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Three new subaerial Achnanthidium (Bacillariophyta) species from a karst landform in the Guizhou Province, China

Three new subaerial species of Achnanthidium are examined and described from a karst landform of central–south China based on light and scanning electron microscopy observations, and are compared with similar taxa worldwide. A. mediolanceolatum sp. nov. is elliptical–lanceolate, not linear–lanceolate as is more typical in this genus, as well as having a bow–tie shaped central area on the raphe valve and a large lanceolate axial area on the rapheless valve, features which together make it easy to distinguish this species from others. A. parvulum sp. nov. and A. guizhouensis sp. nov. are linear–elliptical in shape, which is more common for Achnanthidium and they differ from other species mainly in size, raphe, density of striae and shape of the central area. We compared these three new species with morphologically–similar taxa, and present their ecological settings and distributions as well. The relationship between Achnanthidium and Psammothidium is discussed, and the data from these new species from Guizhou presented herein suggest the two genera are difficult to separate.


Introduction
A generation ago, floristic manuals recognized two genera of monoraphid diatoms occurring in freshwater ecosystems, Cocconeis Ehrenberg and Achnanthes Bory (e.g. Patrick & Reimer 1975;Krammer & Lange-Bertalot 2004). Since that time, morphological evidence has grown to suggest several groups of monoraphid diatoms should be segregated from Achnanthes sensu stricto as typified by A. adnata Bory. The work of  and others either resurrected names once forgotten such as Achnanthidium Kützing and Eucocconeis Cleve ex Meister, or proposed new taxa based on morphological evidence that was distinctive with light and scanning electron microscopy. These new taxa included genera such as Planothidium Round Round. Since this work in the late 1990's establishing new monoraphid genera, studies have been undertaken on the relationships of the groups to one another, resulting in further dissection and circumscription of new taxa from these genera and, in some cases, raising concerns about whether the new taxa were warranted. Phylogenetic analyses have suggested that the monoraphid diatoms represent a grade of organization, not a monophyletic clade of closely related taxa (e.g. Thomas et al. 2016;Kulikovskiy et al. 2016), and that five or more lineages independently secondarily lost the raphe system on one valve (Kociolek et al., accepted). Within Achnanthidium, however, three morphological groups have been identified, recognized by their distinctive features of flexure of the external distal raphe ends and areolar structure (Karthick et al. 2017;Yu et al. 2018;Yu et al. 2019). Lange-Bertalot (in Krammer & Lange-Bertalot 2004) established the genus Platessa for species not easily assigned to Psammothidium or Achnanthidium. Kulikovskiy et al. (2013) established the genus Gliwiczia Kulikovskiy, Lange-Bertalot et Witkowski based on species that have a cavum, like those found in Planothidium, but occurring on both valves of a frustule. On the other hand, some have begun to question the distinctiveness of certain genera, with some species such as Kolbesia suchlandtii (Hustedt) Kingston (Kingston 2000) and K. gessneri (Hustedt) Aboal (Aboal et al. 2003) also being assigned to the genus Karyevia. Monnier et al. (2007) have called into question whether Psammothdium can be separated from Achnanthidum based on species variation and the features used to circumscribe Psammothdium from Achnanthidium.
Achnanthidium species have been found throughout China, with over 60 taxa reported (Liu Y. et al. 2014(Liu Y. et al. , 2015(Liu Y. et al. , 2016Wang & Deng 2017;Yu et al. 2017aYu et al. , 2018Yu et al. , 2019Zhu & Chen 2000 et al.). Only six species of the genus have been described from China, however, including A. sinense Liu et Blanco (Liu B. et al. 2016) from the Wuling Mountains, A. longissimum Yu (Yu et al. 2018) from Jiuzhai Valley, Southwestern China, and another three species from Taiping Lake .
While working on the diatom biodiversity of a karst landform in Guizhou, we observed many interesting diatom species, resulting in the description of one new genus and eleven new taxa (Kociolek et al. 2016a(Kociolek et al. , b, 2019Lowe et al. 2017;You et al. 2016You et al. , 2017You et al. , 2019Yu et al. 2017b). The aim of this article is to describe three new Achnanthidium species, based on light microscopy (LM) and scanning electron microscopy (SEM) of cleaned materials from this same karst landform. We also provide a comparison with morphologically similar taxa, as well as ecological habitats and distribution for each of the new species.

