A new species of Cryptomonas (Cryptophyceae) from the Western Urals (Russia)

A new species, Cryptomonas uralensis Martynenko, Gusev, Kulizin & Guseva sp. nov., is described from western slopes of the Ural Mountains (Russia) based on morphological and molecular data. Phylogenetic relationships inferred from nuclear SSU and LSU rDNA sequences show that the new species forms a clade with C. tetrapyrenoidosa Skuja emend. Hoef-Emden & Melkonian. Comparison of secondary structures of nuclear rDNA ITS2, including analysis of Compensatory Base Changes (CBC), confi rms the separation between C. uralensis sp. nov. and C. tetrapyrenoidosa. Cell morphology and sizes of C. uralensis sp. nov. are very similar to C. tetrapyrenoidosa and C. pyrenoidifera, and C. uralensis sp. nov. may thus represent a species that can only be reliably identifi ed using molecular data.


periplast), and a remnant nucleus of endosymbiotic origin in close association with the plastids (Hoef-Emden & Archibald 2016).
Recent research has shown that even in well-sampled geographical regions and habitats, the true diversity of cryptomonads is often unknown (Hoef-Emden 2007;Lane & Archibald 2008;Shalchian-Tabrizi et al. 2008;Choi et al. 2013). Cryptomonas Ehrenb. (Ehrenberg 1831) is a diverse genus of cryptophytes, with about 70 morphospecies currently accepted (Hoef-Emden & Melkonian 2003). Species in the genus are restricted to freshwater, contain chloroplasts with the biliprotein phycoerythrin 566 or leucoplasts, and are characterized by a dimorphic life cycle (Hoef-Emden & Melkonian 2003). Species display two alternative cell morphotypes, which are related to the life cycle: 1) cryptomorph with an inner periplast composed of hexagonal to polygonal organic plates and a non-keeled posteriorly directed microtubular flagellar root, and 2) campylomorph with a sheet-like inner organic periplast component and keeled rhizostyle (Hill 1991;Hoef-Emden & Melkonian 2003).
Molecular phylogenetic analyses based on nuclear and nucleomorph ribosomal RNA gene sequences demonstrated inconsistencies with traditional Cryptomonas taxonomy based on cell morphology studies by light microscopy (LM). Hoef-Emden & Melkonian (2003) revised the genus by combining phylogenetic analyses with morphological data, emended several species by including molecular data, and described two new species. DNA sequences of additional strains resulted in emendation of five species including one new combination (Hoef-Emden 2007). Identification of most Cryptomonas species may be possible only with molecular data. A total of 14 Cryptomonas species can now be identified by nuclear ITS2/partial LSU rDNA or by nucleomorph SSU rDNA sequences in molecular ecological surveys (Hoef-Emden & Melkonian 2003;Hoef-Emden 2007). It is worth mentioning that these taxa are phylogenetic species, which may not correspond to biological species limits.
Cryptomonads from some freshwater habitats in Russia have been studied morphologically, but molecular data is currently lacking. Data on taxonomic composition and ecology of this group are available mainly from phytoplankton studies. Cryptomonas marssonii Skuja, C. ovata Ehrenb. and C. erosa Ehrenb. have been reported from the Urals based on morphological data (Tretyakova et al. 1988;Tretyakova Fig. 1. Map of the study area; created using SASPlanet (http://www.sasgis.org).
1989; Belyaeva 2013Belyaeva , 2015. A new species of Cryptomonas from Russia is here described based on combination of morphological and molecular data.

Study area
Samples were collected from an unnamed small lake in the Usolka River flood plain, located in Solikamsk town, Permskiy Krai, Russia (59°38′59″ N, 56°44′57″ E, Fig. 1). Permskiy Krai is located on the western slope of the Ural Mountains, in upper and middle reaches of Kama River basin. The climate is cold temperate, with an average January temperature of -18.9°С, and varies from +14.8°С in the north to +18.7°С in the south during July. The annual precipitation also varies, from 450-600 mm in the western plain to 700-1000 mm in eastern mountainous territories of the Northern Urals, with snow cover usually 160-170 days/year (Nazarov 2006).

Samples and collections
Planktonic samples were collected by the first author using a plankton net with 20 μm mesh size. Water mineralization and temperature measurements were performed using the Hanna device (HI 9828, Hanna Instruments, Inc., Woonsocket, RI, USA). Strains were isolated and cultures perpetually transferred to IPPAS (Culture Collection of Microalgae at the K.A. Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences).

Culturing
Monoclonal strains were established by examination of micropipetted single cells under an inverted microscope. Non-axenic unialgal cultures were maintained in WC and Waris-H liquid medium (Andersen 2005;McFadden & Melkonian 1986) at 10°C in a growth chamber with a 12:12 h light:dark photoperiod.

