Department of Entomology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK 2

Transcription

1 Zoological Journal of the Linnean Society, 2008, 153, With 11 figures Termite soldier defence strategies: a reassessment of Prestwich s classification and an examination of the evolution of defence morphology using extended eigenshape analyses of head morphology OLIVIA I. SCHOLTZ 1 *, NORMAN MACLEOD 2 and PAUL EGGLETON 1 1 Department of Entomology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK 2 Department of Palaeontology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK Received 12 June 2007; accepted for publication 2 August 2007 The abundance of termites in many habitats, in particular tropical forests, makes them a valuable source of food for potential predators. Termites have developed a complex system of colony defence that includes the nest, and worker and soldier castes. A classification exists for the mechanical types of soldier defence, which is based on the relationship between morphology and function. This study re-examines the classification system using extended eigenshape analysis as an outline-based morphometric technique. Separate analyses were conducted on the major soldier defence structures: mandibles, head capsule and labrum. Varied support for the defence groups in these structures was demonstrated. Only glue-squirting Nasutitermitinae and asymmetrical snapping mandibles in Termitinae were strongly supported by a selection of head features and had little overlap with the remaining groups. The morphometric descriptions of shape were mapped onto a recent Isopteran phylogeny, and nodal head shapes modelled to examine the evolution of soldier head morphology. Little structural change was observed in the basal nodes and lineages. In contrast, morphological diversification has occurred within the Termitinae subfamily, with several instances of convergent evolution of particular characters, e.g. extended nasus, and mandibles shaped specifically for snapping and piercing The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 153, ADDITIONAL KEYWORDS: character mapping morphometric analysis predation. INTRODUCTION Termites are an abundant and often diverse component of ecological systems, particularly in the forested habitats of the tropical regions (Eggleton et al., 1996, 2002). They are known as ecosystem engineers due to their influence on both the structure, and the biological and chemical components of soil (Lavelle et al., 1997). They also have a very high biomass in ecosystems, particularly in tropical forests (Eggleton et al., 1996), and are eaten by both specialist and non-specialist predators, as varied as birds, primates, amphibians and ants. Termites are relatively easy *Corresponding author. Olivia.scholtz@plymouth.ac.uk, O.Scholtz@nhm.ac.uk prey as they have static colonies with high population densities where most individuals have no defensive abilities. A good example of these predator prey systems is the interaction between termite colonies and raiding termitophagous ants (Dejean & Feneron, 1999; Bayliss & Fielding, 2002). Nesting and foraging life-type strategies (Abe, 1987) have been linked with the defence strategies of the colony, with the colony soil interface influencing predation opportunities (Abe, 1991). One-piece nesters who forage within their (wood) nesting substrate are afforded the greatest inherent protection, while separate-piece nesters are exposed to predation when foraging away from the nest. Mound structure, foraging strategies and investment in soldier defence can reduce predation pressure. Such 631

2 632 O. I. SCHOLTZ ET AL. Table 1. Summary of the Prestwich classification of termite soldier mechanical defence (Prestwich, 1984) Phragmosis (PH) Crushing mandibles (CM) Slashing mandibles (SM) Piercing (BI) Glue squirting (GS) Daubing brush (DB) Symmetrical snapping (SSN) Slashing/snapping Asymmetrical snap (ASN) The head is used to plug entry holes in the colony, creating a physical barrier to predators; cylindrical head, a heavily sclerotized concave rostrum, and shortened mandibles Lower termites, e.g. Cryptotermes. Behaviour also employed by some higher termites, e.g. Cubitermes Mandibles are serrated and robust... The soldiers are low in abundance, sluggish and often phragmotic.... restricted to primitive families of lower termites... the mandibles are more slender, straighter, longer and have a greater angular motion...frequently coupled with injection of materials Higher and lower termites, e.g. Rhinotermitidae, Serritermitidae and Termitidae... slender, inwardly curved mandibles with prominent marginal teeth. Piercing may be accompanied by chemicals... Lower termites, intermediate nasutes and major soldiers of higher rhinotermitines... mandibular regression... developed an ejected terpenoid secretion instead of mandibles. Elongated tunnel-shaped nasus within Nasutitermitinae... mandibular reduction occurred during the development of the labral brush of nasutoid minor soldiers... defence accomplished by topical application of lipophilic contact poisons... storing energy in elastic distortion of the mandibles and releasing it abruptly to strike a percussive blow.... most effective in confined spaces, where the spent soldier blocks an entry hole... mandibles may function in both slashing and snapping modes... thicker, highly elastic mandibles allow lateral blows to be delivered only to the left adaptations include arboreal nesting, a thickened mound with heavily guarded chamber openings, and subterranean and/or covered galleries leading to food sources. It is therefore apparent that colony defence is based on an integrated strategy, involving interactions between nest structure, and soldier and worker morphology and behaviour (Noirot & Darlington, 2000). The contribution of each of these elements is varied and has yet to be quantified or examined in detail across the entire termite clade. The woodnesting (one-piece feeding) species within the lower termites appear to have the most inherent colony protection (Lenz, 1994), while soldier termites appear to contribute to the guarding and protection of gallery entrances rather than of foraging parties in Coptotermes (Rhinotermitidae) (Cornelius & Grace, 1997). Within species that forage outside the nest, soldiers are necessary to guard foraging parties (Noirot & Darlington, 2000). Although workers have important subsidiary roles in colony defence, the soldier caste has a purely defensive role (for rare exceptions see Thorne, Breisch & Muscedere, 2003), and their morphology, chemical ecology and behaviour are highly specialized. Although the role, behaviour and morphology of a sterile soldier caste in colony defence may have arisen secondarily to intercolonial fighting among reproductives (Thorne et al., 2003), the specialized defence traits of soldiers have probably evolved predominantly in response to ant predation. Soldiers use a number of defences both independently and in combination. Two general classifications of functional structures and chemistry have been proposed (Deligne, Quennedey & Blum, 1981; Prestwich, 1984). Deligne et al. (1981) distinguished four major categories of weaponry: phragmotic, mandibular, salivary and frontal. Prestwich (1984) further grouped these categories into mechanical and chemical defences, and included an analytical description of defence mechanisms based on morphological and behavioural observations, and a tabular summary of the literature. Within the mechanical category, partial functional diagnoses relating to termite head morphology were provided (Table 1, Fig. 1). These diagnoses were broadly qualitative. The Prestwich classification has become accepted as a very useful classification scheme for variation in the morphology of the soldier head. It has, for example, been employed to hypothesize a link between habitat and defensibility during the social diversification of termites (Thompson et al., 2000). There is considered to be a close association between termite life-type, diversification and defence strategies (Abe, 1991), although this has not yet been fully quantified. The Prestwich classification is a valuable tool in these types of investigations. There has been

