Description of four new terrestrial diatom species from Luticola and Microcostatus genera from South Africa

Abstract The knowledge about terrestrial diatom assemblages in southern Africa is rather limited, despite a long history of diatom research in this area. Terrestrial habitats are places of characteristic diatom floras, dominated by species resistant to desiccation which are able to thrive in conditions of limited water availability. The presented work expands the knowledge about these unique habitats. During the study on terrestrial moss-inhabiting diatoms from Western Cape Province (South Africa), four taxa with a unique set of valve features have been found and described herein as new species, based on light and scanning electron microscopy. These new species are: Luticolamicrocephala M. Rybak, Peszek & Kochman-Kędziora, sp. nov., Luticolaasymmetrica M. Rybak, Kochman-Kędziora & Peszek, sp. nov., Luticolaterrestris Kochman-Kędziora, M. Rybak & Peszek, sp. nov. and Microcostatusmeridionalis Peszek, M. Rybak & Kochman-Kędziora, sp. nov. Algal community composition had low species richness (9–15 taxa per sample) and samples were dominated by: Humidophilacontenta, Nitzschiabrevissima and Eunotiaaff.pseudominor. The three new Luticola species formed numerous populations, exceeding 10% of the share, whereas Microcostatusmeridionalis reached 5.4%. Basic morphological data of associated diatom flora together with detailed micrographs are also provided.


Introduction
Research on African diatoms has a long tradition, with the first works dating back to the 19th century (i.e. Ehrenberg 1845; Cleve 1881). The dynamic development of diatomological research took place mainly in the second half of the 20th century. Research from diverse habitats was carried out by four scientists: B.J. Cholnoky, M.H. Giffen, F.R Schoeman and R.E.M. Archibald, whose efforts are the foundation of more recent studies (i.e. Cocquyt and Taylor 2019;Ridder and Taylor 2020). The general diatom diversity of inland waters from southern Africa was studied by Cholnoky (e.g. Cholnoky 1960, 1962, who also described many new species (Cholnoky 1954(Cholnoky , 1955(Cholnoky , 1956(Cholnoky , 1957(Cholnoky , 1966. On the other hand, Giffen, in his works from the mid-1960s, dealt mainly with marine and estuarine diatoms (i.e. Giffen 1963Giffen , 1967Giffen , 1970Giffen , 1973Giffen , 1976, as well as some freshwater species from the Eastern Cape Region (Giffen 1966). Undoubtedly, the greatest contribution to understanding both the taxonomy and ecology of diatoms was made by Schoeman and Archibald, who, for over 15 years, published together over 20 works on diatom flora in southern Africa, supplemented by hand-drawings (i.e. Schoeman 1969Schoeman , 1970Archibald 1966) and, in subsequent years, also with microphotographs of the observed species (i.e. Schoeman and Archibald 1986a, 1986b, 1986c, 1986dArchibald andSchoeman 1984, 1985). More recent researchers continued to work on taxonomy (i.e. Cocquyt 2006;Cocquyt et al. 2013Cocquyt et al. , 2014Cocquyt et al. , 2016Taylor et al. 2014a, b), but also concentrated on ecological monitoring (i.e. Bellinger et al. 2006;Taylor et al. 2007b, c). There are two elaborate reports of the most common diatom species from the Congo and Zambezi Basins (Taylor and Cocquyt 2016) and South Africa (Taylor et al. 2007a). In recent years, many new species were described from different parts of Africa. Various studies confirm the presence of a unique diatom diversity, composed of rare and endemic diatoms species (Cocquyt et al. 2013(Cocquyt et al. , 2014Cocquyt and Ryken 2017;Taylor et al. 2014b).
Despite a long history of diatom research in southern Africa, the knowledge about terrestrial diatom assemblages is rather limited (Taylor et al. 2010(Taylor et al. , 2014a; Van de Vijver et al. 2010). Although most of the studies focused on marine, freshwater and brackish diatoms, there are still some unexplored aerial habitats. A study of terrestrial moss-inhabiting diatom communities, conducted by Van de Vijver et al. (2010), resulted in the description of one species, Muelleria taylorii Van de Vijver & Cocquyt from Drakensburg, Freestate Province, South Africa.
