Abstract Based on molecular dating, the origin of insect agriculture is hypothesized to have taken place independently in three clades of fungus-farming insects: the termites, ants or ambrosia beetles during the Paleogene (66–24 Ma). Yet, definitive fossil evidence of fungus-growing behavior has been elusive, with no unequivocal records prior to the late Miocene (7–10 Ma). Here we report fossil evidence of insect agriculture in the form of fossil fungus gardens, preserved within 25 Ma termite nests from southwestern Tanzania. Using these well-dated fossil fungus gardens, we have recalibrated molecular divergence estimates for the origins of termite agriculture to around 31 Ma, lending support to hypotheses suggesting an African Paleogene origin for termite-fungus symbiosis; perhaps coinciding with rift initiation and changes in the African landscape.

Citation: Roberts EM, Todd CN, Aanen DK, Nobre T, Hilbert-Wolf HL, O’Connor PM, et al. (2016) Oligocene Termite Nests with In Situ Fungus Gardens from the Rukwa Rift Basin, Tanzania, Support a Paleogene African Origin for Insect Agriculture. PLoS ONE 11(6): e0156847. https://doi.org/10.1371/journal.pone.0156847 Editor: Faysal Bibi, Museum für Naturkunde, GERMANY Received: December 10, 2015; Accepted: May 20, 2016; Published: June 22, 2016 Copyright: © 2016 Roberts et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by the US National Science Foundation (NSF EAR_0617561, EAR_0854218, EAR 0933619), National Geographic Society (CRE), and funding from Ohio University and James Cook University. TN was supported by a Marie Curie fellowship (FP7-PEOPLE-2012-CIG Project Reference 321725) and by the Portuguese Foundation for Science and Technology (SFRH/BCC/52187/2013). Competing interests: The authors have declared that no competing interests exist.

Introduction Termites are among the most diverse and ecologically important groups of insects in modern ecosystems, playing a critical role as natural decomposers of plant tissues. Termites typically rely on gut symbionts to decompose organic matter. However, members of the subfamily Macrotermitinae have turned to agriculture by developing a highly specialized, symbiotic relationship with fungi of the genus Termitomyces (Basidiomycotina). The fungus-growing termites cultivate fungi in gardens/chambers inside the colony and then exploit the ability of the fungi to convert recalcitrant, nitrogen-poor, plant material into a more easily digestible, protein-rich food source [1, 2]. After ingestion and brief mastication of woody material, modern Macrotermitinae excrete rounded pellets known as primary faeces or mylospheres, composed of concentrated, undigested plant fragments and Termitomyces spores, which germinate and colonize the plant material, thus forming fungal gardens. The critical ecological role of fungus-growing termite colonies as biodiversity and bioproductivity hotspots within African savannah ecosystems has been well documented in recent years [3, 4]. Indeed, much of the decomposition of woody plant material in Africa and Asia takes place as a result of fungus-growing termites [5], with estimates suggesting that more than 90% of dry wood in some semiarid savannahs is reprocessed by members of the Macrotermitinae [6]. A growing body of molecular evidence suggests that termite fungiculture can be traced back to a single origin around 31 Ma (19–49 Ma), when domestication of the ancestor of Termitomyces by the ancestor of the Macrotermitinae occurred [2, 7–10]. Once established, this symbiotic relationship is hypothesized to have remained obligate over its entire evolutionary history, with no evidence of Macrotermitinae ever forming a relationship with any other fungi or abandoning fungus farming [2, 7–9]. Until recently, little fossil evidence has been found to document the antiquity of the termite—fungus mutualism. To date only a single unequivocal report of fossilized termite fungus combs has been described, recovered from a succession of Upper Miocene-Pliocene (≤7 Ma) terrestrial deposits in the northern Chad Basin, Africa [11, 12]. Intriguingly, fossilized termite nests that appear similar to those produced by fungus-farming termites have been reported from continental deposits across Afro-Arabia ranging as far back as the early Oligocene or late Eocene [13–15]. However, diagnostic evidence demonstrating the age and presence of in situ fungus gardens within these fossil termite nests has not yet been clearly confirmed; and hence, the timing of this important evolutionary coupling between termites and fungus (termite fungiculture) is still uncertain. Here we report on the discovery of a new occurrence of fossilized termite nests with in situ fungus gardens from southwestern Tanzania. The new fossils were discovered in a paleosol horizon in a steeply dipping section of the Oligocene Songwe Member of the Nsungwe Formation in the Rukwa Rift Basin [16–18]. The aim of this study is to investigate the paleontology and geologic context of these new trace fossils, and use our findings to recalibrate the molecular phylogeny for fungus farming termites in order to test existing hypotheses regarding the timing and origin of termite-fungus symbiosis in the fossil record.

