Tertiarius minutulus sp. nov. (Stephanodiscaceae, Bacillariophyta) – a new fossil diatom species from Lake Ohrid

1,3,6 Institute of Biology, Faculty of Natural Sciences, Arhimedova 3, 1000 Skopje, R. North Macedonia. 2 Department of Aquatic Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, Netherlands. 4 Institute for Ecology, Evolution and Diversity, Goethe University, Frankfurt, Germany. 4 Department of Animal Ecology and Systematics, Justus Liebig University Giessen, Giessen, Germany. 5 Department of Palaeontology, Stratigraphy and Sedimentology, Geological Institute, Bulgarian Academy of Sciences, Sofi a, Bulgaria.


Introduction
Lake Ohrid, in the Balkan Peninsula, was formed between 1.9 and 1.3 Ma ago (Wagner et al. 2017). It is the oldest existing European lake and one of the few long-lived lakes around the world (Stanković 1960;Radoman 1985;Dumurdžanov et al. 2004;Albrecht & Wilke 2008). The lake has accumulated a wealth of sediments throughout its limnological history, which are well constrained in age and hold well-preserved fossils, of which diatoms (Bacillariophyta Karsten) are one of the most diverse and abundant groups of organisms (e.g., Cvetkoska et al. 2016). In order to investigate in greater details the age and origin of Lake Ohrid, and link its geological and biological evolution, a deep drilling project was performed in 2013 within the frames of the ICDP project "Scientifi c Collaboration on Past Speciation Conditions in Lake Ohrid (SCOPSCO)" . A total of ~ 2100 m of sediment was recovered from four diff erent sites -DEEP, Cerava, Gradište and Peštani. The drilling in the central part of the lake resulted in a recovery of a 569 m long sediment sequence (DEEP-5045-1). Observations of the diatom communities along the entire length of the core revealed the dominance of planktonic diatoms, mostly belonging to genera of the family Stephanodiscaceae Gleser & Markarova Cvetkoska et al. 2016;Jovanovska et al. 2016a). The majority of them belongs to Cyclotella (Kützing) Brébisson s. l., while high abundances of species belonging to Stephanodiscus Ehrenberg and Cribrionella Jovanovska, Cvetkoska, Tofi lovska, Ognjanova-Rumenova & Levkov were also observed in the older parts of the sediment sequence . In previous studies on this core, several new diatom species such as Cribrionella ohridana Jovanovska, Cvetkoska, Tofi lovska, Ognjanova-Rumenova & Levkov (Jovanovska et al. 2016b), Cyclotella cavitata Tofi lovska, Cvetkoska, Jovanovska, Ognjanova-Rumenova & Levkov and Cyclotella sollevata Tofi lovska, Cvetkoska, Jovanovska, Ognjanova-Rumenova & Levkov  were described. Further studies on this core resulted in a record of a new species bearing unique morphological features that is described here as Tertiarius minutulus sp. nov. Additional analyses of core samples from Lake Ohrid are of great importance for improving the understanding of the palaeoecology and biochronology of freshwater diatoms, their evolution and the main environmental factors that shaped the diatom community structure over geological time.

Material and methods
Lake Ohrid (North Macedonia / Albania) is located in a tectonically active graben system in western Macedonia. It is situated in a karstic graben within the Southern Balkan extensional regime (Lindhorst et al. 2010). Earlier studies hypothesized that the age of the lake is between 2 and 10 Ma, thus considering it the oldest European lake (Stanković 1960;Radoman 1985). Most recent results from the analyses of the DEEP site sediment sequence suggested that the lake established between 1.9 and 1.3 Ma (Wagner et al. 2017). The lake is ~ 30 km long and 15 km wide, located at an altitude of 693 m above sea level. The maximum water depth is 289 m, and the total volume of the lake is 55.4 km 3 ). The total water infl ow can be estimated to 37.9 m 3 s -1 , with ca 25% originating from direct precipitation and 25% from riverine infl ow (Wagner et al. 2010). About 50% of the total infl ow derives from karst aquifers, of which ca 8 m 3 s -1 are supposed to come from Lake Prespa (Wagner et al. 2010). Within the SCOPSCO project in 2013, a total of 2100 m of sediments was recovered from four diff erent drilling locations (DEEP, Cerava, Gradište and Peštani). In this study, only core samples from the DEEP site were used. This site is located in the central part of the lake at a water depth of 245 m (Franche et al. 2016), where drilling resulted in a maximum penetration of 569 m below the lake fl oor (Wagner et al.2014). The new species, Tertiarius minutulus sp. nov., was observed at 452 m of the composite sequence that corresponds to an age older than 1.3 Ma (Wagner et al. 2019). The sediment in this part is a mixture of sand, silt and clay  For diatom analyses, ca 0.1 g of wet sediment was sampled and stored in Sterilin tubes at 4°C. Samples were prepared using a modifi cation of Renberg's technique (1990). Diatom samples were acid cleaned by adding a few drops of cold 10% HCl and 35% H 2 O 2 , and left overnight to remove carbonates. The samples were then boiled ca 30 min in 37% HCl to oxidize the organic matter. Samples were rinsed several times with distilled water and subsequently centrifuged. Diatom slides were prepared using Naphrax ® . Slides were observed under oil immersion at 1500 × magnifi cation with a Nikon Eclipse 80i microscope, and diatom images were produced using a Nikon Coolpix P6000 camera. For scanning electron microscopy (SEM), the material was prepared by drying clean diatom suspension onto cover slips that were carbon tape attached to the SEM stubs and coated with gold-palladium (Polaron SC7640 sputter coater, Quorum Technologies, Ashford, UK). SEM observations were performed using a Cambridge S4 Stereoscan at 10 kV (Cambridge Instruments Ltd, Cambridge, UK) at the Friedrich Hustedt Study Centre for Diatoms (BM), Bremerhaven, Germany.

