Click here to view linked References Manuscript File 1 ORIGINAL PAPER 2 3 First Records of ‘Flagship’ Soil Ciliates in North America 4 5 Hunter N. Hinesa,b,1, Peter J. McCarthyb, and Genoveva F. Estebana 6 7 a 8 Environmental Sciences, Poole, Dorset BH12 5BB, UK 9 b 10 Bournemouth University, Faculty of Science and Technology, Department of Life and Harbor Branch Oceanographic Institute, Florida Atlantic University, Fort Pierce, FL 34946, USA 11 12 13 Submitted January 24, 2020; Accepted May 1, 2020 14 Monitoring Editor: Michael Melkonian 15 16 17 18 19 20 Running title: First Records of ‘Flagship’ Soil Ciliates in North America 21 22 23 24 25 26 27 28 1 Corresponding author; e-mail hunter.n.hines@gmail.com (Hunter N. Hines). 29 1 30 ‘Flagship’ ciliates were investigated from soil samples collected in Florida, USA. This was 31 undertaken to determine if species thought to be restricted to a given world region could be 32 uncovered from similar habitats in a novel location, e.g. another continent. Two species of 33 Condylostomides were discovered, and recorded from the North American continent for the 34 first time. Condylostomides etoschensis was known only from Africa, but was found to be 35 thriving in a Florida study site. An 18S rDNA sequence for this species was determined for 36 the first time. Also discovered from the same study site was the ciliate Condylostomides 37 coeruleus, previously known only from Central and South America. These two ‘flagship’ 38 ciliates were found in the same habitat, from a continent well outside of their previously 39 recorded biogeographies. Molecular sequencing and microscopy investigations were 40 conducted to form the baseline for future work within this genus. Soil ciliates can obtain 41 large population numbers and form cysts and are therefore likely able to disperse globally. 42 These new records provide additional evidence that large distances, even between 43 continents, do not hinder microbes from thriving globally. The absence of these 44 conspicuously-colored gold and blue ciliates from previous studies is likely due to 45 undersampling, rather than to any physical barriers. 46 47 Key words: Ciliates; soil; Florida; Condylostomides etoschensis; Condylostomides coeruleus; 48 biogeography. 49 50 51 52 53 54 2 55 Introduction 56 57 Ciliated protozoa are extremely common in soil environments, despite frequently being in a cryptic 58 state (Esteban et al. 2006). Ciliates in soil live within the micro water content surrounding soil 59 particles (Finlay et al. 2001) and many are able to form cysts in order to survive adverse conditions 60 (Bourland 2017). These cysts may remain viable for long periods of time (Foissner 2016), 61 contributing to their dispersal (Finlay et al. 2001). Although a large ciliate population might not at 62 first be readily detected in a given fresh soil sample, when environmental conditions change a 63 dynamic community may develop as the ciliates excyst along with the growth of other protist and 64 prokaryotic communities. Rewetting of soil samples stimulates ciliate excystment (Foissner et al. 65 2002) and reveals a community of ciliates, including cryptic species (Finlay and Fenchel 2001) 66 that emerge as their preferred niche develops. 67 Ciliates in soil are integral members of the microbial loop (Azam et al. 1983) in both 68 directions of trophic levels, acting not only as consumers but also as food for members of the soil 69 community. As grazers on small protists and bacteria, ciliates are fundamentally important in 70 healthy soils (Esteban et al. 2006; Foissner 2016). Ciliates feeding on bacteria within soils release 71 nitrogen (NH4+) which is available as nutrients for plants (Ingham et al. 1985). Ciliates also feed 72 on other protists, regulating these populations and providing additional micronutrients to the 73 community. Ciliates are also important in the mineralization of nutrients in soil (Griffiths 1986) 74 and are therefore beneficial to plant communities. The rates of carbon and nitrogen cycling in soil 75 are stimulated by the ciliates present as grazers on bacterial communities (Finlay et al. 2000). It 76 has been suggested that ciliates could be considered as bioindicators of soil health due to their 77 responses to anthropogenic influences (Li et al. 2010). 