Two genera are mainly considered to occur in terrestrial habitats, like mosses, rocks or soils, the genus Luticola and genus Microcostatus (Round et al. 1990;Johansen and Sray 1998). The genus Luticola D.G. Mann (Round et al. 1990) was distinguished from Navicula to accommodate species included in Naviculae sect. Punctatae with Luticola mutica (Kütz.) D.G. Mann as generitype. So far, over 200 species have been documented worldwide (Guiry and Guiry 2021). A monograph, summarising the entire genus , presented a detailed and extensive revision of the Luticola genus, including more than 20 species observed from Africa. Still, more recent studies show that the diversity of the genus Luticola is underestimated and many new species have been described from Europe , Asia (Liu et al. 2017;Glushchenko et al. 2017;Lokhande et al. 2020), South America (Bąk et al. 2017;Straube et al. 2017;Simonato et al. 2020;Peszek et al. 2021), Madagascar (Bąk et al. 2019) and Antarctic Region (Zidarova et al. 2014;Kohler et al. 2015;Chattová et al. 2017;Kochman-Kędziora et al. 2020). The genus Luticola is highly diverse in valve morphology. Species of this genus are also widespread in brackish, freshwater and terrestrial ecosystems. However, the genus is ecologically characterised as being aerophilous, often noted from sites in splash zones, soil and amongst mosses (Round et al. 1990;Kociolek et al. 2017). It also shows a high degree of endemism Lokhande et al. 2020).
The genus Microcostatus, described by Johansen and Sray (1998), encompasses small naviculoid species with two longitudinal depressions next to the central sternum, a simple raphe system and external microcostae. Until now, this small genus has only 24 species documented worldwide (Guiry and Guiry 2021 The present paper aims to contribute information on the distribution and environmental preferences of terrestrial diatoms in South Africa. This is the first paper providing information about moss-inhabiting diatom assemblages from Western Cape Province, South Africa. Three taxa from the genus Luticola and one from Microcostatus, which cannot be identified using currently available literature, were observed during the study. Additional analysis, based on detailed light and scanning electron microscopy, showed a set of unique features allowing us to describe them as new species. Additional comparison with the most similar taxa is also provided.

Methods
The study area is situated in the Western Cape Province, on the south-western coast of South Africa. The climatological conditions in the Western Cape Province are influenced by both the Indian and Atlantic oceans. Most of the Province is considered to have a Mediterranean climate characterised by cool and wet winters (June-August), whereas summers (December-February) are warm and dry (Ndlovu et al. 2019). The Western Cape is widely-known for its high level of endemism in terrestrial plants, especially fynbos vegetation and naturally-occurring acidic water (Brown and Magoba 2009).
For this study, the moss samples were collected in September 2018 from three different study sites in Western Cape Province, South Africa. Two sites were located on the edge of the forest by the Prince Alfred's Pass. The third site was situated in the Jonkershoek Nature Reserve, where the sample was collected from moss growing on the rock ( Table 1). The samples were collected under permit No: CN35-285316 issued by CapeNature, OP 3570/2018.
In the laboratory, the moss samples were digested in sulphochromic mixture -a mixture of concentrated sulphuric acid and potassium dichromate in order to obtain clean diatom frustules. To remove the sulphochromic mixture, the material was centrifuged at 2500 rpm with distilled water. The cleaned diatom suspension was dried on microscope cover-slips and mounted in the synthetic Naphrax Brunel Microscopes Ltd., Chippenham, UK (refractive index 1.73). Diatoms were identified and counted under a light microscope (LM) Carl Zeiss Axio Imager A2, equipped with a 100× Plan Apochromatic objective with differential interference contrast (DIC) for oil immersion. Diatom images were captured with a Zeiss AxioCam ICc5 camera. For the observations in a scanning electron microscope (SEM), the samples were applied to a polycarbonate membrane filter with a 3 µm mesh, attached to aluminium stubs and sputter-coated with 20 nm of gold, using a turbo-pumped Quorum Q 150T ES coater. Diatoms were analysed using a Hitachi SU8010 SEM. The storage locations of holotype and isotype slides (Diatom Collections) for each of the newly-described species are indicated in the descriptions. Diatom terminology and identification was based on the following references: Round et al. (1990); Taylor et al. (2007aTaylor et al. ( , 2010; Metzeltin and Lange-Bertalot (2002); Levkov et al. (2013); Taylor and Cocquyt (2016); Houk et al. (2017); Lange-Bertalot et al. (2017). Species composition and relative abundance of taxa in diatom assemblages were determined by counting 300 valves in each sample.  Etymology. The specific epithet refers to the size and shape of valve apices. Description. LM (Fig. 1A-V). Valves linear-lanceolate to lanceolate with convex margins and clearly protracted, capitate, small apices, rectangular in girdle view. The width of apices is approximately one third of the valve width. Valve dimensions (n = 25): length 14.0-24.0 µm, width 4.5-6.6 µm. Axial area linear, narrow. An isolated pore present in the central area, located halfway between valve margin and proximal raphe endings. Central area rectangular to slightly bow-tie-shaped and asymmetric, bordered on both sides with 3-4 areolae. Irregularly-scattered areolae and shallow depressions present in the central area. Raphe branches straight, proximal raphe endings deflecting away from isolated pore. Transapical striae radiate throughout, 19-22 in 10 µm.

