Morphology and phylogeny of Nitzschia nandorii sp. nov. (Bacillariophyceae), a new small-celled lanceolate species from a post-mining reservoir Author Olszynski, Rafal M. https://orcid.org/0000-0003-0667-472X University of Lodz, Faculty of Biology and Environmental Protection, Department of Algology and Mycology, ul. Banacha 12 / 16, 90 - 237 Lodz, Poland & UMR Carrtel, INRAE, Universit'e Savoie-Mont Blanc, 75 bis av. de Corzent, FR 74203 Thonon les Bains, France rafal.olszynski@biol.uni.lodz.pl Author Zakrzewski, Piotr K. https://orcid.org/0000-0002-7205-8901 University of Lodz, Faculty of Biology and Environmental Protection, Department of Algology and Mycology, ul. Banacha 12 / 16, 90 - 237 Lodz, Poland Author Rimet, Frederic https://orcid.org/0000-0002-5514-869X UMR Carrtel, INRAE, Universit'e Savoie-Mont Blanc, 75 bis av. de Corzent, FR 74203 Thonon les Bains, France Author Sulkowska, Julia https://orcid.org/0009-0003-8456-1760 University of Lodz, Faculty of Biology and Environmental Protection, Department of Algology and Mycology, ul. Banacha 12 / 16, 90 - 237 Lodz, Poland Author Peszek, Lukasz https://orcid.org/0000-0002-9132-2210 University of Lodz, Faculty of Biology and Environmental Protection, Department of Cytobiochemistry, ul. Pomorska 141 / 143, 90 - 236 Lodz, Poland Author Zelazna-Wieczorek, Joanna https://orcid.org/0000-0003-1044-8151 University of Lodz, Faculty of Biology and Environmental Protection, Department of Algology and Mycology, ul. Banacha 12 / 16, 90 - 237 Lodz, Poland text PhytoKeys 2024 2024-04-04 241 1 26 http://dx.doi.org/10.3897/phytokeys.241.117406 journal article http://dx.doi.org/10.3897/phytokeys.241.117406 1314-2003-241-1 62F62347B4D859878BF48ACEF8D4410E Nitzschia nandorii Olszynski , Zakrzewski & Zelazna-Wieczorek sp. nov. Figs 2 , 3A-AE , 4 -6 Holotype. Slide number: D.BOF2.191022, Algae Collection Department of Algology and Mycology, University of Lodz. The holotype is illustrated in Fig. 3K (designated here). Isotype. Slide number: SZCZ 29106, Szczecin Diatomological Collection, University of Szczecin, Poland. Type locality. Poland. Greater Poland Voivodeship, Bogdalow . Post-mining reservoir Bogdalow . 52°2'53.938"N , 18°35'49.646"E . Morphology description. Nitzschia nandorii sp. nov. possesses two conical-shaped chloroplasts arranged apically with a central, longitudinal groove (Fig. 2 , see: Suppl. material 8). Frustules are small, distended, and broadly lanceolate with short protracted and subcapitate apices. Valves are 9.0-12.0 µm length and 2.5-3.3 µm width. The ratio of length and width is 3.44-4.80 (Fig. 3A -AE). Striae 40-46 in 10 µm (based on SEM analysis) and indiscernible in LM. Fibulae are visible 11-16 in 10 µm . Externally, the valve face is flat and without costae (Figs 4 , 5 ). Striae are uniseriate, and transapically becoming more arched to the apices (Figs 4 , 5 ). Two longitudinal rows of areolae are present along the edge of the valve on the raphe canal with a doublet of pores closest to the raphe and second with a single pore near the junction of the raphe canal and valve face (Fig. 4 ). Distal raphe endings are strongly hooked and deflected in different directions depending on the valve. Polar raphe fissures deflected to the proximal mantle overlap on it (Figs 4 , 5A-D ). The central raphe fissures are missing (Fig. 5A, B, E, F , see: Suppl. material 9). The proximal mantle possesses three rows of areolae (Figs 5C , 6B , see: Suppl. material 9). The first two are arranged to mirror pores on the raphe canal and open to the raphe canal (Fig. 5C ). The third with areolae covered by hymenes (Fig. 6D ) and open to frustule interior (Fig. 5C , see: Suppl. material 9). The distal mantle is narrow with a scalloped edge (Figs 4D , 6C ). Valvocopula is smooth and possesses opened pores (Fig. 4D ). Internally, the fibulae are roughly equidistant so that the central fibulae are not further apart than any of the others (Fig. 6A ). Distal raphe fissures terminate with small helictoglossa internally (Fig. 6E ). Figure 2. Photomicrographs of chloroplast Nitzschia