78 Ciliates are well documented as inhabiting all states of soil oxygenation, from obligate 79 anaerobes to aerophiles (Lynn 2008). This is in contrast to the pervasive beliefs of amateur 80 gardeners found in various blogs and social media outlets such as on Instagram (Hines 2019b), 81 that incorrectly assume the presence of ciliates in soils is indicative of exclusively anaerobic, and 82 allegedly ‘unhealthy’ conditions despite inadequate literature to support this. The community of 83 trophic soil ciliates present in a given area changes over time at small spatial scales and is 84 influenced by factors such as daily fluctuations in water content (Finlay et al. 2000). As such, soil 85 ciliate communities are capable of rapid change, with both total excystment and blooms possible. 3 86 Ciliates are common within all soils (Bamford 1995; Bates et al. 2013) and are important members 87 of microbial communities in all global regions. Soil ciliates are thought to form cysts more readily 88 in areas that experience dryness (Foissner et al. 2002) rather than rainforest habitats which 89 maintain constant moisture (Foissner 1997). It is possible that more saturated soils act in a similar 90 way to freshwater habitats, such that they should be examined immediately after sampling as their 91 community is more active (i.e. not encysted), and vulnerable to change. 92 Although a wealth of ciliate diversity is reported to exist in soils, ciliate biodiversity in 93 general in sparsely recorded (Venter et al. 2018). New species of soil ciliates are still being 94 described from ‘well-searched’ areas such as Europe (Foissner et al. 2005), which confirms that 95 the extent of soil ciliate biogeography and biodiversity is still undetermined. Examples of 96 ‘flagship’ soil ciliates exist in the literature that are described as endemic to a particular region 97 such as Africa (Foissner et al. 2002) or South America (Foissner 2016). 98 Borrowed from the field of wildlife conservation, the term “flagship” refers to ciliates 99 whose morphological distinctiveness is such that their presence should not be missed in any 100 environmental sample. The term as applied to ciliates is used in a unique way, distinct from that 101 commonly used in megafaunal conservation; rather than a species selected to raise conservational 102 awareness, flagship ciliates are used to investigate the potential for restricted biogeographic 103 distributions and endemism (Foissner 2006). As such they have been considered the “ultimate 104 proof” for testing biogeographical theories surrounding microbial endemism (Foissner 2006; 105 Foissner et al. 2008; Segovia et al. 2017) and can be a useful tool for better understanding taxa 106 with unknown spatial distributions (Andelman and Fagan 2000). The idea that flagship ciliates are 107 ‘proof’ for microbial endemism is perhaps a flawed concept: when the size of the globe is 108 considered with the astronomical number of niches compared with the number of microbial 109 ecology researchers present in any given area, it is likely that large parts of the planet remain 110 unexplored for microbial diversity and, even for areas which have been studied, that effort may 111 not be exhaustive. 112 Flagship ciliates represent an ideal target when seeking a better understanding of ciliate 113 biogeography, including soil communities (Bourland 2017). Since Florida had never benefited 114 from an investigation of its soil ciliates, it represents a significant knowledge gap for this group of 115 organisms. A single report of a sample collected from Everglades National Park revealed a new 116 species (Foissner 2016), but it is unclear whether this species is limnetic due to the swamp habitat 4 117 in which it was collected. Florida has not benefited from additional soil ciliate diversity campaigns, 118 despite its interesting geographic location within the subtropics. 119 Due to the vast literature on soil ciliates from global regions (Foissner et al. 2016 and 120 references therein), Florida soil samples were occasionally taken in conjunction with sampling of 121 freshwater habitats during ciliate biodiversity and biogeography surveys within Florida (Hines 122 2019a). When freshwater sites dried up during drought conditions, some sediment from these once 123 aquatic habitats was collected and rewetted. None of the targeted freshwater species were 124 recovered using this technique, however, a different (i.e. soil adapted) community was observed. 125 It should be noted that no soil ‘flagship’ ciliates were recovered from any limnetic sampling during 126 the course of the survey. 