﻿A new species of genus Crenotia (Bacillariophyta) from Tibet, China

﻿Abstract During the investigation of the freshwater diatoms from Tibet, a monoraphid species was observed from a hot spring near Anduo County, located on a plateau in the central portion of Tibet. This species shares the diagnostic features of Crenotia, such as the valve bent along the transapical axis, striae uniseriate to biseriate from centre to the apices and areolae with special structures located at the end of each stria. We compared the morphological characters of this new species with the others in this genus and show it to be new; it is named Crenotiatibetiasp. nov. This species has small valves with slightly protracted ends with nearly capitate apices, lanceolate axial area, central area unilaterally expanded to the margin, striae uniseriate to biseriate, but, in some valves, the striae are only uniseriate. Areolae are round small to irregular in shape and, at the end of each stria, there is a horseshoe-shaped areola present. Observations of developing valves show all the striae begin biseriate, then they become covered by silica to form uniseriate striae. Comparisons are made amongst the species in this genus and with genera assigned to the Achnanthidiaceae.


Introduction
Raphid diatoms that possess a raphe on only one of the valves are very diverse and recent systematic revisions of this group have led to a marked increase in the number of genera, from two (e.g.Hustedt (1930); Patrick and Reimer (1966)) to 27 genera (De Stefano and Marino 2003;Wojtal 2013;Kulikovskiy et al. 2020a;Al-Handal et al. 2021;Ge et al. 2022).Traditionally, these genera have been assigned to one of three families, Achnanthaceae Kützing (1844), Cocconeidaceae Kützing (1844) or Achnanthidiaceae Mann in Round et al. (1990).Recent phylogenetic studies have shown these groups to be unrelated, widely dispersed across the raphid diatom tree of life (Thomas et al. 2016;Kulikovskiy et al. 2019).Even genera within the Achnanthidiaceae have been shown to be non-monophyletic (Kociolek et al. 2019;Kulikovskiy et al. 2019) Crenotia was established in 2013 by Wojtal, when species assigned to it were split from the genus Achnanthidium.The type species of Crenotia, C. thermalis (Rabenhorst) Wojtal, was originally described as a species of Achnanthidium (as A. thermalis Rabenhorst) and then assigned to the genus Achnanthes (as Achnanthes thermalis (Rabenhorst) Schoenfeld).Eight species have been suggested to belong to Crenotia so far.Like other monoraphid diatoms, Crenotia has heterovalvar frustules; however, it is distinguished from other monoraphid genera by lacking a cavum on both valves, having biseriate or uniseriate striae, presence of specialised structures at the end of each stria and no ornamentation on the girdle bands.This genus has been reported to have a worldwide distribution (Rioual et al. 2019), usually being found from benthic or periphytic habitats in lakes, springs and swamps and preferring neutral to alkaline waters (Stockner 1968;Hindáková 2009;Wojtal 2013;Kulikovskiy et al. 2016;Coste et al. 2019;Rioual et al. 2019;Wetzel et al. 2019;Liu et al. 2020).
Tibet is one of the biodiversity hotspots of the world and, in this region, the biodiversity of Crenotia is also relatively high, with five of the eight species of the genus being reported from Tibet.(Rioual et al. 2019;Liu et al. 2020).During the investigation of freshwater biodiversity of the Tibetan Plateau, samples were collected from a hot spring in Anduo County, specifically, Nagqu City.One species was observed with light microscopy (LM) and scanning electron microscopy (SEM), based on its morphological features, demonstrated to be a new species belonging to the genus Crenotia.Herein we describe this Tibetan diatom as new to science.

