Conifer Root Nodules Colonized by Arbuscular Mycorrhizal Fungi in Jurassic

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1 Conifer root nodules colonized by arbuscular mycorrhizal fungi in Jurassic

2 geothermal settings from Patagonia, Argentina

3 Nunes Cristina Isabel1*, Juan Leandro García Massini2, Ignacio Hernán Escapa1,

4 Diego M. Guido3, Kathleen Campbell4

5 1 CONICET-Museo Paleontológico Egidio Feruglio. Av. Fontana 140, Trelew CP

6 9100, Chubut, Argentina.

7 2 Centro Regional de Investigación y Transferencia Tecnológica de La Rioja

8 (CONICET-La Rioja-UNLar-SEGEMAR-UNCa). Entre Ríos y Mendoza s/n, 5301,

9 Anillaco, La Rioja, Argentina.

10 3 CONICET-Facultad de Ciencias Naturales y Museo, Universidad Nacional de La

11 Plata, Instituto de Recursos Minerales (INREMI), Calle 64 y 120, La Plata, (1900),

12 Argentina.

13 4 Geology Programme, School of Environment, University of Auckland, Private Bag

14 92019, Auckland 1142, New Zealand.

15 *corresponding author: [email protected]

17 ABSTRACT

18 Premise of the Research: Despite their ecological significance in modern

19 terrestrial ecosystems, knowledge about the evolution of arbuscular mycorrhizae

20 based on the fossil record is still scarce, especially concerning the case of root

21 nodules harboring arbuscular mycorrhizal fungi as in some extant gymnosperms

22 and angiosperms. Exceptionally preserved conifer nodular roots were found in the

23 Jurassic fossil-bearing chert deposits of the Deseado Massif (Santa Cruz,

24 Argentina), raising the possibility to study them in association with arbuscular

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25 mycorrhizal fungi. The aim of this study is to describe the plant organs and their

26 fungal partners, and to discuss the ecological significance of the interactions

27 observed, particularly with respect to their occurrence in the hot-spring settings.

28 Methodology: Thin sections of chert samples from the ‘Cañadón Nahuel’ locality

29 of the La Matilde Formation, Deseado Massif (Santa Cruz, Argentina) were

30 observed using light microscopy.

31 Pivotal Results: The cortex of the nodules are occupied by several

32 glomeromycotan fungal structures. The structures occur in a specific zone of the

33 cortex - towards its center -, and includes intracellular hyphal coils and arbuscules.

34 Glomoid spores, and coenocytic hyphae possibly penetrating the epidermal cells

35 are also described and analyzed.

36 Conclusions: The root nodules have affinities with the Araucariales, representing

37 the oldest record of such structures for this conifer clade. This is also the first

38 record nodules harboring arbuscular mycorrhizal fungi for the Jurassic; it extends

39 our knowledge of the fossil record of this particular type of fungal association.

41 Keywords

42 Root nodules; arbuscular mycorrhizal fungi; hot-spring settings; Araucariales;

43 Jurassic

46 INTRODUCTION

47 Arbuscular mycorrhizal fungi (AMF) have a deep fossil record (Pirozynski and

48 Dalpé 1989; Remy et al. 1994; Taylor et al. 2015; Walker et al. 2018 and citations

49 therein) and today develop symbiosis with approximately 80% of vascular plants

50 worldwide, including five major groups of living conifers (Brundrett 2004; Smith and

51 Read 2008; Spatafora et al. 2016; Wang and Qiu 2016; Brundrett and Tedersoo

52 2018). In modern conifers, AMF colonize roots of the Taxaceae,

53 Cupressaceae, Sciadopityaceae, Araucariaceae and Podocarpaceae

54 (Brundrett 2009; Wang and Qiu 2006; Dickie and Holdaway 2000). The

55 members of the Araucariales clade (i.e. members of the Podocarpaceae and

56 Araucariaceae) and Sciadopytis harbor the AMF in young fine roots and

57 small spherical differentiated structures called nodules (Spratt 1912; Godoy

58 and Mayr 1989; Breuninger et al. 1999; Mcgee et al. 1999; Russell et al.

59 2002; Leone et al. 2008; Dickie and Holdaway 2011; Padamsee et al. 2016).

60 Although AMF are relatively well-known from the fossil record (Taylor et al.

61 2015; Strullu-Derrien et al. 2018; Walker et al. 2018), the fossil record of

62 arbuscular mycorrhizal associations with conifers and related groups is

63 sparse.

64 Structurally preserved AMF have been described in Carboniferous Cordaites

65 from the Massif Central, France (Strullu Derrien et al. 2009), in voltzialean conifers

66 from the Triassic of Antarctica (Schwendemann et al. 2011; Harper et al. 2015),

67 and in a taxodiaceous Cupressaceae from the Middle Eocene of Canada (Stockey

68 et al. 2001). Nodular roots related to the Araucariales are known from the

69 Cretaceous of Antarctica, Australia and India (Cantrill and Douglas 1988; Cantrill

70 and Falcon-Lang 2001; Banerji and Ghosh 2002), and in the Triassic voltzialean

71 Notophytum krauselli (Schwendemann et al. 2011, Harper et al. 2015). This last

72 record is the only one showing anatomical preservation of the rootlets in which AM

73 fungal colonization has been described.

74 The Middle-Late Jurassic chert deposits of the Bahía Laura Group in the

75 Deseado Massif of Santa Cruz Province, Southern Patagonia, Argentina bear

76 exceptionally preserved organisms in hot-spring settings (Channing et al. 2007;

77 Guido et al. 2010; Channing et al. 2011; García Massini et al. 2012, 2016). The

78 best preserved Jurassic geothermal paleosurfaces can be positively compared

79 based on their structure to hot springs at Yellowstone National Park in the United

80 States (Channing et al. 2009a,b). These are the only geothermal fossiliferous

81 deposits with an ecosystem-level preservation known for the Mesozoic. Preliminary

82 reports on the fossiliferous content and paleoenvironment reconstruction have

83 been carried out on a few localities (Channing et al. 2007; Guido et al. 2010;

84 García Massini et al. 2012; García Massini et al. 2016; Sagasti et al. 2016). Plant

85 remains and fungal and fungus-like vegetative and reproductive structures are

86 abundant (Guido et al. 2010, García Massini et al. 2012, García Massini et al.

87 2013; García Massini et al. 2016, Sagasti et al. 2016, Sagasti et al. 2019), and

88 frequently in association with each other (e.g. García Massini et al. 2016). This

89 provides the opportunity to describe a variety of plant-fungal interactions to

90 characterize the dynamics of the hot-springs associated ecosystems and to

91 broaden our knowledge of fungal evolution and diversity during the Jurassic, and in

92 a broader sense during the Mesozoic.

93 In this contribution we describe particular conifer nodular roots colonized by

94 AM fungi, preserved as inclusions in the matrix of chert blocks collected from the

95 recently discovered ‘Cañadón Nahuel’ locality of the La Matilde Formation, Bahía

96 Laura Group, at the Deseado Massif hot-spring deposits. We discuss possible

97 systematic affinities for these roots based on comparisons with extinct and extant

98 examples of conifer nodular roots. We also address the ecological significance of

99 the arbuscular mycorrizhal interaction between the studied conifers and

100 glomeromycotan fungi in the context of the hot-springs settings. This finding

101 expands our knowledge on fungal diversity and ecology in the Jurassic and fills a

102 gap in the fossil record of arbuscular mycorrhizal symbiosis in conifers.

103

104 MATERIALS AND METHODS

105 Geological setting

106 The studied samples were collected at the ‘Cañadón Nahuel’ locality of the La

107 Matilde Formation, in the Bahía Laura Complex of the Deseado Massif, Santa Cruz

108 Province, Argentina (Fig. 1). The Deseado Massif is an extensive (60,000 km2)

109 region in southern Patagonia, in Santa Cruz Province, Argentina. The geologic

110 history of this region is marked by volcanic activity during the Jurassic.

111 Approximately 178-151 million years ago (Pankhurst et al. 2000), this area was

112 subject to bimodal rhyolitic and andesitic volcanism, which lead to the formation of

113 the Bahía Laura Complex (Guido 2004). Rocks of the Bahía Laura Complex are

114 part of the Chon Aike Silicic Large Igneous Province (which ranges from Patagonia

115 to Antarctica; Pankhurst et al. 1998). This province’s genesis is related to the

116 break-up of Gondwana and birth of the South Atlantic Ocean (Riley et al. 2001;

117 Richardson and Underhill 2002).

118 Widespread hydrothermal activity in the Deseado Massif resulted from the

119 mature (quiescent) volcanic phase in this region during the Late Jurassic, which

120 produced metalliferous epithermal deposits (i.e. formed at shallow depths related

121 to the terrestrial surface, in the upper portion of a hydrothermal system under

122 temperatures between 150 and 300 oC), extensive silicification, and formation of

123 geothermal fields at the surface (Schalamuk et al. 1997; Guido and Campbell

124 2011). Well-preserved fossilized geothermal fields have been found within

125 volcaniclastic fluviolacustrine strata, distributed in a 400 x 250 km area of NNW-

126 SSE and WNW-ESE oriented belts aligned with major regional structures (Guido

127 and Campbell 2011). Twelve of these geothermal fields preserve fossil

128 assemblages, which represent the only known fossiliferous Mesozoic hot-springs.

129 These deposits fill a significant stratigraphic gap between similar Paleozoic and

130 Cenozoic deposits (Channing and Edwards 2013).

131 ‘Cañadón Nahuel’ (GPS coordinates available upon request) is the farthest

132 east of these chert deposits in the Deseado Massif (Fig. 1). The locality is located

133 in the Northern Belt defined by Guido and Campbell (2011). Cherts from ‘Cañadón

134 Nahuel’ are rather randomly distributed over a large area and show textural and

135 fossiliferous characteristics that relate them to formation in the distal parts or

136 fringes of a siliceous sinter geothermal system. The observed distribution of the

137 cherts suggests they have been successively removed by recurrent volcanic and

138 phreatic local eruptions or breccias (i.e. by fragmentation) related to the

139 geothermal activity. In some instances, horizontal bed cherts of variable extension

140 are found and these too are interpreted as having formed in wetlands and shallow

141 saline ponds, in the distal parts of geothermal systems.

142

143 Preparation and imaging of studied materials

144 Thin sections of chert blocks were prepared following standard methodology

145 (Jones and Rowe 1999). Slides were observed using transmitted light microscopy.

146 All images were taken with a Nikon DS-Fi1 camera connected to a Nikon Eclipse

147 80i microscope. General views of the sections were taken using a Canon 7D

148 camera with a Canon macro lens of 60 mm, in a light box with fluorescent tubes. At

149 higher magnifications, multiple images of the same structure of interest were taken

150 at different focal planes to compose an image with greater depth of focus, following

151 an image-stacking technique (Bercovici et al. 2009). We used Helicon Focus

152 software (http://www.heliconsoft.com/heliconfocus.html), with the ‘‘Method B’’

153 (Depth Map) parameter. Several partially overlapping images were merged to

154 create high-quality images of critical features. This technique was applied using

155 Adobe Photoshop CS6, and images were processed after to reveal critical

156 characters. The image-stacking technique was not always applicable because in

157 some cases the stacking of images added too much noise or artefacts to the final

158 composition. In such cases we opted to show different focal planes of the same

159 structure side by side (e.g. Fig. 5).