127 As a result of this limited study of Florida soils, one site was found to be very productive: 128 an abandoned natural wooded area on the Harbor Branch Oceanographic Institute campus. This 129 site yielded two ‘flagship’ ciliate species: one is the first record of the species outside of Africa, 130 and the other is the first record for North America. 131 132 133 Results 134 135 The average water content of soils collected at the sampling site was 18.47%. The remaining solid 136 fraction had an average Total Organic Matter of 8.06%. The average grain size breakdown was: 137 0.62% gravel, 96.95% sand, and 2.42% fines. At a temperature of 23°C the pH was 7.60 and the 138 salinity was 0.06 (PSU), i.e. equivalent to fresh water. 139 Laboratory cultures from freshly collected soils contained diverse ciliate populations 140 including two ciliates which stood out due to their size and color. Based on their morphology these 141 cells were identified as C. etoschensis and C. coeruleus. These laboratory cultures commonly gave 142 densities for Condylostomides etoschensis of 5 cells mL-1 and were stable for at least six months 143 when maintained with water and food at 30 °C. Soil cultures which were over saturated (nearly 144 flooded) and overfed (triple amount of farro wheat grains) and then incubated at 30℃ showed the 145 best results for growth of ‘flagship’ ciliate targets and overall ciliate biomass (e.g. small 146 Hypotrichs and Colpodea) with densities of C. etoschensis reaching 35 cells per mL within the 5 147 first week. 148 149 Condylostomides etoschensis Foissner, Agatha and Berger, 2002 150 C. etoschensis is distinct due to its bright gold coloration and large oral aperture (Fig. 1) which 151 distinguish it from other common soil species. The cells found in Florida samples matched the 152 description of C. etoschensis by Foissner et al. (2002). 153 A large contractile vacuole in the cell’s posterior end was described for the African cells 154 (Foissner et al. 2002), which deforms the cell when full. This was also observed in Florida cells, 155 along with the adoral zone of membranelles (AZM) being long and conspicuous. The oral aperture 156 was wide and occupied nearly 50% of cell length. 157 The type location, and only site of observation in Africa, was within a “highly saline soil” 158 (although no data were given) from an ephemeral pool in Etosha Pan, Namibia (Foissner et al. 159 2002). Conjugation was recorded in the African strains in which two cells lock onto each other at 160 the oral aperture and exchange genetic material. Although rarely observed, this was also recorded 161 in Florida samples (Figure 1B). Cells were observed to stay in this state for over 1 hour. 162 163 Cysts were observed and well documented from the African site. Cysts with a similar appearance were recorded in Florida, however, these were never directly observed to excyst. 164 Based on soil habitat, overall morphology, size, and unusual gold coloration from cortical 165 granules (Table 1) the species was confirmed to be C. etoschensis. No molecular sequence was 166 provided in the diagnostic literature (Foissner et al. 2002) and the species had not been recorded 167 since, including from similar sampling campaigns in South America (Foissner 2016 and references 168 therein). 169 Finding this species in North America is the first record outside of its original African 170 range, at a distance of ~12,000 km from its documented discovery habitat, and suggests that this 171 and other soil ciliate species can overcome barriers to dispersal such as distance. 172 173 Condylostomides coeruleus Foissner, 2016 174 On average, only two C. coeruleus cells per mL could be found in productive samples after 175 thorough searching. 6 176 During investigations of C. etoschensis in Florida (see above), this morphologically-similar 177 but blue-colored species was found within the same subsamples coming from the same cultures. 178 Based on habitat type, morphology and coloration this species was identified as C. coeruleus (Fig. 179 2). Detailed morphometrics (Table 1) were obtained to compare the Florida species to the 180 diagnostic literature (Foissner 2016). 