Materials and methods
Samples were collected from Tibet, during a biodiversity investigation initiated in 2021.Benthic diatoms were collected from Anduo County, Nagqu City, which is located in about the middle of Tibet.Samples were taken from a hot spring located at 31°40′51.24″N,91°51′20.52″Eand 31°40′52.32″N,91°51′20.52″E,at an elevation of 4570 m above sea level.At the time of collection, the water temperature was around 20 °C, pH ranged from 6.55 to 7.77, conductivity ranged from 2790 to 3200 μS•cm -1 (determined by YSI 6920 multiparameter probe).This locality has a cold climate, with dry, windy and cold weather and an annual precipitation of only ca.100-200 mm.
Samples were fixed with 4% formaldehyde in the field.The samples were cleaned with nitric acid (HNO 3 ), then washed and settled using distilled water until the pH was neutral.For LM observations, cleaned diatoms were mounted to make permanent slides with Naphrax.These permanent slides were examined with a Zeiss Imager A2 microscope, equipped with a digital camera (Axio-Cam MRc 5) and observed with DIC (differential interference contrast) optics (Zeiss, Jena, Germany at Harbin Normal University).
For SEM observations, cleaned material was air-dried and coated with gold-palladium and observations made with a Hitachi S-4800 field emission SEM (Hitachi, Tokyo, Japan at Harbin Normal University) at an operating  Round et al. (1990), Kingston (2003) and Kulikovskiy et al. (2022).
Description.LM (Fig. 1).Frustule slightly bent along the transapical axis ("V" shaped), monoraphid, with raphe valve concave, rapheless valve convex.Valve lanceolate with slightly protracted ends, slightly asymmetrical to the apical axis, apices nearly capitate. .Raphe valve: straight raphe positioned in the middle of the valve, axial area lanceolate, with asymmetrical, rectangular to rhombic central area.Striae slightly radiating in the centre, becoming parallel towards the apices.Rapheless valve: Axial area lanceolate, central area expanded unilaterally to the margin.Striae 19-21 per 10 μm on both valves.SEM (Figs 2-5).Raphe valve: Externally, raphe straight, proximal raphe ends slightly deflected to the same side, with distal raphe ends curved to the other side.Axial area lanceolate, nearly 1/3 of the valve width, formed by short striae along the margin.The 3 -4 striae near the apices are biseriate and become uniseriate towards the valve centre.Areolae openings round to elongate, to irregularly-shaped externally.Internally, proximal raphe ends slightly bent to opposite side, helictoglossae slightly elongated.Areolae covered by hymens, forming two rows of "C"-shaped openings for each stria.Along the axial area, at the end of each stria, there is one horseshoe-shaped structure, open with fine radiating slit-like openings.One developing valve was observed; all the striae were biseriate.
Rapheless valve: Internally, axial area lanceolate, narrow at the apices and becoming wider towards the centre, centre area enlarged unilaterally and reaching the margin.Striae uniseriate to biseriate, mostly biseriate at the apices and becoming uniseriate at the centre.Areolae openings round to irregular in shape.Mantle and girdle bands without ornamentation.
Internally, the axial area is lanceolate, wide in the centre and enlarged at one side extending to the margin.Areolae were occluded by hymens with double rows of "C"-shaped openings; at the end of each stria, there is horseshoe-shaped structure, with fine slit-like openings.A developing valve was observed, all the striae were biseriate, with a "C-shaped structure at the end of each stria.
Etymology.Named after the type locality from which it was found.

