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The assembly of -farmed gardens: rspb.royalsocietypublishing.org mutualism specialization following host broadening

Guillaume Chomicki1, Milan Janda2,3 and Susanne S. Renner1 Research 1Systematic and Mycology, University of Munich (LMU), Menzinger Str. 67, 80638 Munich, Germany 2 Cite this article: Chomicki G, Janda M, Biology Centre of the Czech Academy of Sciences, Branisovska 31, 37005 Ceske Budejovice, Czech Republic 3Laboratorio Nacional de Ana´lisis y Sı´ntesis Ecolo´gica, ENES, UNAM, Antigua Carretera a Pa´tzcuaro 8701, Morelia, Renner SS. 2017 The assembly of ant-farmed Mexico gardens: mutualism specialization following GC, 0000-0003-4547-6195; SSR, 0000-0003-3704-0703 host broadening. Proc. R. Soc. B 284: 20161759. Ant-gardens (AGs) are ant/plant mutualisms in which farm epiphytes http://dx.doi.org/10.1098/rspb.2016.1759 in return for nest space and food rewards. They occur in the Neotropics and Australasia, but not in Africa, and their evolutionary assembly remains unclear. We here use phylogenetic frameworks for important AG lineages in Australasia, namely the ant Philidris and domatium-bearing ferns Received: 8 August 2016 (Lecanopteris) and flowering plants in the Apocynaceae (Hoya and Dischidia) Accepted: 19 October 2016 and Rubiaceae (, , Anthorrhiza, Myrmephytum and Squamellaria). Our analyses revealed that in these clades, diaspore dispersal by ants evolved at least 13 times, five times in the Late Miocene and Pliocene in Australasia and seven times during the Pliocene in Southeast Asia, after Philidris ants had arrived there, with subsequent dispersal between these Subject Category: two areas. A uniquely specialized AG system evolved in Fiji at the onset Evolution of the Quaternary. The farming in the same AG of epiphytes that do not offer nest spaces suggests that a broadening of the ants’ plant host spectrum Subject Areas: drove the evolution of additional domatium-bearing AG-epiphytes by evolution selecting on pre-adapted morphological traits. Consistent with this, we found a statistical correlation between the evolution of diaspore dispersal by ants and domatia in all three lineages. Our study highlights how host Keywords: broadening by a symbiont has led to new farming mutualisms. ants, plants, farming mutualism, ant-gardens, evolution, ecology

Author for correspondence: 1. Introduction Guillaume Chomicki Farming mutualisms, wherein an organism promotes the growth of another on e-mail: [email protected] which it depends for food, have evolved in many lineages of the tree of life [1,2]. Examples include bacteria farmed by amoebae [3], fungi [4] or deep-sea crabs [5], and algae farmed by sloths or damselfish [6–8]. More complex forms of farming with active control of the partner’s growth, reproduction and dispersal have evolved in fungus-farming ants [1,9–11], beetles [12,13] and termites [14,15]. Beyond fungi, ants also farm a range of other organisms (see [2] for a review). Control over the dispersal of the ‘crop’ by the ‘farmer’ is a first step required in the evolution of farming mutualisms. In ants, mutualisms between seed-dispersing workers and plants with suitable propagules have been docu- mented or inferred for thousands of species in 77 families of flowering plants [16,17]. Typically, these interactions involve ants gathering seeds to feed on lipid- and protein-rich appendages (elaiosomes) and then abandoning the One contribution to a special feature ‘Ant seeds somewhere nearby [18]. In the Neotropics, ants also disperse certain epi- interactions with their biotic environments’. phytes by placing their seeds inside carton nests, where they germinate and eventually form the so-called ant-gardens (AGs) [19–21]. The epiphytes benefit Electronic supplementary material is available from being dispersed to sites far above the ground and planted in the nutrient- online at https://dx.doi.org/10.6084/m9.fig- rich carton material (often enriched with vertebrate faeces), and the ants benefit through the scaffold formed by epiphyte roots that stabilizes their nests and by share.c.3670204. nutritional rewards, such as extrafloral nectar [22].

