microorganisms Article Tracing the Origin of Planktonic Protists in an Ancient Lake Nataliia V. Annenkova 1,* , Caterina R. Giner 2,3 and Ramiro Logares 2,* 1 Limnological Institute Siberian Branch of the Russian Academy of Sciences 3, Ulan-Batorskaya St., 664033 Irkutsk, Russia 2 Institute of Marine Sciences (ICM), CSIC, Passeig Marítim de la Barceloneta, 37-49, ES08003 Barcelona, Spain; [email protected] 3 Institute for the Oceans and Fisheries, University of British Columbia, 2202 Main Mall, Vancouver, BC V6T 1Z4, Canada * Correspondence: [email protected] (N.V.A.); [email protected] (R.L.) Received: 26 February 2020; Accepted: 7 April 2020; Published: 9 April 2020 Abstract: Ancient lakes are among the most interesting models for evolution studies because their biodiversity is the result of a complex combination of migration and speciation. Here, we investigate the origin of single celled planktonic eukaryotes from the oldest lake in the world—Lake Baikal (Russia). By using 18S rDNA metabarcoding, we recovered 1414 Operational Taxonomic Units (OTUs) belonging to protists populating surface waters (1–50 m) and representing pico/nano-sized cells. The recovered communities resembled other lacustrine freshwater assemblages found elsewhere, especially the taxonomically unclassified protists. However, our results suggest that a fraction of Baikal protists could belong to glacial relicts and have close relationships with marine/brackish species. Moreover, our results suggest that rapid radiation may have occurred among some protist taxa, partially mirroring what was already shown for multicellular organisms in Lake Baikal. We found 16% of the OTUs belonging to potential species flocks in Stramenopiles, Alveolata, Opisthokonta, Archaeplastida, Rhizaria, and Hacrobia. Putative flocks predominated in Chrysophytes, which are highly diverse in Lake Baikal. Also, the 18S rDNA of a number of species (7% of the total) differed >10% from other known sequences. These taxa as well as those belonging to the flocks may be endemic to Lake Baikal. Overall, our study points to novel diversity of planktonic protists in Lake Baikal, some of which may have emerged in situ after evolutionary diversification. Keywords: microbial eukaryotes; phylogeography; marine-freshwater transitions; evolutionary radiation; species flocks; Lake Baikal; sympatry 1. Introduction Even though single cell eukaryotes are fundamental for ecosystem function, a substantial fraction of their diversity remains poorly known. During the last decade, multiple studies have discovered new protist diversity, which was in part possible due to culture-independent molecular approaches using high-throughput sequencing (HTS) and metabarcoding [1,2]. Metabarcoding is highly relevant for pico/nano-sized (~0.8–5 µm, as defined in [3]) unicellular eukaryotes as they are too small to show unambiguous morphological differences and are difficult to culture [3,4]. The key ecological role of these minute eukaryotes has been shown in both oceans [3,5] and lakes [6,7]. Yet, we are still beginning to understand their diversity, evolution, and dispersal. In contrast to marine protists, we know less about the diversity of freshwater counterparts. Pico/nano-sized freshwater protists have been studied using Sanger sequencing in European ponds, lakes [8,9], and Lake George in the USA [10]. More recently, small protists were investigated using Microorganisms 2020, 8, 543; doi:10.3390/microorganisms8040543 www.mdpi.com/journal/microorganisms Microorganisms 2020, 8, 543 2 of 19 metabarcoding in various lakes of Western Europe [11,12], as well as a few lakes in North [13] and South America [14]. Most of these studies pointed to diverse patterns of community structure and novel diversity. Yet, the previous reflects another bias: most studies of freshwater protists correspond mainly to Northern Europe and North America [15]. In contrast, other continents, like Asia, remain underexplored in terms of freshwater protist diversity, apart from a few lakes in China [16] and in the high-mountain Himalayan [17]. Lake Baikal (Siberia, Northern Asia) is the oldest (more than 26 million years), largest (23,615 km3), Microorganismsand deepest 2019 (down, 7, x FOR to 1642PEER REVIEW m depth, with a mean depth of 744 m) freshwater lake in the world3 of 20 (Figure1). As a comparison, the oldest basin of Lake Tanganyika (Africa) is 9–12 million years old andPlankton the Great samples Lakes in were North collected America in beganJuly 2013 to form using around a Niskin 14,000 bottle years from ago. 14 sites Baikal in Lake present-day Baikal, Russiacommunities (Figure inhabit 1). In thethree whole sites water-columnlocated near the and coast include (depth more was than less 3500 than known 40 m), species