Study Area and Fossil Locality The trace fossil locality is located near the southern end of the Rukwa Rift Basin, a segment of the Western Branch of the East African Rift System in southwestern Tanzania [17, 18] (Fig 1). Excellent exposures of fossiliferous Permian to Plio-Pleistocene strata are exposed within the basin, particularly at the southern end in the Songwe Valley [19–25]. The trace fossils described in this study come from steeply exposed type section of the Paleogene Nsungwe Formation, which represents an overall upward fining succession of alluvial fan (Utengule Member) to volcanic-rich fluvial and lacustrine (Songwe Member) facies [17, 18, 23]. The Songwe Member preserves a particularly important and rare window in the late Paleogene of continental sub-equatorial Africa and has produced a rich new fauna, including the earliest records of Old World monkeys and apes [16, 23], along with a diversity of other mammals, crocodiles, birds, lizards, snakes, crustaceans and mollusks [26–31]. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Location and stratigraphy of the trace fossil locality, Tanzania. (A) Location of Tanzania within Africa. (B) Digital elevation model for the study area in the southern end of the Rukwa Rift Basin (white box is shown in C). (C) Geologic map of the Songwe Valley in the southern end of the Rukwa Rift Basin, showing stratigraphy and age of fossil locality. Modified from [18]. (D) Measured section and magnetic stratigraphy through the Nsungwe Formation Type Section, with location of fossil locality shown. Modified from [18]. (E) Photograph of the nest locality in a steeply dipping cliff face along the Nsungwe River. (F, G) Sketch maps of fossil locality showing the orientation and distribution of the termite colonies 1 (RRBP-08248) and 2 (RRBP-15106), with letters corresponding to the different nest chambers in each colony. https://doi.org/10.1371/journal.pone.0156847.g001 The Songwe Member has been precisely dated as late Oligocene, between 26–24 Ma, using a combination of: (1) single-crystal laser fusion Ar/Ar dating of phlogopite; (2) U-Pb LA-ICPMS dating of titanite; and (3) U-Pb LA-ICPMS, SHRIMP and CA-TIMS dating of zircon from multiple volcanic tuffs [17, 23]. The trace fossils reported in this study come from ~265 m above the basal contact of the Songwe Member, along the Nsungwe River Section. Based on radioisotopic dating and magnetostratigraphy, this part of the section is interpreted to fall within chron C7r of the global polarity timescale, indicating an age between ~24.8–24.5 Ma [17, 23]. The trace fossils, representing two discrete termite colonies, were all collected in the same area, but from beds several meters apart in a steeply dipping section of interbedded fluvial channel sandstones and overlying overbank mudrocks. Colony 1 was found near the top of a fine-grained, muddy sandstone complex and with seven chambers clustered in a small area spanning ~90 cm (vertically) x 150 cm (horizontally) (Fig 1). Colony 2 was found ~3 m below Colony 1 in a single 15 cm thick muddy sandstone horizon with six chambers spread out over 1.2 m (Fig 1). The sandstone beds containing Colony 1 fine upward and the densest concentration of nests were found in the finest-grained strata near the top of the bed. Colony 2 was also found in a fine-grained muddy sandstone unit. In both horizons, poorly preserved trough cross-bedding is cross-cut by the nests, associated galleries and root traces, indicating that the trace fossils formed after termination of fluvial flow and subaerial exposure at the top of the channel. Both colonies are overlain by a thin, pale orange to red color-banded sandy mudstone with abundant root mottling, horizontal and vertical burrows and minor CaCO 3 concretions. Considered together, these deposits are interpreted to be the top of an abandoned fluvial channel sequence, which was subjected to several flooding events followed by subaerial exposure and pedogenesis, presumably during the time of nest development and shortly after channel abandonment (see [18] for detailed interpretation of the sedimentology of section). The site includes two termite colonies (Fig 1), each with six to seven fungus chambers, and three of which preserve fungus gardens (also called fungus combs). The trace fossils are interpreted as having formed synchronously with deposition of the Oligocene Songwe Member, rather than being modern constructions associated with recent termite activity, based on the following evidence: 1) the trace fossils are lithified; 2) some of the nests and fungus combs show compaction features, indicating that they were buried after formation; 3) galleries are infilled with similar sediment to the host rock, rather than more recent volcanic ash which is common in the present soil overlying the Oligocene strata; and 4) the nests and fungal combs are oriented parallel to the steeply dipping beds, rather than parallel to the present-day land surface. The gross morphology of the trace fossil, its association inside Vondrichnus, and its peloidal construction of enclosed cells matches the diagnosis of a laminar-type fungus comb, Microfavichnus alveolatus [11]. Upward construction of the comb is evidenced by the concentric form and retention of alveolar form in the upper region. The fossil fungus chamber and fungus comb inside it are comparable to fungus combs produced by extant species of the genera Macrotermes and Odontotermes. However, no large hypogean chambers (calies) were observed with either colony, possibly due to the limited lateral extent of the outcrop.