nandorii sp. nov. A-J light microscope with DIC photomicrographs K-S Confocal Laser Scanning Microscopy photomicrographs, each pair of photomicrographs presents the same chloroplasts from different angles K-N presented single-cell, conical-shaped chloroplasts with visible longitudinal groves O-S presented chloroplasts during cell division. Scale bars: 10 µm ( A-J ); 2 µm ( K-S ). Figure 3. Light microscope photomicrographs of Nitzschia spp. A-L N. nandorii sp. nov. from natural population K type species (marked by an asterisk (*)) M-AE N. nandorii sp. nov. from culture AF-AW N. lacuum AX-BI N. alpinobacillum . Scale bar: 10 µm . Figure 4. SEM photomicrographs of Nitzschia nandorii sp. nov. external valve view A valve view with two rows of areolae on the raphe canal (black arrow), single and double areolae, distal raphe fissures are strongly hooked and deflect (white arrow) B, C valve view with distal raphe fissures deflected to the proximal mantle D decomposed frustule with visible valvocopula with open pores (white arrow). Scale bars: 5 µm . Figure 5. SEM photomicrographs of Nitzschia nandorii sp. nov. external view A, B angle view with visible keel B white arrow indicates uninterrupted raphe C, D apices view with distal raphe fissures deflected to the opposite direction C white arrow indicates rows of areolae on proximal mantle E, F close-up central part of the frustule E white arrow indicates missing of central raphe fissures. Scale bars: 5 µm ( A, B ); 1 µm ( C-F ). Figure 6. SEM photomicrographs of Nitzschia nandorii sp. nov. internal view A valve view with areolae extended on the proximal mantle (white arrow), there is no wider extension between central fibulae (black arrow) B, C central view B areolae on the proximal mantle are arranged the same as on the raphe canal: single and double areolae (white arrow) C doubled areolae are visible from inside the raphe canal (white arrow), narrow mantle with scalloped edge (black arrow) D, E apices view D areolae on the mantle covered by hymenes (white arrow) E distal fissure creates helictoglossa (white arrow). Scale bars: 5 µm ( A ); 1 µm ( B-E ). Gene sequences. sequences were deposited in the GenBank: D.LDZ8 (SSU rDNA: PP082029, rbcL : PP073739, and psbC : PP073738), D.LDZ12 (SSU rDNA: PP082030, rbcL : PP073741, and psbC : PP073740) PhycoBank registration. http://phycobank.org/104257. Molecular phylogeny. The phylogenetic reconstructions based on the ML and BI strategy for the concatenated SSU- rbcL - psbC matrix, as well as separate SSU, rbcL , and psbC analysis, were performed. The three-genes tree placed strains D.LDZ8 and D.LDZ12 (Fig. 7 , green box) within the clade including the Nitzschia Lanceolatae section, which is characterized by double rows of areolae on the raphe canal, with very high node support (BI = 1; ML = 100) (Fig. 7 , grey box). Moreover, the tree topology distinguished the strains described here, i.e., N. nandorii sp. nov., from N. fonticola ( N. cf. romana syn. N. fonticola ) both in BI and ML phylogenetic reconstructions with the strong division support (BI = 1; ML = 100). Phylogenetic trees reconstructed using separate DNA markers, i.e., rbcL (Fig. 8 ), psbC (see Suppl. materials 10, 11), and SSU (see Suppl. materials 12, 13), and the concatenated analysis distinct N. nandorii sp. nov., as a separate clade in Lanceolate section. The rbcL tree topology (Fig. 8 ) was included in the main manuscript here, due to its higher taxon sampling. Figure 7. The phylogenetic tree of the Nitzschia genus with Eunotia bilunaris as the outgroup based on the concatenated nuclear (SSU) and chloroplast ( rbcL and psbC ) DNA markers (total 2236 bp). The tree presents the position of newly identified Nitzschia nandorii sp. nov. The numbers above branches represent posterior probabilities from BI analysis followed by bootstrap values from ML analysis. Asterisk (*) represents BI value = 1, and ML value = 100. En dash (-) represents BI value below 0.95, and ML value below 70. Grey box: Nitzschia Lanceolatae section with double rows of areolae on the raphe canal. The topology