3 MORPHOMETRICS OF TERMITE SOLDIERS 633 Figure 1. The soldier termite mechanical defensive mechanisms described by Prestwich (1984). Reprinted with permission, from the Annual Review of Entomology, Volume by Annual Reviews no previous effort to test the statistical validity of Prestwich s morphologically determined functional groups. Recent advances have been made in understanding the functional diversity, phylogenetic relationships and diversification of termites (Eggleton & Tayasu, 2001; Davies et al., 2003). An accurate statistical description of soldier morphology will add to this framework and allow examination of the evolution and ecology of termite defence strategies. Here we quantify variation in soldier head capsule morphology, using extended eigenshape analysis (EES), in order to examine patterns of morphological variation. We then examine how far this range of morphologies fits the Prestwich classification. We go on to map the morphometric outcomes on to a recently constructed well-corroborated termite phylogeny (Inward, Vogler & Eggleton, 2007) and show that there is a degree of plasticity and convergence in the evolution of termite soldier head capsules, and by extension in soldier defence strategies. METHODS TAXA AND CLASSIFICATION Fifty-two taxa were chosen for the present study, selected on the basis of the Prestwich defence classification (Table 1) and phylogenetic coverage from a

4 634 O. I. SCHOLTZ ET AL. Figure 2. x,y coordinate outline data are converted to a series of f (angular deviation) data points, which have been plotted. The position of the geometric landmark for extended eigenshape analysis is labelled. recent molecular phylogeny of the Termitidae (Inward et al., 2007). All four termitid subfamilies were included: Macrotermitinae, Apicotermitinae, Termitinae and Nasutitermitinae. Four additional families were also included, namely Kalotermitidae, Hodotermitidae, Rhinotermitidae and Serritermitidae, although with fewer representatives due to the known similarity in soldier morphology and defence classification (Appendix 1). In addition, taxa that have not been placed into a classification were included in the analysis. Scanning electron micrographs were available from previous taxonomic research at the Natural History Museum, London (Donovan et al., 2000). Images consisted of a dorsal view of the head orientated in a flattened position with the labrum and mandibles extended on a horizontal plane with the head capsule. These provided the source from which morphometric data were taken. As a result, within-species replication was not possible in this study. Some of these species, however, possess polymorphic soldiers, and all morphs were represented in the analysis. EXTENDED EIGENSHAPE ANALYSIS There are several statistical methods for measuring shapes. Traditionally, multivariate analyses (e.g. Principal Components Analysis, PCA) of distance and angles between landmark points have been used. More recently, outline analysis has been developed as a valuable morphometric tool that can analyse the outlines of shapes. This uses interlandmark curves in order to include biologically important aspects of shape variation. It can be applied to simple shapes (Lestrel & Roche, 1986) and complex shapes (Premoli, 1996). Two methods unite the outline and landmark approaches: landmark-based analyses incorporating outline information (Bookstein & Green, 1992; Bookstein, 1996) and outline-based methods incorporating landmark information (Ray, 1992; MacLeod, 1999). EES (MacLeod, 1999) is the latter approach based on standard eigenshape analysis of outlines (Lohmann, 1983). There are several benefits of both standard and extended eigenshape analysis for morphometric analysis: (1) it handles both simple and complex shapes, and open and closed curves; (2) it can be extended to three-dimensional curves; and (3) it allows the modelling of shapes within the empirical morphometric shape space (i.e. within the principal axes). Eigenshape analysis (Lohmann & Schweitzer, 1990) begins by describing a curve as the angular deviation (f or phi) from the expected direction of a step from the previous step, through a series of equidistant steps (semi-landmarks). The curve, which begins as a series of x,y coordinate points along the outline, is converted into a series of angular deviations between equally spaced points (Fig. 2). The same number of angles represents each object. This means that the length of the steps represents object size, and, during the conversion to f, size is therefore removed from the analysis. The shape functions originate from a common point on each object, thereby geometrically and biologically aligning the outlines with respect to each other. Singular value decomposition is the multivariate method employed in eigenshape analysis (Lohmann, 1983), on the pairwise covariance or correlation matrix for each shape description (f series). Patterns of interobject shape variation are quantified into the fewest number of independent axes. The following are obtained from the analysis. 1. Eigenvalues for each axis of variation: axis-specific vector lengths that describe the amount of shape