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Figure 1. Examples of Australasian AGs. (a) Hydnophytum moseleyanum and Dischidia nummularia cultivated by Philidris cordata in Papua New Guinea. (b) Leca- nopteris carnosa cultivated by a Polyrhachis species from the sericata group, Papua New Guinea. (c) Example of carton nest freshly formed by Philidris myrmecodiae on Calamus ocrea, Papua New Guinea. (d) Myrmecodia platytyrea subsp. antoinii and Dischidia nummularia cultivated by Philidris cordata, Papua New Guinea. (e) Dischidia major, D. nummularia and Myrmecodia cultivated by Philidris cordata, Papua New Guinea. (f) Dozens of Myrmecodia schlechteri on the same tree, cultivated by Philidris myrmecodiae. (Online version in colour.)

The most specialized AGs are farming mutualisms also the largest clade of ant–plants worldwide [29]. because seed planting, cultivation, fertilization or defence To trace the evolutionary history of Australasian against pathogens or [1], as well as harvesting of the and Southeast Asian AGs, we generated phylogenetic crop, all are present. Farming mutualisms require seed recog- frameworks for Lecanopteris, Hoya, Dischidia and the Hydno- nition, here mediated by chemical cues, and convergent phytinae, as well as their most commonly associated ant odours have apparently driven the assembly of Neotropical genus, Philidris. Based on clock-dated phylogenies for the AGs [23–27]. Surprisingly, Neotropical AGs do not involve ants and the plants, we aimed to answer the following domatium-bearing plants, while Australasian and Southeast questions: (i) when, where, and in which sequence did Asian AGs are dominated by such species; in the Neotropics, AG epiphytes and AG-forming Philidris ants originate, and the ants therefore only nest between roots while in Asia, (ii) which trait combinations favored the evolution of AGs they are provided nesting space within their cultivated in Australasia and Southeast Asia? plants. This raises the question of how the assembly of Australasian and Southeast Asian AGs began. Did the assembly start with the cultivation of plants for food (extrafloral nectar or fruits but without domatia) or from 2. Material and methods domatium-based symbioses? Taxon sampling details are provided in the electronic sup- AGs in Southeast Asia and Australasia involve at plementary material, Materials and Methods. least five genera of ants that build carton nests (Philidris, Total genomic DNA was extracted from around 20 mg of leaf tissue, using a commercial plant DNA extraction kit Crematogaster, Pheidole, Camponotus and Diacamma) and 17 (NucleoSpin; Macherey–Nagel, Du¨ ren, Germany) according to genera of plants with together about 78 species ([26], this manufacturer protocols. Polymerase chain reaction (PCR) was study), while Fijian AGs consist only of six plant species performed using Taq DNA polymerase (New England Biolabs, [28]. About 38 of these species belong to the Rubiaceae Cambridge, MA, USA) and a standard protocol (39 cycles, (e.g. Hydnophytum, Myrmecodia and other genera in subtribe annealing temperature 568C). PCR products were purified Hydnophytinae), the Apocynaceae (Hoya, Dischidia) and the using the ExoSap clean-up kit (Fermentas, St Leon-Rot, fern genus Lecanopteris (figure 1). The Hydnophytinae are Germany), and sequencing relied on Big Dye Terminator kits Downloaded from http://rspb.royalsocietypublishing.org/ on March 26, 2017