160 The macro-specimens and thin sections are housed at the Museo Regional

161 Padre Molina of Río Gallegos (Santa Cruz Province, Argentina) under accession

162 numbers MPM-Pb-16033;16039-16045.

163

164 RESULTS

165 Description

166 Fungal specimens occur in association with root nodules that emerge from

167 conifer fine roots. These are preserved in chert and obliquely-cross and

168 longitudinally sectioned (Fig. 2a). A central vascular cylinder with secondary xylem

169 followed by the cortex and an apparent periderm are observed in oblique-cross

170 section of older roots (Fig. 2b). In oblique-longitudinal section, scarce diagnostic

171 characters of the secondary xylem are observed (Fig 2c-e). Pits in the radial walls

172 of the tracheids are contiguous, biseriate or uniseriate with occasional biseriate

173 rows (Fig. 2d-e).

174 The young fine nodular roots are 0.5 to 1 mm in diameter. Some roots are

175 constrained, showing a beaded appearance (Fig. 3a). The nodules emerge at right

176 angle from the parental root (Fig. 3c) and are arranged in variable patterns (Fig 2f),

177 some are densely packed, others scattered, and in some specimens they are

178 alternately arranged (Fig 3a). They are spherical to spheroidal (i.e. longitudinally

179 elongated) (Fig. 2a,f; Fig. 3a-e) and measure 0.8 (0.45-1.18) mm in diameter.

180 Anatomically, the nodules are characterized by a central vascular cylinder of

181 92 (78-120) µm of diameter surrounded by an endodermis of 24 (11-35) µm wide.

182 The endodermis is sorrounded by the cortex, ending in an epidermis, and the

183 cortex is 340 (250-445) µm wide (Fig. 3c-d). Root hairs or their bases are

184 occasionally observed (Fig. 3d,f). Root hairs are approximately 19.5 µm in

185 diameter, they are wider at the base with an approximate diameter of 25 µm.

186 The nodules originate at the protoxylem poles of the roots (Fig. 3b). The

187 vascular strand and the endodermis can be followed from the main root up to the

188 middle portion of the nodule in longitudinal section (Fig. 2f; 3a-e). Cortical tissues

189 are composed of 6-8 (10) layers of cortical cells, cells get flattened towards the

190 epidermis (Fig. 3d). Resurgent growth can be observed in some nodules, where

191 new cortical tissue grows inside the older cortex (Fig. 3e).

192 The cortex of the root nodules is colonized by arbuscular mycorrhizal fungi

193 (Fig. 4, 5, 6, 7). Additionally, AM fungal structures occur in the chert matrix, very

194 close or directly attached to the roots and nodules (Fig. 7). The fungi are mainly

195 restricted to the inner layers of cortical tissue whereas cortical layers below the

196 epidermis remain almost fungal free. In the inner zone of the cortex, the outer

197 layers are occupied by intra-cellular coenocytic hyphae that sometimes form coils

198 (Fig. 4a-f). The hyphae are irregular in diameter, ranging from 3,4 to 14,5 µm, the

199 average diameter is 7 µm. The hyphae also develop knobs along its length (Fig.

200 4b), branch dichotomously (Fig. 4c) and at right angle (Fig. 4d). Some coils display

201 possible circular to ellipsoidal scars and are directly associated to narrower hyphae

202 of an intrusive septate fungus (Fig. 4e-f). However, this scars can be preservation

203 artefacts, rendering the metabolic status of the septate fungus saprothrophic or

204 parasitic.

205 Towards the inner cortex, the hyphae becomes thinner, developing highly

206 ramified structures that resemble arbuscules (Fig. 5a,c). Arbuscules are observed

207 partially occupying the cells lumens (Fig. 5c; 6a-f; see also supplementary

208 material). Trunk hyphae of the arbuscules are less than 4 µm in diameter.

209 Morphologically identical, intra- and extra-rhizal glomoid spores are also

210 observed (Fig. 7a-f). Intra-rhizal spores occur in the cortex of both, young roots and

211 nodules. Extra-rhizal spores are observed dispersed in the chert matrix very close

212 to the roots and nodules. Extra-rhizal mycelium associated to extra-rhizal spores

213 (Fig. 7b,c) extends parallel to the roots and nodule's surface. The hyphae measure

214 1.5 to 4.5 µm in diameter and present Y-branching. In some specimens, coenocytic

215 hyphae emerging from the spores attached to the epidermal surface of nodules

216 (Fig. 7b-c) and possibly penetrating the epidermal cells are observed (Fig. 7d).

217 The spores are terminal, globose to subglobse, variable in size, ranging from

218 26.5 to 43.5 µm in diameter (Fig. 7e-f). The spore wall appears to be comprised of

219 two layers (Fig. 7e-f). The outer layer is hyaline and the inner layer is opaque. The

220 spores are occluded at their attachment point by a fold of the inner layer in some

221 specimens (Fig. 7f), or display a plug in others (Fig. 7e). The spores are attached

222 to simple subtending hyphae. The hyphae are coenocytic and 2.0 (1.2-2.9) µm in

223 diameter.

224

225 DISCUSSION

226 Here we described an ancient plant-fungal association in the root nodules of

227 Jurassic conifers. Coenocytic hyphae forming coils, ramified arbuscules, and

228 spores, attributable to glomeromycotan fungi occur inside the cortical tissues of

229 conifer root nodules. We observe that the AM fungal structures are restricted to the

230 inner layers of the cortex, whereas only a few scattered coenocytic hyphae occur in

231 the outer cortex, close to the entry points below the epidermis. When resurgent

232 growth in the host nodules is present, hyphae are also present in the renewed

233 cortex of the nodules, which positively compares to what has been described in

234 extant Araucariales (Bailys et al. 1963, Russell et al. 2002; Dickie and Holdaway

235 2011). Analogous general distribution of AM fungi in host nodules is also observed

236 in extant Araucariales (Fig. 5b; Table 1; McGee et al. 1999; Russell et al. 2002;

237 Dickie and Holdaway 2011). Similarly, previous records of fossil mycorrhizal plants

238 found identically zoned distribution of AM fungi in the infected root tissues (Remy

239 et al. 1994; Phipps and Taylor 1996; Strullu-Derrien et al. 2009; Strullu-Derrien et

240 al. 2014; Harper et al. 2015).

241 Two morphological types of arbuscular mycorrhizae have been classically

242 distinguished (Gallaud 1905): Arum type, which includes intercellularly hyphal

243 growth in a longitudinal pattern in the cortex, with short side branches that give rise

244 to arbuscules nearly at right angle; and Paris type, characterized by intracellular

245 coiling hyphae with arbuscules forming along the coil (Taylor et al. 2015; Walker et

246 al. 2018). Features of the fungi described here are consistent with the Paris

247 morphological type. However, it has been shown that the morphological types vary

248 in a continnum depending on a series of factors (e.g. host plant tissues, taxa

249 involved; environment; Dickson 2004; Van Aarle et al. 2004; Kubota et al. 2005),

250 rendering this character merely descriptive and of relative use for the classification

251 of AM fungi. Besides, the distinction of these morphologies have been used in

252 fossil comparisons (Harper et al. 2015; Harper et al. 2016; Walker et al. 2018).

253

254 Systematic affinities of the root nodules and the fungi

255 The root nodules described here are characterized by a central vascular

256 cylinder surrounded by an endodermis, followed by several layers of cortex

257 surrounded by an epidermis; spherical to spheroidal three dimensional

258 morphology; and emergence at right angle from the parental root in variable

259 patterns along the main axis. These are features shared by extant Araucariales,

260 which includes the Podocarpaceae and Araucariaceae (see comparisons in Table

261 1). Additional vegetative and reproductive organs assignable to Araucariaceae and

262 Podocarpaceae are found in the La Matilde Formation (e.g., wood, leafy branches,

263 cones; Gnaedinger 2007; Zamuner and Falaschi 2005; Falaschi et al. 2011;

264 Kloster and Gnaedinger 2018; Escapa et al. in prep). Further refinement of the

265 systematic affinities of the nodules based on the morphological characters

266 preserved, especially considering morphological variability known to be present in

267 fossil and extant Podocarpaceae and Araucariaceae, is not possible (Spratt 1912;

268 Dickie and Holdaway 2011; Tables 1,2). Considering the presence of both

269 podocarpaceous and araucariaceous organs in the chert blocks studied and the

270 little morphological variation observed among the mycorrhizal nodules it remains

271 possible that more than one taxa is present.

272 Based on phylogenetic analysis of combined molecular and morphological

273 data, several families have been recognized among the former Glomeromycota

274 (Schüβler et al. 2001; Spatafora et al. 2016): Paraglomeraceae,

275 Archaeosporaceae, Geosiphonaceae, Ambisporaceae, Glomeraceae,

276 Claroideoglomeraceae, Pacisporaceae, Gigasporaceae, Pacisporaceae,

277 Sacculospporaceae, Acaulosporaceae and Diversisporaceae (see Schüβler and

278 Walker 2010; Redecker et al. 2013; http://www.amf-phylogeny.com). Differences

279 mainly in spore type formation, spore wall structure, presence of vesicles, and

280 even ecology, exclude affinities of the fungi described here with several different

281 groups (see Table 3). Particular morphological characters suggest that these

282 fossils most probably affinities are with the Paraglomeraceae. Diagnostic

283 characters supporting affinities with the Paraglomeraceae include arbuscules

284 attached to narrow trunks (<4 µm in diameter), development of intra-rhizal spores

285 and intracellular hyphae frequently coiling. However, Paraglomus spores have a

286 three-layered wall, whereas in our specimens, the wall is comprised by two layers.

287 Nonetheless, the outer wall of the spores in Paraglomus has been mentioned to

288 disintegrate at senescence, resulting in a two-layerd spore wall and thus,

289 comparable to that displayed by the fossils described here (Morton and Redecker

290 2001). Alternatively, affinities of the fossil with Glomeraceae are also possible,

291 being different mainly based on the absence of vesicles; additionally, the

292 Glomeraceae commonly develop straight hyphae, whereas, the fossil most

293 frequently shows coiled hyphae. These same features also make the fossil

294 somewhat comparable to Diversisporaceae and Ambisporaceae, although these

295 two families develop acaulosporoid spores, which have not been observed in the

296 fossil.

297

298 Fossil conifers with arbuscular mycorrhizal fungi

299 The oldest record of nodular roots colonized by arbuscular mycorrhizal fungi

300 is from the Triassic of Antarctica (Schwendemann et al. 2011; Harper et al. 2015).

