Genetic Diversity of Potassium Ion Channel Proteins Encoded by Chloroviruses That Infect Chlorella heliozoae

Keywords: Chloroviruses, potassium ion channels, Kcv channels, algal viruses

Chloroviruses are large, plaque-forming, dsDNA viruses that infect chlorella-like green algae that live in a symbiotic relationship with protists. Chloroviruses have genomes from 290 to 370 kb, and they encode as many as 400 proteins. One interesting feature of chloroviruses is that they encode a potassium ion (K + ) channel protein named Kcv. The Kcv protein encoded by SAG chlorovirus ATCV-1 is one of the smallest known functional K + channel proteins consisting of 82 amino acids. The Kcv ATCV-1 protein has similarities to the family of two transmembrane domain K + channel proteins; it consists of two transmembrane α-helixes with a pore region in the middle, making it an ideal model for studying K + channels. To assess their genetic diversity, kcv genes were sequenced from 103 geographically distinct SAG chlorovirus isolates. Of the 103 kcv genes, there were 42 unique DNA sequences that translated into 26 new Kcv channels. The new predicted Kcv proteins differed from Kcv ATCV-1 by 1 to 55 amino acids. The most conserved region of the Kcv protein was the filter, the turret and the pore helix were fairly well conserved, and the outer and the inner transmembrane domains of the protein were the most variable. Two of the new predicted channels were shown to be functional K + channels. 

Introduction

Chloroviruses (family Phycodnaviridae) are large, plaque-forming, dsDNA viruses that infect certain chlorella-like green algae that live in a symbiotic relationship with protists [1]. Chloroviruses have an internal membrane and they are icosahedral in shape with a spike structure at one of their vertices [2]. They have genomes that are 290 to 370 kb in size and are predicted to encode up to 400 proteins (CDSs) and 16 tRNAs. These viruses are ubiquitous in nature and have been isolated from freshwater ponds, lakes, and rivers across the globe. There are four groups of chloroviruses based on the host they infect: viruses that infect Chlorella variabilis NC64A (referred to as NC64A viruses), viruses that infect Chlorella variabilis Syngen 2-3 (referred to as Osy viruses), viruses that infect Chlorella heliozoae SAG 3.83 (referred to as SAG viruses), and viruses that infect Micratinium conductrix Pbi, (referred to as Pbi viruses). The most studied chlorovirus is the NC64A virus Paramecium bursaria chlorella virus 1 (PBCV-1); its host, C. variabilis NC64A, lives in symbiosis with Paramecium bursaria. 
The PBCV-1 genome is ~331 kb and encodes 416 predicted CDSs and 11 tRNA genes. About half of the identified CDSs resemble proteins of known function, including some that are novel for a virus. One protein that PBCV-1, as well as most of the chloroviruses, encodes is a potassium ion (K + ) channel protein (named Kcv) [3]. When the 94 amino acid Kcv PBCV-1 was discovered, it was the smallest protein known to form a functional K + channel. The Kcv PBCV-1 protein consists of only the basic functional units that are present in all K + channels in that it has a short slide helix, an outer transmembrane helix, a turret, a pore helix, a filter, and an inner transmembrane helix (Figure 1); four of these proteins form a functional K + channel. 
a functional K + channel. 
KcvPBCV-1 is hypothesized to play an important role during infection of its host. After the virus attaches to the host cell wall and degrades the wall at the point of attachment, the PBCV-1 internal membrane fuses with the host's plasma membrane [4]. The Kcv channel is located in the virus's internal membrane [5], and once the two membranes are fused, the Kcv channel becomes part of the host membrane. This allows Kcv to participate in the rapid depolarization of the host cell membrane [6,7] and the release of K + from the cell [8]. 
The rapid loss of K + from the host and associated water fluxes significantly reduce the host turgor pressure, which aids ejection of viral DNA and virion-associated proteins into the host [9]. Host membrane depolarization also inhibits many host secondary transporters [10] and prevents infection by a second virus [11]. Because of the small size of Kcv, it has served as an excellent model for studying K + channels and there are over 60 research publications on Kcv channels. 
Since the discovery of KcvPBCV-1, even smaller chlorovirus-encoded K + channel proteins have been described including an 82 amino acid protein from SAG chlorovirus, ATCV-1, referred to as KcvATCV-1 (Figure 1). Expression studies established that KcvATCV-1 makes a functional, K + selective channel in Xenopus laevis oocytes and in yeast [12]. The objective of this study was to isolate and analyze the sequence diversity of the kcv gene from 103 SAG chloroviruses that come from freshwater collected throughout the world. Ultimately, one can anticipate some physiological differences among the Kcv channels from the SAG viruses. KcvATCV-1 sequence alignment with the sequence of the prototype chlorovirus K + channel KcvPBCV-1 by ClustalW. The slide helix, outer transmembrane (TMD1) turret, pore helix, selectivity filter, and inner transmembrane (TMD2) of the KcvPBCV-1 are highlighted by the horizontal lines. Asterisks indicate positions which have a fully conserved residue. A colon indicates conservation between amino acids with strongly similar properties. A period indicates conservation between amino acids with less similar properties. 

Materials and Methods


Cultures

Water samples were collected from lakes, ponds, and rivers from around the United States, Canada, Guatemala, Brazil, Chile, Germany, and Greenland (Table A1). The samples were passed through a 0.45 µm filter (PES filters, Sartorius, Gottingen, Germany). Chloroviruses were isolated Figure 1. Kcv ATCV-1 sequence alignment with the sequence of the prototype chlorovirus K + channel Kcv PBCV-1 by ClustalW. The slide helix, outer transmembrane (TMD1) turret, pore helix, selectivity filter, and inner transmembrane (TMD2) of the Kcv PBCV-1 are highlighted by the horizontal lines. Asterisks indicate positions which have a fully conserved residue. A colon indicates conservation between amino acids with strongly similar properties. A period indicates conservation between amino acids with less similar properties. 
Kcv PBCV-1 is hypothesized to play an important role during infection of its host. After the virus attaches to the host cell wall and degrades the wall at the point of attachment, the PBCV-1 internal membrane fuses with the host's plasma membrane [4]. The Kcv channel is located in the virus's internal membrane [5], and once the two membranes are fused, the Kcv channel becomes part of the host membrane. This allows Kcv to participate in the rapid depolarization of the host cell membrane [6,7] and the release of K + from the cell [8]. 
The rapid loss of K + from the host and associated water fluxes significantly reduce the host turgor pressure, which aids ejection of viral DNA and virion-associated proteins into the host [9]. Host membrane depolarization also inhibits many host secondary transporters [10] and prevents infection by a second virus [11]. Because of the small size of Kcv, it has served as an excellent model for studying K + channels and there are over 60 research publications on Kcv channels. 
Since the discovery of Kcv PBCV-1 , even smaller chlorovirus-encoded K + channel proteins have been described including an 82 amino acid protein from SAG chlorovirus, ATCV-1, referred to as Kcv ATCV-1 (Figure 1). Expression studies established that Kcv ATCV-1 makes a functional, K + selective channel in Xenopus laevis oocytes and in yeast [12]. The objective of this study was to isolate and analyze the sequence diversity of the kcv gene from 103 SAG chloroviruses that come from freshwater collected throughout the world. Ultimately, one can anticipate some physiological differences among the Kcv channels from the SAG viruses. 

Materials and Methods


Cultures

Water samples were collected from lakes, ponds, and rivers from around the United States, Canada, Guatemala, Brazil, Chile, Germany, and Greenland (Table A1 (Appendix A)). The samples were passed Viruses 2020, 12, 678 3 of 33 through a 0.45 µm filter (PES filters, Sartorius, Gottingen, Germany). Chloroviruses were isolated from the filtered water samples using a plaque assay on a C. heliozoae SAG 3.83 lawn. For the plaque assay, plates were made using Modified Bold's Basal Medium (MBBM) 1.5% agar with tetracycline added at 10 µg/mL [13]. Each plate was filled with about 20 mL of MBBM agar and allowed to solidify. In a tube, 2.5 mL of 0.75% MBBM agar was combined with 1 mL of the filtered water sample and 300 µL of C. heliozoae cells (~1.5 × 10 8 cells/mL). The tube was mixed and poured over the solidified 1.5% MBBM agar plate. Once the top agar layer solidified, the plates were inverted and kept under constant light at 25 • C for a few days. If SAG chloroviruses were present in the water sample, plaques formed on the plates. Two to three unique plaques (e.g., different size and sometimes different shape plaques) were picked from each plate with a sterile toothpick and placed in a 1.5 mL tube filled with C. heliozoae cells. These samples were then placed on a spinning wheel for 1 day to propagate the virus. The resulting lysates were serially diluted to 10 -6 in virus suspension buffer (VSB, 50 mM Tris HCl, 10 mM MgCl 2 , pH 7.8) and 100 µL of the resultant dilution was plaqued. Each virus sample was plaque-purified two or three times to ensure that one had a single virus. For the PCR DNA template preparation, 100 µL of viral lysate was boiled in deionized sterile water for 5-10 min. 

Primer Selection

DNA sequences 450 to 500 bp upstream and 230 to 350 bp downstream from the kcv gene from 13 previously sequenced SAG chloroviruses [14] were used to identify conserved regions for designing degenerate primers. Conserved regions were identified inside the aligned sequences, and four forward primers and five reverse primers (Table 1) were made and tested using known SAG virus DNAs (ATCV-1, BRO604, Can0610, Canal1, GM0701, MN0810, MO0605, NEJV2, NTS1, OR0704, TN603, WI0606). The primers that identified all of the kcv genes were forward primer Kcv8 Frw and reverse primer Kcv6 Rvs as well as forward primer Kcv9 Frw and reverse primer Kcv6 Rvs. As a result, the primer set Kcv9/Kcv6 was selected to amplify the kcv genes. 

Polymerase Chain Reactions (PCR)

The conditions for the PCR reactions followed the recommendations of New England Biolab's (Beverly, MA, USA) Phusion High Fidelity DNA Polymerase kit. The 50 µL PCR reactions contained 10 µL of 5x Phusion High Fidelity buffer, 1 U Phusion DNA polymerase, and 1 µL of DNA template. The final concentration of dNTPs in the PCR reaction was 0.2 mM and the primer final concentration was 0.5 µM forward primer and 0.5 µM reverse primer. The reactions were run for 5 min at 94 • C, followed by 35 cycles of 1 min at 94 • C, 30 s at 56 • C, 1 min at 72 • C, and at the end the final extension 15 min at 72 • C. Deionized water was used as a negative control. 
The PCR products were gel-purified by mixing 20 µL of an amplified sample with a small amount of Ficoll gel loading buffer and loaded on a 1% agarose gel. The New England Biolabs log-2 ladder was used as a molecular weight marker. The gel was run at 5 V/cm for 1 h, then imaged by exposing the gel to ultraviolet light. Amplified kcv genes were excised from the gel and the DNA extracted using the QIAquick Gel Extraction Kit following the manufacturer's instruction (QIAGEN Hilden, Germany). 
The purified DNAs were sequenced using Sanger sequencing by a commercial provider. The accession numbers for the kcv sequences in GenBank are MT560092-MT560194. 

Phylogenetic Analysis

The kcv gene DNA sequences were translated into amino acid sequences, and all of the unique Kcv proteins were aligned. Geneious 11.0.5 software (Biomatters Ltd., Auckland, New Zealand, https: //www.geneious.com) was used for the DNA and protein sequence alignment (Geneious Alignment with the default settings) and the phylogenetic tree was constructed with PhyML (version 3.3.20180621), which is Maximum likelihood, using the default settings. 

