Parallel evolution of termite-egg mimicry by sclerotium ... and Yashiro (2010) Biol J Lin Soc.pdforigin of egg mimicry in the athelioid group (Yashiro & Matsuura, 2007). To elucidate

Parallel evolution of termite-egg mimicry bysclerotium-forming fungi in distant termite groups

KENJI MATSUURA* and TOSHIHISA YASHIRO

Laboratory of Insect Ecology, Graduate School of Environmental Science, Okayama University,Okayama 700-8530, Japan

Received 19 November 2009; revised 17 January 2010; accepted for publication 17 January 2010bij_1444 531..537

Among the great diversity of insect–fungus associations, fungal mimicry of termite eggs is a particularlyfascinating consequence of evolution. Along with their eggs, Reticulitermes termites often harbour sclerotia of thefungus Fibularhizoctonia sp., called ‘termite balls’, giving the fungus competitor-free habitat within termite nests.The fungus has evolved sophisticated morphological and chemical camouflage to mimic termite eggs. To date, thisstriking insect–fungus association has been found in eight temperate termite species, but is restricted to the lowertermite genera Reticulitermes and Coptotermes. Here, we report the discovery of a novel type of termite ball(‘Z-type’) in the subtropical termite, Nasutitermes takasagoensis. Phylogenetic analysis indicated that the Z-typetermite ball is an undescribed Trechisporoid fungus, Trechispora sp., that is phylogenetically distant fromFibularhizoctonia, indicating two independent origins of termite-egg mimicry in sclerotium-forming fungi. Eggprotection bioassays using dummy eggs revealed that Reticulitermes speratus and N. takasagoensis differ inegg-size preference. A comparative study of termite ball size and egg-size preference of host termites showed thatboth fungi evolved a termite ball size that optimized the acceptance of termite balls as a unit investment.Termite-egg mimicry by these fungi offers a model case of parallel evolution. © 2010 The Linnean Society ofLondon, Biological Journal of the Linnean Society, 2010, 100, 531–537.

ADDITIONAL KEYWORDS: brood parasite – egg protection – phylogenetic analysis – termite ball.

INTRODUCTION

Social insect colonies have evolved diverse defensivestrategies to prevent parasite invasion (Schmid-Hempel, 1998; Cremer, Armitage & Schmid-Hempel,2007). However, once these defences are overcome,insect colonies can provide parasites an ideal envi-ronment for homeostatic shelter (Hughes, Pierce &Boomsma, 2008). Mimicry is a common strategy ofsocial parasites, enabling them to steal into the nestsof hosts (Lambardi et al., 2007; Nash et al., 2008;Barbero et al., 2009). One striking example is fungalmimicry of termite eggs, whereby the parasiticfungus, the so-called ‘termite ball’, gains protectionand competitor-free habitat in the termite nest. Thisfungus relies on termites for defense against desicca-tion and other microorganisms, where frequentgrooming by workers keeps the survival rate of thetermite balls at almost 100% (Matsuura, Tanaka &

Nishida, 2000). The evidence obtained to date indi-cates that the interaction is parasitic, in that it isbeneficial for the fungus but costly for the host ter-mites, at least in the short term. In this sense, thetermite-ball fungus is a fungal cuckoo, which takesadvantage of the brood care of host species by mim-icking eggs. Detailed interactions between the egg-mimicking fungus ‘termite ball’ and host termiteshave been described previously (Matsuura, 2006;Matsuura & Yashiro, 2009; Matsuura et al., 2009). Asin avian brood parasitism, egg discrimination by thehost acts as a selection pressure for the evolution ofegg mimicry by the parasite (Brooke & Davies, 1988).The cuckoo fungus performs sophisticated morpho-logical and chemical camouflage so as to be tended bythe termites. However, we still do not understand howselection by host termites facilitated the evolution ofthe sophisticated egg mimicry of the cuckoo fungus.

Termite balls tended by Rhinotermitidae termitesare sclerotia of an Athelioid fungus (Basidiomycota,Agaricomycotina) of the genus Fibularhizoctonia(teleomorph: Athelia; Matsuura et al., 2000). To date,*Corresponding author. E-mail: [email protected]

Biological Journal of the Linnean Society, 2010, 100, 531–537. With 3 figures

© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 531–537 531

egg-mimicking fungi have only been found in temper-ate regions in seven Reticulitermes species(Matsuura, 2005; Yashiro & Matsuura, 2007) and inCoptotermes formosanus (K. Matsuura, unpubl. data).Phylogenetic analysis of termite-ball fungi isolatedfrom different host species has indicated a singleorigin of egg mimicry in the athelioid group (Yashiro& Matsuura, 2007). To elucidate the evolutionaryprocess, a comparative study among similar interac-tions of independent origins could be a powerful tool.Unfortunately, comparative approaches have thus farbeen impossible because all known host speciesharbour termite balls of the same species.