181 182 Molecular Phylogeny 183 We sequenced the 18S rRNA gene from both C. etoschensis and C. coeruleus. The C. etoschensis 184 amplicon was approximately 1510bp: FL1, MK543444 (1,505bp), FL2 MK543442 (1,513bp), and 185 FL3, MK543443 (1,501bp). The three sequences clustered closely (Fig. 3) and were clustered with, 186 but distinct from, the other Condylostomides species in GenBank. The DNA from C. coeruleus 187 amplified poorly and we were only able to sequence the gene in one direction with an amplicon 188 size of 799bp (FL1, MK543445). Despite this, the isolate clusters with the only sequence available 189 in GenBank for C. coeruleus (Fig. 3; [C. coeruleus SLS-2007 AM713188, 98% (784/799) base 190 pairs matched], Schmidt et al. 2007; Foissner, 2016). Our isolate also clusters with a second 191 sequence from a Condylostomides not identified to species level (Fig. 3; KP970236, 98% 192 (785/799) base pairs matched). 193 Many heterotrichs have been sequenced, with several examples of Condylostomides 194 currently available in Genbank. However, since molecular data for C. etoschensis did not exist in 195 the literature, the Florida record is the baseline for future work within this genus and for other 196 global biodiversity studies that may encounter this cell. The Florida cell is related only sequences 197 available for Condylostomides, and also clusters with Linostomella sp. as predicted in the 198 diagnostic literature (Lynn 2008) (Fig. 3). Although, at the morphological level, the gold and blue 199 Condylostomides, respectively, appear closely related, at the molecular level they are related but 200 distinct. 201 202 Observations on Laboratory Cultures 203 To test the response of cultures to adverse conditions, triplicate soil cultures were prepared, 204 examined and found to contain the target flagship soil ciliates. These cultures were left to incubate 205 at 30℃ for 3 months. Without water being added, the cultures were completely dry in less than a 206 week. After 3 months the cultures were restarted and treated as described to stimulate excystment. 7 207 A stable and similar ciliate population developed. This included the population of target flagships 208 at the same densities as previously recorded. A previously productive soil sample ‘forgotten’ in 209 the 30°C incubator was rewetted after being untouched for more than one year, and a similar 210 microbial consortium appeared, including similar densities of the target C. etoschensis despite total 211 desiccation during this time. Similarly, a soil sample frozen at -20 °C immediately after collection 212 and stored, frozen, for 1 year was restarted as previously described. A similar, but less active, 213 microbial consortium developed and the target ciliate C. etoschensis was recorded from this 214 sample. 215 216 217 Discussion 218 219 The ‘Flagship’ soil ciliates investigated here were all isolated from rewetted soil samples and were 220 never found in freshwater samples. The genus Condylostomides has been reported from a wide 221 variety of habitats and geographies, such as the freshwater C. groliere from Europe (Silva Neto 222 1994), and species such as C. vorticella from brackish waters of Africa (Dragesco and Dragesco- 223 Kernéis 1986) and C. nigra from Europe (Lake Geneva), which has a distinct similarity to C. 224 coeruleus including size and color (Dragesco 1960). The new record of these soil flagship 225 representatives in North America further expands the global biogeography for this group. The 226 target ciliate cysts for the species described here were apparently always present in soil samples 227 from the discovery site over the course of sampling for over one year, as the species were always 228 found after rewetting the soil samples. Florida site over the course of sampling for over one year. 229 Gold colored cysts, likely belonging to C. etoschensis, were found in the soil samples, sometimes 230 in numbers > 20 mL-1. Despite numerous attempts these were never directly observed to excyst. 231 The description of the African cysts (Foissner et al. 2002) matches that of the cysts observed in 232 Florida samples. This ciliate was previously described only from Namibia, Africa (Foissner et al. 233 2002), despite numerous soil investigations from other global habitats (Foissner et al. 2008; 234 Foissner 2016 and references therein) leading to the claim that this species was endemic to that 235 world region. 