Phenotypic integration in a series of trophic traits: tracing the evolution of myrmecophagy in spiders (Araneae)

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Phenotypic integration in a series of trophic traits: tracing the evolution of myrmecophagy in spiders (Araneae) c ˇ Stano Pekár a,∗ , Radek Michalko a , Stanislav Korenko a,b , Ondˇrej Sedo , Eva Líznarová a , Lenka Sentenská a , c Zbynˇek Zdráhal a

Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotláˇrská 2, 611 37 Brno, Czech Republic Department of Agroecology and Biometeorology, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Kam´ ycká 129, 165 21 Prague, Czech Republic c Proteomics Core Facility, Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic b

a r t i c l e

i n f o

Article history: Received 22 January 2012 Received in revised form 25 April 2012 Accepted 2 May 2012 Keywords: Ant-eating Trophic adaptations Prey capture Stenophagy Zodariidae

a b s t r a c t Several hypotheses have been put forward to explain the evolution of prey specificity (stenophagy). Yet little light has so far been shed on the process of evolution of stenophagy in carnivorous predators. We performed a detailed analysis of a variety of trophic adaptations in one species. Our aim was to determine whether a specific form of stenophagy, myrmecophagy, has evolved from euryphagy via parallel changes in several traits from pre-existing characters. For that purpose, we studied the trophic niche and morphological, behavioural, venomic and physiological adaptations in a euryphagous spider, Selamia reticulata. It is a species that is branching off earlier in phylogeny than stenophagous ant-eating spiders of the genus Zodarion (both Zodariidae). The natural diet was wide and included ants. Laboratory feeding trials revealed versatile prey capture strategies that are effective on ants and other prey types. The performance of spiders on two different diets – ants only and mixed insects – failed to reveal differences in most fitness components (survival and developmental rate). However, the weight increase was significantly higher in spiders on the mixed diet. As a result, females on a mixed diet had higher fecundity and oviposited earlier. No differences were found in incubation period, hatching success or spiderling size. S. reticulata possesses a more diverse venom composition than Zodarion. Its venom is more effective for the immobilisation of beetle larvae than of ants. Comparative analysis of morphological traits related to myrmecophagy in the family Zodariidae revealed that their apomorphic states appeared gradually along the phylogeny to derived prey-specialised genera. Our results suggest that myrmecophagy has evolved gradually from the ancestral euryphagous strategy by integrating a series of trophic traits. © 2012 Elsevier GmbH. All rights reserved.

1. Introduction Several hypotheses have been proposed to explain the evolution of food specialisation in herbivores, namely utilisation of enemy-free space, interspecific competition avoidance, coevolution, increased physiological efficiency, optimal foraging, neural constraints, phylogeny and trade-offs (Jermy et al., 1990; Singer, 2008), and it seems that these hypotheses apply to predators as well (Pekár et al., 2012a). Some of these hypotheses are based upon mechanisms intrinsic to the predator. These mechanisms are manifested via evolution of adaptations that secure efficient prey search, recognition, capture and handling. The evolution from euryphagy to trophic specialisation can be achieved by a gradual change of traits from pre-existing adaptations. Such a process should be accompanied by increasing

∗ Corresponding author. E-mail address: [email protected] (S. Pekár).

exploitation of the targeted prey and the evolution of adaptations tightly linked to a certain prey, namely cognitive (innate capacity to identify prey), morphological (trophic structures used in capture), behavioural (capture tactics), venomic (peptide and protein composition) and physiological traits (processing of digested food). The parallel evolution of traits securing efficient prey processing gives rise to a complex phenotype. Such phenotypic integration is the result of a common ancestor, natural selection and genetic drift (Pigliucci and Preston, 2004). Ant-eating or myrmecophagy is one of the most frequent trophic specialisations (Richardson, 1987; Hölldobler and Wilson, 1990; Pekár et al., 2012b). Due to their slender bodies and chemical defences, ants are low-quality prey from both the nutritional and energetic points of view (McNab, 1984). Ants are also noxious due to their aggressive behaviour, their tendency to collectively attack intruders, and their production of various defensive secretions (Hölldobler and Wilson, 1990). Predators exploiting ants are, therefore, expected to evolve a variety of specific adaptations that may, however, constrain the utilisation of alternative prey.