Antiquity of Insect Agriculture Only two other insect groups are known to have derived mutualisms with fungi for agriculture: the ambrosia beetles and the leaf-cutter ants. Ants and termites are each considered to have evolved the ability to cultivate fungi for food only once, between 45–65 Ma and 24–34 Ma, respectively [47–49]. However, in ambrosia beetles, this trait may have evolved independently as many as ten different times, probably first around 50 Ma [50, 51]. It is also not clear where fungus gardening developed in ambrosia beetles, although there appears to be a strong evolutionary link to a tropical or sub-tropical forest setting [51]. Unfortunately, no fossil evidence, either in the form of fungal gardens or unequivocal ambrosia beetle borings, exists to validate molecular age estimates for ambrosia beetles or to provide direct geographic evidence on where this symbiosis originated. The oldest evidence of fungus gardening by leaf-cutter ants dates back to the late Miocene of Argentina, between 5.7 and 10 Ma [52]. However, no fungus gardens are preserved, only fossilized ant (Attini) nests interpreted as fungus chambers based on morphology and the presence of fungal hyphae within them [52]. Together, the late Miocene Argentinian leaf-cutter nests and the macrotermitine fungus combs from Chad [11, 12] represent the oldest previously known definitive fossil evidence for insect domestication of fungi, yet both are considerably younger than the Paleogene molecular estimates for the antiquity of agriculture by insects. Hence, the newly discovered Tanzanian trace fossils support a Paleogene record for the important evolutionary partnership between insects and fungi, and more specifically, they confirm recent molecular hypotheses for an African origin of symbiosis between the Macrotermitinae and Termitomyces fungi [9]. Notably, the features observed in the Tanzanian trace fossils lend support to the idea that similar Paleogene trace fossils documented across Afro-Arabia [13–15] may also represent fungus-farming termites. For instance, termite trace fossils Vondrichnus and Termitichnus from Oligocene-Miocene terrestrial ecosystems in Egypt, Ethiopia, Libya and Arabia [13–15] (Fig 5) may have also been produced by fungus farming termites, however these fossils have not been described in detail and so their position on the tree is not clear (see S1 Text, S2 Table and S6 Fig for details on molecular calibrations using these taxa, which do not greatly alter the ages suggested in Simulation 1). The discoveries of fungus combs within termite nests in Chad [11] and now, in Tanzania, confirm these earlier assertions and suggest that fungus-farming termites radiated across Africa early in their evolutionary history. The diversification of the Macrotermitine termites from an African rainforest origin might have been coeval with expansion of savannahs in Africa [8]. Although the expansion of C4 grasses (and hence savannahs) are not well-documented on continental Africa until ~7–8 Ma, the presence of micromammals with crestiform teeth and active-foraging colubroid snakes from well-dated late Oligocene strata in the Rukwa Rift Basin suggest that isolated mixed forest/grassland ecosystems may have been present in ecosystems by 25 Ma, likely reflecting landscape changes associated with the initiation of the East African Rift System [17].