Descriptions of new species
Description. SEM ( Fig. 1W-AD). Externally, striae composed of 1-4 areolae, decreasing from 3-4 in striae next to the central area to only one next to the apices. Areolae elongated, becoming larger towards the valve margin ( Fig. 1W, X, AA, AB). On both sides, the central area bordered by 3 round, isolated areolae. Several ghost areolae present in the central area ( Fig. 1AA, AC). Raphe branches positioned on the slightly raised sternum (Fig. 1Z). Proximal raphe endings shortly bent away from the small, round isolated pore ( Fig. 1 AA, AC). Distal raphe fissures hooked, first deflected towards the same side as the proximal raphe endings, then hooked towards the opposite side, continuing on to the mantle (Fig. 1W, X, AB). Single row of large, usually elongated areolae present on the mantle (Fig. 1Y). Only close to the apices and in the central part of the valve, areolae becoming smaller and rounded (Fig. 1Y, Z). Copulae numerous with 1 to 3 rows of areolae (Fig. 1Z). Internally, areolae occluded by hymenes forming continuous strip (Fig. 1AD). Isolated pore opening rounded, covered by a lipped slit (Fig. 1AD). Longitudinal channel visible internally along valve edges. Fig. 2 Holotype. Slide no. 20-091 stored at the South African National Diatom Collection (SANDC) at North-West University, Potchefstroom, South Africa.  Etymology. The specific epithet refers to the species asymmetry in valve outline and proximal raphe endings.
Description. SEM (Fig. 2S-Z). Striae composed of 2-5 elongated areolae. Areolae close to the valve margin larger (Fig. 2S, T, Z). Usually 3-5 isolated areolae positioned on both sites in central area, close to the valve margin. Small, round isolated pore located in the central area, halfway between the valve margin and proximal raphe endings ( Fig. 2S-U, X, Y). In some specimens, small, irregular depressions present on the valve face producing uneven appearance of the valve face (Fig. 2S, X-Z). Raphe branches straight. Proximal raphe fissures long, unilaterally deflected to the side opposite to stigma and expanded into small pores. In some specimens, proximal raphe endings asymmetrical (Fig. 2T, U, X, Y). Distal raphe fissures hooked towards opposite side, terminating shortly before valve edge (  Etymology. The specific epithet refers to the terrestrial habitat from where the new species is described. Description. LM (Fig. 3A-W). Larger valves lanceolate with weakly-protracted apices; smaller valves rhombic-lanceolate, rectangular in girdle view. Apices usually rounded, in larger valves, slightly subcapitate. Valve dimensions (n = 25): length 8.0-28.5 µm, width 4.4-6.1 µm. Axial area linear, slightly widening towards both the central area and the apices. Central area bow-tie-shaped, often asymmetrical, bordered by shortened striae. One isolated pore present in the central area. Raphe branches straight. Proximal raphe endings unilaterally deflected away from the isolated pore; terminal raphe fissures elongated and hooked. Striae radiate throughout, 20-23 in 10 µm. (Fig. 3X-AF). Striae composed of 2-4 transapically elongated areolae becoming larger towards the valve margins ( Fig. 3X-AA). One elongated stigma present, positioned in between the proximal raphe endings and the valve face margin (Fig. 3X-Z). Ghost areolae often present, mainly on a stigma-bearing site (Fig. 3X, AA). Raphe branches straight. Externally, proximal raphe endings deflected away from the isolated pore-bearing side with small drop-like endings (Fig. 3AB). Terminal raphe fis- sures clearly elongated, first curved to the same side as the proximal raphe fissures, then slightly bent to the isolated pore-bearing side. Terminal raphe fissures are towards the valve apices, terminating on the valve face/mantle junction, well after the final row of areolae (Fig. 3AC). Valve mantle bearing a single row of rounded areolae (Fig. 3Z, AA). Copulae with single row of areolae (Fig. 3AA). Internally, areolae occluded by hymenes, forming a continuous strip across each stria (Fig. 3AD-AF). Internal isolated pore opening rounded, covered by a lipped slit. Proximal and terminal raphe endings weakly deflected towards the pore (Fig. 3AE), the latter terminating on to small helictoglossae (Fig. 3AF). Longitudinal channel visible along valve edges (Fig. 3AE, AF). Etymology. The name refers to the area from where the new species is described (lat. meridional -southern).