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Please cite this article in press as: Pekár, S., et al., Phenotypic integration in a series of trophic traits: tracing the evolution of myrmecophagy in spiders (Araneae). Zoology (2012), http://dx.doi.org/10.1016/j.zool.2012.05.006

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Adaptations for myrmecophagy can differ markedly between predatory vertebrates and invertebrates. While for vertebrates, ants are a tiny prey subdued by oral (trophic) structures, for spiders, ants are large prey subdued by venom. As a result, the selection forces acting on the evolution of traits will differ in predatory vertebrates and invertebrates. While morphological traits that are specialised on the excavation of nests and consumption are prominent in vertebrate species (Richardson, 1987; Reiss, 2001), in invertebrates, such as spiders, behavioural and venomic adaptations that enable the predator to safely handle ants predominate. Physiological traits, i.e., the effective processing of ant flesh, might be similar in both groups. Myrmecophagy in predatory invertebrates will further involve the evolution of traits that prevent a counter-attack by ants. Thompson (1994) suggested that the origin of specialisation might be unravelled only through a combined understanding of the ecology and phylogeny of the species under focus. Here we aim for such a combined approach in considering the evolution of myrmecophagy in the family Zodariidae. A recent study (Pekár et al., 2012b) suggested that stenophagy (capture of certain kinds of prey only) in Zodariidae is derived from euryphagy (capture of various kinds of prey including ants). Species at the base of Zodariidae are euryphagous (Pekár and Lubin, 2009), while species at derived positions of the tree, such as Zodarion, are stenophagous (Pekár et al., 2005a,b). More importantly, the primitive taxa also capture ants, suggesting that the evolution of myrmecophagy did not require a dramatic shift to a novel prey. Instead, the evolution of myrmecophagy within Zodariidae may have followed a gradual process towards a specialised morphotype, which is achieved by a loss of predatory versatility in all adaptations. The analysis of adaptations in stenophagous ant-eating Zodarion spiders showed a high level of specialised cognitive, behavioural and physiological adaptations (Pekár, 2004; Pekár et al., 2008; Pekár and Toft, 2009). Therefore, we predicted that ancestral taxa should possess generalised or versatile adaptations in all trophic traits. In the present study, we focused on behavioural, venomic, morphological and physiological traits in a closely related but more “primitive” spider, Selamia reticulata (Simon, 1870). We studied the fundamental and realised trophic niche of this species to determine its level of euryphagy. Then we performed a study on capture efficiency and venom composition to reveal the level of specificity in behavioural and venomic adaptations. We also reared the spiders on two diets, a pure ant diet and a mixed diet (that included ants), and compared components of fitness in juvenile and adult female spiders to reveal the level of specificity in physiological adaptations. Finally, we performed a comparative analysis of morphological traits related to myrmecophagy in genera of the family Zodariidae. 2. Materials and methods S. reticulata is a zodariid spider occurring in the western Mediterranean (Jocqué and Bosmans, 2001). The spider does not build a web but lives under stones or in leaf litter, hiding in a Yshaped retreat. From its position inside, the spider ambushes prey crawling upon the retreat (see supplementary video S1 in Appendix A). 2.1. Prey analysis To study the natural prey, we collected prey remnants found around the retreats constructed under a stone, as in the laboratory we had previously observed that S. reticulata ejects remnants of the consumed prey from its retreat. However, these remnants could have also been left by other predators. The recording of prey

remnants was performed in southern Portugal at the Alcaria Ruiva site close to Mértola in April 2009 and 2010. At this time of the year both juvenile (3rd to 4th instar) and mature spiders occur. In an area of approx. 2500 m2 we searched for spiders by carefully lifting stones and inspecting the ground underneath. Beside prey remnants we also collected by hand live potential prey under the stones where S. reticulata spiders were found. The collected prey was then identified to the order and family level. The abundance of actual and potential prey was compared using the 2 test. 2.2. Behavioural experiments To investigate the fundamental trophic niche we conducted a series of acceptance experiments under laboratory conditions using a variety of prey (Table 2). In all, 50 individuals (30 juveniles and 20 adult females) of S. reticulata were tested. In these experiments we placed the spiders singly in a Petri dish (diameter 60 mm) one day prior to the trial. The size of the spider (total body length ranging between 6.8 and 11.2 mm) and the prey size (total body length) were measured with callipers before each trial. The prey was offered to the spiders in 2-day intervals, as prey offered earlier was not accepted due to satiation. The different prey items were offered to the spiders in random order. A repeated-measures design was used, because it provides higher test efficiency by using fewer spiders than a completely random design (Mead, 1988). Each S. reticulata spider was used repeatedly for several prey species. If the spider did not attack the prey within 15 min, the prey was replaced by a different one. In each trial we recorded whether the spiders captured the prey or not. The capture success was compared among prey types by means of generalised estimating equations (GEE) with binomial errors within the R environment (R Development Core Team, 2010). GEE were used in order to account for repeatedmeasures and to correct for too high p-values favouring acceptance of the alternative hypothesis (Hardin and Hilbe, 2003). The logit model within GEE was also used to model the effect of size ratio (total body length of prey to total body length of S. reticulata specimens) on capture success. 2.3. Performance experiments Two experiments were conducted, one with S. reticulata juveniles and the other with adult females. Three days after hatching, spiderlings from three egg-sacs produced by females collected in the field were assigned at random to one of two diet treatments, with 33 juvenile individuals in each treatment. In the experiment with adult females there were 27 individuals in each treatment (each mated with two different males). At the beginning of the experiment the length of the prosoma was measured in all females. In each experiment, spiders were housed singly in tubes (diameter 18 mm, length 60 mm) with a layer of gypsum at the bottom. The tubes were plugged with foam rubber and kept in a controlled environmental chamber simulating the outdoor conditions in spring in Portugal (25 ± 1 ◦ C, L:D = 16:8). The gypsum was kept moistened with a few drops of water at 5-day intervals. Mortality and moulting (in the case of juveniles) was recorded daily. Spiders were weighed in fortnightly intervals using a Kern 770 balance (Kern & Sohn GmbH, Balingen, Germany) with a precision of 0.01 mg. The females were weighed until they produced eggs. Two different diets were used in these experiments with juveniles and adult females. The pure ant diet consisted of a variety of ant species; Lasius alienus (average body size 3.5 mm) and Formica rufibarbis (5.2 mm) (both Formicinae) were used in the experiment with juveniles, while Camponotus pilicornis (7 mm), F. rufibarbis (both Formicinae) and Messor barbarus (9 mm; Myrmicinae) were used in the experiment with adult females. Ants were collected in the field prior to feeding. The mixed diet group was fed with