Etymology
The specifi c epithet ʻminutulusʼ refers to its small size.

Isotype
REPUBLIC OF NORTH MACEDONIA • 1 spec.; same collection data as for holotype; BM 81918 (slide).

Type locality
Lake Ohrid core 5045-1, site DEEP, at a depth of 451.92 m of the composite sequence.

Description
Light microscope (LM) Valves circular, 3.5-8.0 μm in diameter, central area 3.0-6.5 μm in diameter (Fig. 1). Valve face uneven with two parts of distinctly diff erent morphology. Marginal area with short, radiating striae exceeding ⁄ -¼ of valve diameter, with ca 24-30 striae in 10 μm. Central area uneven and colliculate with scattered central areolae that are not radially arranged. Frustules rectangular in connective view. (Fig. 2) Valve face uneven and colliculate ( Fig. 2A-C), with small granules in central area ( Fig. 2D-E). Areolae present in central area with simple round openings. Marginal striae short, occluded by cribra, perforated with pores of variable size. Line of bigger pores bordering each costa ( Fig. 2D-E). Marginal fultoportulae situated on costae, close to margin edge ( Fig. 2A-B), situated on every 6 th -7 th costa. Externally marginal fultoportulae with simple, slightly elevated and round opening (Fig. 2C, E). One to three valve face fultoportulae present, with external small openings, round ( Fig. 2A-B). Single solid ligulate girdle band present ( Fig. 2A).  Fig. 3E). One rimoportula present, located at valve face / mantle junction in the middle of costa (arrow in Fig. 3F) or on its side (arrow in Fig 3D). Rimoportula consists of short and narrow labium with oblique slit (Fig. 3D-F).

Distribution
Freshwater fossil species observed only at its type locality, Lake Ohrid.

