1 INTRODUCTION

The diatom genus Nupela was described by Vyverman and Compère (1991: 175) for a biraphid diatom (N. gilwuensis Vyverman and Compère 1991: 178) from Papua New Guinea with external areolae having a thin layer occluding the openings. Since the original description with a single species, the genus has grown to over 85 taxa (Kociolek et al., 2022). The feature thought to unite these taxa is the external hymenate covering over the striae and areolae (Potapova, 2003). Some members of the genus have been transferred to Nupela from genera as diverse as Brachysira Kützing (1836: 3), Achnanthidium Kützing (1844: 75), Navicula Bory (1822: 128), and Neidium Pfitzer (1871: 39), and new species have been described from several continents (e.g., Kulikovskiy et al., 2009, 2020; Potapova, 2011; Buczkó et al., 2013; Tremarin et al., 2015; Yu et al., 2017; Guiry and Guiry, 2021; Kociolek et al., 2022). The genus appears to be unknown from Antarctica and Australia. The first report of the genus in the fossil record is from 40 million years ago during the Eocene Epoch (Siver et al., 2010).

The breadth of morphological forms assigned to the genus has grown dramatically to include biraphid and monoraphid diatoms (Vyverman and Compère, 1991; Lange-Bertalot, 1993; Potapova, 2011), species with symmetrical and asymmetrical central areas (e.g., Siver et al., 2007; Tremarin et al., 2015), with smooth or highly mottled axial areas (Potapova, 2011), being asymmetrical about the transapical axis (Kulikovskiy et al., 2015), the apical axis (Lange-Bertalot, 1993; Metzeltin and Lange-Bertalot, 1998) or symmetrical to both axes (most species), and valves smooth and planar to having deep depressions (Potapova, 2011; Tremarin et al., 2015). The genus has been characterized as occurring in water of low mineral content and pH (Vyverman and Compère, 1991; Sala et al., 2014), but is also known from alkaline environments with high conductivity (Yu et al., 2017).

A single species of Nupela has been previously described from China. Nupela major Yu, Q. M. You & Kociolek (in Yu et al., 2017: 246) was described from a wet wall habitat in the Maolan Nature Reserve, Guizhou Province. This species is the largest described so far (length 20.0–41.5 μm, breadth 6.5–9.5 μm) for the entire genus and has a flat raphe valve and rapheless valve with a deep central trough.

New diatoms of Yunnan Province have been described initially by Skuja (1937), then in a series of papers by Chen (1980) and Li et al.(2010a, b), and more recently by Levkov et al. (2013), You et al. (2015), Liao and Li (2018), Cheng et al. (2018), and Jiang et al. (2018). Yunnan is a province with many microhabitats, and one that harbors significant biodiversity of terrestrial and aquatic environments (e.g., Li, 1993, 1995; Guo and Long, 1998; Zhou and Chen, 2006; Naruse et al., 2018). While the province has a number of new taxa described in the historical and more recent literature, the diverse geography, large lakes, three parallel rivers, hot springs, waterfalls, and other important aquatic environments make it home to most of the endemic freshwater fishes in China (Chen, 2013), and we suppose, potentially the diatoms as well. Our first report on the diatoms of the Shimen wet walls was focused on the valve ultrastucture and transfer of Melosira sinuata-radiata var. yunnanica Chen to the genus Orthoseira Thwaites (Kociolek et al., 2021).

It is the purpose of the present report to present light and scanning electron microscopic observations on five Nupela species collected from wet walls in Yunnan Province, and to formally describe these species as new.

2 MATERIAL AND METHOD

The diatom samples investigated in this study were obtained from Shimen Valley of Ailao Mountain, Yunnan Province, China. Kociolek et al. (2021) describes the ecological setting of this region.

Epilithic samples were collected from a variety of areas of wet wall in Shimen Valley. The individual samples considered herein include: 201904033, 201904055.

Samples were digested with hydrochloric acid to remove organic matter. After repeated washing of the treated material with distilled water, aliquots were dried on cover glasses and mounted in Naphrax (Brunel Microscopes Ltd., Chippenham, UK). All LM observations were performed using Olympus BX-51 light microscopes with a 100× oil immersion objective (numerical aperture 1.40). Specimen images were taken using Olympus DP72 digital cameras at Shanxi University, China, or at the University of Colorado, Boulder, USA.