samples of micro-were takenand macro-eukaryotic at 5 m depth and organisms, in one coastal of which site more (1CC) than samples half seem were to betaken endemic at 1 [and18]. Yet,20 m this depth diversity and mixed.likely represents The rest of only the asites fraction were (perhapslocated in 60%) open of Baikal the total waters diversity and samples of Lake Baikalwere taken [18] asat every1, 15, yearand 50new m speciesdepth. areThese being sampling described. depths Such are biodiversity typically used is the for result surface of a samples complex in process Lake Baikal. including Two in samplessitu evolutionary were taken diversification at 25 m depth as instead well as of immigrations 15 m due to orsampling extinctions logistics. in part Water related samples to geologic were keptactivity in dark and climateand cold change. conditions Few relictbefore groups filtration. are 30–70To collect M years microbial old, while DNA, other up organismsto 5 L of water appeared was pre-filtered3.5–30 M years through ago during a 70 theµm isolation mesh-size of the net Baikal to re depression.move larger Furthermore, organisms variousand then taxa sequentially explosively filtereddeveloped through 0.15–3.5 8 µm M yearsand 0.45 ago µm in response polycarbonate to the most membrane dramatic filters tectonic (Sartorius, changes Göttingen, in the region Germany) coupled usingwith climatea vacuum cooling pump. [19 ].Filters One ofwere the commonimmediately mechanisms frozen at that −20 led°C toand speciation stored at within −70 °C multicellular until DNA extraction.organisms inOnly Lake 0.45 Baikal µm waspore-size adaptive filters radiation, were us whiched for resultedDNA extraction in endemic in order species to flocks focus [18in ,20small]. protists (cell size 0.45–8 µm). FigureFigure 1. 1. A.A. Lake Lake Baikal Baikal position position in inEurasia, Eurasia, B. B.Samp Samplingling sites sites in Lake in Lake Baikal. Baikal. For Foreach each sample, sample, the firstthe firstnumber number and andletter letter indicates indicates the number the number within within one of one the of ba thesins basins (S—Southern, (S—Southern, C—Central, C—Central, N— NorthernN—Northern basins), basins), the thesecond second letter letter indicates indicates coas coastaltal (C; (C; up up to to40 40 m m depth) depth) or or deepwater deepwater (D; (D; more more thanthan 100 100 m m depth) depth) sites, sites, and and number numberss within within brackets brackets indicate indicate the the sa samplemple depth depth (e.g., (e.g., 1CD 1CD (1, (1, 15, 15, 50) 50) meansmeans of of samples samples from from 1, 1, 15, 15, and and 50 50 m m depths depths that that we werere taken taken in in the the first first site site of of the the Central Central basin basin at at aa deep deep site. site. No No depth depth is is shown shown for for the the coastal coastal samples. samples. DNACompared was extracted to its eukaryotic from one macrobiota, filter per site much using less the is knownDNeasy about Plant the Kit diversity, (QIAGEN evolution, GmbH, Hilden,and ecology Germany) of Lake following Baikal eukaryoticthe manufacturer’s microbiota. instructions. Routine monitoring The eukaryotic of Lake universal Baikal planktonprimers TAReukFWD1has been carried (5’-CCAGCASCYGCGGTAAT) out using light microscopy for an mored TAReukREV3 than 60 years (5’-ACTTTCGTTCTTGATYRA- [21–23]. These studies showed 3’) were used for PCR amplification of the 18S RNA gene (V4 region; ~380 bp) [30]. Amplicons from 3 independent PCR reactions for each sample were pooled together. PCR products were sequenced at Fasteris, Switzerland (https://www.fasteris.com) on an Illumina MiSeq (2 × 250 bp). Overall, 23 samples were successfully sequenced. 2.2. V4-Amplicon Data Processing and Statistical Analyses Reads were processed following an in-house pipeline [31]. Briefly, raw reads were corrected using BayesHammer [32] following [33]. Corrected paired-end reads were subsequently merged with PEAR [34] and sequences > 200 bp were quality-checked (maximum expected errors 0.5) and de-replicated using USEARCH [35]. OTUs were delineated at 97% and 99% similarity using UPARSE V8.1.1756 [36]. We used the 97% cut-off to compare the number of OTUs from our study Microorganisms 2020, 8, 543 3 of 19 seasonally recurrent changes in phytoplankton as well as long-term changes that may be explained by global warming. Furthermore, the previous studies pointed to phytoplankton spatial heterogeneity in spring, also indicating a high productivity of picophytoplankton (up to 60% of phytoplankton biomass in some years). Microscopy studies of Baikal protists indicated both cosmopolitan freshwater species as well as examples of endemicity [24,25]. More detailed studies of diatoms [26] and dinoflagellates [27]