Four new species from the diatom (Bacillariophyceae) genus Adlafia Moser, Lange-Bertalot & Metzeltin from waterbodies of Vietnam

Abstract Four species of the diatom genus Adlafia were found from waterbodies of Vietnam and described as new to science. Their formal descriptions are presented herein and they are illustrated by light and scanning electron micrographs. These new species are: A. lamdongiensis Glushch., Kulik. & Kociolek, sp. nov., A. babeiensis Glushch., Kulik. & Kociolek, sp. nov., A. vietnamensis Glushch., Kulik. & Kociolek, sp. nov. and A. dauiensis Glushch., Kulik. & Kociolek, sp. nov. These species are then compared to other similar taxa. Our new findings add to the number of species in this interesting genus and contribute to our understanding of the unique diatom flora found in Vietnam.


Introduction
The genus Adlafia was proposed by Moser et al. (Moser et al. 1998). According to the original description, the genus is overwhelmingly represented by small-cell species (less than 25 µm in length). The raphe is naviculoid; external distal ends are smoothly bent and slightly extend to the mantle externally while the external proximal ones are drop-shaped, slightly bent to the opposite side from the distal ends ). On the inside, the raphe is located on a raised sternum, the distal ends with small helictoglossae, the proximal ends are straight and bent to one side (Morales and Le 2005). A distinctive feature of the genus is the presence of large, often square areolae, closed externally with a hymen and a continuous silica layer (Moser et al. 1998;Lange-Bertalot 2001). Species of the genus are distinguished from those in the genus Kobayasiella Lange-Bertalot in Lange-Bertalot and Genkal (1999) by lacking an "umbilicus", a deflection or nick in the raphe system on the exterior. Currently, the genus belongs to taxa with an unclear taxonomic position (incertae sedis). Molecular studies of the genus require the involvement of more strains .
The genus includes 27 species and infraspecific taxa (Guiry and Guiry 2020). Species of the genus are distributed around the world. Most species are aerophilous, being found mainly on mosses, but others prefer oligotrophic streams and lakes with a slightly higher or lower pH value, but are rare in large rivers (Spaulding and Edlund 2009;Kulikovskiy et al. 2016;Cantonati et al. 2017). Species are also known from fossil sediments (Lange-Bertalot and Metzeltin 1996;Benson and Kociolek 2012).
Southeast Asia is a floristically interesting region, from which many new genera and species of centric and pennate diatoms have been described recently (see Gusev and Kulikovskiy 2014;Glushchenko et al. 2016Glushchenko et al. , 2017Glushchenko et al. , 2018Glushchenko et al. , 2019Kapustin et al. 2017Kapustin et al. , 2019Liu et al. 2018;Kulikovskiy et al. 2019Kulikovskiy et al. , 2020Rybak et al. 2019). Several Adlafia species have been recorded previously from Southeast Asia. In Indonesia, for example, Adlafia bryophila (J. Petersen) Lange-Bertalot in Moser et al. 1998 and Adlafia minuscula (Grunow) Lange-Bertalot in Lange-Bertalot and Genkal 1999 have been reported (Bramburger et al. 2004). Adlafia sinensis Liu & Williams in Liu et al. 2017 was described from south-central China. They also provide a comparison of many Adlafia species. In Vietnam, Adlafia minuscula var. muralis (Grunow) Lange-Bertalot in Lange-Bertalot and Genkal 1999 was reported from reservoirs, but without an image to document the determination (Duong et al. 2006).
The aim of our work was to identify the species diversity of the genus Adlafia in freshwater ecosystems of Vietnam.

Materials and methods
A list of all samples examined in this study with their geographic positions is presented in Table 1. The samples were treated with 10% hydrochloric acid to remove carbonates and washed several times with deionized water for 12 h. The samples were subsequently boiled in concentrated hydrogen peroxide (≈37%) to dissolve organic matter. They were then washed four times with deionized water at 12 h intervals. After decanting and refilling with up to 100 ml deionized water, the suspension was spread onto coverslips and left to dry at room temperature. Permanent diatom preparations were mounted in Naphrax. Light microscopic (LM) observations were performed with a Zeiss Axio Scope A1 microscope equipped with an oil immersion objective (× 100, n.a. 1.4, differential interference contrast [DIC]) and Axiocam ERc 5s camera (Zeiss). Valve ultrastructure was examined by means of a JSM-6510LV scanning electron microscope (IBIW, Institute for Biology of Inland Waters RAS, Borok, Russia). For scanning electron microscopy (SEM), parts of the suspensions were fixed on aluminum stubs after air-drying. The stubs were sputter-coated with 50 nm Au in an Eiko IB 3 sputter coater. Samples and slides are deposited in the public collection of Maxim Kulikovskiy at the Herbarium of the Institute of Plant Physiology Russian Academy of Science, Moscow, Russia. The number of examined valves is indicated in each description of the species. The average value of the valve length, width and striae density, as well as standard deviation were calculated using Microsoft Excel 2020. Terminology of the valve follows Moser et al. 1998;Lange-Bertalot 2001;Morales and Le 2005;Kulikovskiy et al. 2016;Tusset et al. 2017 andCiugulea et al. 2019.   Gusev, 21.06.2012. Description. LM ( Fig. 1A-G). Valves linear with weakly convex margins. Ends are distinctly narrowly-rostrate. Length 9.7-13 µm (11.4 ± 0.9; n = 16), breadth 2.5-2.8 (2.7 ± 0.1; n = 16) µm. Striae and areolae not resolved in LM.
SEM, internal view ( Fig. 2D-F). Raphe slightly lateral, lies in a prominent and raised raphe-sternum. Proximal raphe endings deflected towards primary side of valve. Distal raphe endings terminating in small helictoglossae. Striae continuing onto valve mantle. Short striae alternate with longer striae at valve center. Areolae rounded or rectangular. Openings of apical areolae apically elongated.
Etymology. Epithet refers to the province of Vietnam (Lâm Đồng Province) where the specimens were found.