of the tree is based on BI analysis. Figure 8. The phylogenetic tree of the Nitzschia genus with Eunotia bilunaris as the outgroup based on chloroplast ( rbcL ) DNA marker (1147 bp). The tree presents the position of newly identified Nitzschia nandorii sp. nov. The numbers above branches represent posterior probabilities from BI analysis followed by bootstrap values from ML analysis. Asterisk (*) represents BI value = 1, and ML value = 100. En dash (-) represents BI value below 0.95, and ML value below 70. Grey box: Nitzschia Lanceolatae section with double rows of areolae on the raphe canal. The topology of the tree is based on BI analysis. Etymology. The species name comes from the main character of the TV series " What We Do in the Shadows " Nandor the Relentless, and the name of the authors' cat (RMO, PKZ). Differential diagnosis. Nitzschia nandorii sp. nov. is a species with a small cell size that can be overlooked or misidentified with other taxa, especially when they occur in one sample. The valve shape of the small specimens of N. fonticola can be misidentified with N. nandorii sp. nov., however visible striation and the wider relative spacing between the central fibulae compared to the rest of the fibulae in N. fonticola distinguish these two taxa ( Lange-Bertalot 1976 ; Krammer and Lange-Bertalot 1997 ; Lange-Bertalot et al. 2017 ). The valve outline of N. dealpina Lange-Bertalot & G. Hofmann 1993 is similar to N. nandorii sp. nov., but these taxa can be distinguished due to the lower stria and fibula density per 10 µm in N. dealpina . Furthermore, N. dealpina occurs in an environment with a high concentration of calcium bicarbonate while N. nandorii has so far only been identified in an environment with a moderate concentration of these ions ( Lange-Bertalot 1993 ; Krammer and Lange-Bertalot 1997 ; Rumrich et al. 2000 ; Lange-Bertalot et al. 2017 ; Olszynski et al. 2019 ). The smaller forms of N. alpinobacillum Lange-Bertalot, 1993, which also possess two rows of pores on the raphe canal ( Lange-Bertalot 1993 ; Rumrich et al. 2000 ; Lange-Bertalot et al. 2017 ), have similar outlines to the N. nandorii sp. nov., but have denser striation and fibulae. N. nandorii sp. nov. can be easily misidentified with a small N. lacuum Lange-Bertalot 1980 . Both taxa have similar valve size and stria and fibula density. Neither has a central nodule or a relatively wide gap between central fibulae. However, N. lacuum has different proportions between length and width and, therefore, is more elongated and has more protracted apices than N. nandorii sp. nov. ( Lange-Bertalot 1980 , 1993 ; Lange-Bertalot et al. 2017 ). N. bryophila (Hustedt) Hustedt 1943 has a similar valve outline to N. nandorii sp. nov. However, the frustules of N. bryophila are bigger, apices are more elongated and have clearly visible striations. Fibulae are less dense in N. bryophila than in N. nandorii sp. nov. ( Lange-Bertalot and Metzeltin 1996 ; Krammer and Lange-Bertalot 1997 ). N. bacillum Husted, 1922 has a similar valve outline to N. nandorii sp. nov. In some cases a small form of N. bacillum can be misidentified with newly described species. However, N. bacillum has visible striations and particular areolae can be observed. A character which is visible in SEM and distinguishes this species from N. nandorii sp. nov. is a single row of areolae on the raphe canal ( Lange-Bertalot 1993 ). Another similar species to N. nandorii sp. nov. is N. rosenstockii Lange-Bertalot, 1980. This taxa has specific apices which are short, beak-like and point rounded. In some cases, the valve outline can be similar to N. nandorii sp. nov. However, N. rosenstockii can be easily distinguished in LM from N. nandorii sp. nov. by the valve outline, characteristic valve ends, a higher number of fibulae in 10 µm , and the presence of striae discontinuity in the central part of valves ( Lange-Bertalot 1980 ). For more details see Table 2 . Table 2. Comparison of morphological features of Nitzschia nandorii sp. nov. with similar species. * literature data, n/d - no data.