301 In this case, both the rootlets and the root nodules of Notophytum krauselli are

302 colonized by serveral fungal estructures (i.e. hyphal coils, arbuscules, extra-rhizal

303 hyphae, vesicles and spores; Schwendemann et al. 2011; Harper et al., 2015).

304 However, fungal structures found in the nodules are different from those found in

305 the secondary rootlets, suggesting different arbuscular mycorrhizal symbiosis

306 occurring in different organs of the same plant root system (Harper et al. 2015).

307 Other examples of conifers showing root nodules are from different Cretaceous

308 localities from the Southern Hemisphere (Cantrill and Douglas 1988; Cantrill and

309 Falcon-Lang 2001; Banerji and Ghosh 2002). The lack of anatomical preservation

310 in these nodules found as impressions, compressions and molds prevents any

311 fungal association to be observed. Numerous affinities were discussed for these

312 Cretaceous conifer nodule-bearing roots based on the association with other

313 organs, including affinities with the Podocarpaceae, Araucariaceae,

314 Sciadopityaceae, and even Cupressaceae. This new record of arbuscular

315 mycorrhizal in root nodules with affinities with the Araucariales from the Jurassic of

316 Patagonia fills a gap in the geological history of AM fungi and further documents

317 morphological diversity and ecological preferences of glomeromycetes through

318 time.

319

320 Mycorrhizal nodules and ecology

321 The exceptional preservation of the conifer nodular roots of the ‘Cañadón

322 Nahuel’ locality can be related to the mode of preservation typical of hot-spring

323 settings, where plants and other organisms become immersed in a mineral-

324 saturated solution that gradually transform them into fossils (Channing and

325 Edwards 2004; Hellawell et al. 2015). The presence of fungal structures inside the

326 cortex of these underground organs indicates that the fossilization process of these

327 organisms must have occurred while they were alive or soon after their death

328 (Channing and Edwards, 2004). Such fossilization process consisted in

329 permineralization by silica, which is a characteristic previously observed in ‘La

330 Bajada’ and ‘San Agustín’ hot-spring localities also of the La Matilde Formation

331 (García Massini et al. 2016). Moreover, based on their distribution and textural and

332 fossiliferous content, the plant-rich cherts of the ‘Cañadón Nahuel’ locality are

333 related to the distal areas of siliceous sinter geothermal system, potentially

334 analogue to modern Yellowstone National Park geothermally influenced

335 environments in the USA (Channing et al 2009a,b). Root nodules have also been

336 preliminary described in the above mentioned ‘San Agustín’ and ‘La Bajada’

337 localities (see supplemental material; García Massini et al., 2013, 2016). These

338 root nodules share the general morphology and anatomy of the specimens

339 described here, though the best preserved examples of arbuscular mycorrhizal

340 colonization are those from ‘Cañadon Nahuel’.

341 Presence of arbuscular mycorrhizal fungi is not needed for the development

342 of the root nodules in extant Araucariales, as they are indeed part of the root

343 system ontogeny (Baylis et al. 1963; Baylis 1969), however, these organs are

344 hypothesized to increase the volume of fungal colonization at a lower energy cost

345 than having an extensive root system and therefore maximize mineral nutrient

346 acquisition efficiency (Russell et al. 2002; Dickie and Holdaway 2011). Although

347 the role of the mycorrhizal nodules in the extant Araucariales is not yet completely

348 understood, these organs are thought to be ecologically significant with respect to

349 uptake of water and nutrients, especially phosphorous and nitrogen; additionally,

350 establishment of seedlings in nutrient-poor environments has also been suggested

351 to be influenced by the development of mycorrhizal nodules (Russel et al. 2002;

352 Padamsee et al. 2016; Dickie and Holdaway 2011 and citations therein).

353 Systematic and taxonomical refinement to the family level of the conifers of

354 ‘Cañadón Nahuel’ and further characterization of the paleo-environments where

355 this plants used to live should enrich what can be grasped from these fossils on the

356 evolution and significance of the AM symbiosis in the Jurassic Araucariales of

357 Patagonia.

358

359 CONCLUSIONS

360 We present the first record of structurally preserved arbuscular mycorrhizal

361 fungi colonizing root nodules from the Jurassic, partially filling a gap in the

362 Mesozoic record of conifer mycorrhizal nodules, and extending the record

363 geographically.

364 The mycorrhizal nature of the fungus is demonstrated by the presence of

365 several fungal structures in a delimited zone of the nodule's cortex, especially

366 important is the presence of hyphal coils, arbuscules and glomoid spores.

367 The nodules have affinities with the Araucariales clade of conifers, however, it

368 is not possible to constrain the affinities of these organs further based on the

369 anatomical evidence preserved. Nonetheless, the presence of these structures is

370 important as they expand our knowledge on the morphology and ecology of these

371 conifers during the establishment of the modern conifer families in the Jurassic.

372 They also open the possibility to work on whole-plant concept reconstructions in

373 the future.

374

375 Acknowledgments

376 We kindly thank the anonymous Reviewers for their very detailed and helpful

377 comments and suggestions that considerably improved this manuscript. We thank

378 Dr. Ian Dickie for kindly providing images of root nodules of extant Podocarpaceae.

379 CIN thanks all MEF-CONICET fellows for kind support, and Lic. Ana Andruchow

380 Colombo Andrés Elgorriaga for insightful discussions. We also thank the Culture

381 Bureau of Santa Cruz Province for granting us the research permits to study the

382 geothermal deposits in the Deseado Massiff, and the authorities and personnel of

383 Patagonian Gold and local people for the logistic support. This contribution was

384 founded by CONICET (Res. 2318, P. 202 & 173) (to JLGM) and ANPCyT (PICT

385 2014-3496) (to JLGM & DG).

386

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590

591 TABLES AND FIGURES CAPTIONS

592

593 Table 1. Comparisons of ‘Cañadón Nahuel’ locality specimens of root nodules and

594 the root nodules of extant Podocarpaceae and Araucariaceae species.

595 AMF = arbuscular mycorrhizal fungi; PS = parallel series; LS = longitudinal series;

596 VC = vascular cylinder; MR = main root; En. = endodermis; C. = cortex; E. =

597 epidermis; Ex = exodermis; VS = vascular strand; P = Present; A = absent; Sc. =

598 Scattered; ? = Missing information

599 Information taken from:

600 *a Breuniger et al 2000

601 *b McGee 1990

602 *c Godoy and Mayr 1989

603 *d Padamsee et al 2000

604 *e Russel et al 2002; Dickie and Holdaway 2011

605 *f Leone et al 2008

606

607 Table 2. Fossil record of conifer root nodules. Modified from Schwendemann et al.

608 2011.

609 AMF = arbuscular mycorrhizal fungi; P = preserved; NP = not preserved; ? =

610 missing information

611

612 Table 3. Comparisons within families of the former Glomeromycota that form

613 glomoid spores.

614 G = glomoid; Ac. = acaulosporoid; Occ. = occlusion; P = present; A = absent; ? =

615 missing information or not clear

616

617 *a Schußler and Walker 2010

618 *b Ohel and Sieverding 2004

619 *c Schußler 2002

620 *d Walker et al 2007

621 *e Walker and Schußler 2004

622 *f Morton and Benny 1990

623 *g Morton and Redecker 2001

624 *h Redecker et al 2013

625

626 FIGURE 2. Thin sections of chert blocks from ‘Cañadón Nahuel’ locality, MPM-Pb-

627 16039-16041. – a. General view of chert block showing conifer roots immersed in

628 the matrix. Some nodules in young fine are colored with pink sof-light. – b.

629 Oblique-cross section of conifer diarch root. Note secondary xylem (2ºX), cortex

630 (C) and apparent periderm (Pd). – c. Oblique-longitudinal section of conifer root

631 showing secondary xylem (2ºX) and cortex (C), periderm not observed. – d and –

632 e. Detail of secondary xylem. Note araucarian pitting in the walls of the tracheids. –

633 f. General view of nodular root with spherical to spheroidal nodules, disposed with

634 any apparent pattern. Scale bar for b and c = 0.5 mm; for d and e = 10 µm; for f =

635 100 µm.

636

637 FIGURE 3. Thin sections of chert blocks from ‘Cañadón Nahuel’ locality, MPM-Pb-

638 16033;16039-16040. – a. General view of ‘beaded’ root with nodules, nodules

639 disposed alternately. – b. Cross section of a nodule, showing vascular trace (VT)

640 emerging from the parental root. – c. Oblique-longitudinal section of root, with

641 lateral root nodule emerging at right angle from the main axis. – d. Cross section of

642 a nodule surrounded by the main root cortex, showing general anatomy of the

643 nodule. Note vascular strand (VS), endodermis (En), Cortex (C) and epidermis (E).

644 Detail of vascular strand and endodermis inside box, scale bar = 20 µm. – e. Detail

645 of root nodule showing resurgent growth, new cortex (NC) developing inside the

646 cortex of the older nodule (OC). – f. General view of root hairs. Scale bar for a. 0.5

647 mm; for b, c, d, e and f = 100 µm.

648

649 FIGURE 4. Thin sections of chert blocks from ‘Cañadón Nahuel’ locality, MPM-Pb-

650 16040-16041. – a. General view of root nodule. Pink coloration indicates the

651 presence of hyphal coils. – b. Detail of a coenocytic hyphal coil showing knobs. – c.

652 Detail of coenocytic hyphae showing Y-branching. – d. Detail of coenocytic hyphae

653 showing branching at right angle. – e and f. Detail of possible worn out hyphae

654 intruded by a septate fungus (white arrow heads). Scale bar for a = 100 µm; for b,

655 c, d, e and f = 10 µm.

656

657 FIGURE 5. Thin section of chert block from ‘Cañadón Nahuel’ locality, MPM-Pb-

658 16039, and thin section of extant Podocarpaceae Podocarpus totara root nodule

659 (Images by Larry Peterson and Lewis Melville, from Dickie & Holdaway, 2011) – a.

660 Longitudinal section of fossil root nodule showing zone of the cortex occupied by

661 arbuscules (delimited by dotted line) – b. Longitudinal section of extant root nodule

662 showing zone of the cortex occupied by arbuscules (delimited by dotted line). – c.

663 Detail of arbuscules in fossil root nodule. – d. Detail of arbuscules in extant root

664 nodule. Scale bar for a and b = 100 µm; for c = 20 micras; for d = 50 µm

665

666 FIGURE 6. Thin section of chert block from ‘Cañadón Nahuel’ locality, MPM-Pb-

667 16039. Diversity of arbuscules partially occupying cortical cells of root nodules.

668 Scale bar for all = 10 µm.

669

670 FIGURE 7. Thin section of chert block from ‘Cañadón Nahuel’ locality, MPM-Pb-

671 16039. – a. Root nodule with intra-rhizal glomoid spores. – b. Glomoid spore

672 associated to root nodule. Note connecting hypha (arrowhead). – c. Glomoid spore

673 with multiple hyphal attachments, some of which connect it to the root nodule. – d.