Light microscopy
For light microscopy, living cells were immobilized by embedment in ultra-low gelling agarose (Sigma-Aldrich, A4018, USA) and examined by Nomarski differential interference contrast (DIC) with an oil immersion lens (Plan-Apochromat 100x/1.40 Oil DIC M27; microscope Zeiss AxioScope A1; Carl Zeiss AG, Oberkochem, Germany). Cell shape, size, shape of the furrow-gullet system and cell plastids, and presence, number and position of pyrenoids were examined (88 cells). The morphological description follows the nomenclature of Hoef-Emden & Melkonian (2003) (ventral side corresponds to the opening side of the furrow-gullet system). Calibration of magnification was done with a grated micrometer. Light micrographs were taken with an AxioCam ERc 5s Rev.2.

DNA extraction and amplification
Total DNA was extracted from monoclonal cultures using InstaGeneTM Matrix according to manufacturer's protocol. Fragments of SSU (length 1533 bp) and LSU rDNA (length 946 bp) and ITS2 rDNA (402 bp) were amplified using primers from Choi et al. (2013): 18S_CrN1F, 18S_826F, 18S_956R, 18S_BRK for nuclear SSU rDNA, crLSU_29F and crLSU_942R for partial nuclear LSU rDNA, and crITS_03F and crITS_05R for nuclear ITS2 rDNA. Amplification of all studied fragments was carried out using the premade mix ScreenMix (Evrogen, Russia) for the polymerase chain reaction (PCR). The conditions of amplification for SSU rDNA, LSU rDNA and ITS2 rDNA fragments were: an initial denaturation of 5 min at 95°C, followed by 35 cycles at 94°C for denaturation (30 s), 52°C for annealing (30 s) and 72°C for extension (50-80 s), and a final extension step of 10 min at 72°C. The resulting amplicons were visualized by horizontal agarose gel electrophoresis (1.5%), colored with SYBR Safe (Life Technologies, Carlsbad, CA, USA). Purification of DNA fragments was performed with the ExoSAP-IT kit (Affymetrix, Santa Clara, CA, USA) according to manufacturer's protocol. SSU rDNA, LSU rDNA and ITS2 rDNA fragments were decoded from two sides using forward and reverse PCR primers and the Big Dye system (Applied Biosystems, Foster City, CA, USA), followed by electrophoresis using a Genetic Analyzer 3500 sequencer (Applied Biosystems, Foster City, CA, USA).

Phylogenetic analysis
Electropherograms were verified by eye and sequences were assembled using BioEdit ver. 7.1.3 and MegaX (Kumar et al. 2018). Newly determined sequences and GenBank sequences of 45 other cryptomonads from different morphological groups were included in the alignments. The species Rhodomonas sp. strain M1480 and Chroomonas sp. strain SAG 980-1 were chosen as outgroup taxa. Sequences were aligned using MAFFT ver. 7 with model EINS-i (Katoh & Toh 2010), refined by eye and difficult to align regions were excluded from the analysis. jModelTest ver. 2.1.1 indicated that the GTR model of nucleotide substitution, with Gamma (G) distributed rates across sites and a proportion of invariable sites (I), was the most appropriate evolutionary model for the SSU rDNA and LSU rDNA alignments separately and combined (Posada 2006). Finally, we constructed the concatenated SSU + LSU rDNA alignment of 47 taxa. The SSU and LSU sequences were combined from the same strain. Phylogenies were constructed based on this model for the concatenated alignment using a single partition Bayesian Inference (BI) and Maximum Likelihood (ML) analyses. BI analysis was conducted with MrBayes ver. 3.2.5 (Ronquist & Huelsenbeck 2003). Three "hot" and one "cold" Markov chains were run for 1 × 10 6 cycles in two repetitions with the selection of each 200 th generated tree. Phylogenetic tree and posterior probabilities were obtained after discarding the first 25% to produce estimate parameter models of nucleotide substitutions and likelihood. ML-analysis was performed using MegaX (Kumar et al. 2018). Branch support was estimated using bootstrap analysis with 1000 replicas. Viewing and editing of trees were performed in FigTree ver. 1.4.2 (FigTree 2014) and Adobe Photoshop CC (ver. 19.0).

RNA secondary structure prediction
Nuclear ITS2 sequences were folded using the mfold server (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form) (Zuker 2003) with default values. Hoef-Emden (2007) provided a complete RNA secondary structure graph of the nuclear ITS2 rDNA of Cryptomonas sp. M1634, which was used as a template and assisted in inference of common stems, loops, and bulges.

Molecular phylogeny
The new species, Cryptomonas uralensis sp. nov., represents a separate clade with high statistical support, sister to Cryptomonas tetrapyrenoidosa Skuja emend. Hoef-Emden & Melkonian (Fig. 13). Cryptomonas uralensis sp. nov. differs from the type strain of C. tetrapyrenoidosa by the SSU rDNA, LSU rDNA and ITS2 rDNA sequences as well as in the secondary structure of ITS2 (Fig. 14). For ITS2 sequence of C. uralensis sp. nov. four-helix structures could be inferred with long third helices (Fig.  15). An unpaired U-U was found in Helix II. The length of the helices was different between two taxa (Fig. 15). For the new species, length of ITS2 was 402 nt, while for C. tetrapyrenoidosa was 370 nt. Three Compensatory Base Changes (CBCs) between C. uralensis sp. nov. and C. tetrapyrenoidosa were observed in Helix III (Fig. 15).