Functional Reconstitution of kcv Genes in Planar Lipid Bilayers

K + channel proteins were translated in vitro into nanodiscs (NDs) with the MembraneMax HN Protein Expression Kit (Invitrogen, Carlsbad, CA, USA) as described previously [15]. A His-tag attached to the scaffold protein of the NDs allowed purification of channel/ND-complexes via metal chelate affinity chromatography. To eliminate unspecific binders, the column was washed three times with 400 µL of a 20 mM imidazole solution. Finally, the His-tagged NDs were eluted in three fractions with 200 µL of a 250 mM imidazole solution. All centrifugation steps were performed at 700 g for 2 min. 
Single-channel recordings were done with a vertical bilayer set up (IonoVation, Osnabrück, Germany) as described previously [16]. 
The experimental solution contained 100 mM KCl and was buffered to pH 7.0 with 10 mM HEPES/KOH. As a lipid, we used 1,2-diphythanoyl-sn-glycero-3-phosphocholine (DPhPC) (Avanti Polar Lipids, Alabaster, AL, USA) at a concentration of 15 mg/mL in n-pentane (MERCK KGaA, Darmstadt, Germany). 

Results


kcv Genes Selected for Analysis

In total, kcv genes from 83 SAG chloroviruses were obtained from the PCR experiments. In addition, the kcv genes from the 13 SAG chloroviruses that had been sequenced previously [14] and kcv genes from 7 recently sequenced SAG chloroviruses (not yet in the database) were included in the analysis. Thus, in total we compared kcv genes from 103 SAG chloroviruses (Table A1). Overall, these viruses were from three continents and seven countries. 

Diversity of kcv Genes

Alignment of the nucleotide sequences indicated that the 103 kcv genes had substitutions in 125 of the 249 nucleotides (~50%) (assuming that all the proteins were 82 amino acids long (but see below)) producing 42 unique DNA sequences (Figure A1 (Appendix B)). Nine of the viruses with identical kcv DNA sequences included a virus isolated in Germany in 2002 and viruses isolated in various parts of the United States of America 4 to 16 years later. Twenty of the virus isolates with identical kcv DNA sequences were from ponds near Hohenheim, Germany that were collected on the same day in 2017. It is important to note that the type SAG virus, ATCV-1, was isolated from one of these ponds in 2002. The sequences of the newly isolated viruses differed from ATCV-1 virus by 18-21 amino acids. In these same recent German collections, there were viruses with three additional kcv DNA sequences. The kcv DNA sequence from 18 of the 103 viruses was only found one time. 

Diversity of the Kcv Proteins

The 42 unique kcv DNA sequences produced 26 unique proteins (Figure 2). Of the 26 unique protein sequences, 18 of them were 82 amino acids long, 4 were 84 amino acids, 2 were 85 amino acids, 1 was 87 amino acids, and 1 was 89 amino acids. The proteins with 84 and 85 amino acids had either 2 or 3 extra amino acids at their C-terminal ends. The 87 amino acid Kcv protein from a virus isolate Viruses 2020, 12, 678 5 of 33 from New York state had 5 amino acids added to the N-terminus of the protein. However, this protein has an internal Met that would create an 82 amino acid protein and so we suspect that this internal AUG is the actual translation start site. The 89 amino acid protein from a virus, GLND22, isolated in Greenland has 8 extra amino acids in the turret region between the outer transmembrane domain and the pore region. This turret region also has extra amino acids in the slightly larger Kcv proteins from the NC64A viruses (Figure 1) and the Pbi viruses [17]. 
Viruses 2020, 12, x FOR PEER REVIEW 5 of 23 domain and the pore region. This turret region also has extra amino acids in the slightly larger Kcv proteins from the NC64A viruses (Figure 1) and the Pbi viruses [17]. The KcvCan0610SP only differed from KcvATCV-1 by one amino acid. The Kcvs from viruses collected from Germany in 2017 differed from KcvATCV-1, which originally came from Germany, by 18 to 21 amino acids. The KcvGNLD22 differed the most from KcvATCV-1 with 55 amino acid differences or 56% of the amino acids. The remaining virus Kcvs differed from KcvATCV-1 by 2 to 13 amino acids. 
Alignment of the 26 unique proteins revealed that some areas of the Kcv protein were more conserved than others. The filter domain is typically the most highly conserved domain in K + channel proteins and 20 of the 26 proteins had a TTVGYGDL sequence. Five of the remaining six Kcvs had a TTTGYGDL sequence and the remaining Kcv, KcvGLND22 from Greenland, had a TTTGFGDV sequence (Figure 2). All the SAG chlorovirus Kcvs essentially lack an N-terminal slide helix domain (Figure 1). 
A phylogenetic tree of the 26 Kcv proteins resulted in 3 major clades (Figure 3). The largest clade had 19 Kcv proteins from viruses primarily collected across the United States but also included viruses isolated in Canada, Guatemala, and Brazil, and one from Germany. Kcv proteins isolated from the recent German water samples formed a separate cluster with a distance of 0.4925 substitutions from the rest of the SAG Kcvs (Figure 3). The KcvGNDL22 from Greenland also formed a distinct clade with a distance of 1.518 substitutions from the German samples and a distance of 1.6675 substitutions from all the rest. Interestingly, KcvGNDL22 had more similarity to a Kcv from a Pbi virus MT325 than it did to the other SAG viruses (Figure 3). Virus GNDL22 is also interesting because about 15% of its CDSs are more similar to the MT325 Pbi virus and the other 85% are most similar to SAG viruses. Thus, GNDL22 appears to be some type of hybrid virus. The Kcv Can0610SP only differed from Kcv ATCV-1 by one amino acid. The Kcvs from viruses collected from Germany in 2017 differed from Kcv ATCV-1, which originally came from Germany, by 18 to 21 amino acids. The Kcv GNLD22 differed the most from Kcv ATCV-1 with 55 amino acid differences or 56% of the amino acids. The remaining virus Kcvs differed from Kcv ATCV-1 by 2 to 13 amino acids. 
Alignment of the 26 unique proteins revealed that some areas of the Kcv protein were more conserved than others. The filter domain is typically the most highly conserved domain in K + channel proteins and 20 of the 26 proteins had a TTVGYGDL sequence. Five of the remaining six Kcvs had a TTTGYGDL sequence and the remaining Kcv, Kcv GLND22 from Greenland, had a TTTGFGDV sequence (Figure 2). All the SAG chlorovirus Kcvs essentially lack an N-terminal slide helix domain (Figure 1). 
A phylogenetic tree of the 26 Kcv proteins resulted in 3 major clades (Figure 3). The largest clade had 19 Kcv proteins from viruses primarily collected across the United States but also included viruses isolated in Canada, Guatemala, and Brazil, and one from Germany. Kcv proteins isolated from the recent German water samples formed a separate cluster with a distance of 0.4925 substitutions from the rest of the SAG Kcvs (Figure 3). The Kcv GNDL22 from Greenland also formed a distinct clade with a distance of 1.518 substitutions from the German samples and a distance of 1.6675 substitutions from all the rest. Interestingly, Kcv GNDL22 had more similarity to a Kcv from a Pbi virus MT325 than it did to the other SAG viruses (Figure 3). Virus GNDL22 is also interesting because about 15% of its CDSs are more similar to the MT325 Pbi virus and the other 85% are most similar to SAG viruses. Thus, GNDL22 appears to be some type of hybrid virus. Phylogenetic tree of Kcv proteins from 26 SAG chloroviruses. PhyML, which is Maximum likelihood, with the default settings was used to construct the tree. The branch length shows dissimilarity between strains and the values on the branches are the number of changes. The viruses in blue represent the new Kcv proteins reported in this manuscript. Viruses MT325 representing a Pbi virus and PBCV-1 representing an NC64A virus are in green. Additional information on each of the Kcv proteins from the SAG viruses, including where they were isolated and the number of times that protein sequence appeared, is included in Table A1. 

Functional Reconstitution of Two New Kcv Channels in Planar Lipid Bilayers

To test whether the newly discovered genes were coding for functional K + channels, we selected two representatives for functional testing. The putative channel proteins KcvGNLD22 and KcvCan0610SP were translated in vitro into nanodiscs and after purification reconstituted in planar lipid bilayers. The electrical recordings in Figure 4 show that the two proteins generated typical single-channel fluctuations at positive and negative voltages in the presence of 100 mM KCl on both sides of the membrane. These experiments established that the two genes code for functional ion channels. The overall properties of the two new channels were similar to those of KcvNTS (Figure 4), a well-studied representative of the K + channels from SAG viruses [18]; KcvNTS differed from the reference channel KcvATCV-1 by four amino acids. KcvGNLD22 and KcvCan0610SP were selected because of their large (55 amino Figure 3. Phylogenetic tree of Kcv proteins from 26 SAG chloroviruses. PhyML, which is Maximum likelihood, with the default settings was used to construct the tree. The branch length shows dissimilarity between strains and the values on the branches are the number of changes. The viruses in blue represent the new Kcv proteins reported in this manuscript. Viruses MT325 representing a Pbi virus and PBCV-1 representing an NC64A virus are in green. Additional information on each of the Kcv proteins from the SAG viruses, including where they were isolated and the number of times that protein sequence appeared, is included in Table A1. 

Functional Reconstitution of Two New Kcv Channels in Planar Lipid Bilayers

To test whether the newly discovered genes were coding for functional K + channels, we selected two representatives for functional testing. The putative channel proteins Kcv GNLD22 and Kcv Can0610SP were translated in vitro into nanodiscs and after purification reconstituted in planar lipid bilayers. The electrical recordings in Figure 4 show that the two proteins generated typical single-channel fluctuations at positive and negative voltages in the presence of 100 mM KCl on both sides of the membrane. These experiments established that the two genes code for functional ion channels. The overall properties of the two new channels were similar to those of Kcv NTS (Figure 4), a well-studied representative of the K + channels from SAG viruses [18]; Kcv NTS differed from the reference channel Kcv ATCV-1 by four amino acids. Kcv GNLD22 and Kcv Can0610SP were selected because of their large (55 amino acids) and small (1 amino acid) deviation from the reference channel Kcv ATCV-1 . All three channels exhibited a hallmark of the chlorovirus K + channels with well-resolved channel openings at Viruses 2020, 12, 678 7 of 33 positive voltages and flicker type gating at negative voltages. This flicker type gating resulted from very fast open/close transitions at negative voltages, which cannot be fully resolved by the recording equipment. As a consequence, the unitary channel conductance exhibited an apparent decrease at negative voltages [19,20]. However, it is interesting to note that this fast gating was already apparent in Kcv GNLD22 at voltages negative of 0 mV while the negative slope in the two other channels only occurred at voltages more negative than about -100 mV. A closer scrutiny of the single-channel data also showed additional differences between the three channels. Comparison of the unitary channel conductance showed that this value in Kcv GNLD22 (45 ± 3 pS, n = 7) was only half as big as in Kcv NTS (87 ± 1.4 pS, n = 9) and Kcv Can0610SP (110 ± 3, n = 9). Another striking feature of Kcv GNLD22 was a strong voltage-dependent decrease in open probability at positive voltages, which was not seen in the other two. A peculiar feature of Kcv Can0610SP was long-lived closed states at negative voltages, which explains a voltage-dependent decrease in open probability at negative voltages. These long closures were absent in the two other channels; in Kcv GNLD22 it was even difficult to observe any distinct closure at negative voltages. The data are also in line with a previous study where the kcv gene was sequenced from 41 NC64A viruses, including the prototype chlorovirus PBCV-1. Sixteen of the 94 amino acids in the NC64A Kcv protein differed resulting in six new Kcv channels [20]. The six Kcv-like channels, which differed from KcvPBCV-1 by 4 to 12 amino acids, produced K + selective currents in Xenopus laevis oocytes with altered biophysical properties, including current kinetics, voltage dependency, and inhibition by Cs + [20,21]. The amino acid changes together with the different properties observed in the six Kcv-like channels were used to guide site-directed mutations, either singularly or in combination, to identify key amino acids that confer specific properties to Kcv [20,21]. 
While we assume that the chloroviruses require Kcv activity to replicate, we do have to mention that out of the more than 150 chloroviruses that have been examined for a kcv gene, two NC64A viruses either lack a kcv gene or have a truncated form [1]. We would like to disrupt the kcv gene in some of the chloroviruses to see what effect this has on virus replication, however, currently the technology to do this experiment is not available. 
Compared with the larger KcvPBCV-1, the 82 amino acid KcvATCV-1 lacks a cytoplasmic N-terminus, that is, the slide helix region and a number of charged amino acids in its turret domain (Figure 1) [12]. The only known K + channel proteins smaller than KcvATCV-1 are two channels that are encoded by 