(Applied Biosystems, Foster City, CA, USA) on an ABI 3130 dispersal–extinction–cladogenesis (DEC), dispersal–vicariance 3 automated sequencer (Applied Biosystems, Perkin-Elmer). (DIVALIKE) and BAYAREA (BAYAREALIKE). Founder-event rspb.royalsocietypublishing.org Sequences were edited in SEQUENCHER v. 5.1 (Gene Codes, Ann speciation is modelled via a speciation parameter j that can be Arbor, MI, USA). All new sequences were BLAST-searched in added to each of the models. We selected the best model based GenBank. Sequence alignment was performed in MAFFT v. 7 on log likelihood values as well as the Akaike information criterion in the online server (http://mafft.cbrc.jp/alignment/server) (DAICc). BIOGEOBEARS statistics are shown in the electronic [30] under default parameters except for the internal transcribed supplementary material table S5. spacer region, which was aligned under Q-INS-i optimization, which takes rRNA secondary structure into consideration. Minor alignment errors were corrected manually in MESQUITE (c) Interactions v. 2.75 [31]. Maximum-likelihood (ML) inference relied on Data on interactions between dolichoderine ants and plant RAxML v. 8.0 [32] and the GTR þ G substitution model, with species in our three focal clades came from the field observations empirical nucleotide frequencies and 25 gamma rate categories; of Eva Kaufman [26] and the two first authors. A few additional B Soc. R. Proc. bootstrap support was assessed from 100 replicates under the interaction links came from field observations by Matthew Jebb same model. We also conducted Bayesian inference in MRBAYES in Papua New Guinea (including Indonesian Papua; [42], table v. 3.2 [33] under the substitution model selected by JMODELTEST2 10.2) or from matching geography and morphological traits; for each marker [34] and using the program default of two runs the latter shown as dashed lines in figure 2. When the ant phylo- and four chains (one cold and three heated), with the uniform default geny includes multiple accessions per species, we only added 284 6 priors. We set a 10 10 Markov chain Monte Carlo (MCMC) chain, putative links to a single specimen. 20161759 : sampling trees every 1000th generation. Split frequencies approach- ing zero indicated convergence. We used the 50% consensus tree to assess posterior probabilities for nodes of interest. (d) Correlated evolution of dispersal by ants with other mutualistic traits and ancestral state reconstructions (a) Molecular-clock dating Molecular dating analyses relied on BEAST v. 2 [35] and uncor- of mutualistic traits related lognormal relaxed clock models unless otherwise stated. To test whether the evolution of dispersal by ants correlates We used the GTR þ G substitution model with four rate cat- with other mutualistic traits, we used BAYESTRAITS v. 2 [43], egories and a Yule tree prior. For both our plant and ant trees, which allows detecting correlated evolution between pairs of MCMCs were run for 20 million (Lecanopteris) or 40 million discrete binary traits. We studied the following traits, all treated (Philidris, Hoya-Dischidia, Hydnophytinae) generations, with par- as binary: ant inhabitants (score ‘0’ when no associations with ameters and trees sampled every 10 000 generations. We used ants are formed or only facultative associations with generalist TRACER v. 1.6 [36] to check that the effective sample size (ESS) ants, ‘1’ for consistent association with one or few specialized of all parameters was more than 200, indicating that runs had ant partners), domatium growth (scored ‘0’ for diffuse growth, converged. After discarding 20% as burn-in, trees were summar- and ‘1’ for apical growth), entrance holes (scored ‘0’ when ized in TREEANNOTATOR v. 1.8 (part of the BEAST package) using absent (no tuber) or smaller than 1 cm in diameter (when the option ‘maximum clade credibility tree’, which