8 236 Fresh dry soil may have few active ciliates present, but a vast number may be recorded 237 later as the amount of water increases, due to excystment of ciliates. The large number of cysts 238 present in soils (Foissner et al 2002) ensures the survival of a stable ciliate population under all 239 environmental conditions, and consequently all natural soils contain ciliates. The target flagship 240 soil ciliates were shown to be resistant to unfavorable environmental conditions, with cysts still 241 viable from samples after one year of dry incubation at 30 °C or freezing at -20 °C. 242 The soil communities of Florida were found to contain relatively few species when 243 examined directly from the field, and even after 24 hours only small Colpodea were observed. 244 After two days a more diverse community developed following excystment. At a global level, 245 ciliate soil diversity is unresolved due to undersampling (Chao et al. 2006) which confounds ciliate 246 diversity and biogeographies at all levels. It is likely the natural bacterial and small protist 247 community takes time to develop under incubation, and it is only when these levels have increased 248 that ciliate excystment occurs in high enough numbers to be detected (Foissner et al. 2002). 249 These conditions proved most productive for smaller protists and bacteria to flourish and 250 these serve as the food sources for target ciliates. The literature suggests that the oversaturation of 251 cultures or allowing them to ‘spoil’ negates ciliate species development (Foissner et al. 2002) 252 which is a rule likely true for most samples. The Florida cultures, however, required larger amounts 253 of water and higher food availability to reveal the flagships in greatest density. Standard methods 254 (Foissner 2016) were followed with success, but the two flagship targets were most prevalent when 255 cultures were treated as described above. 256 As reported in the literature, investigations of terrestrial ciliates from South America 257 (Foissner 2016) revealed new species, including the conspicuous species Condylostomides 258 coeruleus. This species was not recorded from similar soil campaigns in African habitats (Foissner 259 et al. 2002). Due to this apparently restricted biogeography this blue ciliate was recently described 260 as a ‘flagship’ with a biogeography limited to the previous discovery sites explored in South and 261 Central America (Foissner 2016). 262 This species has been described as an “endemic Gondwana flagship” (Foissner 2016), this 263 was despite being reported in the same text as Central American areas which were not part of a 264 Gondwana breakup. The new record from Florida, a geologically recently emerged habitat (Watts 265 1969), disprove the alleged restriction. It is surprising though that the gold Condylostomides 9 266 etoschensis was never recorded in South American investigations, but is likely a result of 267 undersampling of ciliates and known difficulty with detection of species even if present. 268 C. coeruleus was always found in subsamples that also contained C. etoschensis. Although 269 appearing blue in color under high-power magnification, when using a dissecting microscope (used 270 for picking of cells and initial observations) they appeared nearly colorless, such that their overall 271 movement type rather than color was used as the indicator for picking cells. No other species of 272 soil Condylostomides were observed during these investigations. The Florida observations of C. 273 coeruleus was smaller than that reported in the literature. Florida measurements were made on 274 cells taken from fresh cultures, and this may not have allowed the species to grow to its full size. 275 All other morphological diagnostics match those described in the literature (Foissner 2016). 276 Molecular comparisons are now possible to further investigate this genus. The ciliate 277 Linostomella sp. was theorized as being the closest relative to Condylostomides (Foissner et al. 278 2002; Lynn 2008). The new sequences and phylogenetic tree for C. etoschensis reported here 279 supports this relationship. 280 It is clear from these results that C. coeruleus and C. etoschensis can thrive within the same 281 ecological habitat. The habitat they require, and the environmental factors that stimulate 282 excystment are evidently present in the Florida soils investigated. The two species were always 283 found together during this project. No cysts were directly observed that match the Venezuelan 284 description of C. coeruleus: bluish and about 100µm in diameter (Foissner 2016). It is possible 285 that even if present in high numbers they were obscured by the soil particles they may attach to, 286 and were therefore overlooked during this investigation. The original description suggests the 287 possibility for this species to be ‘common in slightly to moderately saline habitats’ (although no 288 data values were given) of South and Central America (Foissner 2016). The species was thought 289 to be a litter or limnetic species based on its blunt shape (Foissner 2016); however, the Florida soil 290 habitat was found to be mostly sandy with organic material. This species was never recorded in 291 limnetic samples investigated during this project. 