Measurements of each species were based on 50 specimens. For scanning electron microscope (SEM) observations, the cleaned specimens were air dried onto glass cover slips and attached to copper stubs and coated with approximately 15 nm of gold-palladium. Scanning electron microscope observations were made using a Hitachi SU8010 SEM at a working distance less than 6 mm and accelerating voltage set at 1–2 kV at Shanghai Normal University.

Holotype slides and material are stored at the Freshwater Algal Herbarium of Shanxi University (SXU), Taiyuan, China. Isotype slides are deposited in the Kociolek Collection at the University of Colorado (COLO).

3 RESULT

Nupela shimenensis, Q. Liu, S. L. Xie & Kociolek, sp. nov. Figures 1 (LM) & 2–3 (SEM)

Description: Frustules biraphid, valves elliptical, tapering quickly to the apex, poles barely protracted, rounded. Length 14–24 μm, breadth 5.0–6.5 μm. Axial area narrow at the poles, lanceolate in shape, expanded laterally to form an asymmetrical central area, with one side expanded to the margin, the other side with few striae of irregular length present. Raphe appears filiform, and more or less straight. The proximal raphe ends are dilated. Striae fine, radiate, some of differing lengths, 28–32/10 μm. A small round central nodule is visible.

In the SEM, the valve exterior (Fig. 2) can have varying degrees of silicification. In cells with the smallest amount of silica seen (Fig. 2a–c), individual areolae have coverings over the opening, and there are very small, round openings around the periphery of each areola. The raphe appears straight to slightly undulate, the distal ends of the raphe curve onto the mantle, and there is no siliceous rim around the margin of the valve. In valves with more silica (Fig. 2d–f), the coverings over the striae are less organized, and are thicker, there is a rim of silica at the margin and the raphe is less undulate and the distal ends do not curve onto the mantle.

Internally in the SEM (Fig. 3) the raphe has hooked proximal ends laying at the base of the small, round central nodule (Fig. 3a, c, d, f). The small helictoglossae are positioned at tip of the valve apex (Fig. 3b & e). Areolae are unoccluded internally (Fig. 3b, c, e, f). The valvocopulae appear to have a small septum associated with them (Fig. 3b & e).

Holotype: Slide No. 201904033-1 in collection of SXU, Freshwater Algal Herbarium of Shanxi University, Taiyuan, China, holotype illustrated in Fig. 1a & b.

Type locality: Yunnan Province, Shimen Valley, wet wall, periphyton, China (23°58ʹ22ʺN, 101°31ʹ25ʺE, above sea level (ASL): 1 987.7 m).

Etymology: Named for the region where it was found.

Comments: Nupela shimenensis is similar in outline with N. decipiens (Reimer) Potapova, and the two species also have widened central areas (Reimer and Reimer, 1966; Potapova, 2013). The two species differ in that the specimens from South Carolina, USA, described originally by Reimer and Reimer (1966) and subsequently documented by Potapova (2011) are distinctly heterovalvate, possessing raphe and rapheless valves that are ornamented differently (Potapova, 2013). In N. shimenensis, frustules are not heterovalvate, but rather all valves possess a raphe system and have similar ornamentation.

Nupela yunnanica, Q. Liu, S. L. Xie & Kociolek, sp. nov. Figures 4 (LM) & 5–6 (SEM)

Description: Frustules monoraphid, valves ovoid-elliptical, without protracted apices. Length 11–28 μm, breadth 5.5–8.0 μm. Axial area narrow, narrowly-lanceolate in shape, no distinct central area formed. Axial area mottled with siliceous nodules and depressions. Raphe valve with straight, filiform raphe with dilated proximal ends. Striae radiate, with some striae of differing lengths. Striae 19–23/10 μm.

In the SEM, the exterior of the raphe valve (Fig. 5a–c) has many round to irregular, variously-sized pits or depressions mostly along the axial area but scattered across the valve face. There is a covering over the entirety of each striae that occludes the areolae. The raphe is undulate, with the proximal ends dilated slightly and distal ends curved in the same direction and extending onto the mantle. Internally, the raphe valve (Fig. 5d–f) has distinctly radiate striae and a small, round central nodule. Distal raphe ends terminate as helictoglossae. The proximal ends are anchor-shaped. Areolae are unoccluded internally.