is the tree tuber present), and ‘1’ when larger than 1 cm in diameter), with the highest product of the posterior probability of all its warts, absorptive structures inside Hydnophytinae domatia nodes. The final tree was visualized in FIGTREE v. 1.4 [37]. Cali- [44], (scored ‘0’ when present, and ‘1’ when absent), first doma- brations details are provided in the electronic supplementary tium cavity (scored ‘0’ when it enlarges throughout material, Materials and Methods. development, ‘1’ when its development is determinate), post- anthetic sugar rewards (scored ‘0’ when absent, and ‘1’ when pre- sent), seed dispersal (scored ‘0’ for birds, other or gravity (b) Ancestral area reconstruction alone and ‘1’ for dispersal by ants). Trait states were coded Based on the geological history of Southeast Asia [38,39], we based on Huxley [44], Jebb [42,45], Davidson & Epstein [23], defined nine geographical units relevant to the focal clades: Huxley & Jebb [46–49], Maeyama & Matstumoto [50], Chomicki (i) Fiji and Vanuatu (since there was only a single species from & Renner [51] and Chomicki et al. [52], an unpublished revision Vanuatu); (ii) the Solomon Islands; (iii) the Bismarck archipelago, of Hydnophytum from M. Jebb and C. R. Huxley, personal obser- including New Ireland, New Britain, Normanby Island and vations by G.C. 2014, 2015, personal communications to D’Entrecasteaux Islands; (iv) Northern Papua New Guinea inclus- G.C. from M.H.P. Jebb (March 2015) and C. Huxley-Lambrick ive of and limited by the Maoke range in Indonesian Papua and the (October 2015). We tested the correlation of every pair of Bismarck range in Papua New Guinea; (v) South Papua New traits. We used the maximum clade credibility (MCC) tree Guinea (south of the Maoke range in Indonesian Papua and the from BEAST but pruned the outgroups and first ran a model Bismarck range in Papua New Guinea) and Australia; (vi) Sula- of independent trait evolution and estimated the four-transition wesi, Moluccas and the lesser Indonesian Islands (limited by rate parameters a1, a2, b1, b2, wherein double transitions from Java in the west); (vii) the Philippines; (viii) Sundaland (including state 0,0 to 1,1 or from 0,1 to 1,0 are set to zero. We then ran a Indochina, Sundaland, Borneo, Java, Sumatra that were connected model of dependent trait evolution with eight parameters until the Quaternary [38,39]; we also include continental Asia as far (q12, q13, q21, q24, q31, q34, q42, q43). To compare these west as India since our focal clades are younger than 20 Ma, a time non-nested models, we calculated the Bayes factor score. during which these area were constantly connected; and (ix) Pacific We used stochastic character mapping to infer possible his- islands (Hawaii, Wallis and Futuna, French Polynesia), in which tories of mutualistic traits not only at nodes but also along we also included New Caledonia because only two outgroups branches in the phylogenies. We relied on the function ‘make.sim- are native from this island. map’ in the phytools package (v. 04–60) [53], which implements To infer the ancestral areas of Philidris-inhabited AG plants and the stochastic character mapping approach developed by Bollback to probe whether ancestral areas of Philidris match those of their [54]. We estimated ancestral states using: (i) an equal rate (ER) plant hosts, we used ancestral range reconstruction as implemented model and (ii) an all rate different (ARD) model, and then simu- in the R package BIOGEOBEARS [40,41] on the BEAST chrono- lated 1000 character histories on the MCC trees from BEAST. grams. BIOGEOBEARS infers ancestral geographical ranges and We summarized the 1000 simulated character histories using the permits comparison of three biogeographic models, namely function densityMap (also in phytools). Downloaded from http://rspb.royalsocietypublishing.org/ on March 26, 2017