674 Magnification of previous image showing detail of entry point with penetrating

675 hyphae and development of coil. – e. Detail of intra-rhizal globose spore in

676 decaying root, showing two-layered wall, simple subtending hypha and occlusion

677 plug. – d. Detail of extra-rhizal globose spore, showing two-layered wall, simple

678 subtending hypha, and occlusion.

679

680 SUPPLEMENTAL MATERIAL

681

682 Appendix 1. – A1. Diversity of young fine roots and nodules from different localities

683 of the La Matilde Formation, Deseado Massif, Santa Cruz Province, Argentina. – a,

684 b and c. ‘Cañadón Nahuel’ locality. – d. ‘San Agustín’ locality. – e. ‘La Bajada’

685 locality. Scale bar for a and e = 0.5 mm; b and d = 0.25 mm; c = 0.1 mm

686

687 Appendix 2. – A2. Further images of arbuscules inside cortical cells of root nodules

688 specimens of the ‘Cañadón Nahuel’ locality. Scale bar for all = 10 µm

689

690 Appendix 3. – A3. Sequence of original images used to obtain the final composition

691 of the nodules endodermis. Scale bar for all = 20 µm.

692

693 Appendix 4. - A4I and A4II. Sequences of original images used to obtain the final

694 composition of coiling hypha. Scale bar for all = 10 µm.

695

696 Appendix 5. – A5. Sequence of original images used to obtain the final composition

697 of nodule’s cortical cells occupied by arbuscules. Scale bar for all = 20 µm.

698

699 Appendix 6. - A6I, A6II, A6III. Sequences of arbuscules at different focal planes.

700 Scale bar for all = 10 µm.

701

702 Appendix 7. - A7I. Sequence of original images used to obtain the final composition

703 of a root nodule occupied by intra-rhizal glomoid spores. Scale bar for all = 100

704 µm. – A7II. Sequence of original images used to obtain the final composition of a

705 germinating spore with hyphae getting attached to the epidermis of a root nodule.

706 Scale bar for all = 20 µm. – A7III. Sequence of original images used to obtain the

707 final composition of a hypha penetrating the epidermis of a root nodule. Scale bar

708 for all = 10 µm. – A7IV. Sequence of original images used to obtain the final

709 composition of an intra-rhizal glomoid spore. Scale bar for all = 10 µm.

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1 Conifer root nodules nodules colonized with by arbuscular mycorrhizae

2 mycorrhizal fungi in Jurassic geothermal settings from Patagonia, Argentina

3 Nunes Cristina Isabel1*, Juan Leandro García Massini2, Ignacio Hernán Escapa1,

4 Diego M. Guido3, Kathleen Campbell4

5 1 CONICET-Museo Paleontológico Egidio Feruglio. Av. Fontana 140, Trelew CP

6 9100, Chubut, Argentina.

7 2 Centro Regional de Investigación y Transferencia Tecnológica de Lla Rioja

8 (CONICET-La Rioja-UNLar-SEGEMAR-UNCa). Entre Ríos y Mendoza s/n, 5301,

9 Anillaco, La Rioja, Argentina.

10 3 CONICET-Facultad de Ciencias Naturales y Museo, Universidad Nacional de La

11 Plata, Instituto de Recursos Minerales (INREMI), Calle 64 y 120, La Plata, (1900),

12 Argentina.

13 4 Geology Programme, School of Environment, University of Auckland, Private Bag

14 92019, Auckland 1142, New Zealand.

15 *corresponding author: [email protected]

17 ABSTRACT

18 Premise of the Research: Despite their ecological significance in modern

19 terrestrial ecosystems, knowledge about the evolution of arbuscular mycorrhizae

20 based on the fossil record is still scarce, including very few examples of

21 mycorrhization in conifersespecially concerning the case of root nodules harboring

22 arbuscular mycorrhizal fungi as in some extant gymnosperms and angiosperms.

23 Exceptionally preserved conifer nodular roots were found in the Jurassic fossil-

24 bearing chert deposits of the Deseado Massif (Santa Cruz, Argentina), raising the

25 possibility to study them in association with arbuscular mycorrhizal fungi. The aim

26 of this study is to describe the plant organs and their fungal partners, and to

27 discuss the ecological significance of the interactions observed, particularly with

28 respect to their occurrence in the hot-spring settings.

29 Methodology: Thin sections of chert samples from the ‘Cañadón Nahuel’ locality

30 of the La Matilde Formation, Deseado Massif (Santa Cruz, Argentina) were

31 observed using light microscopy.

32 Pivotal Results: The cortex of the nodules and nodule-bearing roots are occupied

33 by several glomeromycotan fungal structures. The structures occur in a specific

34 zone of the cortex - towards its center -, and includes intracellular hyphal coils and

35 arbuscules. Glomoid spores, and penetrating coenocytic hyphae possibly at entry

36 pointspenetrating the epidermal cells are also described and analyzed.

37 Conclusions: The root nodules have affinities with the Araucariales, comprising

38 representing the oldest record of such structures for this conifer clade. This is also

39 the first record of mycorrhizal nodulesnodules harboring arbuscular mycorrhizal

40 fungi for the Jurassic, ; and one of the very few examples of structurally preserved

41 mycorrhizae in conifersit extends our knowledge of the fossil record of this

42 particular type of fungal association. The presence of this symbiosis may have

43 influenced the distribution of both, plants and fungi in the distal areas of the hot-

44 spring influenced environments of Patagonia.

46 Keywords

47 Root nodules; arbuscular mycorrhizaemycorrhizal fungi; hot-spring settings;

48 Araucariales; Jurassic

51 INTRODUCTION

52 Arbuscular mycorrhizal fungi (AMF) have a deep fossil record (Pirozynski and

53 Dalpé 1989; Remy et al. 1994; Taylor et al. 2015; Walker et al. 2018 and citations

54 therein) and today develop symbiosis with approximately 80% of vascular plants

55 worldwide, including five major groups of living conifers (Brundrett 2004; Smith and

56 Read 2008; Spatafora et al. 2016; Wang and Qiu 2016; Brundrett and Tedersoo

57 2018). In modern conifers, AMF colonize roots of the Taxaceae,

58 Cupressaceae, Sciadopityaceae, Araucariaceae and Podocarpaceae

59 (Brundrett 2009; Wang and Qiu 2006; Dickie and Holdaway 2000). The

60 members of the Araucariales clade (i.e. members of the Podocarpaceae and

61 Araucariaceae) and Sciadopytis harbor the AMF in young fine roots and

62 small spherical differentiated structures called nodules (Spratt 1912; Godoy

63 and Mayr 1989; Breuninger et al. 1999; Mcgee et al. 1999; Russell et al.

64 2002; Leone et al. 2008; Dickie and Holdaway 2011; Padamsee et al. 2016).

65 Although AMF are relatively well-known from the fossil record (Taylor et al.

66 2015; Strullu-Derrien et al. 2018; Walker et al. 2018), the fossil record of

67 arbuscular mycorrhizal associations with conifers and related groups is

68 sparse.

69 Structurally preserved Fungi are important in modern terrestrial ecosystems

70 as they establish a broad spectrum of mutualistic to parasitic symbiotic interactions

71 with many organisms, including plants (Blackwell 2011, Taylor et al. 2015 and

72 citations therein). Mycorrhizal symbiosis is perhaps the most important plant-fungal

73 interaction developed, occurring on approximately 85% of the living plants and

74 involving several clades of fungi (Brundrett 2004; Wang and Qiu 2006; Smith and

75 Read 2008; Brundrett and Tedersoo 2018). This mutualistic interaction results in

76 nutritional benefits for both partners involved, and confer other benefits to host

77 plants, such as stress tolerance and protection against pathogens (Marx 1972;

78 Newsham et al. 1995; Read 1999; Borowicz 2001; Bunn and Zabinski 2003;

79 Hildebrant et al. 2007; Miransari 2010).

80 The most widespread and ecologically important type of mycorrhiza are the

81 arbuscular mycorrhizae (AM), also referred to as endomycorrhizae (Smith and

82 Read 2010). Arbuscular mycorrhizal symbiosis occur between fungi in the former

83 Glomeromycota (Schüßler et al. 2001), now the Glomeromycotina and

84 Mucoromycotina groups (Spatafora et al. 2016), and the roots of the majority of

85 vascular plants (Schüßler et al. 2001, Strullu-Derrien and Strullu 2007; Strullu-

86 Derrien et al. 2018; Taylor et al. 2015; Walker et al. 2018; Brundrett and Tedersoo

87 2018). Arbuscular mycorrhizae play an important role in the uptake of water and

88 soil nutrients, specially phosphorus and nitrogen (Augé 2001; Smith and Read

89 2010; Smith and Smith 2011; Wipf et al. 2019). For this, the AM fungi display an

90 extensive hyphal network in the soil, considerably expanding the absorption

91 function carried out by roots (Taylor et al. 2015; Redecker and Schüßler 2014).

92 Inside roots the exchange between the host plant and the fungus is enhanced by

93 the development of highly ramified hyphal structures called arbuscules (Smith and

94 Read 2010; Redecker and Schüßler 2014, Walker et al. 2018).

95 Both the fossil record and the molecular analyses suggest that AM symbiosis

96 are the ancestral type and have driven colonization of land by plants (Pirozynski

97 and Malloch 1975; Wang et al. 2010; Redecker et al. 2000; Schüßler et al. 2001;

98 Wang and Qiu 2006). From early land plants to modern trachaeophytes,

99 glomeromycotan fungi forming AM and similar symbiosis are present in several

100 lineages of extinct and extant plants, including bryophytes, rhyniophytes, lycopsids,

101 ferns, pteridosperms, cycads, ginkgoales, conifers and angiosperms (Strullu-

102 Derrien and Strullu 2007; Strullu-Derrien et al. 2018; Brundrett and Tedersoo 2018,

103 and citations therein).

104 In modern conifers, arbuscular mycorrhizae are present in the Taxaceae,

105 Cupressaceae, Sciadopityaceae, Araucariaceae and Podocarpaceae, whereas

106 Pinaceae typically develop ectomycorrhizae (Brundrett 2009; Wang and Qiu 2006;

107 Dickie and Holdaway 2000; Brundrett and Tedersoo 2018). The members of the

108 Araucariales clade (i.e. members of the Podocarpaceae and Araucariaceae) and

109 Sciadopytis harbour the AM fungi in young fine roots and small spherical

110 differentiated structures called nodules (Spratt 1912; Godoy and Mayr 1989;

111 Breuninger et al. 1999; Mcgee et al. 1999; Russell et al. 2002; Leone et al. 2008;

112 Dickie and Holdaway 2011; Padamsee et al. 2016). They are suggested to

113 increase the root colonization by AM fungi at a lower cost than developing an

114 extensive root system (Mcgee et al. 1999; Dickie and Holdaway 2001).

115 Preservation of fungi in their original context and three-dimensionally provide

116 a unique way to study their evolution and significance in past terrestrial

117 ecosystems (Taylor and Taylor 2000; Taylor et al. 2015, García Massini et al.