The exterior of the rapheless valves (Fig. 6a–d) show a network of thickened siliceous outgrowths mostly away from the ends and concentrated down the center of the valves. Areolae have thickenings around them. The margin of the valve has a thickened rim. Internally (Fig. 6e–f), the broadly-lanceolate axial area dominates the valve interior of the rapheless valve. Areolae are small, round and without occlusions. A "ghost raphe" can be seen on some valves (Fig. 6f & g).

Holotype: Slide No. 201904055-1 in collection of SXU, Freshwater Algal Herbarium of Shanxi University, Taiyuan, China, holotype illustrated in Fig. 4a & b.

Type locality: Yunnan Province, Shimen Valley, wet wall, periphyton, China (23°58ʹ22ʺN, 101°31ʹ21ʺE, ASL: 2 065.7 m).

Etymology: Named for the province in which it was found.

Comments: Several species of Nupela have been described with the mottled look about the axial area, including N. lapidosa (Krasske) Lange-Bertalot and Genkal (1999) and N. major Yu, You & Kociolek (in Yu et al., 2017). Nupela yunnanica differs from these species by being significantly smaller than N. major and having a valve shape that is more elliptical than what is seen in N. lapidosa (Potapova, 2011)

Nupela magna, Q. Liu, S. L. Xie & Kociolek, sp. nov. Figures 7 (LM) & 8–9 (SEM)

Description: Valves linear-elliptical with produced ends that are broadly-rounded. Length 25–38 μm, breadth 6.0–7.5 μm. Axial area wide, lanceolate, forming a circular central area in most specimens. The central nodule is round. Raphe filiform, with external proximal ends deflected slightly in the same direction. Striae short, fine, composed of small dash-like areolae, radiate, ca. 28–32/10 μm, slightly offset from one another.

In the SEM, the valve exterior (Fig. 8) has areolae covered with occlusions. The raphe is straight to slightly undulate, the external proximal raphe ends are not dilated and the distal ends are hooked in the same direction and extend onto the valve mantle. Areolae are bordered by oblong silica thickenings. Specimens may lack a marginal siliceous border, or the marginal border may be fimbriate or entire (e.g., Fig. 8b & e). Internally (Fig. 9), there is a distinct central nodule at the base of which sit the proximal raphe ends. Internal proximal raphe ends are anchor-shaped, with one side branch larger than the other. Areolae are unoccluded. Distally, the raphe terminates as distinct helictoglossae.

Holotype: Slide No. 201904055-1 in collection of SXU, Freshwater Algal Herbarium of Shanxi University, Taiyuan, China, holotype illustrated in Fig. 7c.

Type locality: Yunnan Province, Shimen Valley, wet wall, periphyton, China (23°58ʹ22ʺN, 101°31ʹ21ʺE, ASL: 2 065.7m).

Etymology: Named for being a large-sized Nupela species.

Comments: Structure of the striae as seen with LM, the areolae in adjoining striae are slightly offset from one another, a feature that is reminiscent of Anomoeoneis or Brachysira (Round et al., 1990). Valves appear as similar to large specimens of Humidophila, but the striae are comprised of more than a single areola.

Nupela sinensis, Q. Liu, S. L. Xie & Kociolek, sp. nov. Figures 10 (LM) & 11–12 (SEM)

Description: Frustules monoraphid, linear-lanceolate in shape, with slightly protracted rounded apices, length 7–25 μm, breadth 2–4 μm. Raphe valve: Axial area straight to slightly lanceolate in shape, expanded unilaterally, rectangular in shape, raphe filiform, straight, slightly swollen at the external proximal ends, distal ends indistinct. Striae radiate, individual areolae not resolved, 27–30/10 μm. Rapheless valve: central area wide, lanceolate in shape. Striae radiate throughout, 27–30/10 μm. Individual areolae not resolved.

In the SEM, the valve exterior of the raphe valve (Fig. 11a–c) has distinct areolae. No occlusions have been observed on the areolae, but there is a distinct rim around the openings. The filiform raphe is distinct, with slightly dilated proximal ends and distal ends that are hooked in the same direction, extending from the valve face onto the mantle. Internally, striae are distinct, radiate and composed of areolae that lack occlusions (Fig. 11d–f). The central nodule is small, circular and is positioned next to the small, asymmetrical, anchor-shaped proximal raphe ends. The raphe terminates at the ends as helictoglossae.