5 MORPHOMETRICS OF TERMITE SOLDIERS 635 Figure 3. Soldier head features independently analysed; straight lines represent the size measurements: dots, landmark points; S, start point; F, finish point. variance contained within each axis relative to the total sample shape variance. For example, samples of similar shapes possess little interobject variance, and therefore a large proportion of the sample variance can be subsumed into the first axis, compared with more varied samples. 2. Eigenfunctions for each axis (termed eigenshape ): the equation that describes the axis-specific modes of shape change and encompass the major aspects of shape variation within the sample. These are vector multiples of the original covariance/ correlation matrix eigenvectors. 3. Axes scores for each object which relate the object s shape to the axis-specific eigenshape functions, and therefore relative position within the morphometric space based on intersample shape variation. The shape specific to a point in morphometric space can be described using the axes score and eigenfunction. Plots of the scores along each eigenshape axis position the objects within the eigenshape space. This represents a linear decomposition of the shapes with respect to interobject shape relationships. Outlines can be constructed by the re-conversion of a f series, which is calculated from the positional relationship (axis score) and eigenshape function. EES allows the specification of landmarks within the shapes that represent biologically or geometrically homologous sites. Constraining the sequence comparisons between the landmark points improves the correspondence of shape descriptive points over the entire outline (Fig. 2). The result is an improved estimate of true shape variance, with corresponding improvements in efficiency of the multivariate decomposition based on (1) improved correspondence between the shape functions on which the analysis is based and (2) differential weighting of more information-rich aspects of the morphology in estimation of the result. OUTLINE DATA CAPTURE For this study a number of soldier head characters were selected for extended eigenshape analysis (Fig. 3). These were characters specifically described in the Prestwich (1984) classification. The left mandible and right mandible were treated separately because there is obvious asymmetry between them in some taxa. Outlines of the mandibles constituted the external mandible sections when still attached to the head, thereby representing the functionally relevant components. A dorsal view of the head capsule shape and the labrum are separate features, in order to avoid analysing outlines that represent biologically different structures (i.e. the anterior shape of the head capsule as against the labrum). The SEM images were traced into simple independent outlines of the features described above (including the scale bar) and saved as digital picture files. Therefore, there were four separate outlines for each taxon, except where the features were reduced, absent or heavily obscured, and were consequently excluded from the analysis (Appendix 1). Mandibles are absent or vestigal, and possess a non-defensive function in some taxa (e.g. Nasutitermes). The labrum similarly can be reduced or obscured by the head capsule (again Nasutitermes). The tpsdig32 program ( sunysb.edu/morph) captures closed outlines of digital shapes, describing them as a specified number of x,y coordinate points. Specification of landmark points allowed open or closed outlines to be captured depending on the requirements, i.e. closed head shape and open mandible and labrum outlines, with the final x,y files for each feature calculated by the conversion program (tpsdigconvert.exe) (Fig. 4). EIGENSHAPE ANALYSIS AND SHAPE MODELLING The conversion of the x,y coordinates to f shape functions in an extended eigenshape xy-phi.exe