118 2012). Despite the importance of mycorrhizae, however, the fossil record of these

119 plant-fungal interactions is still scarce, including very few examples of mycorrhizal

120 conifers (Strullu-Derrien and Strullu 2007; Strullu-Derrien et al. 2018; Walker et al.

121 2018, and citations therein). The oldest fossil record of AMF have been described

122 in Carboniferous Cordaites from the Massif Central, France (Strullu Derrien et al.

123 2009), in inconifers is comprised by voltzialean conifers from the Triassic of

124 Antarctica (Schwendemann et al. 2011; Harper et al. 2015); ), and in further AM

125 symbiosis was inferred in roots related the Araucariales are known from the

126 Cretaceous of Antarctica, Australia and India (Cantrill and Douglas 1988; Cantrill

127 and Falcon-Lang 2001; Banerji and Ghosh 2002); AM fungi were also described

128 from a taxodiaceous Cupressaceae from the Middle Eocene of Canada (Stockey et

129 al. 2001). Nodular roots related to the Araucariales are known from the Cretaceous

130 of Antarctica, Australia and India (Cantrill and Douglas 1988; Cantrill and Falcon-

131 Lang 2001; Banerji and Ghosh 2002), and in the Regarding mycorrhizal root

132 nodules, the only structurally preserved examples are those of the Triassic

133 voltzialean Notophytum krauselli (Schwendemann et al. 2011, Harper et al. 2015).

134 This last record is the only one showing anatomical preservation of the rootlets in

135 which AM fungal colonization has been described.

136 The Middle-Late Jurassic chert deposits of the Bahía Laura Group in the

137 Deseado Massif of Santa Cruz Province, Southern Patagonia, Argentina bear

138 exceptionally preserved organisms in hot-spring settings (Channing et al. 2007;

139 Guido et al. 2010; Channing et al. 2011; García Massini et al. 2012, 2016). The

140 best preserved Jurassic geothermal paleosurfaces can be positively compared

141 based on their structure to hot springs at Yellowstone National Park in the United

142 States (Channing et al. 2009a,b). These are the only geothermal fossiliferous

143 deposits with an ecosystem-level preservation known for the Mesozoic. Preliminary

144 reports on the fossiliferous content and paleoenvironment reconstruction have

145 been carried out on a few localities (Channing et al. 2007; Guido et al. 2010;

146 García Massini et al. 2012; García Massini et al. 2016; Sagasti et al. 2016). Plant

147 remains are abundant in these localities, including completely preserved horsetails

148 (Channing et al. 2011; García Massini et al. 2016), and diverse organs of

149 gleicheniaceous and osmundaceous ferns, cycads, and conifers (Guido et al.

150 2010; Sagasti et al. 2016; García Massini et al. 2016). Among conifer remains,

151 several dispersed organs were identified including wood fragments, leafy twigs,

152 isolated single and multi-veined leaves, isolated ovules, pollen cones, pollen grains

153 in the interior of cones and dispersed in the chert matrix, and roots. All these

154 organs have suggested affinities with the Araucariaceae and Cheirolepidiaceae

155 (Guido et al. 2010; García Massini et al. 2016; Sagasti et al. 2018). Affinities with

156 the Podocarpaceae family are discussed for particular brachyphyllous stems

157 bearing single veined leaves and pollen cones with in situ bisaccate pollen, by

158 Escapa et al. (in prep.). Animal remains (e.g. arthropods, mollusks), algae,

159 bacteria, protists, coprolites and traces have also been preliminarily described

160 (Guido et al. 2010; García Massini et al. 2016).and Fungal fungal and fungus-like

161 vegetative and reproductive structures are also abundant (Guido et al. 2010,

162 García Massini et al. 2012, García Massini et al. 2013; García Massini et al. 2016,

163 Sagasti et al. 2016, Sagasti et al. 2019), and these are frequently in associated

164 association with each other with different plant remains (e.g. García Massini et al.

165 2016). This provides the opportunity to describe a variety of plant-fungal

166 interactions to further characterize the dynamics of the hot-springs associated

167 ecosystems and to broaden our knowledge of fungal evolution and diversity during

168 the Jurassic, and in a broader sense during the Mesozoic.

169 In this contribution we describe particular conifer nodular fine roots colonized

170 by AM fungi, present preserved as inclusions in the matrix in of chert blocks

171 collected from the recently discovered ‘Cañadón Nahuel’ locality of the La Matilde

172 Formation, Bahía Laura Group, at the Deseado Massif hot-spring deposits. We

173 discuss possible systematic affinities for these roots based on comparisons with

174 extinct and extant examples of conifer nodular roots. We also address the

175 ecological significance of the arbuscular mycorrizhal interaction between the

176 studied conifers and glomeromycotan fungi in the context of the hot-springs

177 settings. This finding expands our knowledge on fungal diversity and ecology in the

178 Jurassic and fills a gap in the fossil record of arbuscular mycorrhizal symbiosis in

179 conifers..

180

181 MATERIALS AND METHODS

182 Geological setting

183 The studied samples were collected at the ‘Cañadón Nahuel’ locality of the La

184 Matilde Formation, in the Bahía Laura Complex of the Deseado Massif, Santa Cruz

185 Province, Argentina (Fig. 1). The Deseado Massif is an extensive (60,000 km2)

186 region in southern Patagonia, in Santa Cruz Province, Argentina. The geologic

187 history of this regioón is marked by volcanic activity during the Jurassic.

188 Approximately 178-151 million years ago (Pankhurst et al. 2000), this area was

189 subject to bimodal rhyolitic and andesitic volcanism, which lead to the formation of

190 the Bahía Laura Complex (Guido 2004). Rocks of the Bahía Laura Complex are

191 part of the Chon Aike Silicic Large Igneous Province (which ranges from Patagonia

192 to Antarctica; Pankhurst et al. 1998). This province’s genesis is related to crustal

193 thinning in a diffuse extensional back-arc setting associated with the break-up of

194 Gondwana and birth of the South Atlantic Ocean (Riley et al. 2001; Richardson and

195 Underhill 2002).

196 Widespread hydrothermal activity in the Deseado Massif resulted from the

197 mature (quiescent) volcanic phase in this region during the Late Jurassic, which

198 produced metalliferous epithermal deposits (i.e. formed at shallow depths related

199 to the terrestrial surface, in the upper portion of a hydrothermal system under

200 temperatures between 150 and 300 oC), extensive silicification, and formation of Formatted: Superscript

201 geothermal fields at the surface (Schalamuk et al. 1997; Guido and Campbell

202 2011). Well-preserved fossilized geothermal fields have been found within

203 volcaniclastic fluviolacustrine strata, distributed in a 400 x 250 km area of NNW-

204 SSE and WNW-ESE oriented belts aligned with major regional structures (Guido

205 and Campbell 2011). Twelve of these geothermal fields preserve fossil

206 assemblages, which represent the only known fossiliferous Mesozoic hot-springs.

207 These deposits fill a significant stratigraphic gap between similar Paleozoic and

208 Cenozoic deposits (Channing and Edwards 2013).

209 ‘Cañadón Nahuel’ (GPS coordinates available upon request) is the farthest

210 east of these chert deposits in the Deseado Massif (Fig. 1). The locality is located

211 in the Northern Belt defined by Guido and Campbell (2011). Cherts from ‘Cañadón

212 Nahuel’ are rather randomly distributed over a large area and show textural and

213 fossiliferous characteristics that relate them to formation in the distal parts or

214 fringes of a siliceous sinter geothermal system. The observed distribution of the

215 cherts suggests they have been successively removed by is interpreted as having

216 resulted from explosive recurrent volcanic and phreatic local eruptions or breccias

217 related to the geothermal activitythat successively removed cherts formed by

218 dynamic geothermal activity. In some instances, horizontal bed cherts of variable

219 extension are found and these too are interpreted as having formed in wetlands

220 and shallow saline ponds, in the distal parts of geothermal systems. Formatted: Font color: Black

221

222 Preparation and imaging of studied materials

223 Thin sections of chert blocks were prepared following standard methodology

224 (Jones and Rowe 1999). Slides were observed using transmitted light microscopy.

225 All images were taken with a Nikon DS-Fi1 camera connected to a Nikon Eclipse

226 80i microscope. General views of the sections were taken using a Canon 7D

227 camera with a Canon macro lens of 60 mm, in a light box with fluorescent tubes. At

228 higher magnifications, multiple images of the same structure of interest were taken

229 at different focal planes to compose an image with greater depth of focus, following

230 an image-stacking technique (Bercovici et al. 2009). We used Helicon Focus

231 software (http://www.heliconsoft.com/heliconfocus.html), with the ‘‘Method B’’

232 (Depth Map) parameter. Several partially overlapping images were merged to

233 create high-quality images of critical features. This technique was applied using

234 Adobe Photoshop CS6, and images were processed after to reveal critical

235 characters. The image-stacking technique was not always applicable because in

236 some cases the stacking of images added too much noise or arteifacts to the final

237 composition. In such cases we opted for showingto show different focal planes of

238 the same structure side by side (e.g. Fig. 5).

239 The macro-specimens and thin sections are housed at the Museo Regional

240 Padre Molina of Río Gallegos (Santa Cruz Province, Argentina) under accession

241 numbers MPM-Pb-16033;16039-16045.

242

243 RESULTS

244 Description

245 Fungal specimens occur in association with root nodules that emerge from

246 conifer fine roots. These are preserved in chert and obliquely cross and

247 longitudinally sectioned (Fig. 2a). A Oblique cross and longitudinal sections of

248 anatomically preserved conifer diarch roots with secondary growth and young fine

249 roots bearing nodules immersed in siliceous chert matrix are described (Fig. 2a).

250 A central vascular cylinder with secondary xylem followed by the cortex and an Formatted: Indent: First line: 0"

251 apparent periderm are observed in oblique-cross section of older roots (Fig. 2b). In

252 oblique-longitudinal section, scarce diagnostic characters of the secondary xylem

253 are observed (Fig 2c-e). Pits in the radial walls of the tracheids are contiguous,

254 biseriate or uniseriate with occasional biseriate rows. (Araucarian pattern sensu

255 Phillipe and Bamford (2008)) (Fig. 2d-e).

256 The young fine nodular roots are 0.5 to 1 mm in diameter. Some roots are

257 constrained, showing a beaded appearance (Fig. 3a). The nodules emerge at right

258 angle from the parental root (Fig. 3c) and are disposed arranged in variable

259 patterns (Fig 2f), some are densely packed, others scattered, and in some

260 specimens they are alternately arranged (Fig 3a). They are spherical to spheroidal

261 (i.e. longitudinally elongated), (Fig. 2a,f; Fig. 3a-e) and measure 0.8 (0.45-1.18)

262 mm in diameter.

263 Anatomically, the nodules are characterized by a central vascular cylinder of

264 92 (78-120) µm of diameter surrounded by an endodermis of 24 (11-35)that is

265 seven times thinner than the vascular cylinder µm wide.. The endodermis is

266 followed sorrounded by the cortex, ending in an epidermis, and the cortex is is four

267 times as wide as the vascular cylinder340 (250-445) µm wide (Fig. 3c-d). Root

268 hairs or their bases are occasionally observed (Fig. 3d,f). Root hairs are

269 approximately 19.,5 µm in diameter, they are wider at the base with an

270 approximate diameter of 25 µm.