Diff erential diagnosis
The main diff erential features of T. minutulus sp. nov. that can be observed in LM are its small valve size (diameter = 3.5-8.0 μm) and the presence of central scattered areolae that are not radially arranged. Tertiarius pygmaeus (Pantocsek) Håkansson & Khursevich (1997: 22) is characterized by a valve diameter ranging from 8.0-16.0 μm, and occasionally the areolae are not arranged in radial striae (e.g., Pantocsek 1892: fi gs 2: 22, 4: 59; Houk et al. 2010: fi g. 296: 1, 4). Important diff erences between T. minutulus sp. nov. and T. pygmaeus can be observed with SEM. The marginal striae in T. pygmaeus do not have a complex alveolar structure and are composed of regular rows of fi ne pori. Externally, distinct   spines are present on every costa. Internally, the alveolus is round and small in T. pygmaeus whereas it is larger and more elongated in T. minutulus sp. nov. In T. pygmaeus, the marginal fultoportulae are present on each costa, the number of valve face fultoportulae is one to seven scattered in the central area, and the rimoportula is situated inside of the alvelolar opening on a side of costa. Additionally, the costae are much thicker and with a lower density (8-10 in 10 μm) in T. pygmaeus than in T. minutulus sp. nov.
Another species with small-sized valves is T. mariovensis Ognjanova-Rumenova, Jovanovska,.0 μm), recently described from a diatomite near the village Manastir, Mariovo Neogene Basin, Republic of North Macedonia (Ognjanova-Rumenova et al. 2015: 56, fi gs 36-105). This species was also observed in several sediment samples from Lake Ohrid DEEP-5045-1 core. However, both species can easily be diff erentiated by the structure of the central area.
In T. mariovensis, the areolae in the central area form radially arranged striae. The number of valve face fultoportulae in T. mariovensis is higher (2-9) and the tube of the central fultoportula is always surrounded by three satellite pores. Additionally, the marginal fultoportulae are positioned on every costa, while the rimoportula is located inside and in the middle of the alveolus, but not connected to a costa as in T. minutulus sp. nov. Another small-sized species, Tertiarius distinctus Khursevich & Kociolek (2002: 333, fi gs 1-5, 12-22), has a comparable valve size as T. minutulus sp. nov. (diameter 4.5-14.0 μm), but coarser areolae arranged in short or long radial striae composed of large areolae, while the marginal striae are composed of 4-6 rows of small pori. Marginal fultoportulae are present on every costa, while in the central area there are 1-5 valve face fultoportulae. The rimoportula is located near the base of a costa within the alveolus. Diff erences between T. distinctus and T. minutulus sp. nov. can be noticed in the structure of the marginal and central striae, the number of valve face and marginal fultoportulae, and the position of the rimoportula. Smaller specimens of T. indigenus Khursevich & Kociolek (2002: 336, fi gs 6-11, 23-32) have a valve size comparable to T. minutulus sp. nov. (diameter 5.0-27.5 μm). However, diff erences between these two species can be noticed in the striae structure (longer marginal striae composed of 4-5 regular rows or pori), central area (colliculate central area with 3-5 large areolae and several ʻbumpsʼ), presence of fultoportulae on each costa and one to several rimportulae located on thinner costae within the alveolar chamber. Khursevich & Fedenya (in Khursevich et al. 2003: 306, fi gs 1: 1, 2, 4, 5, 14) is characterized by circular valves with fl at face and diameter of 6.4-16.6 μm, three to 17 valve face fultoportulae arranged in radial rows and one to four sessile rimoportulae located in the submarginal zone of the valve face. Diff erences between T. minutulus sp. nov. and T. baicalensis can be noticed in the ornamentation of the central area (areolae organized in radial striae in T. baicalensis), number and position of valve face fultoportulae, number of satellite pores on marginal fultoportulae (three satellite pores in T. baicalensis), and position of rimoportula(e) that are located in the submarginal zone of the valve face (not connected with costa or within the alveolar chamber).

Tertiarius baicalensis
Another small-celled species, Cribrionella ohridana, was recently described from the same core from Lake Ohrid (Jovanovska et al. 2016b). Both species share several characters such as small size (2.0-7.5 μm in diameter), the presence of areolae in the central area and a single rimoportula situated on a costa, marginal fultoportulae located on each 4 th -5 th costa surrounded by two satellite pores and the presence of inwardly raised circumferential silica trabeculae. However, diff erences between these two species can be noticed in the absence of valve face fultoportula in C. ohridana and areolae internally not occluded with domed cribra as in T. minutulus sp. nov.
One of the dominant and very frequent species in fossil diatom assemblages of Lake Ohrid is Cyclotella minuscula (Jurilj) Cvetkoska. Cyclotella minuscula was recently transferred to Lindavia (Schütt) De Toni & Forti as L. minuscula (Jurilj) Nakov, Guillory, Julius, Theriot & Alverson (Nakov et al. 2015) and later to the genus Pantocsekiella Kiss & Ács as P. minuscula (Jurilj) Kiss & Ács (Ács et al. 2016).   (2020) Both species, Tertiarius minutulus sp. nov. and Cyclotella minuscula, have a similar valve size (diameter 3-7 μm in C. minuscula), a colliculate central area, a small marginal zone, a single valve face central fultoportula, a marginal fultoportula with two satellite pores located on each 4 th -10 th costa and a single rimoportula positioned at a rib, beneath the ring of marginal fultoportulae. Diff erence between these two species can be observed only in a presence / absence of areolae in the central area: in C. minuscula areolae are absent, while in T. minutulus sp. nov. they are present.