6 636 O. I. SCHOLTZ ET AL. Figure 4. Outline data treatment for eigenshape analysis. Original outline described by 300 x,y points: S, start point; F, finish point; LM, landmark point. Converted to 250 equally spaced x,y coordinate points of the open outline. x,y plot of the f points with a 99% tolerance criterion to retain the outline between LM, S and F points. conversion program ( palaeonet/ftp/ftp.html) allowed tolerance criteria (for retaining the observed shapes) to be specified. The number of steps required to recover the actual shape from all the samples to the specified criteria is automatically calculated, and this varies between different landmarks depending on the complexity of the curve in the regions. The tolerance criterion was set for 99% in all data sets. The result is a reduced set of points for analysis without a loss of resolution in shape variation and increased computational efficiency (Fig. 4). EES of the f data was performed in the ExtES.exe program, providing the three sets of information described above. The axes scores obtained were plotted to display the relationships of intersample shape variation along the principal axes of the analysis. The eigenshape functions summarize the principal modes of shape variation within the sample and allow reconstruction of shapes of specified scores. The mode of shape change relating to each axis can be displayed by constructing models pertaining to points distributed along the axis (e.g. Fig. 5C). The ExtESmodel.exe program calculates the representative f series shape description when provided with axes position scores and eigenshape functions. The ExtESphi->xy.exe program converts the f series into its respective x,y coordinates of shape which can then be plotted to view the shape. STATISTICAL TESTING OF THE PRESTWICH CLASSIFICATION We analysed the fit of our EES analysis with the Prestwich classification using Discriminant Function Analysis (DFA) of the EES axes scores tested against the classification. Species with a defence group assigned by Prestwich (1984) were included in the analysis (Appendix 1). Discriminant analysis tests how well the classification discriminates between the axes scores for each feature using MANOVA. This provides statistical tests for each axis of each feature, summarizing the degree of association of each axis with the Prestwich classification. Therefore, a reduced number of axes can be identified that best differentiate the outline shape patterns of defence. This analysis also produces a series of tables indicating misclassifications of taxa based upon their morphometric position in relation to the morphometric variation within each defence groups. PHYLOGENY CHARACTER MAPPING The morphometric analysis provides a quantitative description of intersample morphological relationships, and an objective framework from which to conduct further analyses of morphological patterns. When making use of morphometric descriptors, it should be noted that the axes values are intersample not absolute descriptors of shape (Swiderski, Zelditch & Fink, 2002). In the case of eigenshape analysis, the axes values are relevant only to their respective eigenshape functions of the initial sample analysis. Mesquite (Maddison & Maddison, 2004) was used to map the eigenvector scores provided for each sample onto a phylogenetic tree (Inward et al., 2007). The axis values were treated as continuous variables, using least squared parsimony to map the nodal axes scores. Least squared parsimony minimizes the sum of the square of change on each branch of the phylogeny (Maddison, 1991), and unlike linear parsimony calculates a single node value. The reconstructed scores for the first three axes at each node were recorded and used to model their representative shapes using the ExtESmodel and ExtESphi->xy programs. Repeating this for each head feature enabled the construction of complete modelled soldier heads for each node of the phylogeny. Placing the constructed head shapes onto the phylogeny provides a

7 MORPHOMETRICS OF TERMITE SOLDIERS 637 A B C Figure 5. Head capsule EES plots. A, axis 2 vs. axis 1. B, axis 2 vs. axis 3. C, modelled mode of dorsal head capsule shape change along axes 1, 2 and 3. convenient presentation of morphological change across the phylogeny. In the case of polymorphic soldiers within a species each morph was treated as separate taxa during mapping, forcing the assumption that polymorphism is subsequent to the speciation event of the terminal polymorphic taxa. The alternative would have been to include only one of the morphs, e.g. the major morph across all relevant species, thereby removing the monomorphic species state assumption but sacrificing information on polymorphic adaptation. Axes values for a feature are calculated at common nodes where all the branches themselves possess values. In the case where a feature (e.g. labrum or mandibles) was missing on one of the branches, the common node would lack mapped axes values, therefore implying its loss. To overcome this, the shape pertaining to the apical branch in possession of the feature was modelled onto the nodal head shape conforming with an accelerated transformation optimization (ACCTRAN; Swofford & Maddison, 1987).