271 The nodules originate at the protoxylem poles of the roots (Fig. 3b). The

272 vascular strand and the endodermis can be followed from the main root, generally

273 traversingup to the middle portion of the nodule in longitudinal section (Fig. 2f; 3a-

274 e). Cortex Cortical tissues areis composed of 6-8 (10) layers of cortical cells, cells

275 get flattened towards the epidermis (Fig. 3d). Resurgent growth can be observed in

276 some nodules, where new cortical tissue grows inside the older cortex (Fig. 3e).

277 The cortex of fine roots andthe root nodules are colonized by arbuscular

278 mycorrhizal fungi (Fig. 4, 5, 6, 7). Additionally, AM fungal structures occur in the

279 chert matrix, very close or directly attached to the roots and nodules (Fig. 7). The

280 fungi are mainly restricted to the inner layers of cortical tissue whereas cortical

281 layers below the epidermis remain almost fungal free.

282 The outer layers of cortical tissue, below the epidermis, are mainly devoid of

283 fungal structures. The majority of the fungal structures are restricted to the inner

284 layers of the cortex. In the this specificinner zone of the cortex, the outer layers

285 are occupied by intra-cellular coenocytic hyphae that sometimes form coils (Fig.

286 4a-f). The hyphae are irregular in diameter, ranging from 3,4 to 14,5 µm, the

287 average diameter is 7 µm. The hyphae also develop knobs along its length (Fig.

288 4b), branch dichotomously (Fig. 4c) and at right angle (Fig. 4d) and present

289 dichotomous Y-branching (Fig. 4c). Some coils present display possible circular to

290 ellipsoidal scars and appear to be attacked byare directly associated to narrower

291 hyphaer of an intrusive septate fungus (Fig. 4e-f). However, this scars can be

292 preservation artefacts, rendering the metabolic status of the septate fungus

293 saprothrophic or parasitic. (Fig. 4e-f).

294 Towards the inner cortex, the hyphae becomes thinner, developing highly

295 ramified structures that resemble arbuscules (Fig. 5a,c). Arbuscules are observed

296 partially occupying the cells lumens, and traverse laterally from a cell to the other

297 (Fig. 5c; 6a-f; see also supplementary material). Trunk hyphae of the arbuscules

298 are less than 4 µm in diameter.

299 Morphologically identical, intra- and extra-rhizal glomoid spores are also

300 observed (Fig. 7a-f). Intra-rhizal spores occur in the cortex of both, young roots and

301 nodules. Extra-rhizal spores are observed dispersed in the chert matrix very close

302 to the roots and nodules. Extra-rhizal mycelium associated to extra-rhizal spores

303 (Fig. 7b,c) extends parallel to the roots and nodule's surface. The hyphae measure

304 1.5 to 4.5 µm in diameter and present Y-branching. In some specimens, coenocytic

305 hyphae emerging from the spores attached to the epidermal surface of nodules

306 (Fig. 7b-c) and possibly penetrating the epidermal cells are observed (Fig. 7d).

307 The spores are terminal, globose to subglobse, variable in size, ranging from

308 26.5 to 43.5 µm in diameter (Fig. 7e-f). The spore wall appears to be comprised of

309 two layers (Fig. 7e-f). The outer layer is hyaline and the inner layer is opaque. The

310 spores are occluded at their attachment point by a fold of the inner layer in some

311 specimens (Fig. 7f), or present display a plug in others (Fig. 7e). The spores are

312 attached to simple subtending hyphae. The hyphae are coenocytic and 2.0 (1.2-

313 2.9) µm in diameter.

314

315 DISCUSSION

316 Here we described an ancient plant-fungal association in the root nodules of

317 Jurassic conifers. Coenocytic hyphae forming coils, highly ramified arbuscules, and

318 spores, attributable to glomeromycotan fungi occur inside the cortical tissuesex of

319 conifer root nodules. We observe that the AM fungal structures are restricted to the

320 inner layers of the cortex, whereas only a few scattered coenocytic hyphae occur in

321 the outer cortex, close to the entry points below the epidermis. Further, whenWhen

322 resurgent growth in the host nodules is present, hyphae are also present in the

323 renewed cortex of the nodules, which positively compares to what has been

324 described in extant Araucariales (Bailys et al. 1963, Russell et al. 2002; Dickie and

325 Holdaway 2011). Analogous general distribution of AM fungi in host nodules is also

326 observed in extant Araucariales (Fig. 5b; Table 1; McGee et al. 1999; Russell et al.

327 2002; Dickie and Holdaway 2011). Similarly, previous records of fossil mycorrhizal

328 plants found identicalidentically zoned distribution of AM fungi in the infected root

329 tissues (Remy et al. 1994; Phipps and Taylor 1996; Strullu-Derrien et al. 2009;

330 Strullu-Derrien et al. 2014; Harper et al. 2015).

331 Two morphological types of arbuscular mycorrhizae have been classically

332 distinguished (Gallaud 1905): Arum type, which includes intercellularly hyphal Formatted: Font: Italic

333 growth in a longitudinal pattern in the cortex, with short side branches that give rise

334 to arbuscules nearly at right angle; and Paris type, characterized by intracellular

335 coiling hyphae with arbuscules forming along the coil (Taylor et al. 2015; Walker et

336 al. 2018). Coenocytic hyphae are always intracellular and frequently forming coils.

337 Additionally, the coenocytic hyphae colonize the cortex, spreading directly from cell

338 to cell. These fFeatures of the fungi described here are consistent with the Paris

339 morphological type. However, the presence of highly ramified arbuscules is

340 commonly referred to as a characteristic of the Arum type (Smith and Read, 2010).

341 Morphological variability observed in the AM fungi described is, it has been shown

342 that the morphological types vary in a continnum depending on a series of factors

343 (e.g. host plant tissues, taxa involved; environment; Dickson 2004; Van Aarle et al.

344 2004; Kubota et al. 2005), rendering this character merely descriptive and of

345 relative use for the classification of AM fungi consistent with previously observed

346 morphologies varying in a continuum for a single fungus within the same host plant

347 (Smith and Read 2010; Walker et al. 2018). Besides, the distinction of these

348 morphologies have been used in fossil comparisons (Harper et al. 2015; Harper et

349 al. 2016; Walker et al. 2018).

350

351 Systematic affinities of the root nodules and the fungi

352 The root nodules herein describeddescribed here are characterized by a

353 central vascular cylinder surrounded by an endodermis, followed by several layers

354 of cortex surrounded by an epidermis; spherical to spheroidal three dimensional

355 morphology; and emergence at right angle from the parental root in variable

356 patterns along the main axis. These are features shared by extant Araucariales,

357 which includes the Podocarpaceae and Araucariaceae (see comparisons in Table

358 1). Additional vegetative and reproductive organs assignable to Araucariaceae and

359 Podocarpaceae are found in the La Matilde Formation (e.g., wood, leafy branches,

360 cones; Gnaedinger 2007; Zamuner and Falaschi 2005; Falaschi et al. 2011;

361 Kloster and Gnaedinger 2018; Escapa et al. in prep). Further refinement of the

362 systematic affinities of the nodules based on the morphological characters

363 preserved, especially considering morphological variability known to be present in

364 fossil and extant Podocarpaceae and Araucariaceae, is not possible (Spratt 1912;

365 Dickie and Holdaway 2011; Tables 1,2). Considering the presence of both

366 podocarpaceous and araucariaceous organs in the chert blocks studied and the

367 little morphological variation observed among the mycorrhizal nodules it remains

368 possible that more than one taxa is present.

369 Based on phylogenetic analysis of combined molecular and morphological

370 data, several families have been recognized among the former Glomeromycota

371 (Schüβler et al. 2001; Spatafora et al. 2016): Paraglomeraceae,

372 Archaeosporaceae, Geosiphonaceae, Ambisporaceae, Glomeraceae,

373 Claroideoglomeraceae, Pacisporaceae, Gigasporaceae, Pacisporaceae,

374 Sacculospporaceae, Acaulosporaceae and Diversisporaceae (see Schüβler and

375 Walker 2010; Redecker et al. 2013; http://www.amf-phylogeny.com). Differences

376 mainly in spore type formation, spore wall structure, presence of vesicles, and

377 even ecology, exclude affinities of the fungi described here with several different

378 groups (see Table 3). Particular morphological characters suggest that these

379 fossils most probably affinities are with the Paraglomeraceae. Diagnostic

380 characters supporting affinities with the Paraglomeraceae include

381 Glomeromycotan familes can be distinguished morphologically; however, genera

382 from different families are not always morphologically distinguishable (Schüβler

383 and Walker 2010). Fungi forming arbuscular mycorrhizae in the root nodules herein

384 described are characterized by globose to subglobose glomoid spores, which is

385 shared with the Glomeraceae, Paraglomeraceae, Diversisporaceae,

386 Claroideoglomeraceae, Ambisporaceae, Pacisporaceae and Geosiphonaceae.

387 Affinities with the Claroideglomeraceae, Pacisporaceae and Geosiphonaceae are

388 discarded based on differences in the spore wall structure and spore development

389 and germination (Schüβler 2002; Schüβler and Walker 2010; Ohel and Sieverding

390 2004). The Ambisporaceae are characterized by the development of glomoid or

391 acaulosporoid spores, or both; they also develop vesicles, though some species do

392 not form vesicles (Walker et al. 2007). Species of the Diversisporaceae also

393 develop either glomoid or acaulosporoid spores, sometimes also having vesicles

394 and auxiliary cells (Walker and Schüβler 2004). The Glomeraceae typically develop

395 glomoid spores, but are regarded as producing vesicles (Pirozynski and Dalppé

396 1989; Morton and Benny 1990), which is a feature not observed in the fungi herein

397 described. The Paraglomeraceae are also characterized by the development of

398 glomoid spores and lack of vesicles; vesicles have been observed only in the

399 species Paraglomus brasilianum, although, these have also been regarded as

400 being intra-rhizal spores (Morton and Redecker 2001). Based on the presence of

401 arbuscules attached to narrow trunks (<4 µm in diameter), development of intra-

402 rhizal spores and intracellular hyphae frequently coiling 3-10 µm wide the fossil

403 fungus described here is most similar to the Paraglomeraceaecoiling. However,

404 Paraglomus spores have a three-layered wall, whereas in our specimens, the wall

405 is comprised by two layers. Nonetheless, the outer wall of the spores in

406 Paraglomus has been mentioned to disintegrate at senescence, resulting in a two- Formatted: Font: Italic

407 layerd spore wall and thus, comparable to that displayed by the fossils described

408 here (Morton and Redecker 2001). Alternatively, affinities of the fossil with

409 Glomeraceae are also possible, being different mainly based on the absence of

410 vesicles; additionally, the Glomeraceae commonly develop straight hyphae,

411 whereas, the fossil most frequently shows coiled hyphae. These same features

412 also makesmake the fossil somewhat comparable to Diversisporaceae and

413 Ambisporaceae, although these two families develop acaulosporoid spores, which

414 have not been observed in the fossil.