Discussion

This manuscript demonstrated that the kcv gene is ubiquitous among the 103 chloroviruses that infect C. heliozoae SAG 3.83, which resulted in 42 unique kcv DNA sequences. The 42 unique kcv DNA sequences produced 26 unique proteins or 26 new Kcv channels. Using the Kcv ATCV-1 channel as representative of the SAG viruses, the Kcv ATCV-1 differed from the Kcv GNLD22 channel from Greenland by 55 amino acids or 56% of their amino acids. Other channels differ from the Kcv ATCV-1 channel by 1 to 21 amino acids. 
Due to the role K + channels play in chlorovirus infection and reproduction by depolarizing the host cell membrane, it is not surprising that kcv genes were found in all of the samples [19]. Therefore, we predict that all of the amino acid substitutions in the channels from the SAG viruses will produce functional channels; in fact, the two channels that were tested were functional (Figure 4). Furthermore, we predict that the SAG Kcv channels may have some different biophysical properties, especially the Kcv coded by the GNDL22 virus. This prediction is confirmed by scrutiny of the functional properties of the two new channels and a comparison with the well-studied SAG-type channel Kcv NTS [18]. The mutual comparisons identified differences in the unitary conductance and gating between the different channels. The apparent impact of a few amino acid exchanges between the proteins on functional properties is in good agreement with previous investigations in which we found that mutation of the common Gly at the end of the second transmembrane domain in Kcv NTS to Ser introduces one distinct gate with a long-lived close time [18]. Several of the new channels have the same critical Ser in the same position (90 in Figure 2). Thus, a functional analysis of these channels will be interesting. 
The data are also in line with a previous study where the kcv gene was sequenced from 41 NC64A viruses, including the prototype chlorovirus PBCV-1. Sixteen of the 94 amino acids in the NC64A Kcv protein differed resulting in six new Kcv channels [20]. The six Kcv-like channels, which differed from Kcv PBCV-1 by 4 to 12 amino acids, produced K + selective currents in Xenopus laevis oocytes with altered biophysical properties, including current kinetics, voltage dependency, and inhibition by Cs + [20,21]. The amino acid changes together with the different properties observed in the six Kcv-like channels were used to guide site-directed mutations, either singularly or in combination, to identify key amino acids that confer specific properties to Kcv [20,21]. 
While we assume that the chloroviruses require Kcv activity to replicate, we do have to mention that out of the more than 150 chloroviruses that have been examined for a kcv gene, two NC64A viruses either lack a kcv gene or have a truncated form [1]. We would like to disrupt the kcv gene in some of the chloroviruses to see what effect this has on virus replication, however, currently the technology to do this experiment is not available. 
Compared with the larger Kcv PBCV-1 , the 82 amino acid Kcv ATCV-1 lacks a cytoplasmic N-terminus, that is, the slide helix region and a number of charged amino acids in its turret domain (Figure 1) [12]. The only known K + channel proteins smaller than Kcv ATCV-1 are two channels that are encoded by viruses that infect small marine algae in the Micromonas genus [22]. The channel Kmbv 1 is 79 amino acids long and Kmpv 12T is 78 amino acids long. Expression of Kmbv 1 in HEK293 cells results in currents. However, expression of Kmpv 12T in the same cells does not produce a current, but it does produce a current in a planar lipid bilayer [22]. All of these virus-encoded channels have a long evolutionary history and probably have a common evolutionary ancestor. 
In summary, kcv genes from 103 geographically distinct SAG viruses were sequenced to assess their genetic diversity. Of the 103 kcv genes, there were 42 unique DNA sequences that translated into 26 new Kcv proteins, which we predict will have some different biophysical properties. The amino acid changes together with the expected different properties will be used to guide site-directed mutations to identify key amino acids that confer specific properties to Kcv. 

Appendix A


Figure 1 .

 1 Figure 1. KcvATCV-1 sequence alignment with the sequence of the prototype chlorovirus K + channel KcvPBCV-1 by ClustalW. The slide helix, outer transmembrane (TMD1) turret, pore helix, selectivity filter, and inner transmembrane (TMD2) of the KcvPBCV-1 are highlighted by the horizontal lines. Asterisks indicate positions which have a fully conserved residue. A colon indicates conservation between amino acids with strongly similar properties. A period indicates conservation between amino acids with less similar properties. 


Figure 2 .

 2 Figure 2. Amino acid alignment of SAG chlorovirus unique Kcv proteins using Geneious 11.0.5 software. The number in brackets at the end of each protein is the number of times that the protein had an identical sequence of the 103 viruses examined. Score matrix is Identity. Green color denotes identical amino acids. Other shades of amino acids indicate a level of conservation from olive color as being the most conserved to white color not conserved. 


Figure 2 .

 2 Figure 2. Amino acid alignment of SAG chlorovirus unique Kcv proteins using Geneious 11.0.5 software. The number in brackets at the end of each protein is the number of times that the protein had an identical sequence of the 103 viruses examined. Score matrix is Identity. Green color denotes identical amino acids. Other shades of amino acids indicate a level of conservation from olive color as being the most conserved to white color not conserved. 


Figure 3 .

 3 Figure 3. Phylogenetic tree of Kcv proteins from 26 SAG chloroviruses. PhyML, which is Maximum likelihood, with the default settings was used to construct the tree. The branch length shows dissimilarity between strains and the values on the branches are the number of changes. The viruses in blue represent the new Kcv proteins reported in this manuscript. Viruses MT325 representing a Pbi virus and PBCV-1 representing an NC64A virus are in green. Additional information on each of the Kcv proteins from the SAG viruses, including where they were isolated and the number of times that protein sequence appeared, is included in TableA1. 


Figure 4 .

 4 Figure 4. Two of the newly discovered K + channels are active. Characteristic single-channel fluctuations (a), mean single-channel I/V relations (b), and mean open probabilities (c) of KcvNTS (top row), KcvGNLD22 (middle row), and KcvCan0610SP (bottom row) at ±120 mV. The closed (c) and open (o) levels are indicated along the current traces. Data are means ±s.d. from ≥ 3 independent recordings of channels in the same row. Data were recorded in a DPhPC bilayer with symmetrical 100 mM KCl, 10 mM HEPES, pH 7 in cis and trans chamber. 


Figure 4 .

 4 Figure 4. Two of the newly discovered K + channels are active. Characteristic single-channel fluctuations (a), mean single-channel I/V relations (b), and mean open probabilities (c) of Kcv NTS (top row), Kcv GNLD22 (middle row), and Kcv Can0610SP (bottom row) at ±120 mV. The closed (c) and open (o) levels are indicated along the current traces. Data are means ±s.d. from ≥ 3 independent recordings of channels in the same row. Data were recorded in a DPhPC bilayer with symmetrical 100 mM KCl, 10 mM HEPES, pH 7 in cis and trans chamber. 




  


Table 1 .

 1 Primers tested to isolate the kcv genes. 


		Name	Sequence	Position	GC Content (%)	T m ( • C)
		Kcv6 Frw	CTT TAG YYT TYY TCK GVC	-366	34	49
	Forward	Kcv7 Frw	CTT TAG YYT TYY TCK GVC G	-366	38	54
	Primers	Kcv8 Frw	GAA GCA GGY ACC ACT TTA G	-379	47	53
		Kcv9 Frw	GCA GGY ACC ACT TTA G	-376	50	47
		Kcv6 Rvs	CRC RGM ATR TRT CAT TTG WCC C	+256	48	64
	Reverse Primers	Kcv7 Rvs Kcv8 Rvs Kcv9 Rvs	CTT ACR CRG MAT RTR TCA TTT G CTT ACR CRG MAT RTR TC HKB YMC GAT CTT ATA CAC	+259 +264 +292	39 44 39	56 44 45
		Kcv10 Rvs	CAT TTC TTA CRC RGM ATR TRT C	+264	39	55

Table A1 .

 A1 Source and sequence of SAG chlorovirus encoded potassium ion channel (Kcv) genes 1 . 


	Viruses 2020, 12, 678					
	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGTTGCTGCTTCTCATACATCTCAGCATTTTGGT				
		ACTTTTCACTACCATATACAAGATGCTCCCCGGTGGC				
	5th_CW_MN_s1a (2) [2]	ATGTTCTCGAACACGGATCCGTCCTGGGTCGATTGCC TGTACTTTTCGGCATCAACGCACACCACCGTGGGGTA CGGGGACCTCACGCCAAAATCACCCGTGGCAAAACT	5th Crow Wing Lake; Nevis, Minnesota	5/4/2017	5th_CW_MN_s1a	5th_CW_MN_s1a
		CACGGCCACGGCACACATGCTGATCGTATTCGCGATC				
		GTCATTTCTGGCTTCACGTTCCCGTGGTAA				
		ATGTTGCTGCTTCTCATACATCTCAGCATTTTGGTA				
		CTTTTCACTACCATATACAAGATGCTCCCCGGTGGCAT				
	5th_CW_MN_L4	GTTCTCGAACACGGATCCGTCCTGGGTCGATTGCCTGT ACTTTTCGGCATCAACGCACACCACCGTGGGGTACGGG GACCTCACGCCAAAATCACCCGTGGCAAAACTCACGGC	5th Crow Wing Lake; Nevis, Minnesota	5/4/2017	5th_CW_MN_s1a	5th_CW_MN_s1a
		CACGGCACACATGCTGATCGTATTCGCGATCGTCATTTCT				
		GGCTTCACGTTCCCGTGGTAA				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATA				
		GTGTTCACTGCCATCTACAAGATGCTCCCCGGC				
		GGCATGTTCTCGAACACAGACCCTACTTGGGTT				
	ATCV-1 (8) [9]	GATTGCCTGTACTTTTCGGCATCGACGCACACC ACCGTGGGGTACGGAGATCTCACGCCCAAATC	Germany	2002	ATCV-1	ATCV-1
		ACCCGTGGCAAAACTCACGGCAACGGCACAC				
		ATGTTGATCGTATTCGCGATCGTCATTTCTGGCT				
		TCACGTTTCCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATA				
		GTGTTCACTGCCATCTACAAGATGCTCCCCGGCG				
		GCATGTTCTCGAACACAGACCCTACTTGGGTTGA				
	MO0605SPH	TTGCCTGTACTTTTCGGCATCGACGCACACCACC	Missouri	2006	ATCV-1	ATCV-1
		GTGGGGTACGGAGATCTCACGCCCAAATCACCCG				
		TGGCAAAACTCACGGCAACGGCACACATGTTGAT				
		CGTATTCGCGATCGTCATTTCTGGCTTCACGTTTCCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATA				
		GTGTTCACTGCCATCTACAAGATGCTCCCCGGCG				
		GCATGTTCTCGAACACAGACCCTACTTGGGTTGA				
	WI0606	TTGCCTGTACTTTTCGGCATCGACGCACACCACC	Madison, Wisconsin	2006	ATCV-1	ATCV-1
		GTGGGGTACGGAGATCTCACGCCCAAATCACCCG				
		TGGCAAAACTCACGGCAACGGCACACATGTTGAT				
		CGTATTCGCGATCGTCATTTCTGGCTTCACGTTTCCGTGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGTTGCTGCTTATCATACATATCATCATTCTG				
		ATAGTGTTCACTGCCATCTACAAGATGCTCCCCGGC				
		GGCATGTTCTCGAACACAGACCCTACTTGGGTTGAT				
	Drexel	TGCCTGTACTTTTCGGCATCGACGCACACCACCGTG	Drexel, Missouri	Jun-17	ATCV-1	ATCV-1
		GGGTACGGAGATCTCACGCCCAAATCACCCGTGGC				
		AAAACTCACGGCAACGGCACACATGTTGATCGTATT				
		CGCGATCGTCATTTCTGGCTTCACGTTTCCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATA				
		GTGTTCACTGCCATCTACAAGATGCTCCCCGGCGGC				
	NPRLb	ATGTTCTCGAACACAGACCCTACTTGGGTTGATTG CCTGTACTTTTCGGCATCGACGCACACCACCGTG GGGTACGGAGATCTCACGCCCAAATCACCCGTGG	North Platte River; Highway 27, Nebraska	10/23/2017	ATCV-1	ATCV-1
		CAAAACTCACGGCAACGGCACACATGTTGATCGT				
		ATTCGCGATCGTCATTTCTGGCTTCACGTTTCCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGAT				
		AGTGTTCACTGCCATCTACAAGATGCTCCCCGG				
		CGGCATGTTCTCGAACACAGACCCTACTTGGGT				
	SPRma	TGATTGCCTGTACTTTTCGGCATCGACGCACAC CACCGTGGGGTACGGAGATCTCACGCCCAAAT	South Platte River; Big Spring, Nebraska	10/23/2017	ATCV-1	ATCV-1
		CACCCGTGGCAAAACTCACGGCAACGGCACA				
		CATGTTGATCGTATTCGCGATCGTCATTTCTGG				
		CTTCACGTTTCCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTG				
		ATAGTGTTCACTGCCATCTACAAGATGCTCCCCGG				
		CGGCATGTTCTCGAACACAGACCCTACTTGGGTT				
	SPRsb	GATTGCCTGTACTTTTCGGCATCGACGCACACCA CCGTGGGGTACGGAGATCTCACGCCCAAATCAC	South Platte River; Big Spring, Nebraska	10/23/2017	ATCV-1	ATCV-1
		CCGTGGCAAAACTCACGGCAACGGCACACATG				
		TTGATCGTATTCGCGATCGTCATTTCTGGCTTCA				
		CGTTTCCGTGGTAG				

Table A1 .