8 638 O. I. SCHOLTZ ET AL. Table 2. Discriminant Function Analysis results for the three principal axes from each eigenshape analysis. Significant results in bold DFA score F P Related shape change Head capsule (ax1) > 0.1 Overall shape axis Head capsule (ax2) < 0.01 Convex nasus to concave hollow Head capsule (ax3) < Rostrum with concave base to convex point Right mandible (ax1) > 0.1 Mandible wide to narrow Right mandible (ax2) < Flat broad blade to hooked Right mandible (ax3) < 0.05 Medial tooth to medial notch Left mandible (ax1) > 0.1 Mandible wide to narrow Left mandible (ax2) < Twisted left to hooked right Left mandible (ax3) > 0.1 Medial notch to medial tooth Labrum (ax1) > 0.5 Extent of anterior development of labrum Labrum (ax2) < 0.01 Bi-lobed to single central lobe Labrum (ax3) > 0.05 Narrow extended to wide with central lobe RESULTS THE MORPHOLOGICAL RELATIONSHIPS Head capsule The analysis of head capsule shape demonstrates a clear division between termites that possess an elongated nasus and those that do not (Fig. 5A, C). This separation occurs along axis 2, and partly in axis 1. In the absence of morphometric analysis, identifying termites with an elongate nasus is, of course, straightforward. However, the analysis does demonstrate the absence of a continuous morphological distribution from short to elongate. This distinct cluster at low axis 2 values largely comprised the glue-squirting (GS) group (Table 1), which have either no mandibles or only vestigial points (Fig. 1). Two biting/piercing and injecting classified (BI) taxa are present within the nasute cluster, but possess functional mandibles. Unlike the GS soldiers, these have direct predator contact, using a combination of mandible action and chemical secretion from the nasus. The remaining major cluster in the upper region of axis 2 is continuously distributed along axis 3, with no clear morphometric clustering according to the defence groups. Support for the classification is found along axis 2 and 3 (Table 2), which encompass variation within the frontal projection, and a subtle shape change also in the anterior region of the head, respectively (Fig. 5B, C). Mandibles Mandibles in some cases are strongly asymmetrical, resulting in different patterns of shape variation and distribution between the left and right mandible analyses. Analysis of the left mandible clearly separates taxa with very outwardly bent mandibles along axis 2, which when modelled displays a change from an outward to inward curvature (Fig. 6A, C), with the morphometric values strongly supporting the Prestwich classification (Table 2). There is no apparent functional grouping along axis 1 or 3 (Fig. 6B). The major patterns and modes of axis shape variation are similar in the left and right mandible analyses, with the degree of mandible curvature captured in axis 2 supporting the classification (Table 2), as well as toothing described by axis 3. The distinctiveness of the asymmetrical snapping (ASN) has been lost in the right analysis (Fig. 7A). The right mandible of ASN taxa is robust and straight, and is responsible for the strong lateral blow when released from the outwardly bent left mandible. The symmetrical snapping (SSN) mandibles are morphologically more similar to the ASN, and less closely associated with the remaining groups. Labrum A distinct gap is evident along axis 1 (Fig. 8A), between a small group of broad and shallow structure in lower axis 1, and the remaining more prominent shapes (Fig. 8C). None of the shallow shapes belongs to classified taxa. However, their shape and distribution may be an artefact of SEM image capture, in the event that the head capsule was partially obscuring the labrum. No clear morphometric clustering of the three daubing brush (DB) representatives is apparent; instead they are widely distributed in the upper half of axis 2, and mid to lower region of axis 3 (Fig. 8B). Support for the classification is found in how the labrum is lobed (Fig. 8C, Table 2), which does not, however, have a clear defensive function. PHYLOGENETICALLY MAPPED HEAD SHAPES The head shapes of the deep nodes and branches of the non-termitidae families show little change from a

9 MORPHOMETRICS OF TERMITE SOLDIERS 639 A B C Figure 6. Left mandible EES plots. A, axis 2 vs. axis 1. B, axis 2 vs. axis 3. C, modelled mode of mandible shape change along axes 1, 2 and 3. large head capsule and relatively short robust mandibles (Fig. 9). The most significant shift is seen at node a within the family Rhinotermitidae where the minor morphs of Dolichorhinotermes and Schedorhinotermes possess a strongly extended labrun for daubing chemicals onto predators. Apical to the Termitinae/Nasutitermitinae common node morphological changes become common and varied. A tendency towards asymmetric mandibles arises twice at nodes b and c in polyphyletic Termitinae clades. It is interesting that at node b mandibular asymmetry precedes symmetrical mandibles, while at node c asymmetry is more derived. The projection of the nasus in combination with mandibles arises within the Termitinae (node d ) and Syntermitinae (node e ) subfamilies. The most prominent projected nasus occurs at the base of the Nasutitermitinae, where the mandibles are reduced and vestigal. A shift to a

10 640 O. I. SCHOLTZ ET AL. A B C Figure 7. Right mandible EES plots. A, axis 2 vs. axis 1. B, axis 2 vs. axis 3. C, modelled mode of mandible shape change along axes 1, 2 and 3 piercing-type structure of inwardly curved mandibles is apparent in the Syntermitinae (node e) and two Termitinae (nodes f and g ) lineages. DISCUSSION MORPHOMETRIC TESTING OF THE PRESTWICH CLASSIFICATION Phragmotic soldiers Many Kalotermitidae are phragmotic, and the habit is found sporadically across many other families. The blunt and often heavily sclerotized heads are used as an effective barrier to block gallery entrances, predominantly in wood. The mandibles are generally short, and the antennae are often protected by ridges on the head capsule (Deligne et al., 1981). Cryptotermes dudleyi Banks (Kalotermitidae) was the only one of our study taxa that Prestwich classified as phragmotic. However, in the EES analysis we could not differentiate it from other taxa that are not classified as phragmotic. Consideration of both head and mandible shape together may be necessary to reveal structural patterns associated with phragmotic behaviour (i.e. sclerotized, ridged head and short mandibles combined). Some species soldiers show phragmotic behaviour despite lacking the convex