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Miocene Pliocene Quat. Quat. Pliocene Miocene Ma Ma 20 15 10 05 0 5 10 15 Figure 2. Dated phylogenies and ancestral area reconstructions for Philidris ants (left), and Hoya-Dischidia, Lecanopteris and Hydnophytinae plant hosts. Colour- coding on the branches indicates ancestral areas as explained on the inset map. Photos show Philidris myrmecodiae (left), an important AG-forming ant; and domatium-forming AG epiphytes Dischidia nummularia (top right), Lecanopteris carnosa (middle right), and Myrnecodia longifolia (lower right). Photographic credit: M. Janda except lower right, U. Bauer. (Online version in colour.)

3. Results chrysacantha/A. caerulea clade (60/0.55), probably because of missing data for A. chrysacantha. (a) Phylogenetic relationships in Australasian and Southeast Asian dolichoderines and their host (b) Times of origin of ant-dispersed epiphytes plants and their dispersers The 5-gene phylogeny of ants recovered the Our molecular-clock dating estimates are congruent with pre- genus Philidris as monophyletic with maximal statistical sup- vious estimates for the Apocynaceae [57], Rubiaceae [58] and port while the internal topology had little statistical support leptosporangiate ferns [59]. For the ants, no earlier molecular- (electronic supplementary material, figure S1), with the clock dating is available; our secondary calibrations came exception of a few nodes, importantly the Philidris nagasau from a 10-gene Dolichoderinae phylogeny calibrated with six clade from Taveuni Island (Fiji), which was strongly fossils [60]. The most recent common ancestor of Philidris origi- supported (maximum-likelihood bootstrap support (ML nated at 13.2 + 7 Ma, older than the age of the oldest ant- BS) ¼ 81%, Bayesian posterior probability (pp) ¼ 1). Never- dispersed Hydnophytinae lineage (Myrmecodia)(6.3+ 3 Ma), theless, we found strong biogeographic signal. Phylogenetic but Philidris clades of younger ages also inhabit Myrmecodia relationships in Lecanopteris and Hoya-Dischidia are discussed (figure 2). The ant-dispersed Anthorrhiza clade dates to 5.8 + in previous studies [55,56]. Our 6-gene Hydnophytinae 2 Ma. The Fijian P. nagasau clade dates from 3.5 + 2 Ma, match- phylogeny yielded a strongly supported tree (electronic ing the age of its obligate plant host species in the genus supplementary material, figure S2) and implies a single Squamellaria dating to 2.5 + 1.5 Ma (figures 2 and 3). The Hyd- origin of the characteristic modified hypocotyl tuber nophytum moseleyanum group originated some 4.9 + 2Ma. domatium (figure 1a,d,f). The ant-dispersed lineages are Epiphyte seed dispersal independently evolved in Anonycho- recovered with high support: Myrmecodia (ML BS ¼ 89%, myrma more recently, some 2.6 + 2 Ma (figures 2 and 3). The pp ¼ 0.99), H. formicarum (71/0.99), H. moseleyanum (100/1), crown age of Lecanopteris is 4.8 + 2 Ma, and that of ant-dis- Myrmephytum arfakianum (99/1), Philidris nagasau-inhabited persed Dischidia 4.9 + 3 Ma. A minimum of four AG Hoya Squamellaria clade (85/1), except for the Anthorrhiza lineages originated during the last 2 Myr (figures 2 and 3). Downloaded from http://rspb.royalsocietypublishing.org/ on March 26, 2017

ant inhabitants domatium growth entrance holes warts first cavity post-anthetic seed dispersal 5 sugar rewards rspb.royalsocietypublishing.org n naiat oaimgot nrnehlswarts entrance holes domatium growth ant inhabitants

86.62 –27.17 2.74 43.81 39.34 24.82

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2.73 –27.35 10.96 is aiypost-anthetic first cavity

64.93 59.96 sugar rewards

81.10 seed dispersal

Figure 3. Correlates of dispersal by ants in the Hydnophytinae. Binary correlations using Pagel’s (1994) approach (Material and methods) between seven plant traits, numbers in the boxes are Bayes factors (BF) deriving from the comparison of non-correlated and correlated models of trait evolution. Support from Bayes factor is reported by the following colour coding: very strong support (BF . 10): red, strong support (5 , BF , 10): orange, positive support (BF . 2): yellow, no support (BF , 2): white. Phylogenies show ancestral state reconstructions for these seven traits using stochastic mapping (phytools), under equal rate and all rate different models of evolution. For all analyses, 1000 character simulations were computed. (Online version in colour.)