 A1 Cont. 


	Viruses 2020, 12, 678					
	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGTTGCTGCTTATCATACATATCATCATTCTG				
		ATAGTGTTCACTGCCATCTACAAGATGCTCCCCGG				
		CGGCATGTTCTCGAACACAGACCCTACTTGGGTT				
	Smith-Lake_ Large-Plaque	GATTGCCTGTACTTTTCGGCATCGACGCACACCA CCGTGGGGTACGGAGATCTCACGCCCAAATCAC	Smith Lake, Western Nebraska	2018	ATCV-1	ATCV-1
		CCGTGGCAAAACTCACGGCAACGGCACACATG				
		TTGATCGTATTCGCGATCGTCATTTCTGGCTTCA				
		CGTTTCCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGA				
		TAGTGTTCACTGCCATCTACAAGATGCTCCCCGGCG				
		GCATGTTCTCGAACACAGACCCTACTTGGGTTGATT				
	OH-S (1)	GCCTGTACTTTTCGGCATCGACGCACACCACCGT GGGGTACGGAGATCTCACGCCCAAATCACCCGT	Ohio	Jul-17	OH-S	ATCV-1
		GGCAAAACTCACGGCAACGGCACACATGTTGAT				
		CGTATTCGCGATCGTCATTTCTGGCTTCACATTT				
		CCGTGGTAG				
		ATGTTGCTGCTTCTCATACACCTCTGTATTCTGAT				
		AATTTTTACTACCATATACAAGATGTTGCCCGGAGG				
		CATGTTCTCTAACACGGACCCGTCGTGGGTCGATTG				
	BRO604 (1) [1]	CCTGTACTTCTCGGCATCAACGCACACCACCGTGGG	Brazil	2006	BRO604	BRO604
		GTACGGGGATCTCACGCCCAAATCACCCGTGGCAA				
		AACTCACAGCAACGGCACACATGCTGATCGTATTCG				
		CGATCGTAATAACTGGCTTCACATTCCCGTGGTAA				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATA				
		GTGTTCACTACCATCTACAAGATGCTCCCCGGCGG				
		CATGTTCTCGAACACAGACCCTACTTGGGTTGATT				
	Can0610SP (2) [18]	GCCTGTACTTTTCGGCATCGACGCACACCACTGT GGGGTACGGAGATCTCACGCCCAAATCACCCGT	British Columbia, Canada	2006	Can0610SP	Can0610SP
		GGCAAAACTCACGGCAACGGCACACATGTTGA				
		TCGTATTCGCGATCGTCATTTCCGGCTTCACG				
		TTCCCGTGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGTTGCTGCTTATCATACATATCATCATTCT				
		GATAGTGTTCACTACCATCTACAAGATGCTCCCCGGC				
	Or0704	GGCATGTTCTCGAACACAGACCCTACTTGGGTTGAT TGCCTGTACTTTTCGGCATCGACGCACACCACTGTG GGGTACGGAGATCTCACGCCCAAATCACCCGTGGC	Willamette River; Corvallis, Oregon	Jul-07	Can0610SP	Can0610SP
		AAAACTCACGGCAACGGCACACATGTTGATCGTA				
		TTCGCGATCGTCATTTCCGGCTTCACGTTCCCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATA				
		GTGTTCACTACCATATACAAGATGCTCCCCGGCGGCATG				
		TTCTCGAACACAGACCCTACTTGGGTTGATTGCCTGTACT				
	Chile_7s (1)	TTTCGGCATCGACGCACACCACTGTGGGGTACGGAGATCT	Chile	Jan-19	Chile_7s	Can0610SP
		CACGCCCAAATCACCCGTGGCAAAACTCACGGCAACGGCA				
		CACATGTTGATCGTATTCGCGATCGTCATTTCCGGCTT				
		CACGTTCCCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGTTC				
		ACTACCATCTACAAGATGCTCCCCGGTGGCATGTTCTCGAACA				
	K2 (5)	CGGACCCGACTTGGGTTGATTGCCTGTACTTTTCGGCATCGA CGCACACCACCGTGGGGTACGGAGATCTCACGCCCAAATC	Missouri River; Atchison, Kansas	5/31/2017	K2	Can0610SP
		ACCCGTGGCAAAACTCACGGCAACGGCACACATGTTGAT				
		CGTATTCGCGATCGTCATTTCCGGCTTCACGTTCCCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGT				
		TCACTACCATCTACAAGATGCTCCCCGGTGGCATGTTCTCGA				
	K2s	ACACGGACCCGACTTGGGTTGATTGCCTGTACTTTTCGGCAT CGACGCACACCACCGTGGGGTACGGAGATCTCACGCCCAA	Missouri River; Atchison, Kansas	5/31/2017	K2	Can0610SP
		ATCACCCGTGGCAAAACTCACGGCAACGGCACACATGTTG				
		ATCGTATTCGCGATCGTCATTTCCGGCTTCACGTTCCCGTGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGTTC				
		ACTACCATCTACAAGATGCTCCCCGGTGGCATGTTCTCG				
	NPRma	AACACGGACCCGACTTGGGTTGATTGCCTGTACTTTTC GGCATCGACGCACACCACCGTGGGGTACGGAGATCTC ACGCCCAAATCACCCGTGGCAAAACTCACGGCAACG	North Platte River; Highway 27, Nebraska	10/23/2017	K2	Can0610SP
		GCACACATGTTGATCGTATTCGCGATCGTCATTTCCG				
		GCTTCACGTTCCCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGTTC				
		ACTACCATCTACAAGATGCTCCCCGGTGGCATGTTCTCGA				
	NPRsb	ACACGGACCCGACTTGGGTTGATTGCCTGTACTTTTCGG CATCGACGCACACCACCGTGGGGTACGGAGATCTCACG CCCAAATCACCCGTGGCAAAACTCACGGCAACGGCAC	North Platte River; Highway 27, Nebraska	10/23/2017	K2	Can0610SP
		ACATGTTGATCGTATTCGCGATCGTCATTTCCGGCTTC				
		ACGTTCCCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGTTC				
		ACTACCATCTACAAGATGCTCCCCGGTGGCATGTTCTCGAA				
	Pl_R_Lma	CACGGACCCGACTTGGGTTGATTGCCTGTACTTTTCGGCAT CGACGCACACCACCGTGGGGTACGGAGATCTCACGCCCA AATCACCCGTGGCAAAACTCACGGCAACGGCACACATGT	Platte River; Louisville, Nebraska	10/26/2017	K2	Can0610SP
		TGATCGTATTCGCGATCGTCATTTCCGGCTTCA				
		CGTTCCCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAG				
		TGTTCACTACCATCTACAAGATGCTCCCCGGCGGCATG				
	KS3-S (1)	TTCTCGAACACAGACCCTACTTGGGTCGATTGCCTGTA CTTTTCGGCATCGACGCACACCACCGTGGGGTACGGAG ATCTCACGCCCAAATCACCCGTGGCAAAACTCACGGCA	Delaware River; Valley Fall, Kansas	Jun-17	KS3-S	Can0610SP
		ACGGCACACATGTTGATCGTATTCGCGATCGTCATTT				
		CCGGCTTCACATTTCCGTGGTAA				

Table A1 .

 A1 Cont. 