-	Valve outline	Valve length [µm]	Valve width [µm]	Number of striae in 10 µm	Number of fibulae in 10 µm	Length/width ratio	Additional features	Reference
N. nandorii sp. nov.	Distended, widely lanceolate with short protracted subcapitate ends	9.0-12.0 (n = 19)	2.6-3.3 (n = 19)	40-46 (n = 17)	14-16 (n = 19)	3.44-4.80 (n = 19)	All fibulae are relatively equidistant, and central fissures missing. Two rows of striae on raphe canal	This paper
N. lacuum	Lanceolate with capitate to acutely rounded ends	13.0-22.0 (n = 48) 10.0-20.0*	2.0-3.5 (n = 48) 2.0-3.0*	36-38 (n = 11) 35-40*	13-18 (n = 48)	4.38-10.00 (n = 48)	All fibulae are relatively equidistant, and central fissures missing.	This paper; Krammer and Lange-Bertalot 1997 ; Lange-Bertalot et al. 2017
N. alpinobacillum	Lanceolate with elongated and capitate ends	15.0-22.0 (n = 16) 14.0-24.0*	3.0-4.0 (n = 16) 3.0-4.0*	23-24 (n = 16) 25-27*	10-14 (n = 16) 9-11*	4.21-6.41 (n = 16)	All fibulae are relatively equidistant. Two rows of striae on raphe canal	This paper; Lange-Bertalot 1993 ; Lange-Bertalot et al. 2017
N. dealpina	Relatively short, appearing more widely lanceolate with short protracted and acutely rounded	8.0-13.0*	3.2-4.2*	26-28*	12-14*	n/d	No wider extension between central fibulae. One row of striae on raphe canal	Lange-Bertalot and Metzeltin 1996
N. fonticola	Widely or narrowly lanceolate with drawn-out, subcapitate ends	7.0-46.0*	2.5-5.5*	24-33*	9-14*	n/d	Relatively wide gap between central fibulae	Krammer and Lange-Bertalot 1997 ; Lange-Bertalot 1976
N. bryophila	Lanceolate to linear-lanceolate with short or slightly capitate ends	15.0-26.5*	4.0-5.0*	30-32*	9-10*	n/d	No wider extension between central fibulae.	Lange-Bertalot and Metzeltin 1996 ; Krammer and Lange-Bertalot 1997
N. bacillum	Lanceolate with pointed or slightly capitate ends	12.0-20.0*	2.0-3.5.0*	27-32*	12-16*	n/d	No wider extension between central fibulae. One row of striae on raphe canal	Lange-Bertalot 1993 ; Krammer and Lange-Bertalot 1997
N. rosenstockii	Lanceolate with short, beak-like, pointed rounded ends	8.0-16.0*	3.0-4.0*	44-46	17-20	n/d	No wider extension between central fibulae. Hyaline area in centre of the valve	Lange-Bertalot 1980 ; Krammer and Lange-Bertalot 1997