An updated list of Serbian diatom flora: new recorded taxa

Diatoms are a widespread group of organisms with well-known ecological preferences. Knowledge of their diversity is of great importance for assessing the environmental status of different aquatic ecosystems. The present study was based on 55 different localities that included rivers, streams, channels, accumulations and salt marshes throughout Serbia. The results of this study expanded the diatom checklist by 80 taxa, including two new recorded genera (Fistulifera and Microfissurata) for Serbia. The data were obtained by combining light microscopy (LM) and scanning electron microscopy (SEM) that provided reliable identification, which is very important in diatom diversity studies.


IntRoduCtIon
Species diversity is one of the main factors in assessing the environmental state of various aquatic ecosystems.It also has an important role in ecosystem processes and functioning, food chains and ecosystem integrity [1].Widespread in different environments, well-known ecological preferences and standardized identification methods point to diatoms as an important group of algae for species diversity studies that can serve to evaluate ecosystems health and environmental changes [2,3].
Diatom diversity have been relatively well studied across Europe and is presented in various books [4][5][6][7][8][9], monographs [10,14] and articles [11][12][13]15].Compared to previously cited data, diatom records and literature in Serbia are scattered.The first diatom data in Serbia date from 1883 [16].However, it was only in the second half of the 20 th century that extensive research into diatoms began [17][18][19][20][21], and in the last twenty years the number of new recorded diatom taxa for Serbian flora has increased [20][21][22][23][24].The aim of this paper was to report new records of diatom taxa from different sites throughout Serbia in order to supplement the diatom checklist.

Study area
The study areas were located in different parts of Serbia (Fig. 1).Samples of diatoms were collected at different localities, in rivers, streams, channels, accumulations and salt marshes.The list of the 55 localities is presented in Table 1.

Sample preparation
Diatoms were collected from different types of substrate (mosses, macrophytes, mud, stones) and communities (benthos, plankton) and poured into 100-mL bottles.Epiphytic and epilithic diatom samples were collected by squeezing out and scraping off the surface with a toothbrush, respectively.Epipelic diatom samples were collected from the sediment surface using a corer (Ø 1 cm), and phytoplankton samples were collected using a plankton net (Ø 25 μm) drawn through open water.All samples were fixed with formaldehyde to a final concentration of 4%.

Sample analysis
In the laboratory, the algological samples were treated with a standard method with concentrated acid (H 2 SO 4 ) and a KMnO 4 solution in order to remove organic matter; the samples were then washed several times with distilled water until pH 7 [25].Permanent slides were prepared by air-drying of the material on cover glasses and mounting in Naphrax ® mounting medium.Microscopic examinations were done using a Zeiss AxioImagerM.1 light microscope (LM) with DIC optics and AxioVision 4.8 software.Scanning electron microscopy (SEM) observations were made at the Institute of Physics, University of Belgrade, using a TESCAN MIRA 3 scanning electron microscope with maximum accelerating voltage of 30 kV.Sample surfaces were sputtered with gold using Quorum Technologies Mini Sputtercoater SC7620 for enhanced conductivity.
The terminology of valve morphology is based on Krammer and Lange-Bertalot [25] and Hofmann et al. [26].Taxa were identified according to different literature sources, indicated next to each type.

ReSultS
A total of 80 diatom taxa belonging to 33 genera have been noted for the first time in Serbian diatom flora.Among the 33 genera, two, Microfissurata and Fistulifera, are new to Serbian diatom flora.Species names as well as their dimensions and distribution in Serbia are given below.31))
Nitzschia vermicularoides Lange-Bertalot (Fig. 3(2)) Basionym: Nitzschia vermicularoides Lange-Bertalot dISCuSSIon On the territory of Serbia, c. 800 diatom taxa have been recorded to date (unpublished database of Serbian diatom flora, Krizmanić).The diversity of diatoms in Serbia is relatively low as compared to other European countries, e.g.only in two lakes in Macedonia (Lakes Ohrid and Prespa) 919 taxa were recorded [14].The number of diatom taxa in Serbia is very similar to the number of recorded taxa in Turkey (more than 800), although Turkey is much larger than Serbia [41].The highest number of recorded taxa in Serbia is widespread and common in European, Asian, North and South American freshwaters [7,[41][42][43].The results of this study are based on 55 differnet localities (rivers, streams, channels, accumulations and salt marshes) and have expanded the diatom cheklist with 80 new recorded taxa.The reasons for the constant increase in the number of recorded diatom taxa are numerous, such as improved light microscope resolution and digital cameras with accompanying software, and the increasing use of scanning electron microscopy (SEM), which have enabled the description of a great number of new taxa as well as the separation of new taxa from sensu lato species.
One third of our identified taxa are diatoms with a small cell size, and belong to the genera Achnanthidium, Adlafia, Fistulifera, Humidophila, Mayamaea and Psammothidium.The recently described new taxa, Achnanthidium crassum and Achnanthidium druartii [27,28], are separated from the A. pyrenaicum group.A. pyrenaicum was described Achnanthidium for the first time by Obušković [44], but it is only now these taxa have provided new records.This example illustrates how a better understanding of SEM is important in diatom diversity investigations.Also, SEM has led to a marked distinction of the genus Humidophila from Diadesmis and the description of new taxa, H. irata and H. brekkaensis [45], which are new records for Serbian diatom flora.
The small-sized genus, Microfissurata, with its unique combination of morphological characteristics, was described using both light and SEM.It includes only two species, M. paludosa and M. australis [36].M. australis was recorded at Ročnjak stream in an epilithic community, a small-discharge stream affected by seasonal desiccation.However, literature data indicate epiphytic and epipelic as the community types, but not epilithic [36].Distinguishing between the Fistulifera pelliculosa form and F. saprophila is hardly possible without the use of SEM due to its very small dimensions (8.9-12.5 µm, breadth 4-6.2 µm) and specific morphological valve features (numerous copulae of the girdle) [7].Our findings of Fistulifera correlate with literature data.
In almost all investigated Serbian freshwaters from 1984 to 2016, Reimeria sinuata has been recorded [22,44,46,47].Although more than 15 years ago a related species, R. uniseriata, was identified [40], it has not been recorded on the territory of Serbia until now.The widespread use of high-resolution light microscopes in Serbia is probably the reason leading to an increased insight into the main characteristics of this species, its uniseriate striae, in contrast to R. sinuata, which has biseriate striae.
This study has contributed a diatom checklist and provides a fuller insight of diatom diversity in Serbia.As the potential diversity of diatoms in Serbia is probably higher, this type of research is very important.Also, further detailed investigation, especially with SEM, may identify new interesting taxa for the diatom flora of Serbia.

table 1 .
Investigated localities with UTM coordinate.