292 293 Conclusions 294 The diversity of ciliates in any habitat is still poorly investigated, with both new species awaiting 295 discovery, and ‘flagship’ ciliates being recorded from new biogeographies. The discovery of two 296 flagship soil ciliates in Florida, with minimal sampling effort revealed the first record outside of 10 297 Africa for Condylostomides etoschensis, which is further evidence for the ability of ciliates to 298 disperse globally. The first record for North America of Condylostomides coeruleus is additional 299 evidence that species thought to be restricted to South and Central America can overcome this 300 geographic barrier and thrive within Florida, and likely other habitats at a global level. Sequences 301 for flagship ciliates alleged to have restricted biogeography (Foissner et al. 2008) simply do not 302 exist in databases (Schmidt et al. 2007), with only a handful present at the time of writing. 303 Deposition of the three C. etoschensis sequences will allow for future researchers to compare their 304 study sites to the Florida baseline. The ability of soil ciliates to readily form cysts, as well as exhibit 305 conspicuous coloration makes them good candidates to test for ciliate biogeography. As sampling 306 efforts increase, these and other soil ciliates will probably have their biogeographic distributions 307 expanded. 308 Florida has been shown to harbor freshwater flagship species originally proposed to be 309 restricted to a given biogeography (Hines 2019a; Hines et al. 2016), and species once thought to 310 be restricted often are found in further regions as sampling efforts increase (Hines et al. 2018; 311 Esteban et al. 2001; Finlay 2002). Soil samples were taken sporadically in addition to intensive 312 sampling of freshwater habitats. As such, these results although novel, are by no means exhaustive, 313 and likely many other flagship soil taxa await discovery in Florida. This investigation of soils 314 suggests that Florida is both capable of harboring a diverse ciliate community, and that soil 315 ‘flagships’, like freshwater ‘flagships’, can spread to global regions wherever they find their 316 preferred ecological niche. 317 318 319 Methods 320 321 Study site: The sampling location site surrounds a wild growing Citrus tree resembling in 322 appearance and taste Citrus aurantium (known commonly as “bitter orange” or “Seville orange”) 323 located within an old, unmanaged, wooded area with the fruit falling and rotting back into the 324 ground. Numerous smaller orange trees were found to be germinating within several meters. The 325 tree is within a densely wooded area and the site has been untouched for at least 50 years. The site 326 is rich with insects of the family Culicidae (Mosquitos) confirming that it is chemically untreated. 327 The site is located at 27°31'53.1"N 80°21'18.3"W in St. Lucie County, Florida. 11 328 Soil samples: The soil is largely sandy (white ‘sugar sand’) with dense organic material 329 mixed throughout, and some leaf litter present. Top soil layers down to 1.5 cm were collected 330 using a sterile metal scoop and transferred into sterile 125 mL Nalgene bottles. 331 Soil pH and salinity: Standard methods were followed to obtain soil data (Finlay et al. 332 2000). Samples were freshly collected and dried overnight at 60˚C. This material was then sieved 333 (2mm) to remove large organics. A 1:5 soil/water suspension was made with 60g soil to 300mL 334 deionized water (DI H2O) and stirred for 30 minutes. The sample was allowed to settle for 15 335 minutes. The pH and salinity of the solution were determined using a YSI probe (four port “Digital 336 Professional Series”, Xylem, Yellow Springs OH, USA). 337 Soil type: Soil samples were collected in triplicate and processed for soil characteristics 338 within an hour of collection using the following techniques (modified from Folk 1974; Dean 1974). 