	Viruses 2020, 12, 678					
	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAG				
		TGTTCACTACCATCTACAAGATGCTCCCCGGTGGCA				
	LP_CO_F1m (7)	TGTTCTCGAACACGGACCCGACTTGGGTTGATTGCC TGTACTTTTCGGCATCGACGCACACCACCGTGGGGT ACGGAGATCTCACGCCCAAATCACCCGTGGCAAAA	Cache la Poudre River, Colorado	5/29/2017	LP_CO_F1m	Can0610SP
		CTCACGGCAACGGCACACATGTTGATCGTATTCGCGA				
		TCGTCATTTCCGGCTTCACGTTTCCGTGGTAA				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATA				
		GTGTTCACTACCATCTACAAGATGCTCCCCGGTGGCAT				
	LP_CO_F2L	GTTCTCGAACACGGACCCGACTTGGGTTGATTGCCTGT ACTTTTCGGCATCGACGCACACCACCGTGGGGTACGGA GATCTCACGCCCAAATCACCCGTGGCAAAACTCACGGC	Cache la Poudre River, Colorado	5/30/2017	LP_CO_F1m	Can0610SP
		AACGGCACACATGTTGATCGTATTCGCGATCGTCATTTC				
		CGGCTTCACGTTTCCGTGGTAA				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGTT				
		CACTACCATCTACAAGATGCTCCCCGGTGGCATGTTCTCGA				
	LP_CO_F2m	ACACGGACCCGACTTGGGTTGATTGCCTGTACTTTTCGGCA TCGACGCACACCACCGTGGGGTACGGAGATCTCACGCCCA	Cache la Poudre River, Colorado	5/31/2017	LP_CO_F1m	Can0610SP
		AATCACCCGTGGCAAAACTCACGGCAACGGCACACATGTTG				
		ATCGTATTCGCGATCGTCATTTCCGGCTTCACGTTTCCGTGGTAA			
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGTT				
		CACTACCATCTACAAGATGCTCCCCGGTGGCATGTTCTC				
	LP_CO_F2s	GAACACGGACCCGACTTGGGTTGATTGCCTGTACTTTT CGGCATCGACGCACACCACCGTGGGGTACGGAGATCT CACGCCCAAATCACCCGTGGCAAAACTCACGGCAAC	Cache la Poudre River, Colorado	6/1/2017	LP_CO_F1m	Can0610SP
		GGCACACATGTTGATCGTATTCGCGATC				
		GTCATTTCCGGCTTCACGTTTCCGTGGTAA				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGTT				
		CACTACCATCTACAAGATGCTCCCCGGTGGCATGTTCTC				
	LP_CO_F3L	GAACACGGACCCGACTTGGGTTGATTGCCTGTACTTTT CGGCATCGACGCACACCACCGTGGGGTACGGAGATCT CACGCCCAAATCACCCGTGGCAAAACTCACGGCAAC	Cache la Poudre River, Colorado	6/2/2017	LP_CO_F1m	Can0610SP
		GGCACACATGTTGATCGTATTCGCGATCGTCATTTC				
		CGGCTTCACGTTTCCGTGGTAA				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGTTCA				
		CTACCATCTACAAGATGCTCCCCGGTGGCATGTTCTCGAAC				
	LP_CO_F3m	ACGGACCCGACTTGGGTTGATTGCCTGTACTTTTCGGCATC GACGCACACCACCGTGGGGTACGGAGATCTCACGCCCAAA TCACCCGTGGCAAAACTCACGGCAACGGCACACATGTTG	Cache la Poudre River, Colorado	6/3/2017	LP_CO_F1m	Can0610SP
		ATCGTATTCGCGATCGTCATTTCCGGCTTCACGTTTCC				
		GTGGTAA				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGT				
		TCACTACCATCTACAAGATGCTCCCCGGTGGCATGTTCTC				
	LP_CO_F4s	GAACACGGACCCGACTTGGGTTGATTGCCTGTACTTTTCG GCATCGACGCACACCACCGTGGGGTACGGAGATCTCACG CCCAAATCACCCGTGGCAAAACTCACGGCAACGGCACA	Cache la Poudre River, Colorado	6/4/2017	LP_CO_F1m	Can0610SP
		CATGTTGATCGTATTCGCGATCGTCATTTC				
		CGGCTTCACGTTTCCGTGGTAA				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATA				
		GTGTTCACTACCATATACAAGATGCTCCCCGGCGGC				
	WR_DE (2)	ATGTTCTCGAACACAGACCCTACTTGGGTCGATTGC CTGTACTTTTCGGCATCGACGCACACCACCGTGGGG TACGGAGATCTCACGCCCAAATCACCCGTGGCAAAA	Wilson Run: Winterhur, Delaware	Jul-17	WR_DE	Can0610SP
		CTCACGGCAACGGCGCACATGTTGATCGTATTCGCG				
		ATCGTCATTTCTGGATTCACGTTCCCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGT				
		GTTCACTACCATATACAAGATGCTCCCCGGCGGCAT				
	WR_DE_s2cr2	GTTCTCGAACACAGACCCTACTTGGGTCGATTGCC TGTACTTTTCGGCATCGACGCACACCACCGTGGGG TACGGAGATCTCACGCCCAAATCACCCGTGGCAAA	Wilson Run, Winterthur; Delaware	Jul-17	WR_DE	Can0610SP
		ACTCACGGCAACGGCGCACATGTTGATCGTATTCG				
		CGATCG TCATTTCTGGATTCACGTTCCCGTGGTAG				
		ATGTTGCTGCTCCTTATACACGTTGGTATTTTGGTATTTT				
		TCACCACCGTATACAAGATGCTCCCCGGTGGCATGTTC				
	Canal1 (2) [2]	TCGAATACGGACCCTAGCTGGGTAGATTGCTTATACTTC TCAGCGTCAACTCACACCACCGTTGGGTACGGAGATC TCACGCCCAAATCACCCGTGGCGAAACTCGTGGCGAC	Canal exiting Smith Lake, Nebraska	2008	Canal1	Canal1
		GGCGCATATGATGATCGTGTTCGCGATCGTTGTATCTAG				
		CTTCACGTTTTCGTGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGTTGCTGCTCCTTATACACGTTGGTATTTTGGTATTTT				
		TCACCACCGTATACAAGATGCTCCCCGGTGGCATGTTCTCGAA			
	Smith-Lake_ Small-Plaque	TACGGACCCTAGCTGGGTAGATTGCTTATACTTCTCAGCGT CAACTCACACCACCGTTGGGTACGGAGATCTCACGCCCAA ATCACCCGTGGCGAAACTCGTGGCGACGGCGCATATGAT	Smith Lake, Western Nebraska	2018	Canal1	Canal1
		GATCGTGTTCGCGATCGTTGTATCTAGCT				
		TCACGTTTTCGTGGTAG				
		ATGTTGCTGCTTCTCATACATCTCAGCATTTTGGTACTTTT				
		CACTACCATATACAAGATGCTCCCCGGCGGCATGTTC				
		TCGAACACGGACCCATCCTGGGTCGATTGCCTGTAC				
	GM0701 (1) [1]	TTTTCAGCATCAACGCACACGACCGTGGGGTACGG	Guatemala	2007	GM0701	GM0701
		GGACCTCACGCCAAAATCGCCCGTGGCAAAACTC				
		ACAGCAACGGCACACATGCTGATCGTATTC				
		GCGATCGTAATAACTGGCTTCACATT CCCGTGGTAA				
		ATGCTGCGGTCAATATTGCCTCATATCATAGTGTTCAC				
		GTTTTTCGTTGTTCTTTACAAATTTTTCCCCGGGGGG				
		TTTGAAGACTCATTCAAACGAGGAGACGGGTCCCGCA				
	GNLD22 (1) [1]	GAAAGGCGACGTGGATGGACTGCATCTATTTCGCGACG GCAACGCACACCACCACCGGGTTTGGTGATGTAGTCC	Lake Sisimiut, Greenland	2012	GNLD22	GNLD22
		CCGACAACGACGCCGCAAGAACAGCTGTCACGATGC				
		ACATGCTCATAGTTTTCGCGATCGTAGTATT				
		GGGGATAAAACTCTAA				
		ATGTTGCTGCTCCTTATACACATCGGTATTTTGGTATTT				
		TTCACTATCGTGTACAAGCTGCTCCCTGGTGGCATGTT				
	Hale-L (2) [6]	CTCGTACGCAGACCCGACCTGGGTCGACTGCTTGTATT TTTCGGCATCAACGCACACCACCGTGGGGTATGGGGAT CTCACGCCCAAATCACCCGTGGCAAAACTCACGGCC	North branch of Elk Creek; Hale, Wisconsin	7/4/2017	Hale-L	Hale-L
		ACGGCACACATGCTGATTGTATTCGCGATCGTTGTCTC				
		TAGCTTTACGCTCCCCTGGTAA				
		ATGTTGCTGCTCCTTATACACATCGGTATTTTGGTATTTTT				
		CACTATCGTGTACAAGCTGCTCCCTGGTGGCATGTTCTCGT				
	Hale_WI_m4	ACGCAGACCCGACCTGGGTCGACTGCTTGTATTTTTCGGCA TCAACGCACACCACCGTGGGGTATGGGGATCTCACGCCCA AATCACCCGTGGCAAAACTCACGGCCACGGCACACATGCT	North branch of Elk Creek; Hale, Wisconsin	7/4/2017	Hale-L	Hale-L
		GATTGTATTCGCGATCGTTGTCTCTAGCTTTAC				
		GCTCCCCTGGTAA				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGTTGCTGCTCCTTATACACATTGGTATTTTGGTATTTTTC				
		ACTATCGTGTACAAACTGCTCCCTGGTGGCATGTTCTCGT				
	TX3-L1 (4)	ACGCAGATCCGACCTGGGTCGACTGCTTGTATTTTTCGGC ATCAACGCACACCACCGTGGGGTATGGGGATCTCACGCC CAAATCACCCGTGGCAAAACTCACGGCCACTGCACACA	Colorado River Pond; Austin, Texas	Jul-17	TX3-L1	Hale-L
		TGCTGATTGTATTCGCGATCGTTGTCTCTAGCTTTAC				
		GCTCCCCTGGTAA				
		ATGTTGCTGCTCCTTATACACATTGGTATTTTGGTATTTTT				
		CACTATCGTGTACAAACTGCTCCCTGGTGGCATGTTCTC				
	TX3-L2	GTACGCAGATCCGACCTGGGTCGACTGCTTGTATTTTTCG GCATCAACGCACACCACCGTGGGGTATGGGGATCTCACG CCCAAATCACCCGTGGCAAAACTCACGGCCACTGCACAC	Colorado River Pond, Austin, Texas	Jul-17	TX3-L1	Hale-L
		ATGCTGATTGTATTCGCGATCGTTGTCTCTAGCTTTA				
		CGCTCCCCTGGTAA				
		ATGTTGCTGCTCCTTATACACATTGGTATTTTGGTATTT				
		TTCACTATCGTGTACAAACTGCTCCCTGGTGGCATGTTC				
	TX3m	TCGTACGCAGATCCGACCTGGGTCGACTGCTTGTATTTT TCGGCATCAACGCACACCACCGTGGGGTATGGGGATCTC ACGCCCAAATCACCCGTGGCAAAACTCACGGCCACTGC	Colorado River Pond; Austin, Texas	Jul-17	TX3-L1	Hale-L
		ACACATGCTGATTGTATTCGCGATCGTTGTCTCTAGCTTT				
		ACGCTCCCCTGGTAA				
		ATGTTGCTGCTCCTTATACACATTGGTATTTTGGTATTTTT				