Discussion
Earlier studies have described 17 taxa (16 species and one variety) which belong to the genus Tertiarius (Guiry & Guiry 2019), Table 1. The initial description of the genus Tertiarius is based on sediment samples of Miocene and Pliocene age from Köpecz (Transylvania, Romania). Furthermore, new species of Tertiarius have been described from deposits in the western USA (Khursevich & Kociolek 2002), Pliocene sediments from Mexico (Caballero et al. 2009) and Middle Pliocene sediments from Lake Baikal in Asia (Khursevich et al. 2003). In addition, two species from this genus, viz., T. jurijlii Ognjanova-Rumenova, Jovanovska, Cvetkoska & Z.Levkov and T. mariovensis, are described from sediments of Pliocene age from Mariovo Basin, North Macedonia (Ognjanova-Rumenova et al. 2015).
The presence of Tertiarius mariovensis is also recorded from sediments of Lake Ohrid, at ca 630 ka ). According to Khursevich & Kociolek (2012), the biostratigraphic range of species from the genus Tertiarius in the Northern Hemisphere extends into the Miocene to Pliocene periods. Interestingly, both T. mariovensis and T. minutulus sp. nov. have been observed in Middle Pleistocene up to Quaternary sediments. These two recent records of Tertiarius probably represent the latest occurrence of the genus in freshwater habitats, again supporting the notion of Lake Ohrid being refugium for many species throughout its existence.
SEM observations indicate that T. minutulus sp. nov. shares characteristics with the genus Lindavia (Schütt) De Toni & Forti (= Handmania M.Peragallo = Puncticulata Håkansson). The genus Tertiarius is considered as fossil with a biostratigraphic range from Miocene to Pliocene, while Lindavia (= Handmannia M.Peragallo) has a longer range from Middle Eocene to present. Tertiarius is characterized by laterally positioned rimoportula on a fultoportula-bearing costa, while in Lindavia, the rimoportula is located on the valve face (Nakov et al. 2015). In the latter study, the authors proposed a broad concept of the genus Lindavia, but Ács et al. (2016) narrowed the concept with a description of the newly erected genus Pantocsekiella Kiss & Ács (in Ács et al. 2016: 62). Both Lindavia and Pantocsekiella have the rimoportula situated on the valve face. However, the position of the rimoportula can be variable (Houk et al. 2010. Similarly, in Tertiarius, the position of the rimoportula is variable: in some species it is located in the alveolus (e.g., T. mariovensis), on the side of costa (e.g., T. pygmaeus), or in the marginal zone (T. baicalensis). In most species of Lindavia, the rimoportula is located on the valve face, but in L. thienemannii (Jurilj) Nakov, Guillory, Julius, Theriot & Alverson, it is associated with a costa and situated near the valve face / mantle junction (Levkov unpubl. data). Another important feature of T. minutulus sp. nov. is the arrangement of marginal fultoportulae (on every 6 th -7 th costa). In all species of Tertiarius so far observed with SEM, marginal fultoportulae are located on every costa whereas it is on every 3 rd -7 th costa in species of Lindavia (Houk et al. 2010).
In general, species of Tertiarius and Lindavia can be diff erentiated mainly by the position of the rimoportula. Tertiarius minutulus sp. nov. shares the features of both genera and might be placed in any of these two genera. Based on the number and position of fultoportulae, T. minutulus sp. nov. can be related to Lindavia, but based on the position of rimoportula it is closer to Tertiarius. Since the occurrence of this fossil species is in relatively deeper geological time, molecular analyses that would uncover its phylogenetic status are yet rather impossible (see for example ancient DNA limitation in Wilke et al. 2016 (Round & Håkansson 1992). Tofi lovska et al. (2016: 230) discussed in detail the validity of the transfer of Cyclotella minuscula to Lindavia, pointing out the variability of a main synapomorphic character, the position of the rimoportula, that is quite variable in this species (see also Levkov et al. 2007 andCvetkoska et al. 2014). Similarly, as in Cribrionella ohridana and T. minutulus sp. nov., the rimoportula in Cyclotella minuscula is located at the base of costa. Having in mind that the position of rimoportula should be considered as a main synapomorphic character, then these three species (Cyclotella minuscula, Cribrionella ohridana and Teriarius minutulus sp. nov.) might represent a natural group and could be placed in the same genus. This raises another important question about the stability of characters considered as synapomorphic for any particular genus. As was pointed out, the position and location of rimoportula, the presence of valve face fultoportulae, the number of satellite pores on the fultoportulae, the presence of central lamina, etc. are variable characters within each genus. In such case, the only possible and obviously accepted solution in diatom systematic is to use a combination of characters that is unique for each genus. However, this approach can result in the future in separation and description of new cyclotelloid genera, based on already noted and diff erentiated morphological groups by Khursevich & Kociolek (2012). There is a general consensus that the number of diatom genera is underestimated, but the main task is to fi nd appropriate and stable morphological characters that are synapomorphic for all members of the genus. There are several recent studies that resulted in synonymization of newly described genera It seems that in the older species of Tertiarius, the rimoportula is located near the valve margin (within the alveolus or connected with the costa) while in T. minutulus sp. nov., it is located on the costa. In contemporary species of Lindavia, the rimoportula is located on the valve face distantly from the costa. It might be hypothesized that during the evolution, the rimoportula moved from the valve margin to the valve face. A similar pattern can be noticed in Cyclotella vs Pantocskiella, where in Cyclotella s. str. the rimoportula is located on the valve mantle while in Pantocsekiella it is on the valve face. In some fossil species of Cyclotella (e.g., Cyclotella iris Brun & Héribaud-Joseph) the rimoportula is located on the transition of valve the mante to the valve face (e.g., Houk et al. 2010: fi g.s 183: 5). However, additional SEM observations especially on fossil species of Lindavia and Pantocsekiella are necessary to obtain a precise answer.