Cryptomonas uralensis Martynenko, Gusev, Kulizin & Guseva sp. nov. Figs 2-12 Diagnosis
Cells (absolute minimal and maximal values of strain UR168) 15-26 µm long, 8-17 µm wide, 8-12 µm thick. Cells more or less flattened in dorso-ventral plane, in ventral view elliptical to ovoid. In lateral view with oblique or rounded apex and rounded antapex. One plastid with two to four pyrenoids. Species differs from other species of the genus by the order of nucleotides in nuclear ITS2, LSU and SSU rDNA gene sequences.

Etymology
The species name derived from "Ural", the region from which this taxon was described.

Geographical distribution
To date, Cryptomonas uralensis sp. nov. has only been observed at the type locality. At the time of collection, the pH was 6.5, temperature 18°C.

Discussion
Cryptomonas uralensis sp. nov. represents a distinct clade with high statistical support, sister to C. tetrapyrenoidosa, but no obvious morphological characters distinguishing the two taxa were found. Initially C. tetrapyrenoidosa was described based on LM studies of populations from Sweden and Latvia (Skuja 1948). Later, the strain M1092 from Germany (Remscheid) was chosen as the epitype and sequenced (Hoef-Emden & Melkonian 2003). To date, C. tetrapyrenoidosa has not been recorded in the Urals, thus areals of this species and C. uralensis sp. nov. do not overlap. Mention should be made that there were no previous intensive molecular studies of cryptophytes in the Urals. Recent studies of cryptomonads of South Korea, region remote from Europe, revealed several strains from the Cryptomonas tetrapyrenoidosa clade that showed SSU rDNA, LSU rDNA and ITS2 rDNA nucleotide differences from the type specimen and formed two subclades (Choi et al. 2013). However, no formal taxonomic descriptions have been made. In our opinion, these are very likely a new species. Thus, the C. tetrapyrenoidosa clade requires further study. Previous studies have suggested that the nuclear internal transcribed spacer 2 (ITS2) is a good marker to identify biological species using degree of conservation. Coleman (2000) and Müller et al. (2007) have proposed that CBCs in the ITS2 correlate with speciation and thus can be used to distinguish species. The CBC species concept states that two organisms/strains whose ITS2 sequences differ by even a single CBC in the conserved regions of Helix II and Helix III represent two different biological species (Coleman 2000). According to Müller et al. (2007) and Wolf et al. (2013) the presence of any CBC in the whole ITS2 molecule is sufficient for distinguishing species. Later, Caisová et al. (2013) identified consensus structure model of the ITS2 molecule that can be applied to two classes of green algae. Also was reported that CBCs in the ITS2 of four orders of the green algal class Chlorophyceae Wille in Warming were mostly associated with higher order taxonomic levels like genera, families and orders, and CBC concept for species delimitation have some constraints in chlorophycean algae (Caisová et al. 2013). Dimorphic strains and a few reports of sexual reproduction indicate the presence of biological species also in cryptophyte taxa (Hoef-Emden 2007). The nuclear ITS2 was successfully used to predict putative biological species in the last revision of Cryptomonas (Hoef-Emden 2007).
Here, comparison of the secondary structures of ITS2 confirms separation between C. uralensis sp. nov. and C. tetrapyrenoidosa.

Fig. 14.
Predicted secondary structure of the nuclear internal transcribed spacer 2 of the strain UR168 (from 5' to 3' terminus in clockwise direction). Cryptomonas pyrenoidifera Geitler emend. Hoef-Emden & Melkonian was found to be sister to the clade containing C. uralensis sp. nov. and C. tetrapyrenoidosa. Cryptomonas pyrenoidifera is known from Germany, United Kingdom, Czech Republic and Australia based on molecular data. The morphology and cell sizes of C. uralensis sp. nov. are very similar to cryptomorphs of C. pyrenoidifera, as well as C. tetrapyrenoidosa. Cryptomonas curvata Ehrenberg emend. Hoef-Emden & Melkonian is one more species which has cryptomorph cells with the same morphological features (shape, size, number of pyrenoids), but placed distantly on phylogenetic tree from C. uralensis sp. nov. Incongruences between classical morphospecies and molecular phylogenetic trees have previously been reported for many species in the genus Cryptomonas (Hoef-Emden 2007). Morphological species descriptions of this genus are often ambiguous because of microscopic cells and only few visible characters. The most recent revisions of the genus show that correct identification of most Cryptomonas species may be possible only with molecular data (Hoef-Emden & Melkonian 2003, Hoef-Emden 2007. In the case of C. uralensis sp. nov., no sufficiently distinct morphological characters could be identified. The species can only be distinguished by molecular characters.