Please cite this article in press as: Pekár, S., et al., Phenotypic integration in a series of trophic traits: tracing the evolution of myrmecophagy in spiders (Araneae). Zoology (2012), http://dx.doi.org/10.1016/j.zool.2012.05.006

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larvae of Tribolium destructor (10 mm; Tenebrionidae), larvae of Tenebrio molitor (13 mm; Tenebrionidae), Acheta domestica (7 mm; Gryllidae), flies Drosophila melanogaster (3 mm), adult termites Reticulitermes sp. (5 mm) (both Isoptera) and F. rufibarbis. Except for ants, all prey came from laboratory cultures. In the mixed group, one type of prey was given to spiders on each feeding date in a regular systematic order. In the experiment with juveniles, prey was offered on feeding dates twice a week until satiation. Different numbers of prey items were used to compensate for weight differences (for example, either a single Formica or two Lasius ants). When feeding with ants, the head was crushed with a pincer to prevent the ant from killing the spider. All prey remnants were removed the following day. In the experiment with females, individuals were fed on each feeding date (every second day) until satiation. The feeding continued until egg sacs were laid. The juveniles were reared for a period of three months, while females were reared for a period of six weeks. The survival of spiders on different diets was compared with the Cox proportional hazard (CPH) model, as the hazard function was constant (Tableman and Kim, 2004). Generalised least squares (GLS) were used to model weight change over time, as the errors were autocorrelated and heteroscedastic, and to compare the size of offspring (due to the use of a nested design) (Pinheiro and Bates, 2000). The duration of instars was compared using the Welch test (due to heteroscedastic variance). Linear models (ANOVA, ANCOVA) were used for the comparison between diets in cases when the response variable was continuous and errors were homoscedastic (final weight, weight change). Generalised linear models (GLM) were used in cases when the response variable was assumed to come from a Poisson (GLM-p), gamma (GLM-g) or binomial (GLM-b) distribution (Pekár and Brabec, 2009). All these analyses were performed in R.

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Table 1 Relative frequency of the actual (prey remnants) and potential prey of Selamia reticulata in the field, including the estimation of prey diversity. Prey order

% Actual

% Potential

Araneae Isopoda Chilopoda Diplopoda Blattodea Dermaptera Heteroptera Hymenoptera – Formicidae Hymenoptera – others Coleoptera Diptera Total N Simpson–Yule index

0 0 0 2.8 0 0 2.8 33.3 5.6 52.8 2.8 36 2.53

9.6 2.6 0.9 17.5 50.0 0.9 0 10.5 0 7.9 0 114 3.25

was prepared on a Milli-Q plus 185 water purification apparatus (EMD Millipore, Billerica, MA, USA). All chemicals were of analytical grade. Signals reproduced in at least 11 out of the total of 15 spectra obtained from each sample were taken into account. Peptides/proteins from different samples were considered homologous if the masses were within 0.3% of molecular weight and in this case were combined (Sanz et al., 2006). The spectra were compared using the Simpson–Yule diversity index and the coefficient of dispersion (CD), which is the ratio of sample variance to sample mean (Southwood and Henderson, 2000). The spectra were split into categories by 200 Da and for each category the sum of intensity was computed. The values of the Simpson–Yule index and the CD were compared between species using the Wilcoxon rank test. 2.5. Morphological traits