11 MORPHOMETRICS OF TERMITE SOLDIERS 641 A B C Figure 8. Labrum EES plots. A, axis 2 vs. axis 1. B, axis 2 vs. axis 3. C, modelled mode of labrum shape change along axes 1, 2 and 3. sclerotized rostrum commonly associated with this defence (Deligne et al., 1981), confounding easy identification of this strategy based on morphology. It is apparent therefore that a phragmotic defensive behaviour is not strongly associated with a specific morphology. Reticulitermes (Rhinotermitidae) combines mandibular action with phragmotic behaviour, and displays intercolony stabilization of head size corresponding with gallery openings to the nest (Matsuura, 2002). Prestwich suggests that symmetrically snapping soldiers may block galleries after striking, thereby providing an additional defensive function. In support of this, the protruding vertex of Dihoplotermes major and minor and of Spinitermes is reflected in their lower position along axis 2 of the head capsule analysis along axis 2 (Fig. 5B); however, this is not demonstrated for Termes, which also possesses a raised vertex. Mandible biting Crushing (CM) is considered a plesiomorphic condition, often in association with phragmosis, and with and without chemical association (Prestwich, 1984). However, the mandibles classified as crushing display little morphological homogeneity (Figs 6, 7), and are not as consistently well supported as crushing in the DFA (Table 3). Although the slashing mandibles (SM) loosely group together in the left mandible EES analysis, there is little support for this group in the DFA even when only considering the mandible results (Table 3). Slashing behaviour has been associated with a biting/ piercing mode and symmetrically snapping (Mill, 1982) and in combination with chemical secretions (Prestwich, 1979; Deligne et al., 1981), suggesting that a discrete slashing structure based on mandible shape may not exist.

12 642 O. I. SCHOLTZ ET AL. Figure 9. Mapped and reconstructed soldier head shapes positioned across a Termitidae phylogeny. Nodes a g are referred to in the text. The broad functions within the biting, piercing and injecting group (BI) are demonstrated by the dispersed distribution of the BI taxa within the EES plots. Marked exceptions are Curvitermes and Armitermes (Syntermitinae), which possess an elongated nasus used to secrete chemicals, positioning them with the glue-squirting group in the head capsule analyses, although their curved mandibles may perform a piercing function (Fig. 6B). Nasus development associated with mandibular regression in the glue-squirting group, confined to the Nasutitermitinae, is clearly supported by the head capsule analyses (Table 3). Due to the distinctive nasute character, Syntermitinae was included within the Nasutitermitinae until quite recently (Engel & Krishna, 2004). Mandibular regression is also associated with the development of the labrum as a daubing brush (DB) to deposit chemicals secreted from the frontal gland pore onto predators. This is true in its most extreme form (e.g. Dolichorhinotermes minors; mandibulate morphs are completely absent in Acorhinotermes), though in several species with daubing brushes, mandibles are present and functional. The labrum, however, is not a well-supported character and is therefore not effective at distinguishing the DB taxa (Table 3). In Rhinotermitidae an evolutionary path within this defensive strategy has been proposed (Deligne et al., 1981) from prominent mandibles and simple apical brush, to vestigial mandibles, elongated clypeolabrum and bushy brush. Morphs within a species include the extremities of this pathway (e.g. Dolichorhinotermes major and minor), which possibly relates to the division of social (foraging and defensive) tasks that has been observed in the soldier caste (Kaib, 1987).

13 MORPHOMETRICS OF TERMITE SOLDIERS 643 Table 3. Discriminant Function Analysis supported defence categorization of each head feature analysis, with a final category (shown as the new category) assigned based on majority support, from the informative features (i.e. mandibles and head capsule) P classification Labrum Left Right Head capsule Maj Acanthotermes acanthothorax int BI BI BI BI BI BM Acanthotermes acanthothorax maj BI BI BI BI CM BM Acanthotermes acanthothorax min BI BI BI BI BI BM Amitermes amifer BI BI BI BI BI BM Angularitermes nasutissimus GS * * * GS GS Armitermes cerradoensis BI * BI BI GS PI Basidentitermes aurivilli SM SM BI BI BI BM Capritermes sp. ASN DB ASN ASN ASN ASN Cavitermes tuberosus SSN DB SSN SSN BI SSN Cephalotermes rectangularis BI BI BI BI BI BM Coptotermes sjoestedti BI BI BI BI BI BM Cornitermes cumulans BI BI BI BI BI BM Crenetermes albotarsalis BI SM BI BI BI BM Cryptotermes dudleyi CM BI BI BI CM BM Cubitermes falcifer SM SM BI BI BI BM Cubitermes fungifaber SM ASN BI BI BI BM Curvitermes odontognathus BI BI BI BI GS PI Cylindrotermes parvignathus SM BI BI BI BI BM Dihoplotermes inusitatus maj SSN/ASN * SSN ASN SSN SSN Dihoplotermes inusitatus min SSN/ASN * SSN ASN SSN SSN Dolichorhinoterms longilabius maj CM BI BI CM BI BM Dolichorhinoterms longilabius min DB BI * * DB DB Globitermes globosus BI BI BI BI BI BM Heterotermes platycephallus BI BI BI BI BI BM Hodotermes mossambicus CM CM CM CM BI CM Hypotermes xenotermitis BI BI BI BI BI BM Labiotermes labrallis BI BI BI BI BI BM Longipeditermes longiceps maj GS * * * GS GS Longipeditermes longiceps min GS * * * GS GS Macrotermes malaccensis maj BI BI BI BI BI BM Macrotermes malaccensis min BI BI BI BI BI BM Microcerotermes boreus SM BI BI BI BI BM Nasutitermes corniger GS * * * GS GS Neocapritermes araguaia ASN * ASN ASN ASN ASN Occasitermes occasus GS * * * GS GS Pericapritermes nitobei ASN SM ASN ASN ASN ASN Planicapritermes planiceps ASN SM ASN ASN BI ASN Schedorhinotermes intermedius maj DB BI BI BI DB BM Schedorhinotermes intermedius min DB SSN CM CM BI CM Serritermes serrifer CM BI SSN BI BI BM Spinitermes trispinosus SM SM BI BI BI BM Termes hospes SSN BI SSN SSN BI SSN Termitogeton planus BI BI BI BI CM BM Trinervitermes trinervius maj GS * * * GS GS Trinervitermes trinervius min GS * * * GS GS Mandibular snapping ASN is a complex interaction between the opposing mandibles and musculature action, and asymmetry is required for the right mandible to deliver a violent blow. The ASN and SSN groups are supported in both the left and the right mandible analysis (Table 3). ALTERATIONS TO THE CLASSIFICATION TO EXAMINE EVOLUTIONARY TRENDS It is apparent that there is varied support for the Prestwich taxonomy based on the EES and DFA results; only the glue squirting and snapping