		CACTATCGTGTACAAACTGCTCCCTGGTGGCATGTTCTC				
	TX3s	GTACGCAGATCCGACCTGGGTCGACTGCTTGTATTTTTC GGCATCAACGCACACCACCGTGGGGTATGGGGATCTCA CGCCCAAATCACCCGTGGCAAAACTCACGGCCACTGC	Colorado River Pond; Austin, Texas	Jul-17	TX3-L1	Hale-L
		ACACATGCTGATTGTATTCGCGATCGTTGTCTCTAGCTTT				
		ACGCTCCCCTGGTAA				
		ATGTTGCTGCTCCTTATACACATCGGTATTTTGGTATTTTT				
		CACTATCGTGTACAAGCTGCTCCCTGGTGGCATGTTCTCG				
	Hale-s1 (2) [2]	TACGCAGACCCGACCTGGGTCGACTGCTTGTATTTTTCGG CATCAACGCACACCACCGTGGGGTATGGGGATCTCACGC CCAAATCACCCGTGGCAAAACTCACGGCAACGGCACAC	North branch of Elk Creek; Hale, Wisconsin	7/4/2017	Hale-s1	Hale-s1
		ATGCTGATCGTACTCGCGATCGTCATTTCTGGCTTCACGT				
		TCCCGTGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGTTGCTGCTCCTTATACACATCGGTATTTTGGTATTTTTC				
		ACTATCGTGTACAAGCTGCTCCCTGGTGGCATGTTCTCGT				
	Hale-s2	ACGCAGACCCGACCTGGGTCGACTGCTTGTATTTTTCGG CATCAACGCACACCACCGTGGGGTATGGGGATCTCACGC CCAAATCACCCGTGGCAAAACTCACGGCAACGGCACA	North branch of Elk Creek; Hale, Wisconsin	7/4/2017	Hale-s1	Hale-s1
		CATGCTGATCGTACTCGCGATCGTCATTTCTGGCTTCAC				
		GTTCCCGTGGTAG				
		ATGTTGCTGCTTCTCATACATCTCAGCATTTTGGTAATT				
		TTCACTACCATATACAAGATGCTCCCCGGCGGCATGT				
		TCTCGAACACGGATCCGTCCTGGGTCGATTGCCTGTA				
	IL-1 (1) [6]	CTTTTCGGCATCAACGCACACCACCGTGGGGTACGG	Lake Zurich, Illinois	6/27/2017	IL-1	IL-1
		GGACCTCACGCCAAAATCACCCGTGGCAAAACTCA				
		CGGCAACGGCACACATGCTGATCGTATTTGCGATCGT				
		CATTTCTGGCTTCACGTTCCCGTGGTAA				
		ATGTTGCTGCTTCTCATACATCTCAGCATTTTGGTA				
		ATTTTCACTACCATCTACAAGATGCTCCCCGGCGGC				
		ATGTTCTCGAACACGGACCCATCCTGGGTCGATTGC				
	Dismal_NE (1)	CTGTACTTTTCGGCATCAACGCACACCACCGTGGG	Dismal River, Nebraska	5/26/2017	Dismal_NE	IL-1
		GTACGGGGACCTCACGCCAAAATCACCCGTGGCA				
		AAACTCACGGCAACGGCACACATGCTGATCGTATT				
		CGCGATCGTCATTTCTGGCTTCACGTTCCCGTGGTAG				
		ATGTTGCTGCTTCTCATACATCTCAGCATTTTGGTAATTTTCA				
		CTACCATCTACAAGATGCTCCCCGGCGGCATGTTCTCGAAC				
		ACGGACCCATCCTGGGTCGATTGCCTGTACTTTTCGGCAT	Moodna Creek;			
	NY2s1 (2)	CAACGCACACCACCGTGGGGTACGGGGACCTCACGCCCA	Washingtonville, New	6/21/2017	NY-2s1	IL-1
		AATCACCCGTTGCAAAACTCACGGCAACGGCACACATGC	York			
		TGATCGTATTCGCGATCGTCATTTCTGGCTTCACGTT				
		CCCGTGGTAA				
		ATGTTGCTGCTTCTCATACATCTCAGCATTTTGGTAAT				
		TTTCACTACCATCTACAAGATGCTCCCCGGCGGCAT				
		GTTCTCGAACACGGACCCATCCTGGGTCGATTGCCT	Moodna Creek;			
	NY-2s2	GTACTTTTCGGCATCAACGCACACCACCGTGGGGT	Washingtonville, New	6/21/2017	NY-2s1	IL-1
		ACGGGGACCTCACGCCCAAATCACCCGTTGCAAAA	York			
		CTCACGGCAACGGCACACATGCTGATCGTATTCGC				
		GATCGTCATTTCTGGCTTCACGTTCCCGTGGTAA				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGTTGCTGCTTCTCATACATCTCAGCATTTTGGTA				
		ATTTTCACTACCATCTACAAGATGCTCCCCGGCGG				
	Pl_R_OLa (1)	CATGTTCTCGAACACGGACCCATCCTGGGTCGAT TGCCTGTACTTTTCGGCATCAACGCACACCACCGT GGGGTACGGGGACCTCACGCCAAAATCACCCGTG	Platte River; Odessa, Nebraska	10/23/2017	Pl_R_Ola	IL-1
		GCAAAACTCACGGCAACGGCACACATGCTGATCG				
		TATTCGCGATCGTCATTTCTGGCTTCACGTTCCCATGGTAG				
		ATGTTGCTGCTTCTCATACATCTCAGCATTTTGGTA				
		ATTTTCACTACCATATACAAGATGCTCCCCGGCGGC				
	Somers_MT_m1 (1)	ATGTTCTCAAACACGGATCCGTCCTGGGTCGATTGC CTGTACTTTTCGGCATCAACGCACACCACCGTGGG GTACGGGGACCTCACGCCAAAATCACCCGTGGCAA	Flathead Lake; Somers, Montana	6/27/2017	Somers_MT_m1	IL-1
		AACTCACGGCAACGGCACACATGCTGATCGTATTC				
		GCGATCGTCATTTCTGGCTTCACGTTCCCGTGGTAA				
		ATGTTGCTGCTTATCATACATCTCAGCATTTTGGTAA				
		TTTTCACTACCATATACAAGATGCTCCCCGGCGGCA				
		TGTTCTCGAACACGGATCCGTCCTGGGTCGATTGCC				
	IL-M (1) [1]	TGTACTTTTCGGCATCAACGCACACCACCGTGGGGT	Lake Zurich, Illinois	6/27/2017	IL-M	IL-M
		ACGGGGACCTCACGCCAAAATCACCCGTGGCAAAA				
		CTCACGGCAACGGCACACATGCTGATCGTATTTGCG				
		ATCGTCATTTCTGGCTTCACATTTCCGTGGTAG				
		ATGTTGCTGCTCCTTATCCACGTGTGTATTTTGACAGTC				
		TTCACGATTGTTTACAAGATGCTCCCTGGCGGCATGTTC				
	Island-Lake_ Medium (1) [1]	TCTAACGCGGACCCGTCGTGGGTAGACTGCTTATACTTT GCCGCGTCGACTCACACCACAGTGGGGTACGGGGACC TCACCCCCAAATCGCCAGTGGCAAAGCTCACGGCGAC	Island Lake, Western Nebraska	2018	Island-Lake_Medium	Island-Lake_Medium
		GGCCCACATGTTGATCGTGTTCGCGATCATTATATC				
		TAGCTTCACACTGCCATGGTAG				
		ATGTTGCTGCTTATCATACATATCGTCATTCTTATAGTG				
		TTCACTACCATCTACAAGATGCTCCCCGGCGGCATGTT				
	Island-Lake_Small (1) [1]	CTCGAACACGGACCCGACTTGGGTTGATTGCCTGTACT TTTCGGCATCGACGCACACCACCGTGGGGTACGGAGA TCTCACGCCCAAATCACCCGTGGCAAAGCTCACGGCA	Island Lake, Western Nebraska	2018	Island-Lake_Small	Island-Lake_Small
		ACGGCACACATGCTGATCGTATTCGCGATCGTCATTTCT				
		GGCTTCACGTTTCCGTGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGAAGCTGCTACTTTCACATATTGTTATTCTAATATGTT				
		TCACCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTC				
		TCGGAAGCAGACCCGTCGTGGGTTGACTGTCTGTATTT				
	mid_1.1 (1) [20]	CTCGACGGCAACACACACGACAACGGGCTACGGCGAT	Germany	Oct-17	mid_1.1	mid_1.1
		CTAACGCCAGAAACCCCGGTGGCAAAACTCGTGACAA				
		CGGTGCACATGTTAACCGTGTTCATCATCGTTATTTCCG				
		GCTTCACTGGCTTCGCATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTT				
		CACCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCT				
		CGGAAGCAGACCCGTCGTGGGTTGACTGTCTGTATTTC				
	up_1.2m1 (19)	TCGACGGCAACACACACGACAACGGGCTACGGCGATC	Germany	Oct-17	up_1.2m1	mid_1.1
		TAACGCCAGAAACCCCGGTGGCAAAACTCGTGACAAC				
		GGTGCACATGTTAACCGTGTTCATCATCGTTATTTCCGG				
		CTTCACTGGCTTCGCATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTT				
		TCACCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCT				
		CGGAAGCAGACCCGTCGTGGGTTGACTGTCTGTATTTCT				
	mid_1.2m	CGACGGCAACACACACGACAACGGGCTACGGCGATCTA	Germany	Oct-17	up_1.2m1	mid_1.1
		ACGCCAGAAACCCCGGTGGCAAAACTCGTGACAACGG				
		TGCACATGTTAACCGTGTTCATCATCGTTATTTCCGGCTTC				
		ACTGGCTTCGCATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTC				
		ACCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTCGGA				
		AGCAGACCCGTCGTGGGTTGACTGTCTGTATTTCTCGACG				
	mid_1.2s2	GCAACACACACGACAACGGGCTACGGCGATCTAACGCCA	Germany	Oct-17	up_1.2m1	mid_1.1
		GAAACCCCGGTGGCAAAACTCGTGACAACGGTGCACATG				
		TTAACCGTGTTCATCATCGTTATTTCCGGCTTCACTGGCTT				
		CGCATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTC				
		ACCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTCGG				
		AAGCAGACCCGTCGTGGGTTGACTGTCTGTATTTCTCGAC				
	mid_10.1L	GGCAACACACACGACAACGGGCTACGGCGATCTAACGCC	Germany	Oct-17	up_1.2m1	mid_1.1
		AGAAACCCCGGTGGCAAAACTCGTGACAACGGTGCACAT				
		GTTAACCGTGTTCATCATCGTTATT				
		TCCGGCTTCACTGGCTTCGCATTATGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTC				
		ACCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTCGG				
		AAGCAGACCCGTCGTGGGTTGACTGTCTGTATTTCTCGAC				
	mid_10.1s1	GGCAACACACACGACAACGGGCTACGGCGATCTAACGCC	Germany	Oct-17	up_1.2m1	mid_1.1
		AGAAACCCCGGTGGCAAAACTCGTGACAACGGTGCACA				
		TGTTAACCGTGTTCATCATCGTTATTTCCGGCTTCACTGGC				
		TTCGCATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTC				
		ACCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTCG				
		GAAGCAGACCCGTCGTGGGTTGACTGTCTGTATTTCTCG				
	mid_10.1s2	ACGGCAACACACACGACAACGGGCTACGGCGATCTAAC	Germany	Oct-17	up_1.2m1	mid_1.1
		GCCAGAAACCCCGGTGGCAAAACTCGTGACAACGGTG				
		CACATGTTAACCGTGTTCATCATCGTTATTTCCGGCTTCA				
		CTGGCTTCGCATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTT				
		CACCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTCGGA				
		AGCAGACCCGTCGTGGGTTGACTGTCTGTATTTCTCGACGG				
	mid_11.1m	CAACACACACGACAACGGGCTACGGCGATCTAACGCCAG	Germany	Oct-17	up_1.2m1	mid_1.1
		AAACCCCGGTGGCAAAACTCGTGACAACGGTGCACATGT				
		TAACCGTGTTCATCATCGTTATTTCCGGCTTCACTGGCTTCG				
		CATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTC				
		ACCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTCGG				
		AAGCAGACCCGTCGTGGGTTGACTGTCTGTATTTCTCGAC				
	mid_13.1L1	GGCAACACACACGACAACGGGCTACGGCGATCTAACGCC	Germany	Oct-17	up_1.2m1	mid_1.1
		AGAAACCCCGGTGGCAAAACTCGTGACAACGGTGCACA				
		TGTTAACCGTGTTCATCATCGTTATTTCCGGCTTCACTGGC				
		TTCGCATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTT				
		CACCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTC				
		GGAAGCAGACCCGTCGTGGGTTGACTGTCTGTATTTCTC				
	mid_13.1L2	GACGGCAACACACACGACAACGGGCTACGGCGATCTAA	Germany	Oct-17	up_1.2m1	mid_1.1
		CGCCAGAAACCCCGGTGGCAAAACTCGTGACAACGGT				
		GCACATGTTAACCGTGTTCATCATCGTTATTTCCGGCTTC				
		ACTGGCTTCGCATTATGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTT				
		CACCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTC				
		GGAAGCAGACCCGTCGTGGGTTGACTGTCTGTATTTCTC				
	mid_13.1s1	GACGGCAACACACACGACAACGGGCTACGGCGATCTAA	Germany	Oct-17	up_1.2m1	mid_1.1
		CGCCAGAAACCCCGGTGGCAAAACTCGTGACAACGGTG				
		CACATGTTAACCGTGTTCATCATCGTTATTTCCGGCTTCAC				
		TGGCTTCGCATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTC				
		ACCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTCG				
		GAAGCAGACCCGTCGTGGGTTGACTGTCTGTATTTCTCG				
	mid_5.1L1	ACGGCAACACACACGACAACGGGCTACGGCGATCTAA	Germany	Oct-17	up_1.2m1	mid_1.1
		CGCCAGAAACCCCGGTGGCAAAACTCGTGACAACGGT				
		GCACATGTTAACCGTGTTCATCATCGTTATTTCCGGCTTC				
		ACTGGCTTCGCATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTT				
		TCACCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCT				
		CGGAAGCAGACCCGTCGTGGGTTGACTGTCTGTATTTCT				
	mid_5.1L2	CGACGGCAACACACACGACAACGGGCTACGGCGATCTA	Germany	Oct-17	up_1.2m1	mid_1.1
		ACGCCAGAAACCCCGGTGGCAAAACTCGTGACAACGG				
		TGCACATGTTAACCGTGTTCATCATCGTTATTTCCGGCTT				
		CACTGGCTTCGCATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTC				
		ACCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTCGG				
		AAGCAGACCCGTCGTGGGTTGACTGTCTGTATTTCTCGAC				
	mid_5.1s1	GGCAACACACACGACAACGGGCTACGGCGATCTAACGCC	Germany	Oct-17	up_1.2m1	mid_1.1
		AGAAACCCCGGTGGCAAAACTCGTGACAACGGTGCACAT				
		GTTAACCGTGTTCATCATCGTTATTTCCGGCTTCACTGGCT				
		TCGCATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTCACCG			
		TCGTGTACAAGATGCTCCCCGGTGGCATGTTCTCGGAAGCAGAC			
	mid_7.2	CCGTCGTGGGTTGACTGTCTGTATTTCTCGACGGCAACACACAC GACAACGGGCTACGGCGATCTAACGCCAGAAACCCCGGTGGC	Germany	Oct-17	up_1.2m1	mid_1.1
		AAAACTCGTGACAACGGTGCACATGTTAACCGTGTTCATCATC				
		GTTATTTCCGGCTTCACTGGCTTCGCATTATGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTCAC				
		CGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTCGGAAGC				
	up_1.2m2	AGACCCGTCGTGGGTTGACTGTCTGTATTTCTCGACGGCAAC ACACACGACAACGGGCTACGGCGATCTAACGCCAGAAACCC	Germany	Oct-17	up_1.2m1	mid_1.1
		CGGTGGCAAAACTCGTGACAACGGTGCACATGTTAACCGTG				
		TTCATCATCGTTATTTCCGGCTTCACTGGCTTCGCATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTCAC				
		CGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTCGGAAGC				
	up_4.1L1	AGACCCGTCGTGGGTTGACTGTCTGTATTTCTCGACGGCAAC ACACACGACAACGGGCTACGGCGATCTAACGCCAGAAACCC	Germany	Oct-17	up_1.2m1	mid_1.1
		CGGTGGCAAAACTCGTGACAACGGTGCACATGTTAACCGTG				
		TTCATCATCGTTATTTCCGGCTTCACTGGCTTCGCATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTCAC				
		CGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTCGGAAG				
		CAGACCCGTCGTGGGTTGACTGTCTGTATTTCTCGACGGCA				
	up_4.2	ACACACACGACAACGGGCTACGGCGATCTAACGCCAGAAA	Germany	Oct-17	up_1.2m1	mid_1.1
		CCCCGGTGGCAAAACTCGTGACAACGGTGCACATGTTAAC				
		CGTGTTCATCATCGTTATTTCCGGCTTCACTGGCTTCGCATT				
		ATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTCA				
		CCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTCGGA				
		AGCAGACCCGTCGTGGGTTGACTGTCTGTATTTCTCGACG				
	up_7.2	GCAACACACACGACAACGGGCTACGGCGATCTAACGCCA	Germany	Oct-17	up_1.2m1	mid_1.1
		GAAACCCCGGTGGCAAAACTCGTGACAACGGTGCACAT				
		GTTAACCGTGTTCATCATCGTTATTTCCGGCTTCACTGG				
		CTTCGCATTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTCA				
		CCGTCGTGTACAAGATGCTCCCCGGTGGCATGTTCTCGGAA				
		GCAGACCCGTCGTGGGTTGACTGTCTGTATTTCTCGACGGC				
	up_8.1	AACACACACGACAACGGGCTACGGCGATCTAACGCCAGA	Germany	Oct-17	up_1.2m1	mid_1.1
		AACCCCGGTGGCAAAACTCGTGACAACGGTGCACATGTT				
		AACCGTGTTCATCATCGTTATTTCCGGCTTCACTGGCTTCG				
		CATTATGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTCA				
		CCGTTATTTACAAGATGCTCCCCGGTGGCATGTTCTCGGAT				
		GCGGACCCGTCGTGGTTTGACTGTCTGTATTTCTCGACGG				
	mid_3.1s (4) [4]	CGACGCATACGACAACAGGCTACGGCGATCTAACGCCTAA	Germany	Oct-17	mid_3.1s	mid_3.1s
		GACGCCGGTGGCAAAACTCGTGACCACAGCGCATATGTTA				
		ACCGTTTTCGCGATCGTTATTTCCGGTTTCGCTGGCTTCAA				
		GTTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTC				
		ACCGTTATTTACAAGATGCTCCCCGGTGGCATGTTCTCGGA				
		TGCGGACCCGTCGTGGTTTGACTGTCTGTATTTCTCGACGG				
	mid_14.1	CGACGCATACGACAACAGGCTACGGCGATCTAACGCCTAA	Germany	Oct-17	mid_3.1s	mid_3.1s
		GACGCCGGTGGCAAAACTCGTGACCACAGCGCATATGTTA				
		ACCGTTTTCGCGATCGTTATTTCCGGTTTCGCTGGCTTCAA				
		GTTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTCA				
		CCGTTATTTACAAGATGCTCCCCGGTGGCATGTTCTCGGATG				
		CGGACCCGTCGTGGTTTGACTGTCTGTATTTCTCGACGGCGA				
	mid_14.2	CGCATACGACAACAGGCTACGGCGATCTAACGCCTAAGAC	Germany	Oct-17	mid_3.1s	mid_3.1s
		GCCGGTGGCAAAACTCGTGACCACAGCGCATATGTTAACC				
		GTTTTCGCGATCGTTATTTCCGGTTTCGCTGGCTTCAAGTTA				
		TGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTC				
		ACCGTTATTTACAAGATGCTCCCCGGTGGCATGTTCTCGGA				
		TGCGGACCCGTCGTGGTTTGACTGTCTGTATTTCTCGACGG				
	mid_14.3	CGACGCATACGACAACAGGCTACGGCGATCTAACGCCTA	Germany	Oct-17	mid_3.1s	mid_3.1s
		AGACGCCGGTGGCAAAACTCGTGACCACAGCGCATATG				
		TTAACCGTTTTCGCGATCGTTATTTCCGGTTTCGCTGGC				
		TTCAAGTTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTC				
		ACCGTTATTTACAAGATGCTCCCCGGCGGCATGTTCTCGG				
		ATGCAGACCCGTCGTGGTTTGACTGTCTGTATTTCTCGAC				
	mid_6.1 (1) [1]	GGCGACGCATACGACAACAGGCTACGGCGATCTAACGC	Germany	Oct-17	mid_6.1	mid_6.1
		CCAAGTCGCCGGTGGCAAAACTCGTTACCACGGTGCAT				
		ATGTTAACCGTGTTCGCGATCGTTATTTCCGGGTTCGCTG				
		GCTTCAAGNTTCCATGGTAG				