Materials and Methods
Diatom samples were collected from two sampling areas, Daqikong (Big Seven holes) scenic spot and Maolan Nature Reserve, in Libo county, Guizhou Province on Oct. 3th, 4th, 2015. This region is located in the middle of the subtropical zone and is typical for karst landforms. Daqikong is famous for its steep hills and natural canyon, the Mengtang (Dagou) River flows through canyon, there are many tourists throughout the year. Maolan Nature Reserve is protected by the Chinese government and international agreements through inclusion as part of International Man and Biosphere Reserve Network (UNESCO) , and consequently it is less affected by human factors. These two areas are adjacent, and the distance between sampling sites is ca. 30 km. The collection habitats in this study are mainly on wet stonewalls at the base of hills, wet walls near caves, rock scrapes from a variety of lentic habitats and other subaerial environments. Elevation at the localities is ca. 400-500 a.s.l. Details on the collections used in this study can be found in Table 1. In the laboratory, samples were processed as described by You et al. (2016). Terminology for the morphological description follows Round et al. (1990), , Monnier et al. (2007), Manoylov (2007) and Potapova (2010Potapova ( , 2012.   rounded apices. Valve length 11.5-18.5 µm, width 4-5 µm (n=100). On the raphe valve, axial area narrow linear, centrally expanded to form a large, bow-tieshaped central area, symmetrical, and extending to the margins. Raphe filiform, with external proximal raphe ends straight, slightly expanded, external distal fissures obviously hooked in same direction. Transapical striae radiate throughout, straight, 18-20 in 10 µm. Areolae fine, not distinguishable. Striae near the apices on one side of the valve appear on a different focal plane compared to striae on the opposite side. Rapheless valve with short striae, resulting in axial areas large and wide, lanceolate in shape, occupying 2/3 of the valve breadth. Transapical moderately radiate throughout, 16-19 in 10 µm. Incompletely filled-in raphe slit commonly observed.

Remarks:
The valve of A. mediolanceolatum is lanceolate-elliptical, this outline is not common in this genus and makes this new species easy to distinguish from others. This new Achnanthidium species is similar to Psammothidium helveticum (Hustedt) Bukhtiyarova et Round , P. marginulatum and P. grischunum in size and outline, especially for smaller specimens in the populations studied. However the new species can be easily distinguished by striae density and structure of the axial area on the rapheless valve. The striae of the new species are more coarse, less than 20 in 10 µm, as compared to ca. 18-30 in 10 µm in others (Hustedt 1933;Manoylov 2007). The axial area on the rapheless valve is lanceolate-elliptical and occupies the most valve, it is much larger than others. A comparison of these species is provided in Table 2. On the raphe valve: external views show straight raphe with proximal raphe endings straight, dilated, distal ends obvious curved in same direction on the valve face, not reaching the margins (Figs 117, 124). Central area narrow, forming a transverse rectangle, not reaching the margins (Fig. 117). Striae uniseriate, continuing to the valve face/valve mantle junction. Areolae not uniform, elliptical in the central part of the valve, 1-4 (usually 2-3) per stria, with one or two rows of slit-like areolae near the margin and one row of slit-like areolae on the mantle (Figs 117, 121, 122). Internally, raphe straight, with proximal raphe endings slightly bent in opposite directions, distal raphe endings terminate as helictoglossae. Areolae large, elliptical or subelliptical in shape, occluded with fine hymenate structures that include small openings around the periphery (Figs 118, 123). On the rapheless valve: both exterior and interior, axial area linear to narrowly-lanceolate, striae uniseriate, areolae round to elliptical, 27-32/10 µm, hymenate occlusions Distribution: This species was present in collections GZ1510049, GZ1510063 and GZ1510068. Detailed ecological information is presented in Table 1.