Here, we report a novel termite-egg mimickingfungus (the ‘Z-type’ termite ball) from the subtropicaltermite Nasutitermes takasagoensis. We conducted aphylogenetic analysis of termite-ball fungi to investi-gate the phylogenetic relationship between the Z-typeand currently known termite-ball fungi. Based onphylogenetic evidence of two distinct origins, wefurther explored the evolutionary selections ontermite-ball size in egg-mimicking fungi by examininghow selection is related to the egg size and sizepreference of different host termites.

MATERIAL AND METHODSSAMPLING

We collected 13 nests (nine arboreal and four moundnests) of N. takasagoensis from Iriomote Island, south-ern Japan, in February 2008 (Fig. 1). Locations of eachcollection site were recorded using a GarminGPSMAP � 60CSx handheld GPS unit (Garmin Inter-national, Olathe, KS, USA; Table 1). To determine thefactors affecting the distribution of termite balls in

host termite nests, we generated a logistic regressionmodel with three explanatory variables: nest type(mound or arboreal), nest size, and colony stage (ergo-nomic or reproductive). Model results were furtheranalysed with a likelihood ratio test using JMP v6.0.3(SAS Institute, Cary, NC, USA). Carton nests werecarefully dissected to extract egg piles from nurserychambers. Eggs and termite balls extracted from eachnest were placed in a Petri dish (90 mm in diameter)lined with moist 100% pulp unwoven cloth, along with200 workers, before transporting to the laboratory. Werandomly chose 50 eggs and 50 termite balls from eachof two representative colonies for size analysis.Termite workers always grasp the short side of ovaleggs while carrying them, and thus use the shortdiameter as a morphological cue in egg recognition(Matsuura, 2006). The short diameter of each egg andthe diameter of each termite ball were measured to thenearest 0.001 mm under a stereomicroscope, using thedigital imaging system FLVFS-LS (Flovel, Tokyo,Japan). Size data were analysed using nested analysisof variance (ANOVA) with colony and host species asindependent variables.

PHYLOGENETIC ANALYSIS

Detailed descriptions of fungal isolation, DNA extrac-tion and the phylogenetic analysis were described inprevious papers (Matsuura, 2006; Yashiro & Mats-uura, 2007). The five strains of Z-type termite-ballfungus were isolated from the egg piles of N. takasa-goensis colonies (#3, 5, 8, 9, and 12) from IriomoteIsland, Okinawa, Japan. To isolate termite-ball fungi,c. 20 termite balls were extracted from each colony.They were rinsed with sterilized distilled water and

Table 1. Nest profile and distribution of termite balls

Colony no. LocationAltitude(m)

Nesttype*

Nestsize (L) Nymph

Termiteball

1 24°25.809′N, 123°46.901′E 6 M 26.5 + -2 24°25.160′N, 123°47.361′E 92 M 82.5 + -3 24°24.332′N, 123°49.986′E 12 A 30.8 + +4 24°23.819′N, 123°48.176′E 26 M 38.2 - -5 24°23.598′N, 123°47.757′E 40 A 49.4 + +6 24°22.910′N, 123°53.718′E 60 A 48.2 - -7 24°22.854′N, 123°53.646′E 76 A 1.7 - -8 24°20.347′N, 123°54.955′E 29 A 12.5 + +9 24°20.323′N, 123°54.968′E 25 A 69.7 + +

10 24°20.314′N, 123°54.959′E 34 A 10.2 - -11 24°20.293′N, 123°55.032′E 35 A 9.0 - -12 24°20.277′N, 123°54.982′E 24 A 17.5 + +13 24°16.012′N, 123°50.939′E 31 M 55.5 + -

*A, arboreal nest; M, mound nest on the ground. See Figures 1A and B.

532 K. MATSUURA and T. YASHIRO

© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 531–537

then arranged on a plain agar plate soon after collec-tion in the field. They germinated on agar within aweek. After germination, each termite ball was inocu-lated on a potato dextrose agar plate (PDA; BectonDickinson and Company, Sparks, MD, USA) contain-ing 200 ppm tetracycline, and incubated at 25 °C for 3weeks.

DNA was extracted from vegetative hyphae of eachfungal isolate using CTAB (cetyltrimethyl-ammonium

bromide) extraction. The entire region of ITS1-5.8S-ITS2 was amplified by polymerase chain reaction(PCR) using the primer set ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), designed by White et al. (1990), andsequenced by ABI 3100 (Applied Biosystems, FosterCity, CA, USA). Sequence data were analyzed usingPAUP4.0b10 (Swofford, 2002). Tremella mesenterica(AY463475) was used as the out-group. Maximum

Figure 1. Two distinct nest types in Nasutitermes takasagoensis. A, an arboreal nest (colony 10). B, a mound nest (colony2). C, Z-type termite balls in a termite egg pile of an arboreal nest (colony 12). Termite eggs are transparent and oval,whereas Z-type termite balls are brown and spherical. D, locations of arboreal nests (circles) and mound nests (triangles)are indicated on the map with colony identification numbers. The nests that had Z-type termite balls in egg piles wereindicated by open circles, whereas the nests without termite balls were indicated by closed circles. No mound nest hadtermite balls.