The exterior of the rapheless valve (Fig. 12a–c) has areolae bordered by siliceous rims. On the mantle, each stria opens with a single, elongated areola. In some specimens, there is an isolated opening at the terminus of the valve, at the end of the axial area (Fig. 12c). Internally (Fig. 12d–f) the areolae are unoccluded, and there is a small but distinct septum present each apex (Fig. 12d & f).

Holotype: Slide No. 201904055-1 in collection of SXU, Freshwater Algal Herbarium of Shanxi University, Taiyuan, China, holotype illustrated in Fig. 10e & f.

Type locality: Yunnan Province, Shimen Valley, wet wall, periphyton, China (23°58ʹ22ʺN, 101°31ʹ21ʺE, above sea level ASL: 2 065.7 m).

Etymology: Named for this species being found in China.

Nupela comperei, Q. Liu, S. L. Xie & Kociolek, sp. nov. Figrues 13 (LM) & 14 (SEM)

Description: Frustules biraphid. Valves linear-lanceolate to lanceolate in shape, with apices rounded. Length 8–17 μm, breadth 4–5 μm. Axial area linear, expanded laterally at the center to form a small, rectangular central area. Raphe straight to slightly undulate, filiform. Striae very fine, radiate, 35–40/10 μm, individual puncta indistinct.

In the SEM, the valve exterior (Fig. 14a–d) shows the striae comprised of 2–3 areolae, interrupted along the apical axis. Areolae are occluded externally. The valve may have a thickened margin, which appears wavy in some specimens. The thickened margin may be lacking. Raphe with proximal ends dilated slightly, with distal ends hooked and extending onto the valve mantle. Internally (Fig. 14e–h), a small, round, raised central nodule is evident. The central nodule bears the external proximal raphe ends, which are straight. The raphe terminates at the apices as helictoglossae. The areolae are unoccluded and are irregularly aligned from stria to stria.

Holotype: Slide No. 201904055-1 in collection of SXU, FreshwaterAlgal Herbarium of Shanxi University, Taiyuan, China, holotype illustrated in Fig. 13a.

Type locality: Yunnan Province, Shimen Valley, wet wall, periphyton, China (23°58ʹ22ʺN, 101°31ʹ21ʺE, ASL: 2 065.7 m).

Etymology: Named for Pierre Compère, former research scientist at the National Botanic Garden in Meise, Belgium, and one of the original describers of the genus Nupela.

4 DISCUSSION

The concept of Nupela has broadened dramatically since its original description as a monotypic genus over 30 years ago (Vyverman and Compère, 1991). One interesting aspect of the genus as currently circumscribed is that species can be biraphid with a raphe system that extends from the central area to the apices, to biraphid taxa where the raphe may be much shorter than a "typical" raphe might be, to frustules that are heterovalvate, and there is one valve with a raphe and the other without. Potapova et al. (2003) even describe the situation in one heterovalvate species where there are degrees of differences. Work on the raphe number suggests that secondary loss of the raphe has happened 5–6 times (Kociolek et al., 2019; Kulikovskiy et al., 2020), and that even within one taxon, a species can produce biraphid, monoraphid or even araphid frustules (Liu et al., 2018). Lange-Bertalot (in Krammer and Lange-Bertalot, 1991) hinted at this possibility in species that ultimately were transferred to Nupela from Achnanthidium and Navicula (Potapova, 2011), and suggested that despite being in two different genera, the taxa might be the same taxon. However, these species, while both were transferred to Nupela, were kept separate in part due to raphe number. In the cases of the species we present here as new, three are biraphid and the other two are monoraphid. However, two species of biraphid diatom have similar structure on both valves (N. magna sp. nov. and N. comperei sp. nov), and in the third the frustule can be heterovalvate, with the length of the raphe branches differing between valves on the same frustule (N. shimenensis sp. nov.). One species (N. sinensis sp. nov.) has predominately monoraphid frustules, but in a few cases it appears to produce biraphid frustules as well, while the other monoraphid species (N. yunnanica sp. nov.) is clearly heterovalvate in terms of structure, with the raphe valve being much more rugose than the rapheless valves. Plasticity in raphe number within a species has been documented by Liu et al. (2018), where a single species can have 0, 1 or 2 raphe systems on a frustule.