Table A1 .

 A1 Cont. 


	Viruses 2020, 12, 678					
	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTC				
		ACCGTTATTTACAAGATGCTCCCCGGCGGCATGTTCTCGG				
		ATGCAGACCCGTCGTGGTTTGACTGTCTGTATTTCTCGAC				
	mid_6.2 (5) [5]	GGCGACGCATACGACAACAGGCTACGGCGATCTAACGCC	Germany	Oct-17	mid_6.2	mid_6.2
		CAAGTCGCCGGTGGCAAAACTCGTTACCACGGTGCATAT				
		GTTAACCGTGTTCGCGATCGTTATTTCCGGGTTCGCTGGC				
		TTCAAGTTTCCATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTT				
		CACCGTTATTTACAAGATGCTCCCCGGCGGCATGTTCTC				
		GGATGCAGACCCGTCGTGGTTTGACTGTCTGTATTTCTC				
	mid_12.1	GACGGCGACGCATACGACAACAGGCTACGGCGATCTA	Germany	Oct-17	mid_6.2	mid_6.2
		ACGCCCAAGTCGCCGGTGGCAAAACTCGTTACCACG				
		GTGCATATGTTAACCGTGTTCGCGATCGTTATTTCCGG				
		GTTCGCTGGCTTCAAGTTTCCATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATAT				
		GTTTCACCGTTATTTACAAGATGCTCCCCGGCGGCA				
		TGTTCTCGGATGCAGACCCGTCGTGGTTTGACTGT				
	mid_12.3L	CTGTATTTCTCGACGGCGACGCATACGACAACAGG CTACGGCGATCTAACGCCCAAGTCGCCGGTGGCAA	Germany	Oct-17	mid_6.2	mid_6.2
		AACTCGTTACCACGGTGCATATGTTAACCGTGTTCG				
		CGATCGTTATTTCCGGGTTCGCTGGCTTCAAGTTT				
		CCATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATG				
		TTTCACCGTTATTTACAAGATGCTCCCCGGCGGCAT				
		GTTCTCGGATGCAGACCCGTCGTGGTTTGACTGTCT				
	mid_12.3s	GTATTTCTCGACGGCGACGCATACGACAACAGGCT ACGGCGATCTAACGCCCAAGTCGCCGGTGGCAAA	Germany	Oct-17	mid_6.2	mid_6.2
		ACTCGTTACCACGGTGCATATGTTAACCGTGTTCG				
		CGATCGTTATTTCCGGGTTCGCTGGCTTCAAGTTT				
		CCATGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence		Location collected	Date collected	DNA Group	Amino Acid Group
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATAT				
		GTTTCACCGTTATTTACAAGATGCTCCCCGGCGGC				
		ATGTTCTCGGATGCAGACCCGTCGTGGTTTGACTG				
	mid_8.1	TCTGTATTTCTCGACGGCGACGCATACGACAACA GGCTACGGCGATCTAACGCCCAAGTCGCCGGTGG		Germany	Oct-17	mid_6.2	mid_6.2
		CAAAACTCGTTACCACGGTGCATATGTTAACCGTG				
		TTCGCGATCGTTATTTCCGGGTTCGCTGGCTTCAA				
		GTTTCCATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAAT				
		ATGTTTCACCGTTATTTACAAGATGCTCCCCGGTG				
		GCATGTTCTCGGATGCGGACCCGTCGTGGTTTGA				
	mid_9.1 (1) [1]	CTGTCTGTATTTCTCGACGGCGACGCATACGACA ACAGGCTACGGCGATCTAACGCCTAAGTCGCCG		Germany	Oct-17	mid_9.1	mid_9.1
		GTGGCAAAACTCGTGACCACGGTGCATATGTTA				
		ACCGTTTTCGCGATCGTTATTTCCGGGTTCGCTG				
		GCTTCAAGTTATGGTAG				
		ATGCTGCTTCTCCTGATACACATTGCCATATTGACATTC				
		TTTACGGTCGTGTACAAGATGCTCCCCGACGGCGTG				
		TTCTCGAACGGGGACCCGTCGTGGGTAGACTGCTTAT				
	MN08101 (1) [1]	ACTTTTCCGCGTCCACTCACACCACCGTGGGATACGG		Minnesota	2008	MN08101	MN08101
		GGACCTCACCCCCAAATCACCCGTGGCAAAACTCAC				
		GGCAACGGCCCATATGATGATCGTGTTCGCGATTGTAG				
		TGTCTAGCTTCACGTTCCCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGT				
		TCACTACCATCTACAAGATGCTCCCCGGTGGTATGTTC				
	NEJV2 (2) [6]	TCGAACACGGACCCGACTTGGGTTGATTGCCTGTACT TTTCGGCATCGACGCACACCACTGTGGGGTACGGAGA TCTCACGCCCAAATCACCCGTGGCAAAACTCACGGC	Rowe Bird Sanctuary; Gibbon, Nebraska	2008	NEJV2	NEJV2
		AACGGCACACATGTTGATCGTATTCGCGATCGTCATTT				
		CCGGCTTCACGTTTTCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTG				
		TTCACTACCATCTACAAGATGCTCCCCGGTGGTATGTTCTCGAA			
	Dismal_NE_s3	CACGGACCCGACTTGGGTTGATTGCCTGTACTTTTCGGCATCGA CGCACACCACTGTGGGGTACGGAGATCTCACGCCCAAATCAC	Dismal River, Nebraska	5/26/2017	NEJV2	NEJV2
		CCGTGGCAAAACTCACGGCAACGGCACACATGTTGATCGTAT				
		TCGCGATCGTCATTTCCGGCTTCACGTTTTCGTGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGTTCACT				
		ACCATCTACAAGATGCTCCCCGGCGGCATGTTCTCGAACACA				
	NEJV3 (1)	GACCCGACTTGGGTTGATTGCCTGTACTTTTCGGCATCGACG CACACCACTGTGGGGTACGGAGATCTCACGCCCAAATCACC	Gudmundsen Ranch, NE	2008	NEJV3	NEJV2
		CGTGGCAAAACTCACGGCAACGGCACACATGTTGATCGTAT				
		TCGCGATCGTCATTTCCGGCTTCACGTTTTCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGTTCAC				
		TACCATCTACAAGATGCTCCCCGGCGGCATGTTCTCGAACA				
	MO3 (3)	CAGACCCTACTTGGGTTGATTGCCTGTACTTTTCGGCATCGA CGCACACCACCGTGGGGTACGGAGATCTCACGCCCAAATC	Lake Lotawana, Missouri	5/31/2017	MO3	NEJV2
		ACCCGTGGCAAAACTCACGGCAACGGCACACATGTTGATC				
		GTATTCGCGATCGTCATTTCCGGCTTCACGTTTTCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGTTCA				
		CTACCATCTACAAGATGCTCCCCGGCGGCATGTTCTCGAA				
		CACAGACCCTACTTGGGTTGATTGCCTGTACTTTTCGGCA	Moodna Creek;			
	NY2m	TCGACGCACACCACCGTGGGGTACGGAGATCTCACGCC	Washingtonville, New	6/21/2017	MO3	NEJV2
		CAAATCACCCGTGGCAAAACTCACGGCAACGGCACACA	York			
		TGTTGATCGTATTCGCGATCGTCATTTCCGGCTTCACGTT				
		TTCGTGGTAG				
		ATGTTGCTGCTTATCATACATATCATCATTCTGATAGTGT				
		TCACTACCATCTACAAGATGCTCCCCGGCGGCATGTTC				
		TCGAACACAGACCCTACTTGGGTTGATTGCCTGTACTTT	South fork of the			
	Verbena_VA_s3	TCGGCATCGACGCACACCACCGTGGGGTACGGAGATCT	Shenandoah River;	7/1/2017	MO3	NEJV2
		CACGCCCAAATCACCCGTGGCAAAACTCACGGCAACG	Verbena, Virginia			
		GCACACATGTTGATCGTATTCGCGATCGTCATTTCCGGC				
		TTCACGTTTTCGTGGTAG				
		ATGTTGCTGCTTCTCATACATCTCAGCATTTTGGTAATTTT				
		CACTGCCATCTACAAGATGCTGCCCGGCGGCATGTTCTC				
	NTS1 (1) [1]	AAACACAGACCCGACTTGGGTCGATTGCCTGTACTTTTC GGCATCAACGCACACCACCGTGGGGTACGGGGACCTC ACGCCAAAATCACCCGTGGCAAAACTCACGGCAACGG	Next to Smith Lake, Western Nebraska	2008	NTS1	NTS1
		CACACATGCTGATCGTATTCGCGATCGTCATTTCTGGCTT				
		CACGTTCCCGTGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGTTGCTGCTTCTCATACATCTCAGCATTTTGGTAATTTTC				
		ACTACCATCTACAAGATGCTTCCCGGCGGCATGTTCTCGAA				
		CACGGACCCATCCTGGGTCGATTGCCTGTACTTTTCGGCAT	Moodna Creek;			
	NY-2 (1) [4]	CAACACACACCACCGTGGGGTACGGGGACCTCACGCCAA	Washingtonville, New	6/21/2017	NY-2	NY-2
		AATCACCCGTGGCAAAACTCACGGCAACGGCACACATGC	York			
		TGATCGTTTTCGCGATCGTCATTTCTGGCTTCACGTT				
		CCCGTGGTAG				
		ATGTTGCTGCTTCTCATACATCTCAGCATTTTGGTAATTTTC				
		ACTACCATCTACAAGATGCTCCCCGGCGGCATGTTCTCGA				
	Somers_MT_L3 (1)	ACACGGACCCATCCTGGGTCGATTGCCTGTACTTTTCGGC ATCAACGCACACCACCGTGGGGTACGGGGACCTCACGCC AAAATCACCCGTGGCAAAACTCACGGCAACGGCACACA	Flathead Lake; Somers, Montana	6/27/2017	Somers_MT_L3	NY-2
		TGCTGATCGTATTCGCGATCGTCATTTCTGGCTTCACGT				
		TCCCGTGGTAA				
		ATGTTGCTGCTTCTCATACATCTCAGCATTTTGGTAATTTT				
		CACTACCATCTACAAGATGCTTCCCGGCGGCATGTTCTCG				
	Verbena_VA_L4 (2)	AACACGGACCCATCCTGGGTCGATTGCCTGTACTTTTCG GCCAAAATCACCCGTGGCAAAACTCACGGCAACGGCA GCATCAACGCACACCACCGTGGGGTACGGGGACCTCAC	South fork of the Verbena, Virginia Shenandoah River;	7/1/2017	Verbena_VA_L4	NY-2
		CACATGCTGATCGTTTTCGCGATCGTCATTTCTGGCTTC				
		ACGTTCCCGTGGTAG				
		ATGTTGCTGCTTCTCATACATCTCAGCATTTTGGTAATTTT				
		CACTACCATCTACAAGATGCTTCCCGGCGGCATGTTCTC				
		GAACACGGACCCATCCTGGGTCGATTGCCTGTACTTTTC	South fork of the			
	Verbena_VA_m1	GGCATCAACGCACACCACCGTGGGGTACGGGGACCTC	Shenandoah River;	7/1/2017	Verbena_VA_L4	NY-2
		ACGCCAAAATCACCCGTGGCAAAACTCACGGCAACGG	Verbena, Virginia			
		CACACATGCTGATCGTTTTCGCGATCGTCATTTCTGGCT				
		TCACGTTCCCGTGGTAG				
		ATGTTGCTGCTCCTTATCCACATGTGTATTTTGACATTCTT				
		CACAGTTGTTTACAAGATGCTCCCTGGCGGCATGTTCTC				
	Smith_L_La (3) [3]	TAACGCGGACCCGTCGTGGGTAGACTGCTTATACTTTGC CGCGTCGACTCACACCACGGTGGGGTACGGGGACCTCA CCCCCAAATCGCCAGTGGCAAAGCTCACGGCGACGGC	Smith Lake, Western Nebraska	10/22/2017	Smith_L_La	Smith_L_La
		CCACATGTTGATCGTGTTCGCGATCATTATATCTAGCTTC				
		ACACTCCCATGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGTTGCTGCTCCTTATCCACATGTGTATTTTGACATTCT				
		TCACAGTTGTTTACAAGATGCTCCCTGGCGGCATGTTC				
	Smith-Lake_ Medium-Plaque	TCTAACGCGGACCCGTCGTGGGTAGACTGCTTATACTT TGCCGCGTCGACTCACACCACGGTGGGGTACGGGGAC CTCACCCCCAAATCGCCAGTGGCAAAGCTCACGGCGA	Smith Lake, Western Nebraska	2018	Smith_L_La	Smith_L_La
		CGGCCCACATGTTGATCGTGTTCGCGATCATTATATCTA				
		GCTTCACACTCCCATGGTAG				
		ATGTTGCTGCTCCTTATCCACATGTGTATTTTGACATTCTTC				
		ACAGTTGTTTACAAGATGCTCCCTGGCGGCATGTTCTCTA				
	Island-Lake_ Large-Plaque	ACGCGGACCCGTCGTGGGTAGACTGCTTATACTTTGCCGC GTCGACTCACACCACGGTGGGGTACGGGGACCTCACCC CCAAATCGCCAGTGGCAAAGCTCACGGCGACGGCCCAC	Island Lake, Western Nebraska	2018	Smith_L_La	Smith_L_La
		ATGTTGATCGTGTTCGCGATCATTATATCTAGCTTCACACT				
		CCCATGGTAG				
		ATGTTGCTGCTCCTTATACACATCGGTATTTTGGTATTTTTC				
		ACTATCGTGTACAAGATGCTCCCCGGCGGCATGTTCTCGA				
	SPRLa (1) [1]	ACACAGACCCTACTTGGGTCGATTGCCTGTACTTTTCGGC ATCGACGCACACCACCGTGGGGTACGGAGATCTCACGCC CAAATCACCCGTGGCAAAACTCACGGCAACGGCACACAT	South Platte River; Big Spring, Nebraska	10/23/2017	SPRLa	SPRLa
		GTTGATCGTATTCGCGATCGTCATTTCCGGCTTCACGTTTC				
		CGTGGTAG				
		ATGTTGCTGCTTCTCATACACCTCTGTATTTTGATAATTTTTA				
		CTACAATATACAAGATGTTGCCCGGAGGCATGTTCTCGAAC				
		ACGGACCCGTCATGGATAGATTGCCTGTACTTCTCGGCATC				
	TN603 (1) [1]	AACGCACACCACCGTGGGGTACGGGGATCTCACGCCCAAA	Tennessee	2006	TN603	TN603
		TCGCCCGTGGCAAAACTCACAGCAACGGCACACATGCTG				
		ATCGTATTCGCGATCGTAATAACTGGCTTCACATTCCCG				
		TGGTAA				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTT				
		CACCGTTATTTACAAGATGCTCCCCGGTGGCATGTTCTCG				
		GATGCGGACCCGTCGTGGTTTGACTGTCTGTATTTCTCGA				
	up_3.1 (4) [4]	CGGCGACGCATACGACAACAGGCTACGGCGATCTAACG	Germany	Oct-17	up_3.1	up_3.1
		CCTAAGTCGCCGGTGGCAAAACTCGTGACCACAGCGCA				
		TATGTTAACCGTTTTCGCGATCGTTATTTCCGGTTTCGCTG				
		GCTTCAAGTTATGGTAG				

Table A1 .

 A1 Cont. 


	Sample ID	DNA Sequence	Location collected	Date collected	DNA Group	Amino Acid Group
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTTC				
		ACCGTTATTTACAAGATGCTCCCCGGTGGCATGTTCTCGG				
		ATGCGGACCCGTCGTGGTTTGACTGTCTGTATTTCTCGAC				
	up_5.2L	GGCGACGCATACGACAACAGGCTACGGCGATCTAACG	Germany	Oct-17	up_3.1	up_3.1
		CCTAAGTCGCCGGTGGCAAAACTCGTGACCACAGCGC				
		ATATGTTAACCGTTTTCGCGATCGTTATTTCCGGTTTCGC				
		TGGCTTCAAGTTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTT				
		CACCGTTATTTACAAGATGCTCCCCGGTGGCATGTTCTC				
		GGATGCGGACCCGTCGTGGTTTGACTGTCTGTATTTCTC				
	up_5.3m	GACGGCGACGCATACGACAACAGGCTACGGCGATCTA	Germany	Oct-17	up_3.1	up_3.1
		ACGCCTAAGTCGCCGGTGGCAAAACTCGTGACCACAG				
		CGCATATGTTAACCGTTTTCGCGATCGTTATTTCCGGTT				
		TCGCTGGCTTCAAGTTATGGTAG				
		ATGAAGCTGCTACTTTCACATATCGTTATTCTAATATGTTT				
		CACCGTTATTTACAAGATGCTCCCCGGTGGCATGTTCTC				
		GGATGCGGACCCGTCGTGGTTTGACTGTCTGTATTTCTC				
	up_5.3s2	GACGGCGACGCATACGACAACAGGCTACGGCGATCTAA	Germany	Oct-17	up_3.1	up_3.1
		CGCCTAAGTCGCCGGTGGCAAAACTCGTGACCACAGC				
		GCATATGTTAACCGTTTTCGCGATCGTTATTTCCGGTTTC				
		GCTGGCTTCAAGTTATGGTAG				

1Sequences submitted to the GenBank. The accession numbers for the kcv sequences in GenBank are MT560092-MT560194. 
Funding: This work was funded in part by the University of Nebraska-Lincoln's Undergraduate Creative Activities and Research Experience (UCARE) grant (C.R.M.), funding from the National Science Foundation under grant No. 1736030, (J.L.V.E.), and the European Research Council (ERC; 2015 Advanced Grant 495 (AdG) n. 695078 noMAGIC (G.T.). 


Conflicts of Interest:

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
Carter R Murry, Irina V Agarkova, Jayadri S Ghosh, Fiona C Fitzgerald, Roger M Carlson, Brigitte Hertel, Kerri Kukovetz, Oliver Rauh, Gerhard Thiel, James L Van Etten
Genetic Diversity of Potassium Ion Channel Proteins Encoded by Chloroviruses That Infect Chlorella heliozoae