Species composition and diversity of moss-inhabiting diatom communities
A total of 20 diatom taxa were observed in all samples. Amongst them, four were described as a new species (Table 2, Figs 5-9).

Discussion
The vast majority of research on diatoms in Africa was conducted in the second half of the twentieth century and many species descriptions are based on sketches and the classification of species is based on broad approaches to genera (Kociolek and Williams 2015). Many of them still wait for their verification using modern-day taxonomy focusing on detailed microscopic analysis.
Amongst other species described from South Africa, Navicula submutica var. capitata also shows some degree of similarity to L. microcephala sp. nov., but there is a lack of detailed microscopic pictures of this species. However, based on the description and the line drawing of this taxon (Fusey 1964, p. 27, pl. 3, fig. 41), Navicula submutica var. capitata differs from L. microcephala sp. nov. in having more lanceolate valve outline, wider central area and lower striae density (16 in 10 µm versus 19-22 in L. microcephala sp. nov.).
Based on light microscopy observations, the Microcostatus meridionalis sp. nov. is the most similar to Microcostatus egregius (Hustedt) (Johansen and Sray 1998). All species share similar valve outline and invisible or very difficult to observed striae in LM. Only in two species -M. meridionalis sp. nov. and M. werumii, apart from a raphe and valve outline, no other morphological features are discernible in LM. In other species, some additional features (for example, depressions on both sides of the sternum, visible as arch-shaped shadows or striation pattern) can be observed in LM. Microcostatus meridionalis sp. nov can be separated, based on its cuneate valve ends, whereas M. werumii has slightly rostrate valve ends. Additionally, proximal raphe endings are different in both species -in M. werumii, they are are poorly separated and located close to each other (Metzeltin et al. 2009, p. 225, figs 1-16). The drop-like proximal raphe endings of M. meridionalis are clearly visible, located on the asymmetrically constructed sternum. Under SEM, M. meridionalis sp. nov. poses a set of unique features that allows it to be easily distinguished from other representatives of the genus Microcostatus. Microcostatus meridionalis sp. nov. has areolae occluded only in the central area, not on the entire valve face. Moreover, it has no pseudoconopeum/conopeum in contrast to M. aerophilus and M. egregius (Stanek-Tarkowska et al. 2016, p. 166, figs 13-15). Internally, in the aspect of striae pattern and the shape of the central area, the described species is similar to Microcostatus schoemanii Taylor, Levanets, S. Blanco & Ector (Taylor et al. 2010, p. 180, fig. 8) and M. cholnokyi Taylor, Levanets, S. Blanco & Ector (Taylor et al. 2010, p. 182, fig. 25), but, based on the different shape of raphe endings and valve outline, are easy to distinguish. Regarding valve dimensions and striae density, Microcostatus meridionalis sp. nov. resembles M. krasskei. Both taxa overlap their dimensions (5-14 µm length, 3-4 µm width and 35-45 striae in 10 µm in M. krasskei versus 7.5-14 µm length, 3.5-4.5 µm width and 36-42 striae in 10 µm in M. meridionalis), but can be separated, based on the striae pattern and present of central area in. M. meridionalis (Johansen and Sray 1998, p. 99, figs 17, 19). The others of the aforementioned most similar species, although they have a similar shape, differ in striae density. Microcostatus egregius has 33-36 striae in 10 µm, whereas Microcostatus aerophilus up to 40-50 in 10 µm (Metzeltin et al. 2009;Stanek-Tarkowska et al. 2016), unlike to Microcostatus meridionalis which has 36-42 striae in 10 µm. Microcostatus aerophilus (Stanek-Tarkowska et al. 2016) fully matches in the aspect of valve dimensions, but usually has higher striae density (40-50 in 10 µm).