(c) Biogeographic analysis [38,39]. From Australia-Southern New Guinea, Philidris The biogeographic model comparison yielded the DEC þ J colonized Sundaland around 10 Ma (figure 2), including model as best fitting for Philidris (236.47 versus 244.17 today’s Borneo, peninsular Malaysia, Sumatra, Java and for DEC), the Hoya-Dischida data (279.44 versus 291.46 continental Asia [38,39]. Fiji was apparently colonized by for DEC), the Hydnophytinae (LnL ¼ 2130.53 versus long-distance dispersal also from Australia-Southern New 2170.05 for DEC) and Lecanopteris (225.53 versus 227.07) Guinea (figure 2), and since P. nagasau is endemic from (electronic supplementary material table S5). Philidris likely Vanua Levu and Taveuni, the maximal age of the coloniza- originated in Australia because during the Mid-Miocene, tion is 4 Ma; Vanua Levu and Taveuni only emerged 0.8 Ma Southern New Guinea was still submerged [38,39]. The [61,62]. Alternatively, the colonization of Fiji could have decrease in sea level in the Late Miocene resulted in the con- happened as early as 6.5 + 3Ma (P. nagasau stem age, nection of Australia to the emerging Southern New Guinea figure 2) on Viti Levu (the oldest, and largest Fijian [38,39], enabling Philidris to expand its range by ca 10 Ma island), and then Philidris would have gone extinct on this and providing a stepping-stone for the colonization of island. Northern New Guinea between 10 and 5 Ma (figure 2), The Hoya–Dischidia clade apparently originated in conti- which at the time formed several disconnected landmasses nental Asia, and underwent at least two long-distance Downloaded from http://rspb.royalsocietypublishing.org/ on March 26, 2017

(a) MIOCENE PLIOCENE PLEISTOCENE 6

New Guinea and Australia Philidris crown group rspb.royalsocietypublishing.org

Anonychomyrma scrutator group Myrmecodia

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Southeast Asia Southeast Asian Philidris clade 284 20161759 : lineage colour legend ant-dispersed Dischidia clade AG Dolichoderinae ants (Philidris and anonychomyrma) Hoya multiflora Hoya spartioides AG Apocynaceae (Dischidia and Hoya) Hoya lacunosa Hoya imbricata

Lecanopteris Hoya darwinii clade Southeast Asian Lecanopteris clade 2000 km AG Hydnophytinae Myrmecodia tuberosa group (Anthorrhiza, Myrmecodia, Myrmephytum, Hydnophytum, and Squamellaria) Hydnophytum formicarum group

(b) Fiji Philidris nagasau group Vanua Levu New Guinea Southeast Australia Asia Taveuni 50 50 44 Taveuni island subclade 34 Fiji 25 25 100 km

0 0 ant-dispersed Squamellaria clade 50

25 6 Fiji 0 2000 km

15.0 12.5 10.0 7.5 5.0 2.5 0 million years ago Figure 4. (a) Temporal assembly of ant gardens (AGs) in the three main biogeographic areas. We simplified the areas shown in figure 2 into three main areas where Asian/Australasian AGs originated. Triangles represent clades (crown ages), while single lines represent singletons (thus stem age). The grey error bars show the 95% CI from BEAST. Bars connecting to clades depict stem branches. (b) Directions and intensities of the dispersal of AG lineages within the three main areas (Papua New Guinea and Australia, Southeast Asia and Fiji), with a bar graph showing the number of AG plant species per area. (Online version in colour.)

