﻿Achnanthidiumbratanense sp. nov. (Bacillariophyceae, Achnanthidiaceae), a new diatom from the Lake Bratan (Bali, Indonesia)

﻿Abstract A new species, Achnanthidiumbratanense, is described from Lake Bratan, located on the island of Bali (Indonesia). The morphology of this species was analyzed with light (LM) and scanning electron microscopy (SEM). A.bratanense is characterized by linear-elliptic to nearly elliptic valves with convex margins and rounded, broadly subcapitate apices. The striae of this species are hardly discernable under LM; they are weakly radiate throughout the valve and composed of one to four large transapically elongated areolae of different length and shape. The most similar taxon to A.bratanense is A.macrocephalum, a species described from Sumatra, another Indonesian island. The differences of A.bratanense from similar taxa are discussed.


Introduction
The genus Achnanthidium Kützing, 1844 is one of the largest genus among monoraphid diatoms. Although it had been described as a separate genus it was considered as a subgenus of Achnanthes Bory, from 1822 until the 90s (Round et al. 1990; Round and Bukhtiyarova 1996). Currently, Achnanthidium includes, according to different estimates, between 139 (Guiry and Guiry 2017) and nearly 200 species ). Revision of the genus continues up to now. Recently, two genera, namely Gogorevia (Kulikovskiy et al. 2020b) and Gomphothidium (Kociolek et al. 2021b) have been segregated from Achnanthidium.
The Achnanthidium taxa are common in different climatic zones all over the world (e.g., Ponader and Potapova 2007;Wojtal et al. 2011;Novais et al. 2015;Karthick et al. 2017;Marquardt et al. 2017;Krahn et al. 2018;Yu et al. 2019). However, their identification is challenging because of the small size of these diatoms, often requiring examination using electron microscopy, and significant variability of diagnostic features (Ponader and Potapova 2007;Hlúbiková et al. 2011).
The number of publications dealing with freshwater diatoms from Indonesia is still rather low. The most comprehensive treatment was made by Hustedt (1937Hustedt ( , 1942. Some of his new taxa have been re-examined (e.g., Hamsher et al. 2014;Kapustin et al. 2017Kapustin et al. , 2020Kapustin and Kulikovskiy 2018;Wetzel et al. 2019;Kulikovskiy et al. 2020a). Also, a lot of new diatom species were described from Indonesian freshwaters over the last two decades (Bramburger et al. 2006;Kociolek et al. 2018;Kapustin et al. 2019Kapustin et al. , 2021Kulikovskiy et al. 2019;Rybak et al. 2019), including two new Achnanthidium species (Tseplik et al. 2021a,b). The aim of this paper is to describe a new monoraphid species, Achnanthidium bratanense sp. nov., from Lake Bratan located on the island of Bali, Indonesia.

Materials and methods
A benthic sample containing Achnanthidium was collected from a volcanic Lake Bratan on 14 November 2010 (08°16.579'S, 115°09.985'E). For general characteristics of this lake see Green et al. (1978). Environmental variables were measured with a Hanna multiparameter probe meter (HANNA HI98128).
The sample was boiled in concentrated hydrogen peroxide (~37%) to dissolve the organic matter. It was then washed with deionized water four times at 12 h intervals. After decanting and filling with deionized water up to 100 ml, the suspension was spread on to coverslips and left to dry at room temperature. Permanent diatom slides were mounted in Naphrax. Light microscopic (LM) observations were performed with a Zeiss Scope A1 microscope equipped with an oil immersion objective (100×/n.a.1.4, differential interference contrast [DIC]) and Zeiss Axio-Cam ERc 5s camera. Valve ultrastructure was examined with a JSM-6510LV scanning electron microscope (Papanin Institute for Biology of Inland Waters RAS, Borok, Russia), operated at 10 kV and 11 mm distance. 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 of gold.
The original sample preserved with Lugol's solution, as well as cleaned material preserved with 96% ethanol, are housed at the Laboratory of Molecular Systematics of Aquatic Plants, K.A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences (Moscow, Russia).
Distribution. So far, this species is known from its type locality only.

Discussion
Our new species is closely related to Achnanthidium macrocephalum (Hustedt) Round & Bukhtiyarova, 1996. This taxon was described by Hustedt (1937) Table 1). In contrast to A. macrocephalum, the striae of A. bratanense is composed of 1-4 transapically elongated areolae. In A. macrocephalum the striae composed of 1 (smaller valves) or two (rarely 3) areolae (Wetzel et al. 2019). Also A. bratanense has weakly radiate striae throughout the valve whereas in A. macrocephalum the striae become parallel towards the valve ends. It should be noted that Hustedt (1942) reported Achnanthes minutissima var. macrocephala from Lake Bratan on Bali and suggested that it might be widely distributed in the Indo-Malayan region. Unfortunately, he gave neither description nor images to support the written statement. It is very likely he actually observed A. bratanense instead of A. macrocephalum. Wetzel et al. (2019) supposed that Hustedt (1937) included in his description of A. macrocephalum two morphotypes.
Also A. bratanense is similar to several other taxa including A. rosenstockii (Lange-Bertalot) Lange-Bertalot var. rosenstockii, 2004, A. rosenstockii var. inareolatum Lange-  Table 1). Traditionally, three morphological groups are recognized within Achnanthidium (e.g. Novais et al. 2015;Karthick et al. 2017;Krahn et al. 2018;Yu et al. 2019): 1) A. minutissimum complex which is characterized by having straight external distal raphe ends, and striae density that increase towards the apex; 2) A. pyrenaicum complex which is characterized by having external distal raphe ends that deflect or hook to one side of the valve, and 3) A. exiguum complex have external distal raphe ends curved in opposite directions. Recently, the latter complex has been segregated into a new genus, Gogorevia (Kulikovskiy et al. 2020a). Although, A. bratanense, A. macrocephalum and A. rosenstockii can be placed in A. minutissimum complex based on the raphe structure, they have completely different striae structure and represent a separate morphological group. Interestingly, Pinseel et al. (2017) revealed 12 distinct lineages within A. minutissimum complex and one of them was described as the new species, A. digitatum Pinseel et al., 2017. Recently, Tseplik et al. (2021b described from the ancient Lake Matano (island of Sulawesi, Indonesia) the new species, A. gladius Tseplik et al., 2021b, which was closely related to the latter taxon. Thus, further detailed study of the pore apparatus ultrastructure as well as molecular studies of A. bratanense and allied taxa will help to better understand the taxonomic status and phylogenetic placement of this morphological group.