﻿Cocconeiscrisscrossis sp. nov., a new monoraphid diatom (Bacillariophyta) from southern China

﻿Abstract A novel monoraphid diatom species, Cocconeiscrisscrossis You, Yu, Kociolek & Wang, sp. nov. is examined and described from the Qingyi River and Maolan Nature Reserve of southern China. The morphological description is based on light microscopy and scanning electron microscopy observations and the new species is compared with similar taxa in this genus. The characteristics unique to Cocconeiscrisscrossissp. nov. include its central area extending irregularly to both sides, it having closed valvocopulae with heavily silicified fimbriate margins and poles of the valvocopulae have ‘sword-shaped’ siliceous extensions. These features differentiate this new species from others in the genus. This new species was found in alkaline waterbodies, including streams, waterfall and ponds. It was usually found as an epiphyte on the stones; however, it was present on other substrates such as mosses.


Introduction
The first described genus of Cocconeidaceae Kützing was Cocconeis Ehrenberg (1838).In his early compendium book "Infusionsthierchen", Ehrenberg (1838) described the morphology, growth habit and ecology of the genus in French and Latin.The name of the genus is derived from its growth habit, adnate on filamentous algae.Cocconeis Ehrenberg is a monoraphid diatom genus widely distributed in marine and freshwater environments, with more than 200 described species, with the main taxa being marine species (Guiry 2023;Kociolek et al. 2023).The frustules of this genus are heterovalvar, with one raphe valve (RV) and one rapheless or sternum valve (SV), where the former is typically less convex than the latter (Jahn et al. 2009;Stancheva 2018;Mora et al. 2022).Solitary cells of Cocconeis are attached to filamentous algae or other substrates by their RV, while the SV is exposed to the environment.Valves of Cocconeis are elliptical or slightly linear-elliptical in shape and this genus has distinctly different areolae structures on both valves (Round et al. 1990).The areolae of the RV are fine and often have a semicircular hyaline area near the valve ends, while the areolae of the SV are relatively coarse extending to the valve edge and lacking a hyaline area.Striae are usually uniseriate in freshwater species and the poroids are closed by hymenes (Costa et al. 2020).The frustule structure of Cocconeis is diverse and complex, species delineation requires detailed observations of the RV morphology, the SV and their connecting elements including valvocopulae and the cingulum (Potapova and Spaulding 2013;Stancheva 2022).
In China, 18 freshwater Cocconeis taxa have been recorded, including ten species and eight varieties (Skvortsov 1935;Chin 1951;Zhu and Chen 1989, 1994, 2000;Lin and Wang 1992).China remains a poorly-researched area, with few studies of this genus having gained the attention of researchers.In recent years, we conducted an extensive biodiversity investigation of monoraphid diatoms in China and one new Cocconeis species was found, which is distributed in the Qingyi River and Maolan Nature Reserve.This paper describes the frustule morphological characteristics of C. crisscrossis sp.nov.using LM and SEM and compares this new species with similar species of the genus.

Materials and methods
Diatom samples were collected from two sites: Qingyi River (Yi County, Anhui Province) and Maolan Nature Reserve (Libo County, Guizhou Province).Samples were collected using tweezers or turkey baster.The samples were preserved using formalin solution before being stored in sealed plastic bottles.Sample information is listed in Table 1, including location of samples, longitude and latitude, habitat, pH, conductivity and collection date.
In the laboratory, the samples were processed with concentrated nitric acid using the Microwave Accelerated Reaction System (Model MARS, CEM Corporation, Charlotte, USA); the specific processing and observation steps are described in You et al. (2021).Images were compiled with Adobe Photoshop 2023.Morphological terminology follows Donadel et al. (2018) and Riaux-Gobin et al. (2021).The holotype image of the single specimen is a circled specimen on the type slide.The samples and permanent slides are preserved in Lab of Algae and Environment, College of Life Sciences, Shanghai Normal University.Description.Light microscopy (LM) (Fig. 1A-X).Valve elliptical to slight round-elliptical, apices are obtusely rounded; 12.5-42.0µm long, 9.5-25.0µm wide.The number of striae on both valves is similar, with parallel orientation at the centre and radiate towards the apices.Both valves display striae with a density of 18-22 rows per 10 µm.Raphe valves (RV) have a thickened hyaline rim on the margin and striae composed of small, nearly round areolae.The raphe is straight, the axial area is narrow, the central area is irregularly narrow, cuniform and the axial area and central area form an irregular cross-like structure.Sternum valve (SV) areolae are rounded and form 7-9 undulating longitudinal lines per hemivalve (increasing in number with increased valve width).The sternum is narrow, linear.The valvocopula has irregularly-spaced digitate fimbriae.
Scanning electron microscopy (SEM) (Figs 2-6) shows RV face is flat with a weakly concave mantle and very narrow linear axial area; the central area extends irregularly to both sides (Figs 3A-C, E, 4A-D).Externally, the raphe is straight, filiform and proximal raphe endings are slightly drop-like and expanded (Fig. 3E).Internally, the proximal raphe fissures are bent to opposite sides (Fig. 4D); the distal raphe endings are straight and end in weakly-elevated helictoglossae on the hyaline rim (Fig. 4E).Striae are made up of round to slightly elliptical uniseriate areolae which are small and regularly spaced, increasing in size from the axial area towards the valve margin and the apices (Fig. 3A-C).Internally, the areolae are occluded by round lattice-structured hymens with short slits around the margin and tiny perforations in the middle (Fig. 4F).SV has a narrow and straight sternum (Fig. 5A, B).In the SEM, it can be seen that, on the SV, the areolae differ greatly between the internal and the external sides (Figs 5C, 6C).Externally, striae are composed of irregular, variably-sized and mostly round areolae, differentiated to the valve edge (Fig. 5A).Areolae are occluded by lattice-structured hymens of marginal slits (Fig. 5C).Internally, the areolae are like puncta.There is a linear and straight axial area (Fig. 6A).Valvocopulae with well-developed digitate fimbriae on both valves are characteristics typical of the C. crisscrossis, which are close and heavily silicified fimbriae which seem to be attached to the hyaline rim and are irregularly spaced (Fig. 2A-F).The poles of valvocopulae possess unique 'sword-shaped' siliceous extensions that extend towards the interior of the valve and may vary in length (see arrows in Fig. 2A-F).
Etymology.Species was named for its crisscross-shaped central area of the raphe valve.
Distribution and ecology.So far, the new species has been collected at Qingyi River and Maolan Nature Reserve.The Qingyi River population of the new species appears to have a higher abundance in that location than populations from the Maolan Nature Reserve.The habitat of the new species is characterised by circumneutral to alkaline pH (7.5-8.5) and conductivity (120-305 µs/cm) and temperature range 18.0-24.0°C.