The studied assemblages consisted of diatoms with various geographic distributions. Cosmopolitan species, such as: Humidophila contenta, Hantzschia amphioxys, Nitzschia brevissima, Pinnularia borealis (Krammer and Lange-Bertalot 1986;Krammer 2000;Lange-Bertalot et al. 2017) occurred together with species with a wide distribution in the tropics -Luticola distinguenda and L. intermedia Glushchenko et al. 2017;Straube et al. 2017;Da Silva-Lehmkuhl et al. 2019). On the other hand, several of the noted species have not been reported from Africa so far or their occurrence in Africa has not been confirmed. Luticola permuticoides was originally described by Metzeltin and Lange-Bertalot (2007) from South America. So far, the occurrence of this species in Africa has not been confirmed. However, in the original description, the authors indicated the presence of a similar taxon (Metzeltin and Lange-Bertalot 2002, plate 27, fig. 17) in Madagascar, which probably is, indeed, Luticola permuticoides. Another species that has not been recorded in Africa so far is Orthoseira circularis, a taxon originally described from South America, but observed also in materials from Java (Houk et al. 2017). Together with other species identified in our study, O. circularis is probably a typical inhabitant of the terrestrial environment. Based on the presented results and literature data, both mentioned taxa can be considered as having pantropical distribution in terrestrial habitats.
In the studied material, some species were scarce and their exact identification was not entirely possible. In the literature data, there is a lack of information about valve dimensions of Stauroneis pygmaea f. undulata -the species originally described from Asia (Hustedt 1942). For this reason, despite the high level of morphological similarity in the valve outline and striae pattern, the observed species were noted as Stauroneis cf. pygmaea f. undulata. Two populations of Eunotia, observed in samples 2018/424 and 2018/426, show a highly similar pattern to Eunotia pseudominor Pavlov & Levkov. Both of our populations have similar dimensions: 12-35.6 µm length, 3.6-5.3 µm width and 11-15 striae in 10 µm in E. pseudominor ) and 9.9-33.4 µm length, 3.2-4.1 µm width and 13-16 striae in 10 µm in our populations of Eunotia aff. Pseudominor; however, smaller valves were observed in our material. Moreover, the taxa observed in this study have less straight valve outline and more bent apices. Both taxa are also associated with moss vegetation. All of the observed differences in morphology can result from both the possible presence of two distinct species or only morphological differences within a single widelydistributed species. Therefore, we decided to identify it as similar to E. pseudominor (Eunotia aff. pseudominor).
Many diatom taxa develop various adaptations to changes in humidity of aerial habitats, such as: production of thickened valves, reduction of areolae number and occlusion of areolae with silica (Lowe 2011;Lowe et al. 2007;Round et al. 1990;Dodd and Stoermer 1962;Main 2003). Newly-described species represent genera commonly noted in various terrestrial habitats (Johansen and Sray 1998;Lange-Bertalot et al. 2017). Amongst them, the highest level of adaptation is shown in M. meridionalis, which has fully covered areolae in the central part of the valve. Accompanying Pinnularia and Hantzschia taxa also show the presence of external areolae occlusions. This type of structure is also present on the surface of Eunotia cf. pseudominor, whereas the genus Eunotia does not create hymen or other occlusions (Round et. al 1990). The development of diatoms as epiphytes on mosses, which are able to accumulate water to provide a favourable environment for many organisms, can by also considered as form of adaptation to local environmental conditions (Round 1957;Lindo and Gonzales 2010;Glime 2017).
The investigated moss samples were characterised by a small diversity of diatom species (from 9 to 15 species per sample). This low species richness is quite typical for terrestrial environments, such as soils, rock crevices or clumps of terrestrial bryophytes. Especially in the Southern Hemisphere, terrestrial diatom assemblages are still poorly known, both in taxonomic and ecological aspects. The present study showed that these environments are often "hot spots" for the occurrence of potentially new and rare taxa. and efforts towards improving our manuscript. The authors thank especially Jonathan Taylor -Curator of the South African National Diatom Collection at North-West University and Prof. Andrzej Witkowski from University of Szczecin (Poland). The work was supported by the program of the Minister of Science and Higher Education named "Regional Initiative of Excellence" in the years 2019-2022, project number 026/RID/2018/19.