Remarks:
This new small Achnanthidium species is similar in appearance to Psammothidium curtissimum (Carter) Aboal (Aboal et al. 2003), P. semiapertum (Hustedt) Aboal (Aboal et al. 2003), P. levanderi (Hustedt) Bukhtiyarova et Round  and P. pennsylvanicum Potapova (Potapova 2012), the length of these species do not exceed 13 µm. These species, however, are differentiated from one another mainly by features of valve width, striae density, and extent and shape of the axial and central areas. A comparison of these small species is provided in Table 3.
Both proximal raphe endings and distal endings straight, barely expanded (Figs 126, 127). Central area small, variable, asymmetrical, sometimes not obvious. Striae uniseriate, straight, mostly not continuing beyond the valve face/valve mantle junction, with a distinct hyaline area at the margin (Figs 125-127). Areolae not uniform, 2-5 per stria, elliptical or subelliptical on most of the valve, occlusions in the interior of the valve can be seen from the exterior (Figs 125-128), with one or two rows of small, rounded areolae near the margin and one row small rounded areolae in the mantle (Figs 126, 127). Internal raphe straight, proximal endings slightly bent in opposite directions, distal raphe endings terminate as helictoglossae (Figs 129-131). Areolae elliptical or subelliptical in shape and occluded with fine hymenate structures that include small openings around the periphery (Figs 129-132). On the rapheless valve, on both the exterior and interior sides, striae uniseriate, not terminating on the valve face/valve mantle junction, with a narrow hyaline area at the margin (Figs 133-135, 137-139). The structure and arrangement of areolae similar to the raphe valve (Figs 136,140  , P. marginulatum  and P. chlidanos  in terms of size and outline of the valves, but it is easy to differentiate them from each other based on striae density and the shape and extent of the axial and center areas. These species are compared in terms of their valve features in Table 4.

Discussion
With the advent of better electron microscopes and the discovery and description of new species, we are beginning to more fully document the morphological diversity exhibited by species of Achnanthidium. In our samples, for example, the three new species possess similar marginal regions, e.g. striae do not continue across the valve face/valve mantle junction, but instead are separated by a distinct marginal hyaline area. There are, however, some differences between the species. For example, there are two types of raphe morphology and areolae. The external raphe valves of A. mediolanceolatum sp. nov. and A. parvulum sp. nov. have straight raphes, with proximal endings straight and expanded, and distal endings curved in same direction on the valve face and not reaching the margins. In A. guizhouensis sp. nov., however, the raphe as well as the external proximal and distal endings are all straight. In terms of areolae, in A. mediolanceolatum sp. nov., the areolae are small, rounded or slit-like openings in external view, but internally the areolae are large, elongate ovals, occluded by individual hymenes. In A. parvulum sp. nov. and A. guizhouensis sp. nov., the external areolae are relatively large and elliptical along most of the valve, and the internal hymenate occlusions can be seen from the exterior with high magnification. The fine hymenate structures appear as small granules that include small openings around the periphery. These diagnostic characters are important for species Achnanthidium (Karthick et al. 2017;Yu et al. 2018Yu et al. , 2019, and two of these three groups have representatives among the new taxa described here. A. mediolanceolatum sp. nov. and A. parvulum sp. nov. belong to the A. pyrenaicum complex, which have external distal raphe fissures deflected in the same direction, but areolae of A. parvulum sp. nov. are elliptical in shape, not lineate, which is the typical type of areolae found in this group. A. guizhouensis sp. nov. belongs to A. minutissimum complex, members of which have straight distal raphe endings and round to elliptical areolae. Similarities in valve morphology between these Achnanthidium and Psammothidium taxa are striking, and provide more data to support Monnier et al.'s (2007) opinion that Achnanthidium and Psammothidium cannot be unambiguously separated on the basis of morphological criteria. The species described here lack most of the features proposed by  to diagnose Psammothidium, including convex curvature of the raphe valve and raphe fissures in channels. Even the subjective feature of valve shape used to distinguish between these genera (linear valves representing Achnanthidium species, more elliptical valves representing Psammothidium species), seems less helpful in distinguishing between the genera, given these two genera cannot be ditinguished by morphological characters, eg. frustule outline, valve length, valve width, striae arrangement and density, and raphe structure. Therefore, given this situation, we have not assigned these new species to Psammothidium. Potapova (2012) thought the boundaries of recently established freshwater monoraphid genera should be questioned. It may be necessary to review the distribution of morphological features among the genera with smaller species and, through formal analyses of morphology and molecular data in a taxonomic and systematic revision and recognition of monophyletic groups (Kociolek & Williams 2015), consider the redrawing of generic boundaries among these taxa.