415

416 Fossil conifers with arbuscular mycorrhizal fungi conifers

417 Fossil examples of mycorrhization in conifers have been reported from the

418 Triassic to the Eocene, including very scarce examples of arbuscular mycorrhizae

419 in voltzialean, taxodiaceous and araucarialean conifers, and one example of

420 ectomycorrhizal symbiosis in pinales (Strullu-Derrien and Strullu 2007 and citations

421 therein; Schwendemann et al. 2011; Harper et al. 2015). The oldest record of

422 nodular roots colonized by arbuscular mycorrhizal conifers fungi is comprised by

423 nodular roots and rootlets of voltzialean conifers from the Triassic of Antarctica

424 (Schwendemann et al. 2011; Harper et al. 2015). In this case, both the rootlets and

425 the root nodules of Notophytum krauselli are colonized by serveral fungal

426 estructures (i.e. Hyphal hyphal coils, and arbuscules, extra-rhizal hyphae, occur in

427 the cortical tissue of rootlets and nodules of Notophytum, as well as extra-rhizal

428 hyphae, vesicles and spores; (Schwendemann et al. 2011; Harper et al., 2015).

429 However, fungal structures found in the nodules are different from those found in

430 the secondary rootlets, suggesting different arbuscular mycorrhizal symbiosis

431 occurring in different organs of the same plant root system (Harper et al. 2015).

432 Other examples of conifers showing root nodules apparent mycorrhization in

433 conifers come are from different Cretaceous localities from the Southern

434 Hemisphere (Cantrill and Douglas 1988; Cantrill and Falcon-Lang 2001; Banerji

435 and Ghosh 2002). The lack of anatomical preservation in these nodules found as

436 impressions, compressions and molds prevents any fungal association to be

437 observed.Symbiosis is inferred based on the presence of root nodules preserved

438 as impressions, compressions and molds, but none fungal structure is actually

439 preserved (Cantrill and Douglas 1988; Cantrill and Falcon-Lang, 2001; Banerji and

440 Ghosh, 2002). Numerous affinities were discussed for these Cretaceous conifer

441 nodule-bearing roots based on the association with other organs, including

442 affinities with the Podocarpaceae, Araucariaceae, Sciadopityaceae, and even

443 Cupressaceae. Also, anatomically preserved arbuscular mycorrhizae were

444 described in roots of Metasequoia from the Middle Eocene of Princeton Chert,

445 British Columbia (Stockey et al. 2001). Therefore, tThis new record of arbuscular

446 mycorrhizal in root nodules with affinities with the Araucariales from the Jurassic of

447 Patagonia fills a gap in the geological history of AM fungi and further documents

448 morphological diversity and ecological preferences of glomeromycetes through

449 time.

450

451 Mycorrhizal nodules and ecology

452 The exceptional preservation of the conifer nodular roots of the ‘Cañadón

453 Nahuel’ locality can be related to the mode of preservation typical of hot-spring

454 settings, where plants and other organisms become immersed in a mineral-

455 saturated solution that slowly gradually transform them into fossils (Channing and

456 Edwards 2004; Hellawell et al. 2015). The presence of fungal structures inside the

457 cortex of these underground organs indicates that the fossilization process of these

458 organisms must have occurred while they were alive or soon after their death

459 (Channing and Edwards, 2004). Such fossilization process consisted in

460 permineralization by silica, which is a characteristic previously observed in ‘La

461 Bajada’ and ‘San Agustín’ hot-spring localities also of the La Matilde Formation

462 (García Massini et al. 2016). Moreover, based on their distribution and textural and

463 fossiliferous content, the plant-rich cherts of the ‘Cañadón Nahuel’ locality are

464 related to the distal areas of siliceous sinter geothermal system, potentially

465 analogue to modern Yellowstone National Park geothermally influenced

466 environments in the USA (Channing et al 2009a,b). Root nodules have also been

467 preliminary described in the above mentioned ‘San Agustín’ and ‘La Bajada’

468 localities (see supplemental material; García Massini et al., 2013, 2016). These

469 root nodules share the general morphology and anatomy of the specimens

470 described here, though the best preserved examples of arbuscular mycorrhizae

471 mycorrhizal colonization are those from ‘Cañadon Nahuel’.

472 The distribution of organisms, especially of the vegetation, in the geothermal

473 wetland and distal environments of hot-spring systems is limited by the unusual

474 chemical and physical characteristics (Walter and Des Marais 1993; Channing et

475 al. 2011; Channing and Edwards 2013; Channing 2017). These environments are

476 often dominated by high temperatures, elevated salinity, extreme pH values, and

477 presence of phytotoxic elements such as heavy- and transition-metals and

478 metalloids (Channing and Edwards 2009; Channing 2017). In the distal areas of

479 hot-spring systems, osmotic and chemical stresses also limit organisms’

480 establishment (Channing and Edwards 2013; Channing 2017). In hot springs,

481 where specialized organisms inhabit, fungal symbioses are probed to confer host

482 plants stress tolerance to extreme physico-chemical parameters and, at the same

483 time, positively influence fungal distribution along the geothermal gradient

484 (Redman et al 2002; Rodriguez and Redman 2008; Rodriguez et al. 2008; Bunn

485 and Zabinsky 2003). Examples of arbuscular mycorrhizal symbiosis in plants

486 growing in geothermal soils have been reported for Yellowstone National Park

487 (Bunn and Zabinsky 2003; Appoloni et al. 2008; Bunn et al. 2009). This stress

488 tolerance through symbiosis in plants has also been reported for other

489 environments (e.g., salt marshes) with similar chemical stresses (Rodriguez et al.

490 2004; Rodriguez et al. 2008; Wilde et al. 2009).

491 Presence of arbuscular mycorrhizal fungi is not needed for the development

492 of the root nodules in extant Araucariales, as they are indeed part of the root

493 system ontogeny (Baylis et al. 1963; Baylis 1969), however, these organs are

494 hypothesized to increase the volume of fungal colonization at a lower energy cost

495 than having an extensive root system and therefore maximize mineral nutrient

496 acquisition efficiency (Russell et al. 2002; Dickie and Holdaway 2011). Although

497 the role of the mycorrhizal nodules in the extant Araucariales is not yet completely

498 understood, these organs are believed thought to be ecologically significant with

499 respect to uptake of water and nutrients, especially phosphorous and nitrogen;

500 additionally, establishment of seedlings in nutrient-poor environments has also

501 been suggested to be influenced by the development of mycorrhizal nodules

502 (Russel et al. 2002; Padamsee et al. 2016; Dickie and Holdaway 2011 and

503 citations therein). Presence of arbuscular mycorrhizal fungi in the root nodules of

504 the Araucariales of the ‘Cañadón Nahuel’ locality may have played a key role for

505 the colonization of the hostile distal areas of the hot-spring environments. Further,

506 the presence of mycorrhizal nodules might have been crucial for the establishment

507 conifers in the Jurassic waterlogged hot-spring settings. Similarly, it has been

508 hypothesized that AM fungi are crucial for the development of podocarps in hot-

509 spring related wetlands (Russel et al. 2002). Systematic and taxonomical

510 refinement to the family level of the conifers of ‘Cañadón Nahuel’ and further

511 characterization of the paleo-environments where this plants used to live should

512 enrich what can be grasped from these fossils on the evolution and significance of

513 the AM symbiosis in the Jurassic Araucariales of Patagonia.

514

515 CONCLUSIONS

516 We present the first record of structurally preserved arbuscular mycorrhizal

517 fungi colonizing root nodulese from the Jurassic, partially filling a gap in the

518 Mesozoic record of conifer mycorrhizal nodules, and extending the record

519 geographically.

520 The mycorrhizal nature of the fungusnodules is demonstrated by the

521 presence of several fungal structures in a delimited zone of the nodule's cortex,

522 especially important is the presence of hyphal coils, arbuscules and glomoid

523 spores. These mycorrhizal organs would have played a crucial role in the

524 establishment of conifers in the distal areas of the hot-spring systems.

525 The nodules have affinities with the Araucariales clade of conifers, however, it

526 is not possible to constrain the affinities of these organs further based on the

527 anatomical evidence preserved. Nonetheless, the presence of these structures is

528 important as they expand our knowledge on the morphology and ecology of these

529 conifers during the establishment of the modern conifer families in the Jurassic.

530 They also open the possibility to work on whole-plant concept reconstructions in

531 the future.

532

533 Acknowledgments

534 We kindly thank the anonymous Reviewers for their very detailed and helpful

535 comments and suggestions that considerably improved this manuscript. We thank

536 Dr. Ian Dickie for kindly providing images of mycorrhizal root nodules of extant

537 Podocarpaceae. CIN thanks all MEF-CONICET fellows for kind support, and Lic.

538 Ana Andruchow Colombo and Andrés Elgorriaga for insightful discussions. We

539 also thank the Culture Bureau of Santa Cruz Province for granting us the research

540 permits to study the geothermal deposits in the Deseado Massiff, and the

541 authorities and personnel of Patagonian Gold and local people for the logistic

542 support. This contribution was founded by CONICET (CONICETRes. 2318, P. 202

543 & 173) (to JLGM) and ANPCyT (PICT 2014-3496) (to JLGM & DG).

544

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829

830

831 TABLES AND FIGURES CAPTIONS

832

833 Table 1. Comparisons of ‘Cañadón Nahuel’ locality specimens of root nodules and

834 the root nodules of extant Podocarpaceae and Araucariaceae species.

835 AMF = arbuscular mycorrhizal fungi; PS = parallel series; LS = longitudinal series;

836 VC = vascular cylinder; MR = main root; En. = endodermis; C. = cortex; E. =

837 epidermis; Ex = exodermis; VS = vascular strand; P = Present; A = absent; Sc. =

838 Scattered; ? = Missing information

839 Information taken from:

840 *a Breuniger et al 2000

841 *b McGee 1990

842 *c Godoy and Mayr 1989

843 *d Padamsee et al 2000

844 *e Russel et al 2002; Dickie and Holdaway 2011

845 *f Leone et al 2008

846

847 Table 2. Fossil record of conifer root nodules. Modified from Schwendemann et al.

848 2011.

849 AMF = arbuscular mycorrhizal fungi; P = preserved; NP = not preserved; ? =

850 missing information

851

852 Table 3. Comparisons within families of the former Glomeromycota that form Formatted: Line spacing: Double

853 glomoid spores.

854 G = glomoid; Ac. = acaulosporoid; Occ. = occlusion; P = present; A = absent; ? =

855 missing information or not clear

856

857 *a Schußler and Walker 2010

858 *b Ohel and Sieverding 2004

859 *c Schußler 2002

860 *d Walker et al 2007

861 *e Walker and Schußler 2004

862 *f Morton and Benny 1990

863 *g Morton and Redecker 2001

864 *h Redecker et al 2013

865

866

867 FIGURE 2. Thin sections of chert blocks from ‘Cañadón Nahuel’ locality, MPM-Pb-

868 16039-16041. – a. General view of chert block showing conifer roots immersed in

869 the matrix. Some nodules in young fine are colored with pink sof-light. – b.