Discussion
Based on the morphological features of the valve and striae structure, this new species appears to be best placed in the genus Crenotia.This small genus currently is known to have nine species, including eight previously-described taxa.In comparing this new species with other known taxa (Table 1), C. angustior differs by its small frustule and capitate ends.Crenotia hedinii was formally transferred to Crenotia by Rioual et al. (2019); it has more slender valves and more acutely-rounded ends than our new species.Crenotia grimmei, first reported by Krasske (1925), was originally named Achnanthes grimmei Krasske; however, Lange-Bertalot and his colleagues rechecked the type and lectotype (Lange-Bertalot and Ruppel (1980), tafel 2, figs  A, B, C, E, F); 500 nm (D).46-50;Lange-Bertalot et al. (1996), tafel 4, figs 8-13) and suggested it is synonymous with A. thermalis.Crenotia.gibberula was described originally as Achnanthes gibberula Grunow in Cleve & Grunow.Lange-Bertalot and Ruppel (1980) suggested that A. grimmei and its varieties should be considered synonymous with A. gibberula and illustrated A. gibberula as a morphologically variable taxon (Håkansson 1982).Although Lange-Bertalot and Ruppel (1980) presented the type material of Crenotia grimmei and C. gibberula, it is hard to suggest that they belong to the LM and SEM images were also presented by Håkansson (1982, plate I: 3-6) for A. gibberula, but the striae showed a "macroareolae"-like structure on both valves, similar to those shown in the genus Madinithidium Witkowski, Desrosiers & Riaux-Gobin (Desrosiers et al. 2014) or, probably, similar to developing valves of Achnanthidium species.Okuno (1974, pl. 855-856), also showed a SEM of A. grimmei, but, based on the stria pattern and areola structure, the specimen presented was more similar to Achnanthidium rather than Crenotia.
Compared with the former three species, C. tibetia is morphologically most similar to C. grimmei, but C. tibetia has a more highly deflected frustule about the transapical axis, the raphe valve is more concave and is larger than C. grimmei (Lange-Bertlaot et al. (1996) report length 13.0-16.7 μm, breadth 3.6-4.7 μm, striae 16-19/10 μm, for this taxon), striae are denser on both valves, with shorter capitate ends and axial area are wider on the rapheless valves.
Crenotia grimmei and C. gibberula were also recorded in Tibet by Zhu and Chen (2000), as Achnanthes grimmei and A. gibberula, respectively, plate 46: 11-14), but, based on the published line drawings, their specimens do not match well either of these species.The morphology of C. rumrichorum was observed in detail by Hindáková (2009) as Achnanthes thermalis var.rumrichorum.Based on the structure, this species was found to belong to the genus Crenotia.Crenotia rumrichorum has more acute apices, distinguishing it from C. tibetia.
Within the genus, C. distincta and C. oblonga are endemic to Tibet so far and these two species have chambered and multiseriate striae on both valves, which easily distinguish them from our new species.However, these two species do not share the typical features of Crenotia and the valve structure resembles the genus Haloroundia Diaz & Maidana (2006), a monotypic genus described from Chile.The differences between Crenotia and Haloroundia can be seen in terms of striae structure, raphe system and degree of flexure of the frustules, but further investigations on the relationships between these two genera are warranted.
The work from Lake Baikal (Kulikovskiy et al. 2013(Kulikovskiy et al. , 2020a, b) , b) established many new genera within Achnanthidiaceae.In research on monoraphid diatoms, curvature of the valve, valve shape and raphe system have been considered as critical features used to separate genera within this family (e.g.Yu et al. (2019); You et al. (2021)).However, molecular data showed the raphe number of frustules does not play such an important role in diatom taxonomy and its reduction or loss occurred many times during the evolution of raphid diatoms (Kulikovskiy et al. 2016).Features such as the "cavum" seem to have played important roles in the evolution of this group.Both morphological features and molecular data were used to identify this group and recognise: 1) species with a sinus; 2) species with a cavum; 3) species without these features (Kulikovskiy et al. 2022).Since more and more "intermediate species" between genera have been observed (You et al. 2021), the relationships between some uniseriate genera, such as Achnanthidium, Gomphothidium and Psammothidium (Round et al. 1990;Bukhtiyarova and Round 1996;Kociolek et al. 2021); and multiseriate genera such as Platebaikalia, Lemnicola and Haloroundia (Diaz and Maidana 2006;Kulikovskiy et al. 2020b) and those with macroareolae, such as Scalariella, Madinithidium, Karayevia and Kolbesia (Riaux-Gobin et al. 2012;Desrosiers et al. 2014;Kulikovskiy et al. 2022), appear to be in need of revision.Investigations with formal analyses of both morphological and molecular data may clarify the systematic position and diagnostic features amongst these genera.

Figure 1 .
Figure 1.Crenotia tibetia sp.nov., LM.Raphe and rapheless valves from the type population.A, A' illustrations of the holotype."=" means the raphe valve and rapheless valve are from the same frustule.Scale bar: 10 μm.

Figure 2 .
Figure 2. Crenotia tibetia sp.nov.SEM, external view of the raphe valve A external view of the whole valve B, C apices of the valve D magnification of areolae E central area of the valve F girdle view of the valve.Scale bar: 1 μm (A, B, C, E, F); 500 nm (D).

Figure 3 .
Figure 3. Crenotia tibetia sp.nov.SEM internal view of the raphe valve A internal view of the whole view B, C apices of the valve D central area of the valve internally E magnification of the areolae, showing the horseshoes areola at the end of striae F internal view of a developing valve.Scale bar: 1 μm (A, B, C, D, F); 500 nm (E).

Figure 4 .
Figure 4. Crenotia tibetia sp.nov.SEM external view of the rapheless valve A, B external view of whole valve C, D apices of the valve, showing the areolae.Scale bar: 1 μm.

Figure 5 .
Figure 5. Crenotia tibetia sp.nov.SEM internal view of the rapheless valve A internal view of the whole valve B apex of the developing valve C apex of the valve D magnification of the areolae, showing the horseshoe-shaped structure at the end of the areolae.Scale bar: 1 μm.

Table 1 .
Comparison of morphological characteristics of Crenotia tibetia sp.nov.and closely related taxa. C.