14 644 O. I. SCHOLTZ ET AL. Figure 10. The EES axes and values of morphometric variation that support a revised soldier defence classification. a, Planicapritermes; b, Dihoplotermes; c, Cavitermes; d, Termes. strategies are well supported due to their discrete morphology pertaining to defensive behaviour. In order to determine whether there is statistically significant support for the remaining functional groups, further analysis may be necessary that considers both the three-dimensional head shape, and a single analysis of all head features to include information on their relative size and orientation. For the purposes of examining the evolution of defensive strategies here, alterations to the classification have been made where necessary using the statistically significant axes identified in the DFA (Table 2). The BI, SM and CM categories have been sunken into a single biting mandible (BM) group, due to limited support across the head features in the DFA (indicated where there is a change in the group membership, Table 3) and continuous distribution within a three-dimensional plot of three axes most relating to defensive function (Fig. 10). Two exceptions are: Hodotermes, which has majority support to retain a CM category (Table 3), and Dolichorhinotermes minor as DB due to an extended nasus and lack of functional mandibles. As expected, the glue-squirting group clearly cluster together although an artificial mandible axis value of 0 has been assigned due to the lack of this feature (Fig. 10). The BI taxa with an extended nasus also cluster here, although the retention of functional mandibles separates them and they have been placed into an introduced piercing/injecting (PI) group. Mapping this temporary classification onto the phylogenetic tree (Fig. 11) shows a basal state of BM, with subsequent evolution of ASN (twice), SSN (once), PI (twice), DB (once) and GS (once). The BM strategy is retained within Termitinae and Syntermitinae lineages. However, due to the variation of structures yet lack of differentiation between them in this study, there may be quite distinct defensive behaviour occurring in BM taxa across the phylogeny. THE DIVERSIFICATION OF SOLDIER MORPHOLOGY Due to the lack of morphometric distinction in several of the groups, mapping the eigenshape analysis results across the phylogeny allowed an additional yet unconstrained consideration of soldier head evolution (Fig. 9). The shapes modelled on the basal nodes support the suggestion that ancestral termite soldiers had large heads with robust crushing-type mandibles (Prestwich, 1984). The single-piece nesting structure of primitive termites is thought to be the most significant factor in colony defence (Lenz, 1994). The direct colony defence function of primitive soldiers has been questioned, although they do seem to provide an intrinsic cost-benefit by improving the reproductive output of the colony (Roisin, 2000; Roux & Korb, 2004). The less extensive defensive role in primitive soldiers may diminish selective pressures on morphological adaptations directly for defence. Indeed, observations of Cryptotermes secundus (Hill)

15 MORPHOMETRICS OF TERMITE SOLDIERS 645 Figure 11. Revised classification mapped on to the termite phylogeny. (Kalotermitidae) revealed few behavioural differences between soldiers and workers, which may have been due to low predation and competitive pressures (Roux & Korb, 2002). However, in other Kalotermitidae, a corresponding shift in habitat type and defence to phragmosis by soldiers (employing Prestwich s classification) has been proposed (Thompson et al., 2000). Although this supports the hypothesized link in defence and habitat during termite diversification (Abe, 1991), it also demonstrates the complexity of interdependent defence strategies (e.g. life-type ) and soldier function, which have yet to be fully explored. The probable sister group to Termitidae, Rhinotermitidae, includes members with greater morphological variability such as polymorphic soldiers that display defensive polyethism within the colony (Kaib, 1987). This defensive variation may be attributable to a change in lifetype from single-piece to intermediate- and separate-piece nesting (Eggleton & Tayasu, 2001). Foraging away from the nest will inevitably increase predation pressure and change the selective nature of such pressure. The apparent retention of a plesiomorphic morphology in Apicotermitinae soldiers could be related to the life-type of most species: soil dwelling and feeding with a lack of a defined nest boundary (Eggleton et al., 1996, 1997). If one equates this with the singlepiece nest strategy of the basal termite families, a reduced predatory pressure may arise, corresponding to the colony surface area exposed to attack (Maki & Abe, 1986). Apicotermitinae workers often contribute to colony defence, including phragmotic behaviour and autothysis (gland rupture) (Sands, 1972; Costa- Leonardo, 2004). The secondary loss of sterile soldiers