dispersal events to Southern New Guinea/Australia in the to a few widespread species (figure 2) for which phylogeo- Pliocene and Quaternary (figure 2). The Philippines were graphic work would be required. recurrently colonized from Sundaland during the same period (figure 2). The Hydnophytinae clade apparently origi- nated in northern New Guinea some 15.5 Ma, at a time when it was separated from Australia [38]. An early dispersal event (e) Correlated evolution of mutualistic traits occurred from New Guinea to the South Pacific archipelago Of the 21 pairwise correlations between each of the seven of Fiji and Vanuatu (figure 2, [51]). The Solomon Islands binary traits (ant inhabitants, domatium growth type, entrance were colonized by dispersal, not vicariance, since the Solomon hole size, wart presence or absence, first cavity enlargement, arc had drifted from the Fiji–Vanuatu arc some 12 Ma [61,62]. post-anthetic sugar rewards presence or absence, dispersal While most of the Hydnophytinae radiation (ca 75%) is ende- type), 11 were very strongly correlated (Bayes factor . 10), mic from New Guinea, a number of dispersal events to one was strongly correlated (5 , Bayes factor , 10), three islands off Moluccas, Philippines, Sulawesi and Sundaland were positively correlated (Bayes factor . 2) and six were occurred during the last 5 Myr. For the fern Lecanopteris,we not correlated (figure 4). Dispersal type (presence or absence were unable to infer its detailed biogeographic history owing of dispersal by ants) was correlated with all mutualistic traits Downloaded from http://rspb.royalsocietypublishing.org/ on March 26, 2017

except the size of entrance hole. The plots summarizing the implies the earlier existence of AGs without domatium-bearing 7 stochastic mapping of ancestral states are shown in figure 4. plants or at least without the domatium-bearing lineages rspb.royalsocietypublishing.org studied here. That dispersal by ants and rewards for ants (in the form of nectar) are correlated in the oldest domatium-bear- 4. Discussion ing lineage here studied, the Rubiaceae clade Hydnophytinae (figure 4), suggests that food rewards may be a pre-adaptation (a) The spatio-temporal assembly of Australasian for AG lineages, as they are more generally for farming mutual- isms [1]. Other pre-adaptations for the convergent evolution of ant-gardens AG plants and ants may include chemical compounds in the The assembly of the 13 AG plant lineages studied here occurred seeds [25–27], making them attractive to ants, and polydomy independently in three areas, two of which already harboured [22], an ant species’ ability to form several interlinked nests,