In addition to the number of raphe systems per frustule, within and between species, there is also variability between Nupela taxa with regards to the structure of the raphe, in particular the internal proximal raphe ends. Some Nupela species have anchor-shaped internal proximal raphe ends, where the hooked elements are of equal proportions (e.g., N. yunnanica) while in others (such as N. magna) the anchor-shape is asymmetrical, with one side longer than the other. These anchor-shaped internal proximal raphe ends are similar to those seen in the genus Gomphosphenia Lange-Bertalot (1995; see also Kociolek et al., 1988). In some species the internal proximal raphe ends are recurved to one side only, typical of species among the freshwater gomphonemoid diatoms (e.g., Kociolek and Stoermer, 1993). Finally, N. comperei seems to have straight internal proximal raphe ends. Sala et al. (2014), Tremarin et al. (2015), and Kulikovskiy et al. (2020) showed other species with straight, not recurved, internal proximal raphe ends in Nupela species.

Nupela magna sp. nov. is quite large relative to most species assigned to the genus; it is approximately the same size as N. major Yu et al. (2017) (valve length 25–38 μm versus 20.0–41.5 μm, breadth 6.0–7.5 μm versus 6.5–9.5 μm). Taxa septa (N. sinensis and N. shiwanensis) are the first taxa in the genus Nupela reported to have these structures, further expanding the known morphological diversity of the genus.

The diagnostic feature of the genus Nupela is the presence of hymenate occlusions on the exterior of the areolae, which differentiates the genus from many naviculoid and monoraphid genera which have occlusions on the interior. Though Cox (2015) has suggested Nupela may be closely related to Brachysira (echoing the original describers of the genus (Mann in Round et al., 1990; Vyverman and Compère, 1991)); the genus has internal hymenate occlusions over the areolae (Lange-Bertalot and Moser, 1994; Anderson et al., 2013). The same is true for many other biraphid genera symmetrical about the apical and transapical axes, including others classified close to Brachysira (Round et al., 1990). In monoraphid genera such as Achnanthidium, areolae also have internal hymenes (e.g., Yu et al., 2018; You et al., 2019). Authors such as Potapova et al. (2003) have pointed to the presence of an external covering ("coalescing hymens") across the striae externally as support in their placement of species in Nupela (whether that covering was visible or not, and whether it was demonstrated in the generitype, N. gilwuensis). They discuss the placement of areolar coverings as a fundamental difference between lineages of naviculoid diatoms (Potapova et al., 2003). Kulikovskiy et al. (2020) have shown Nupela to be closely related Diploneis (Ehrenberg) P. T. Cleve (1894: 76), as part of a distinct monophyletic group with Biremis D. G. Mann & E. J. Cox in Round et al. (1990: 664) and quite separate from Brachysira. It is not clear, however, if the diversity of valve morphologies expressed in species assigned to Nupela represent a single monophyletic group and the genus is plastic in terms of raphe number and in the morphological diversity represented, or if these different morphological groups may reflect a more complex set of phylogenetic relationships. To date, only two taxa have any molecular data generated from them, and both species have straight, internal proximal raphe ends (Kulikovskiy et al., 2020). The morphological diversity seen from the wet wall in Yunnan has not yet been investigated with molecular tools.

The new species described herein from a wet wall in Yunnan Province provide further evidence that this province harbors a diversity of endemic diatom taxa. Skuja (1937) was the first to document unique species from Yunnan. Species described as the province, include "centric" and pennate diatoms, and in the latter group, across a wide range of lineages. And while some of Skuja's species are still present (Li et al., 2018; Liu et al., 2020), it appears they may be declining in numbers (Tao et al., 2016). In these works, only a small number of aquatic habitats have been investigated, and almost no high elevation lakes, none of the largest rivers ("3 Parallel Rivers") and habitats from the high mountainous regions have been studied in detail. Further research on the diatoms from Yunnan Province is needed to document a baseline of biodiversity present, so that the effects of human disturbance can be assessed.

5 DATA AVAILABILITY STATEMENT

All data included in this study are available upon request by contact with the corresponding author.

6 ACKNOWLEDGMENT

We are grateful to Shanxi Academy of Advanced Research and Innovation in acquiring SEM images.