EVOLUTION OF TERMITE-EGG MIMICKING FUNGI 533


parsimony analysis was conducted using a heuristicsearch with equal weighting of all characters. Toassess the robustness of the relationship, 500 boot-strap replicates were performed with 100 replicationsof random addition sequence. All nucleotide sequencedata of the termite-ball fungus isolates were depos-ited in the DDBJ/EMBL/GenBank nucleotidesequence database. Accession numbers are given inFigure 2. Some sequences were drawn from GenBankfor comparison. A phylogenetic tree including data onthe host N. takasagoensis colonies was reconstructedby the method described in a previous paper (Yashiro& Matsuura, 2007).

EGG PROTECTION BIOASSAY

Dummy-egg bioassays were performed in Petri dishes(35 mm in diameter) using workers from two Reticuli-termes speratus colonies collected in Okayama, Japan,and two N. takasagoensis colonies. To determine theegg-size preference of workers, we used dummy eggsmade of glass beads of 22 different size classes (0.10–1.20 mm at 0.05-mm intervals). Extraction of crudeegg-recognition pheromone from eggs was conductedusing published methods (Matsuura et al., 2007).Glass beads were coated with the crude pheromone ofeach species at a concentration of 1.0 mg per bead. Werandomly arranged ten eggs and 20 dummy eggs onmoist unwoven cloth in a Petri dish (35 mm in diam-eter), and ten workers were released into the dish.Dishes were maintained at 25 °C in the dark. After24 h, the number of dummy eggs carried into egg pileswas counted. The size of glass beads accepted byworkers was compared with the egg size and termite-ball size using Mann–Whitney U-tests. Based on thedistribution of the size of accepted dummy eggs, weestimated the optimal termite-ball size that maxi-mized the number of termite balls tended by termitesat a unit resource investment. The relative probabilityof being tended by termites, i.e. size preference func-tion, is given by the probability density function:

P xx

( ) =− −( )⎧

⎨⎩

⎫⎬⎭

ασ

μσ2 2

2

2πexp ,

where parameters m and s are the mean and standarddeviation of the size of accepted dummy eggs, respec-tively, and a is a standardized coefficient to make themaximum preference (Pmax) equal to 1. The productionof larger termite balls requires more hyphae, as scle-rotium is a tightly connected hypha. The cost ofproducing a termite ball of size x is represented as:

C xx

( ) =( )4 23

3π.

Thus, the relationship between termite-ball size andfitness can be modelled as:

F x P x C x( ) = ( ) ( )β ,

where b is a standardized coefficient to makemaximum fitness (Fmax) equal to 1 at the optimal size.We estimated the termite-ball size to achieve optimalpreference, and the size to gain optimal fitness. fromthe pooled data of two colonies of each host species.The expected optimal preference size and the optimalfitness size were compared with actual termite-ballsize and egg size using two-tailed Student’s t-tests.

RESULTS

The nest type and colony stage of N. takasagoensissignificantly affected the distribution of Z-typetermite balls (nest type, likelihood ratio, c2 = 9.36,P < 0.01; colony stage, c2 = 10.59, P < 0.01), whereasnest size did not (likelihood ratio, c2 = 5.27 ¥ 10-12,P > 0.05). Z-type termite balls were only found incolonies at the reproductive stage in arboreal nests(Table 1). The altitude of nest location did not affectthe nest type (likelihood ratio, c2 = 0.012, P > 0.05).

Phylogenetic analysis of the fungi based on internaltranscribed spacer (ITS) sequences indicated that theZ-type termite-ball fungus is an undescribed Trechis-poroid fungus, Trechispora sp. (Basidiomycota,Agaricomycotina), which is most closely related toTrechispora incise (Fig. 2). Phylogenetic analysis ofhost termites showed no significant molecular differ-ences between arboreal and mound nesters in N.takasagoensis.