339 Approximately 60g of soil from each replicate was dried for 1 hour at 60°C and clumps 340 were broken apart using a mortar and pestle. Samples were then sieved through 2 mm and 0.063 341 mm sieves to separate the gravel (> 2mm), sand (2mm to 0.063 mm), and fines (< 0.063 mm) into 342 fractions (Folk 1966). The sieves were shaken by hand for ten minutes and each of the fractions 343 was rinsed into separate pre-weighed beakers using deionized water. Samples were dried in a 60°C 344 oven for 48 hours. Each fraction’s absolute weight was divided by the total of all three fractions 345 to calculate the percentage. 346 Water content was determined by drying ~30g of soil in glass Petri dishes for 48 hours at 347 60 °C. The dried sediment was ground briefly using a mortar and pestle and sieved to remove the 348 fraction above 2mm which was used for total organic matter analysis: One gram of the fraction 349 was put into ceramic crucibles and heated for 4 hours in a 550 °C pre-heated muffle furnace. The 350 organic content was determined from weight loss and reported as % Total Organic Matter. 351 Soil cultures: Soil cultures were started within 1 hour of collection by placing ~50g soil 352 in sterile 9 cm glass Petri dishes (Pyrex) and wetting with ~25 mL sterile deionized H2O. The dish 353 was swirled to mix in the overlaying floating soil particles. Grains of farro wheat (Triticum sp.) 354 were prepared by boiling in deionized H2O for ~15 minutes and then allowing them to cool for 10 355 minutes in fresh sterile deionized H2O. Grains were squashed by hand and were added to the 356 cultures with one at the edge and one in the center of the Petri dish, such that each grain was half 357 submerged and half above water/sediment line to encourage fungal growth. The lid was placed on 358 the Petri dishes and cultures were incubated at 25 °C, 30 °C, and 37 °C. After 24 hours of 12 359 incubation the enriched cultures were examined every 24 to 72 hours for periods up to several 360 weeks. Water was added as needed as incubation caused drying. New farro grains were added 361 when breakdown (e.g. consumed by bacteria, fungi and worms) had occurred. 362 In order to sample the enriched cultures, they were held at a slight tilt and a sterile pipette 363 was used to transfer the top runoff water at an edge onto an observation chamber. Due to the 364 relatively low amount of water in these concentrated soil cultures, after observation this liquid was 365 returned to the culture, with additional deionized H2O added as needed. 366 Microscopy: Individual ciliate cells were picked using a micropipette under a dissecting 367 microscope for DNA extraction, culture, or onto welled slides for further examination under higher 368 powered microscopy. Initial observations were made using a 1 mL Sedgewick-Rafter counting 369 chamber which allowed observation, enumeration and photography. 370 A fully equipped Olympus BX-53 microscope was used for detailed observation and 371 photomicroscopy. An Olympus DP72 camera and its associated software (cellSens v1.17) was 372 used to record images. 373 DNA extraction: REDExtract-NAmp PCR ReadyMix (Sigma Aldrich) was used for both 374 extraction and amplification of the single cell samples. The method followed the ‘saliva’ protocol 375 described by Kim and Min (2009). Samples were either amplified immediately or stored at -20 °C. 376 Amplification used the Euk-82F and EukB primers (Elwood et al. 1985; Medlin et al. 1988; 377 Integrated DNA Technologies (Coralwood, IA, USA)). Sequences obtained from these single cell 378 samples were deposited into GenBank. 379 Sanger sequencing was conducted by MCLab (South San Francisco, CA, USA). Analysis 380 was performed using the software packages within the DNAStar Lasergene 12 Core Suite which 381 allowed editing of sequences and the creation of contigs. Sequences were aligned using MEGA 382 version 10.0.5. 383 Phylogenetic analysis: The evolutionary history was inferred by using the Maximum 384 Likelihood method and Tamura-Nei model (Tamura and Nei 1993). The tree with the highest log 385 likelihood (-10674.67) was used. The percentage of trees in which the associated taxa clustered 386 together is shown next to the branches. Initial tree(s) for the heuristic search were obtained 387 automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise 388 distances estimated using the Maximum Composite Likelihood (MCL) approach, and then 13 389 selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch 390 lengths measured in the number of substitutions per site. This analysis involved 27 nucleotide 391 sequences. Codon positions included were 1st+2nd+3rd+Noncoding. There were a total of 2602 392 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al. 393 2018) and the phylogenetic tree was edited using Interactive Tree of Life (iTOL) version 5 (Letunic 394 and Bork 2019). 