and \u003cem\u003eWitkowskia\u003c/em\u003e. The results of performed molecular investigation are supported by morphological analysis. Morphological diagnoses of the new families are based on a combination of features: type of valve symmetry, raphe structure, presence and position of stigmata or stigmoids, number and location of apical pore fields and morphology of chloroplast. In addition, we discuss the phylogeny of selected genera of the order Cymbellales. In addition, based on their morphology, 3 and 5 genera are assigned with provisionary position in Encyonemataceae fam. nov. and Witkowskiaceae fam. nov., respectively.\u003c/p\u003e","manuscriptTitle":"Description of Encyonemataceae fam. nov. and Witkowskiaceae fam. nov. 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