Philidris (New Guinea/Australia and Sundaland) and one which allows negotiating the lag-time between seed-planting B Soc. R. Proc. where the inferred time of colonization by Philidris coincides and rewards (domatia or food), essential in any farming mutu- with the inferred evolution of AG plant lineages (Fiji) (figures 2 alism. The recurrent recruitment of new epiphyte lineages into and 3). Surprisingly, the Philidris crown age predates that of the the AGs probably promoted the evolution of domatia, which oldest AG lineages by 6 Myr. Perhaps Philidris plant farming enhance uptake of ant-derived nutrients [44,64,65]. This leads 284 only evolved in the very Late Miocene, about 6.3 Ma, when to the, perhaps counterintuitive, conclusion that the broadening the oldest domatium-bearing lineage, Myrmecodia, first diversi- of host ranges by the ants led to the specialization of new plant 20161759 : fied on New Guinea. This would imply that Philidris lineages hosts. initially did not nest in plant domatia. Indeed, years of field observations across Papua New Guinea (2004–2014) by M.J. have revealed much variation in these ants in terms of nest for- 5. Conclusion mation and acceptable nest spaces, mostly with low plant host specificity. Alternatively, previous AG communities dissolved Our study reveals the assembly of tropical Asian AGs and their plant lineages went extinct or are not sampled here. through time and space. We inferred a minimum of 13 The second-oldest domatium-bearing lineage in our sample, independent origins of AG plants during the last 6–7 Myr. Dischidia, is from Sundaland and dates to 3–5 Ma, followed by Our dating and biogeographic analyses revealed that AGs the2–5 MaoldSquamellaria clade from Fiji, which today is exclu- evolved independently in Australasia and Southeast Asia sively and obligatorily inhabited by P. nagasau [51] and whose where the often domatium-nesting Philidris ants were already crown age matches that of Fijian Philidris (figures 2 and 3). present, with subsequent dispersal between these two areas New AG lineages were repeatedly recruited following and a uniquely specialized AG system evolving in Fiji. The broadening of the host range. This appears to have occurred host broadening by Philidris ants resulted in the recurrent in New Guinean Hydnophytinae (H. moseleyanum species entry of diverse (probably pre-adapted) plant lineages into group) and also among distantly related groups, such as Hyd- the AG ‘adaptive zone’ (sensu Simpson [66]), resulting nophytinae and Lecanopteris ferns, although it remains unclear in the specialization of additional hosts. That the ants may whether ants disperse the spores of this fern [26]. The non- be the key driver both in AGs (this study) and in terrestrial domatium-bearing orchid Dendrobium insigne has also been ant/plant symbioses [67] matches the overall asymmetry of recruited into AGs [63]. In Sundaland, host broadening also ant/plant symbioses worldwide. Such symbioses involve led to multispecies gardens with non-domatium-bearing 113 species of ants and 684 species of vascular plants [29], species, including orchids, Araceae, Apocynaceae, Gesneria- highlighting that generalists can drive mutualism specializ- ceae, Melastomataceae, Moraceae, Piperaceae, Polypodiaceae, ation, consistent with the asymmetric specialization found Urticaceae and Zingiberaceae [26], growing together with in mutualistic networks where a subset of specialists interacts domatium-bearing species from the three clades studied here with generalists [68,69]. (figures 2 and 3). Dispersal events must be responsible for Data accessibility. Data are available in TreeBase accessions http://purl. the AG lineages now found in Sundaland, New Guinea and org/phylo/treebase/phylows/study/TB2:S1476 (Dischidia and Australia (figures 2 and 3b) and led to the origin of an Hoya) and Dryad accession http://dx.doi.org/10.5061/dryad.4tv0s additional epiphyte-farming lineage within the Dolichoderi- [70] (Philidris, Hydnophytinae and Lecanopteris). nae genus Anonychomyrma (figures 2 and 3). Anonychomyrma Authors’ contributions. G.C. designed study and analysed the data; G.C. and M.J. generated the data; G.C. and S.S.R. wrote the manuscript and Philidris have different niches (dry vegetation versus rain- with edits from M.J.; all authors provided reagents. forest, lowlands versus high altitude areas [42,44], M. Janda Competing interests. We declare we have no competing interests. 2014, personal observation), which probably helped the Funding. This work was supported by a grant from the German expansion of AGs in New Guinea. Research Foundation (DFG), RE 603/20, and grants from the Society of Systematic Biologists and the American Association of Plant to G.C. M.J. was supported by a grant from Czech Science (b) Mutualism specialization following host broadening Foundation: P505/12/2467 and the Marie Curie IOF PIOFGA2009- 25448. onto pre-adapted plant lineages Acknowledgements. We thank Eva Kaufmann for Philidris samples, The correlation between the evolution of ant domatia and seed Matthew Jebb and Camilla Huxley-Lambrick for discussion, Andreas dispersal by ants (figures 2 and 4), together with the inference Wistuba for samples of cultivated plants, Jeremy Aroles for proof- that the oldest Australasian ant-dispersed epiphyte lineages reading the manuscript, and William Baker for identifying were domatium bearing, suggests that these AGs evolved the palm in figure 1c. M.J. extends his gratitude to the staff of the New Guinea Binatang Research Center for field assistance, from domatium-based symbioses, not food-only-based mutual- to V. Novotny and N. Pierce for assistance with the Papua New isms, answering our second research question. Nevertheless, the Guinea project and to M. Borovanska and P. Matos-Maravi for older age of the Philidris lineage than any of the plant lineages assistance with molecular data. Downloaded from http://rspb.royalsocietypublishing.org/ on March 26, 2017

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