Methods Permits The Tanzanian Commission for Science and Technology (COSTECH) and the Tanzanian Antiquities Unit granted us permission to carry out our field studies and to take samples. Our field studies did not involve endangered or protected species. Specimens Portions of two fossilized termite colonies containing 13 individual trace fossil termite nest structures (Vondrichnus planoglobus), three of which contained in situ fungus combs (Microfavichnus alveolatus), were assigned specimen numbers: RRBP 08248a-g and RRBP 15106a-f. Due to the fragile nature of these trace fossils, only five nest structures were collected for further study. These include samples RRBP08248a, c, f, and g and RRBP 15106c. The other trace fossils were too fragile to collect and remain in situ or have since weathered out of the outcrop. Specimens included in the contribution are accessioned with RRBP (Rukwa Rift Basin Project) identifiers and are permanently housed through the Antiquities Division of the Republic of Tanzania (Dar es Salaam, Tanzania). Paleontological approaches In order to obtain a better understanding of the internal architecture of the trace fossils, three samples were cross-sectioned through a vertical mid-plane passing from the upper to lower surface using a lapidary saw with no water. One side was cross-sectioned through the equator, perpendicular to the first cut. All cut surfaces were polished for higher-resolution observation. Scanning electron microscopy (SEM) was used to image the internal structure of the trace fossils and micro-CT analysis was unsuccessfully employed to observe internal architecture of one of the trace fossils due to a lack of density differences between the different materials. One of the cross-sectioned para-types was also vacuum impregnated with epoxy and polished to observe internal structures in better detail and to construct a microprobe mount. Element concentrations were measured using EDS and BSE images of the sample were taken to examine preservation patterns of the nests and fungus combs (Figs 2 and 4). This work was conducted on an electron probe microanalyser (EPMA; Jeol JXA8200 “superprobe”) at the Advanced Analytical Centre (AAC) at James Cook University. Fungus-growing termite dating Data and methodology used for the phylogeny calibration were the same as in Nobre et al. [9] (also see S1 and S2 Tables; S1–S7 Figs; S1 Text). Briefly, we used DNA sequences of the mitochondrial genes COI and COII and the nuclear ribosomal gene ITS2 for the 19 species of fungus-growing termites from all genera (except for the genera Allodontermes, Synacanthotermes and Protermes) and three outgroups [9]. Divergence dates were determined using the Bayesian relaxed-clock uncorrelated exponential approach implemented in BEAST 1.54 [53]. In the phylogeny reconstruction, the topology of the resulting tree was constrained to the genus-level phylogeny as estimated previously ([2, 7, 9] drawn schematically in Fig 6), and three Markov chain Monte Carlo searches were run for 10 000 000 generations each. Convergence was assessed using the log likelihood distributions of individual chains, and the burn-in level was assessed graphically in Tracer v1.4. In all simulations, the Odontotermes node was constrained to a minimum age of 7 Ma (based on the age of fossilized fungus comb associated with Odontotermes nest trace fossils from Chad [7]) following a lognormal distribution going as far back as the new fossil encountered (ca. 25 Ma) [8] [lognormal mean = 1.9, lognormal SD = 2.9, zero offset = 7]; and the ancestor of Macrotermes jeanneli was constrained to a minimum age of 3.4 Ma (based on the age of trace fossils reported from Tanzania [41]) [lognormal mean = 1.2, lognormal SD = 3.1, zero offset = 3.4]. We used the new fossils as a third node constraint of 25 Ma [lognormal mean = 3.2, lognormal SD = 2.7, zero offset = 25]. Because we inferred that the new fossils most likely belong to the clade composed of all genera except Pseudacanthotermes and Acanthotermes, the main approach applied this constraint to node b (simulation 1; the estimates from this analysis were used for the schematic Fig 6). Since the classification of the fossilized combs based on comparison with extant fungus combs is not unambiguous, we did four additional simulations (Table 1; Fig 6), using alternative calibration points of the newly discovered fossils: the most recent common ancestor of fungus-growing termites (node a; simulation 2); the most recent common ancestor of Pseudacanthotermes and Acanthotermes (node d; simulation 3); the most recent common ancestor of Microtermes and Ancistotermes (node e; simulation 4) and the most recent common ancestor of the genus Microtermes (simulation 5) (see S1–S6 Figs and S2 Table). Typically, the first 10% of the trees were discarded as burn-in, prior to results being pooled in LogCombiner v1.5.4 and tree visualization (see S1–S7 Figs; S1 Table for details concerning the details on the DNA analysis and files with raw trees produced in BEAST 1.54 [53]).

Acknowledgments We thank D. Kamamba, F. Ndunguru, and J. Temu (Tanzania Antiquities Unit), P. Msemwa (Tanzania Museum of House of Culture), and N. Boniface (University of Dar es Salaam), and the Tanzania Commission for Science and Technology (COSTECH) for logistical and administrative support; M. Gottfried, M. Hendrix, Z. Jinnah and the RRB excavation teams for field assistance. J. Hammerli, C. Pirard and K. Blake assisted with making polished slabs and microprobe mounts and imaging. We thank Termite Web for giving us permission to use photo shown in Fig 2F. We are grateful to the editor F. Bibi and two reviewers (M. Schuster and J. Scott) for their constructive feedback, which greatly improved the manuscript.

Author Contributions Conceived and designed the experiments: EMR CNT DKA TN HLHW PMO LT CM NJS. Performed the experiments: EMR CNT DKA TN HLHW. Contributed reagents/materials/analysis tools: EMR CNT DKA TN HLHW PMO NJS. Wrote the paper: EMR CNT DKA TN HLHW PMO LT CM NJS.