Adlafia babeiensis
Etymology. Epithet refers to the lake of Vietnam where the new species was found. Distribution. Vietnam. Known only from the type locality.   Distal raphe endings positioned on the valve mantle, hooked and curved in the same direction, and terminating at the junction valve face. Striae uniseriate. Areolae rounded or rectangular, occluded by hymenes. Slit-like opening of apical areolae invisible. Areolae 50-55 in 10 µm (52.5 ± 1.0; n = 20). SEM, internal view ( Fig. 6D-F). Raphe straight, lying in a prominent and raised raphe-sternum. Proximal raphe endings deflected towards primary side of valve. Distal raphe endings terminating in small helictoglossae. Striae continuing onto valve mantle. Short striae alternate with longer striae at the center of the valve. Areolae rounded or rectangular. The openings of apical areolae apically elongated.

Adlafia vietnamensis
Etymology. Epithet refers to the country where the new species was found. Distribution. Vietnam. Slides no. 00325 (type locality) and no. 04633.   SEM, internal view ( Fig. 8D-F). Raphe straight, lying in a prominent and raised raphe-sternum. Proximal raphe endings deflected towards primary side of valve. Distal raphe endings terminating in small helictoglossae. Striae continuing onto valve mantle. Short striae alternate with longer striae at the center of the valve. Areolae rounded or rectangular. The openings of apical areolae apically elongated.

Adlafia dauiensis
Etymology. Epithet refers to the river of Vietnam where the new species was found. Distribution. Vietnam. Known only from the type locality.

Discussion
The four new species described here from Southeast Asia are morphologically similar to each other, but can be differentiated on the basis of valve shape, valve ends and striae density. All species share the morphological features typical for the genus Adlafia. A comparison of species to each other and with previously-described taxa shows that the new species from Southeast Asia are easily distinguished, unique taxa (Table 2). Adlafia lamdongiensis sp. nov. resembles specimens identified by Lee as Kobayasiella venezuelensis Metzeltin & Lange-Bertalot (2007, p. 155, pl. 141, figs 10-23) specimens as illustrated with light micrographs (Lee 2012, fig. 15, K-M) on the basis of valve outline. Moreover, the valve identified by Lee in the SEM (Lee 2012, fig. 15, N) would appear to belong to the genus Kobayasiella, since there is a characteristic break of the raphe (the "umbilicus") inherent to representatives of this genus. The valve has noticeably convex edges (Lee 2012, fig. 15, N), while in our material, and the light micrographs of Lee, valves are slightly convex. In our opinion, the light micrographs and a scanning image of Lee (2012) belong to species from different genera.
These new species were found in different water ecosystems of Vietnam that show this genus is widespread in this country, especially in acidic ecosystems. Morales and Le (2005) suggested Adlafia is a monophyletic group but they did not perform any formal analysis or present data to support their conclusion. Based only on a single species, Thomas et al. (2016) suggested Adlafia is part of a monophyletic group that could be considered the Cymbellales. No other analysis was forthcoming on this taxon, so this work did not address whether Adlafia is a monophyletic genus. Several authors, including in the original description of Adlafia, have made comparisons with Kobayasiella Lange-Bertalot in Lange-Bertalot and Genkal 1999(as Kobayasia Lange-Bertalot, 1996, non Kobayasia S. Imai & A. Kawamura, 1958; see also Morales and Le 2005;Monnier et al. 2012;Van de Vijver et al. 2017). The two genera have fine striae, external distal raphe ends that are distinctly curved and external hymenate occlusions on the areolae. The difference between the two genera is usually suggested to be the absence (in Adlafia) or presence (in Kobayasiella) of a deflection (umbilicus) in the raphe system. However, this distinction has not always been applied consistently. For example, Le Cohu and Azémar (2010, figs 12, 13) showed specimens of K. jaagi (Meister) Lange-Bertalot, 1999 without the umbilicus. Liu et al. (2017) highlighted areas of the girdle that might help diagnose Adlafia as a monophyletic group, but these observations await formal analysis.