16 646 O. I. SCHOLTZ ET AL. is found in the Apicotermitinae (and at least one Termitinae lineage), underlining the fact that retaining a caste solely for defence may be both unnecessary, in those cases where predation pressure is reduced due to soil dwelling, and/or impossible given energetic limitations imposed by feeding on soil, which is a highly recalcitrant energy source (Eggleton, Davies & Bignell, 1998). The fungus-growing Macrotermitinae display polymorphism and a morphological shift towards long and slender mandibles while maintaining a prominent labrum similar to that found in Rhinotermitidae for depositing chemicals (Fig. 9). Intense predatory pressure from termitophagous ants has been described in Macrotermitinae, with defensive polyethism (i.e. they have up to three soldier morphs) among soldiers in a colony (Konate et al., 2000). Beyond the Apicotermitinae, the prolific speciation within the Termitinae, Syntermitinae and Nasutitermitinae subfamilies is accompanied by structural diversification in soldier morphology. There are several instances of convergent evolution of characters. However, these rarely occur in combination with a similar suite of other characters. The loss of functional mandibles and acquisition of a prominent nasus at the base of the Nasutitermitinae has been stable, with little discernible change seen in subsequent nodes and apical branches. The other instances of a developed frontal projection ( nasus ) are within the Syntermitinae, where the projection is used to deposit chemicals on to predators, and at node d in Termitinae possibly for a phragmotic purpose in combination with snapping mandibles. The polyphyletic origin of asymmetrical snapping is supported in this study (Noirot, 2001). The evolution of strongly curved mandibles, possibly for piercing, is polyphyletic within Termitinae, and is found in some members of the Syntermitinae (Fig. 9). CONCLUSIONS The EES conducted on soldier head features demonstrated the plasticity in morphology within and between groups defined in the Prestwich classification. No single head structure can be used to differentiate between all the groups. The anterior region of the head capsule and degree of curvature of the mandibles were the most informative of functional associations. The identification of a group requires considering several characters, e.g. nasute development on the head capsule and presence/absence of mandibles for the GS and PI, and degree of curvature of the mandibles for the snapping mandible groups. Morphological variation was largely continuous between the taxa that lack these more distinctive features. Due to the ambiguity of assigning taxa to these groups (crushing, slashing, biting and piercing mandibles) a large combined group was assigned for the consideration of evolutionary trends. A limitation of this study was the need to consider each head feature independently to avoid capturing apparently homologous data for non-homologous structures due to the two-dimensional nature of the outline data. This reduced the strength of the outcomes as relative size and configuration of the shapes was lost, and therefore the mandibulate nasutes and the glue-squirting taxa grouped together on the head capsule analysis. It also imposed the need to assimilate the separate results in a subjective manner to determine group membership. A single analysis of the complete head shape which can capture variation in the head capsule, mandibles and labrum in three dimensions would resolve this problem. Extended eigenshape analysis on three-dimensional data is possible, but a suitable data capture method on termite heads has yet to be found. The morphometric outcomes mapped onto a Termitidae phylogeny both unconstrained and as an emended classification demonstrate the polyphyletic origins of some defensive structures. Interestingly, these are not necessarily accompanied by similar characteristics of other structures, e.g. an extended nasute for ejecting chemical secretions is associated with a lack of functional mandibles in the Nasutitermitinae, but is combined with mandibles in the Syntermetinae. The complexity in soldier defensive mechanisms implies a strong selective pressure during the diversification of Termitidae, resulting in the convergent evolution of particular defensive structures and strategies. A selective arms race has been suggested between ant predator strategies and termite defence strategies over the last 50 million years (Noirot & Darlington, 2000). There is no simple predator prey trajectory between ants and termites, and contemporary ant termite interactions within communities are highly varied and complex. The trophic interaction between ants and termites within tropical rain forests, where they often dominate the faunal biomass and diversity (Fittkau & Klinge, 1973; Eggleton et al., 1996), is a particularly important but poorly studied area. Employing morphometric analyses such as EES, delineating statistically supported functional groups and determining phylogenetic relationships can provide a means to explore these interactions. ACKNOWLEDGEMENTS The scanning electron micrographs were supplied by the Termite Research Group at the Natural History Museum, London, from a previous study conducted by

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