Egg size significantly differed between R. speratusand N. takasagoensis (nested ANOVA; species, F1,196 =35.69, P < 0.0001; colony, F2,196 = 4.38, P = 0.014;Fig. 3). Termite-ball size also significantlcy variedbetween host species (nested ANOVA; species,F1,196 = 2266.0, P < 0.0001; colony, F2,196 = 7.59, P <0.001). In R. speratus, termites preferred dummy eggsthat were significantly larger than both the actual eggsize (Mann–Whitney U-test, Z = 9.269, P < 0.0001)and the termite-ball size (Z = 8.9708, P < 0.0001),although the sizes of eggs and termite balls did notsignificantly differ (Z = 0.777, P = 0.437). Similarly, inN. takasagoensis, the preferred size of dummy eggswas significantly larger than the actual egg size(Z = 14.605, P < 0.001) and the size of Z-type termiteballs (Z = 7.900, P < 0.0001). In this species, the sizesof eggs and Z-type termite balls were significantlydifferent (Z = 12.21, P < 0.001).

The mean termite-ball size (0.34026 ± 0.0375 mm,SD) matched the expected optimal fitness size of0.344 mm (t99 = 0.525, P > 0.05), but significantly dif-fered from the expected optimal preference size(0.4304 mm) of the host R. speratus (two-tailed Stu-dent’s t-test: t99 = 25.25, P < 0.0001; Fig. 3). Similarly,in N. takasagoensis, the mean size of Z-type termite



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DISCUSSION

The evolution of mimicry for brood parasitism reflectsthe recognition system and preference of the hosts(Brooke & Davies, 1988; Alvarez, 2000; Barbero et al.,2009). In egg protection, preference for egg sizeslarger than the normal egg size is common in birds(Tinbergen, 1953; Alvarez, Arias de Reyna & Segura,1976; Haartman, 1981). The general attraction oflarge egg sizes may partially explain why cuckoo eggsare usually slightly larger than the eggs of their hosts

(Alvarez, 2000). We found that dummy eggs that werelarger than true eggs were more attractive to termiteworkers. In termites, the egg size depends not only onthe initial investment to each egg but also on embry-onic development (Matsuura & Kobayashi, 2007).Therefore, a larger egg size indicates a greater benefitat a lower remaining cost of egg care during hatching.The strong large-size preference in egg protection inN. takasagoensis adequately explains why Z-typetermite balls are larger than the eggs of its host.

In addition to morphological mimicry, chemicalcamouflage is needed for the fungus to mimic termiteeggs and thus be tended by termites. The termiteegg-recognition pheromone consists of the antibacte-rial protein lysozyme (Matsuura et al., 2007) and thedigestion enzyme b-glucosidase (Matsuura et al.,

Figure 3. Comparison of egg size (A), size preference (B), and termite-ball size (C) in Reticulitermes speratus (left) andNasutitermes takasagoensis (right). In the photos, eggs are oval and transparent, whereas termite balls are spherical andlight brown. Preference P(x) and fitness F(x) functions of termite-ball size are indicated in (C) (R. speratus, m = 0.430,s2 = 0.00983, Of = 0.344; N. takasagoensis, m = 0.7187, s2 = 0.0258, Of = 0.587). Optimal fitness size Of is the termite-ballsize to gain the optimal fitness Fmax.



2009). Both enzymes are major salivary compounds intermites and are also produced in termite eggs. Ter-mites produce b-glucosidase for cellulose digestion asa primary function, and also use it for egg recognitionas a secondary function. The termite-ball fungus in R.speratus nests mimics termite eggs by producingb-glucosidase (Matsuura et al., 2009). The enzymeb-glucosidase has many distinct biological roles infungi, including essential functions in cellulose diges-tion in decay fungi, and in the pathogenicity of phy-topathogenic fungi (Kinghorn & Turner, 1992). Whenconsidering the evolutionary process of chemicalmimicry, it is noteworthy that both Fibularhizoctoniaand Trechispora termite-ball fungi originally had thepotential to produce b-glucosidase, as both fungalgroups are decay fungi, and some Athelia fungi areplant pathogens. Production of b-glucosidase togetherwith sclerotium formation would have served as animportant pre-adaptation for the evolution of termite-egg mimicry by these fungi.

The overlap of the cellulose digestion niche betweentermites and the fungus sharing the same chemicalsprovided the opportunity for the origin of termite-eggmimicry by the fungus. Egg mimicry, through whichthe fungus can easily gain access to the centre of thenest, seems to be an evolutionary detour around anti-parasite defence in termites. Our findings suggestthat termite-egg mimicry by sclerotium-forming fungievolved at least twice independently under similarselection pressures from host termites, thus providingan ideal system to study parallel evolution.

ACKNOWLEDGEMENTS

This research was funded by grants from the JapanSociety for the Promotion of Science (JSPS) to KM(grant no. 09001407) and The Program for Promotionof Basic Research Activities for Innovative Bio-sciences (PROBRAIN) to KM (grant no. 07051747).

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Parallel evolution of termite-egg mimicry by sclerotium ... and Yashiro (2010) Biol J Lin Soc.pdforigin of egg mimicry in the athelioid group (Yashiro & Matsuura, 2007). To elucidate