Discussion
The morphological differences between species of Cocconeis are limited (Stancheva 2022).Cocconeis crisscrossis sp.nov.was compared with other related species (e.g. C. pediculus Ehrenberg, Cocconeis chernobyliensis Genkal, Shcherbak & Semenyuk and C. molesta Kützing) which are structurally similar under the light microscope: they all have an elliptical valve outline, concave valve curvature, as well as the size and the arrangement of the striae which are almost parallel in the centre, radiate and slightly curved towards the apices.While the most obvious differences are mainly with respect to the central area under the light microscope.The central area of C. crisscrossis sp.nov.extends irregularly to both sides.That feature is absent or not as distinct in morphologically similar taxa. it is one characteristic that distinguishes our new species from other similar species (Table 2).The morphology of the RV, the SV and their connecting elements, such as the valvocopulae and the cingulum, are essential for delimiting species within the genus (Riaux-Gobin et al. 2013;Stancheva 2022).The valvocopula system and arrangement show significant variation amongst Cocconeis taxa, ranging from intricate fimbriae to no ornamentation; however, the morphology of the valvocopulae is thought to be diagnostic for each taxon (Holmes et al. 1982; Kobayasi and Nagumo 1985;Round et al. 1990;Riaux-Gobin et al. 2021;Stancheva 2022).As shown in Table 2, C. crisscrossis was compared with three similar taxa which are structurally different in ultrastructure, mainly in the valvocopulae.Cocconeis crisscrossis and C. pediculus possess close valvocopulae with fimbriate margins, while the fimbriae margins of C. pediculus are short and present only in the central part of the valve, never at the poles.In contrast  During our observation of this new species, we have discovered that the fimbriate margins of the valvocopula, which occur in the central part of the valve, display considerable morphological stability, while the size and length of the fimbriae exhibit variation.In addition, the size and length of the 'sword-shaped' siliceous extensions at the end of valvocopula also vary.

Figure 2 .
Figure 2. A-F SEM micrographs, the valvocopula of C. crisscrossis sp.nov.from Qingyi River with well-developed digitate fimbriae, which is close and heavily silicified clamp-like fimbriae seem to be attached to the hyaline rim and are irregularly spaced; the poles of valvocopulae possess unique 'sword-shaped' siliceous extensions (arrows in A-F) A valvocopula of the RV B-F valvocopula of the SV.Scale bars: 5 µm (A-D); 1 µm (E, F).

Figure 3 .
Figure 3. A-E SEM micrographs, RV external views of C. crisscrossis sp.nov.from Qingyi River A-C complete internal valve D proximal raphe endings are slightly drop-like expanded E irregular hyaline central area.Scale bars: 5 µm (A-C); 1 µm (D, E).

Figure 4 .
Figure 4. A-F SEM micrographs, RV internal views of C. crisscrossis sp.nov.from Qingyi River A-C complete internal valve D irregular hyaline central area and the proximal raphe fissures are bent to opposite sides E weakly elevated helictoglossae F the areolae are occluded by round lattice-structured hymens.Scale bars: 5 µm (A-C); 1 µm (D-F).

Figure 6 .
Figure 6.A-C SEM micrographs, SV internal views of C. crisscrossis sp.nov.from Qingyi River A complete internal valve B, C detail of the internal valves ends.Scale bars: 5 µm (A); 1 µm (B, C)).

Table 1 .
Locality data and habitat for samples studied.