870 Oblique-cross section of conifer diarch root. Note secondary xylem (2ºX), cortex

871 (C) and apparent periderm (Pd). – c. Oblique-longitudinal section of conifer root

872 showing secondary xylem (2ºX) and cortex (C), periderm not observed. – d and –

873 e. Detail of secondary xylem. Note araucarian pitting in the walls of the tracheids. –

874 f. General view of nodular root with spherical to spheroidal nodules, disposed with

875 any apparent pattern. Scale bar for b and c = 0.5 mm; for d and e = 10 µm; for f =

876 100 µm.

877

878 FIGURE 3. Thin sections of chert blocks from ‘Cañadón Nahuel’ locality, MPM-Pb-

879 16033;16039-16040. – a. General view of ‘beaded’ root with nodules, nodules

880 disposed alternately. – b. Cross section of a nodule, showing vascular trace (VT)

881 emerging from the parental root. – c. Oblique-longitudinal section of root, with

882 lateral root nodule emerging at right angle from the main axis. – d. Cross section of

883 a nodule surrounded by the main root cortex, showing general anatomy of the

884 nodule. Note vascular strand (VS), endodermis (En), Cortex (C) and epidermis (E).

885 Detail of vascular strand and endodermis inside box, scale bar = 20 µm. – e.

886 Detail of root nodule showing resurgent growth, new cortex (NC) developing inside

887 the cortex of the older nodule (OC). – f. General view of root hairs. Scale bar for –

888 a. 0.5 mm; for b, c, d, e and f = 100 µm.

889

890 FIGURE 4. Thin sections of chert blocks from ‘Cañadón Nahuel’ locality, MPM-Pb-

891 16040-16041. – a. General view of root nodule. Pink coloration indicates the

892 presence of hyphal coils. – b. Detail of a coenocytic hyphal coil showing knobs. – c.

893 Detail of coenocytic hyphae showing Y-branching. – d. Detail of coenocytic hyphae

894 showing branching at right angle. – e and f. Detail of worn out hyphael intruded by

895 a septate fungus (white arrow heads). Scale bar for a = 100 µm; for b, c, d, e and f

896 = 10 µm.

897

898 FIGURE 5. Thin section of chert block from ‘Cañadón Nahuel’ locality, MPM-Pb-

899 16039, and thin section of extant Podocarpaceae Podocarpus totara root nodule

900 (Images by Larry Peterson and Lewis Melville, from Dickie & Holdaway, 2011) – a.

901 Longitudinal section of fossil root nodule showing zone of the cortex occupied by

902 arbuscules (delimited by dotted line) – b. Longitudinal section of extant root nodule

903 showing zone of the cortex occupied by arbuscules (delimited by dotted line). – c.

904 Detail of arbuscules in fossil root nodule. – d. Detail of arbuscules in extant root

905 nodule. Scale bar for a and b = 100 µm; for c = 20 micras; for d = 50 µm

906

907 FIGURE 6. Thin section of chert block from ‘Cañadón Nahuel’ locality, MPM-Pb-

908 16039. Diversity of arbuscules partially occupying cortical cells of root nodules.

909 Scale bar for all = 10 µm.

910

911 FIGURE 7. Thin section of chert block from ‘Cañadón Nahuel’ locality, MPM-Pb-

912 16039. – a. Root nodule with intra-rhizal glomoid spores. – b. Glomoid spore

913 associated to root nodule. Note connecting hypha (arrowhead). – c. Glomoid spore

914 with multiple hyphal attachments, some of which connect it to the root nodule. – d.

915 Magnification of previous image showing detail of entry point with penetrating

916 hyphae and development of coil. – e. Detail of intra-rhizal globose spore in

917 decaying root, showing two-layered wall, simple subtending hypha and occlusion

918 plug. – d. Detail of extra-rhizal globose spore, showing two-layered wall, simple

919 subtending hypha, and occlusion.

920

921

922 SUPPLEMENTAL MATERIAL

923

924

925 Appendix 1. – A1. Diversity of young fine roots and nodules from different localities

926 of the La Matilde Formation, Deseado Massif, Santa Cruz Province, Argentina. – a,

927 b and c. ‘Cañadón Nahuel’ locality. – d. ‘San Agustín’ locality. – e. ‘La Bajada’

928 locality. Scale bar for a and e = 0.5 mm; b and d = 0.25 mm; c = 0.1 mm

929

930 Appendix 2. – A2. Further images of arbuscules inside cortical cells of root nodules

931 specimens of the ‘Cañadón Nahuel’ locality. Scale bar for all = 10 µm

932

933 Appendix 3. – A3. Sequence of original images used to obtain the final composition

934 of the nodules endodermis. Scale bar for all = 20 µm.

935

936 Appendix 4. - A4I and A4II. Sequences of original images used to obtain the final

937 composition of coiling hypha. Scale bar for all = 10 µm.

938

939 Appendix 5. – A5. Sequence of original images used to obtain the final composition

940 of nodule’s cortical cells occupied by arbuscules. Scale bar for all = 20 µm.

941

942 Appendix 6. - A6I, A6II, A6III. Sequences of arbuscules at different focal planes.

943 Scale bar for all = 10 µm.

944

945 Appendix 7. - A7I. Sequence of original images used to obtain the final composition

946 of a root nodule occupied by intra-rhizal glomoid spores. Scale bar for all = 100

947 µm. – A7II. Sequence of original images used to obtain the final composition of a

948 germinating spore with hyphae getting attached to the epidermis of a root nodule.

949 Scale bar for all = 20 µm. – A7III. Sequence of original images used to obtain the

950 final composition of a hypha penetrating the epidermis of a root nodule. Scale bar

951 for all = 10 µm. – A7IV. Sequence of original images used to obtain the final

952 composition of an intra-rhizal glomoid spore. Scale bar for all = 10 µm.

953 FIGURE 1. Diversity of young fine roots and nodules from different localities of the

954 La Matilde Formation, Deseado Massif, Santa Cruz Province, Argentina. – a, b and

955 c. ‘Cañadón Nahuel’ locality. – d. ‘San Agustín’ locality. – e. ‘La Bajada’ locality.

956 Scale bar for a and e = 0.5 mm; b and d = 0.25 mm; c = 0.1 mm

957

958 FIGURE 2. Further images of arbuscules inside cortical cells of root nodules

959 specimens of the ‘Cañadón Nahuel’ locality. Scale bar for all = 10 µm

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Nodules AMF Conifer species Arrangement Morphology Size mm General anatomy Root hairs Outer cortex Inner cortex Araucaria Arbuscules, ? Spheroidal ? ? ? Penetration hyphae angustifolia *a vesicles Araucaria Spherical and Central VC connected to ? ? ? Scattered hyphae Arbuscules cunninghami *b conical MR. / En. / C. / E. Central VC connected to Abundant Araucaria araucana P (young PS Spheroidal 0.55 MR. / En. / C. / E. Ex (in Mainly free. arbuscules, *c nodules) older nodules) vesicles Central VC connected to Coiled hyphae, Spherical. Beaded MR. / En. / C. / E. Agathis australis *d Irregularly spaced ≤1 ? Scattered hyphae arbuscules, nodules. VS = half length of the vesicles nodule Spherical and Central VC connected to Agathis robusta*b ? ? ? Scattered hyphae Arbuscules conical MR. / En. / C. / E. Spherical and Central VC connected to Wollemia nobilis *b ? ? ? Scattered hyphae Arbuscules conical MR. / En. / C. / E. Central VC connected to Mainly free. Scattered Dacrycarpus Arbuscules, 2 or 4 LS Spherical ? MR. /En. / C. / E. VS = A hyphae (below entry dacrydioides *e vesicles length of the nodule point) Central VC connected to Mainly free. Scattered Dacrydium Arbuscules, 2 or 4 LS Spherical ? MR. / En. / C. / E. VS = half A hyphae (below entry cupressinum *e vesicles length of the nodule point) Abundant Dacrydium fonckii ? Spherical ? ? ? ? arbuscules, *c scattered vesicles Podocarpus Abundant PS Spherical 1.2 ? P ? nubigena *c arbuscules Podocarpus Arbuscules, ? Spherical ? ? ? ? parlatorei *f vesicles Podocarpus Arbuscules, 2 or 4 LS Spherical 0.7 ? P ? salignus *c vesicles Central VC connected to Mainly free. Scattered Prumnopitys Arbuscules, 2 or 4 LS Spherical ? MR. / En. / C. / E. VS = half P hyphae (below entry ferruginea*e vesicles the length of the nodule point) Central VC connected to Mainly free. Scattered Prumnopitys Arbuscules, 2 or 4 LS Spherical ? MR. / En. / C. / E. VS = half P hyphae (below entry taxifolia *e vesicles the length of the nodule point) Prumnopitys andina Arbuscules, ? ? 1.1-1.5 ? Sc. ? *c vesicles Saxegothaea Arbuscules, PS ? 0.5-1.0 ? P ? conspicua *c vesicles Central VC connected to Mainly free. Scattered ‘Cañadón Nahuel’ Spherical or P Coiled hyphae, Variable 0.8 MR. / En. / C. / E. VS = half hyphae (below entry specimens Spheroidal (occasionally) arbuscules, spores the length of the nodule point)

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Age Locality Preservation Nodules Affinities AMF Prolate, Middle Antarctica Permineralization spheroidal Voltziales P Triassic 1.1 x 0.8 mm Spherical, Middle Patagonia, Permineralization spheroidal Araucariales P Jurassic Argentina 0.8 mm Australia Compresions/Impressions ? ?Taxodiaceae NP Spherical Antarctica Compresions/Impressions ?Podocarpaceae NP Cretaceous ≤ 2 mm Spherical, oval ?Podocarpaceae India Molds NP 1-2.5 x 1-2 mm ?Araucariaceae

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Family Spore Glomoid spore Vesicles Mycelium Observations type wall structure and occlusion Claroideoglomeraceae G 2 layers ? ? ? *a,h (apparent ‘endospore’). Occ.: Septum Pacisporaceae G Two wall P (some ? ? *a,b,h groups. species) Occ.: Septum Geosiphonaceae G 3 layers ? Sparsely Association with *c,h Occ.: septate at cyanobacteria amorphous senescence plug. Ambisporaceae G/Ac. 2 layers P/A ? ? *d,h Occ.: Open- pored or Septum Diversisporaceae G/Ac. 3 layers P/A ? With or without *e,h auxiliary cell

Glomeraceae G 3 layers P Narrow ? *f,h Occ.: variable trunks. Coiling rare Paraglmoeraceae G 3 layers P?/A Narrow Intra-rhizal *g,h Occl.: Septum? trunks. spores? Coiling frequent

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