395 396 'Declarations of interest: none'. 397 398 399 400 References 401 402 403 Andelman S, Fagan W (2000) Umbrellas and flagships: efficient conservation surrogates or 404 expensive mistakes? 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Mol Biol Evol 10:512-526 496 Venter P, Nitsche F, Scherwass A, Arndt H (2018) Discrepancies between molecular and 497 morphological databases of soil ciliates studied for temperate grasslands of central europe. 498 Protist 169:521-538 499 18 Figure 1 Click here to access/download;Figure;Gold ciliate figure 600 tiff1.tiff Figure 2 Click here to access/download;Figure;Blue soil ciliate figure 600 tiff.tiff Tree scale: 0.01 Tree Condylostomides sp. AM713188 Condylostomides sp. KP970236 100 Condylostomides coeruleus MK543445 Condylostomides etoschensis MK543443 81 Condylostomides etoschensis MK543442 Condylostomides etoschensis MK543444 100 100 Linostomella sp. LN869952 Condylostentor auriculatus DQ445605 Condylostentor auriculatus KP970235 100 Condylostoma minutum DQ822482 100 Condylostoma spatiosum DQ822483 100 Condylostoma elongatum KJ866148 83 80 Condylostoma sp. FJ868178 Fabrea salina DQ168805 74 Fabrea salina KM222110 100 Blepharisma americanum AM713182 100 Blepharisma japonicum AM713185 Stentor coeruleus AM713189 97 100 Stentor polymorphus AM713190 Spirostomum ambiguum AM398201 Spirostomum minus AM398200 100 89 Spirostomum semivirescens MH295830 Loxodes rex MK507765 100 97 Loxodes magnus L31519 Loxodes striatus U24248 Legends text Location Cell length (µm) Cell width (µm) Moniliform macronucleus Number of micronuclei Macronucleus size Nodule number Nodule length (µm) Contractile vacuole Kineties Color Molecular sequence Condylostomides etoschensis Africa Florida 160- 300 (mean 165-310 (mean 240) 225) 70- 150 (mean 70-150 (mean 110) 110) 1 1 Condylostomides coeruleus South America Florida 150-315 (mean 110-220 (mean 235) 160) 85-155 (mean 40-64 (mean 120) 55) 1 1 ~21 ND ND ND ⅔ cell length ~8 ~25 Present 37 Gold No ⅔ cell length ~8 ~25 Present ~40 Gold Yes ⅔ cell length ~9 ~25 Present 39 Blue Yes ⅔ cell length ~9 ~25 Present ~40 Blue Yes Table 1. Morphometrics for Condylostomides etoschensis discovered in Florida compared to the original description recorded in Africa (Foissner et al. 2002) and for Condylostomides coeruleus discovered in Florida compared to the original description from South America (Foissner 2016). The Florida cell matches to that described from the literature (Schmidt et al. 2007; Foissner 2016). Figure 1. Flagship soil ciliate Condylostomides etoschensis from Florida (USA). A: in vivo image. The ciliate is swimming and the natural gold color is clear in brightfield microscopy. Scale bar 100 µm. B: the two cells are joined in conjugation at the mouth to exchange genetic material. Scale bar 100 µm. C: a close up of the cell’s cytoplasm showing the ciliary rows and cortical granules which cause the gold coloration. Scale bar 10 µm. D: the large oral aperture at upper right is conspicuous in this in vivo image, as well as the long Adoral Zone of Membranelles. Scale bar 100 µm. Figure 2. Condylostomides coeruleus in vivo from Florida (USA). A: brightfield microscopy showing distinct blue green coloration of a swimming cell. Oral aperture at upper left. Scale bar 40 µm. B: the cell is feeding off bacteria surrounding soil particles. Scale bar 40 µm. C: view of oral aperture (top) and ciliary rows leading down to terminal vacuole of C. coeruleus. Long visible above oral aperture. The blue hue of the cell’s coloration is obvious under DIC microscopy. Scale bar 40 µm. Figure 3. Phylogenetic tree of the Heterotrichea inferred from nuclear small subunit (SSU) r DNA sequences using the Maximum Likelihood method and Tamura-Nei model. The karyorelctean species Loxodes striatus, Loxodes magnus, and Loxodes rex were chosen as the outgroup. The Condylostomides coeruleus and Condylostomides etoschesis sequences generated during this project are indicated in blue. The phylogeny of these species is: Eukaryota; Alveolata; Ciliophora; Postciliodesmatophora; Heterotrichea; Heterotrichida; Condylostomatidae; Condylostomides. Conflict of Interest Author conflict of interest: NONE