2.4. Capture efficacy and venom composition To determine the capture efficiency for different prey, each one of 20 female S. reticulata individuals collected in the field was offered three prey types of a similar body size of up to 7 mm: M. barbarus ants, Reticulitermes sp. termites, and T. destructor beetle larvae. The spider was placed in a Petri dish of 5 cm diameter 2 h before the trial. The prey was offered randomly over a oneday period. We recorded the time between the bite and complete immobilisation (when the prey did not move appendages after touching it with a pincer) for each prey. The latencies were compared between prey types using GEE with a gamma error structure (due to repeated measures and heteroscedasticity). The analysis of venom gland contents was performed for five individuals of S. reticulata collected in Alcaria Ruiva and five individuals of strictly myrmecophagous Zodarion styliferum (Simon, 1870) collected in Mitra (Portugal). Pairs of venom glands were dissected and placed into 50 ␮l physiological saline (0.9% NaCl). The glands were squeezed and the sample was stored at −20 ◦ C prior to analysis. The samples were subjected to MALDI-TOF mass spectrometric analysis with an Ultraflex III instrument (Bruker Daltonik, Bremen, Germany) operated in a linear positive arrangement under FlexControl 3.0 software. External calibration of the mass spectra was performed using Escherichia coli DH5 alpha standard peaks. Samples were deposited on three positions (wells) of the sample plate at a volume of 0.3 ␮l and, after drying at room temperature, overlain with 0.3 ␮l of saturated alpha-cyano-4-hydroxycinnamic acid (Bruker Daltonik, Leipzig, Germany) in acetonitrile:water:TFA (50:47.5:2.5, v/v) mixture (Merck, Darmstadt, Germany). Five independent spectra, each comprising 1000 laser shots, were acquired from each of the wells. Mass spectra were processed using flexAnalysis (version 3.0; Bruker Daltonik, Leipzig, Germany). Water

We selected six morphological traits that are expected to be related to ant-eating behaviour from a total of 79 known characters (Gertsch, 1961; Jocqué, 1988, 1991) that are expected to be related to ant-eating behaviour. Their apomorphic state is related to myrmecophagy as follows: (1) the presence of short cheliceral fangs, which are assumed to be short to avoid stabbing through the ant’s thin legs; (2) the presence of cheliceral fusion, which is expected to enhance the precision of an attack on an ant leg; (3) the presence of a femoral organ that emits a volatile substance (Pekár ˇ and Sobotník, 2007) probably used by the spider as defence against ants; (4) the presence of flattened indented hairs that imitate the setosity of ants and are used in defence against ants (Pekár and Král, 2002); (5) the absence of spines on all legs which are probably used to dig burrows by burrowing spiders (Jocqué, 1991); and (6) slender legs that enable the spider to run fast and are therefore used for quick attack (Pekár, 2004). We also gathered information on the diet of zodariid spiders from published sources (see Pekár et al., 2012b). Using this information we performed a regression analysis with GEE from the APE package (Paradis, 2006) within R. The model was based on a binomial error structure and a phylogenetic variance–covariance matrix (Paradis, 2006). The phylogeny was constructed on the basis of morphological characters (Jocqué, 1991), the only phylogeny of Zodariidae available. 3. Results 3.1. Prey In the field, the prey of S. reticulata was composed mainly of beetles and ants (Table 1), whereas the potential prey was mainly composed of cockroaches and diplopods. Thus, the potential prey

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0.8

39 8 32 6 20 50 11 20 20 7 45 8 45 20

Survival 0.4 0.6

N

66.7 12.5 12.5 0 100 52.4 45.5 100 100 85.7 75.6 50 75.6 100

Ants Mixed

0.2

% Captured

Araneae Oniscoidea Diplopoda Collembola Blattodea Ensifera Isoptera Heteroptera Lepidoptera – caterpillars Lepidoptera – imagoes Coleoptera – larvae Coeoptera – imagoes Hymenoptera –Formicidae Diptera

0.0

Prey order

1.0

Table 2 Comparison of the relative frequency of acceptance of different prey species by Selamia reticulata females in the laboratory.

(syntopically occurring with the spider) was significantly different from the actual prey (210 = 102.2, P < 0.0001). The diversity of the potential prey was higher than that of the actual prey, suggesting selective capture behaviour (Table 1). The proportion of ants was significantly higher in the actual than in the potential prey (21 = 8.9, P = 0.003). 3.2. Capture behaviour

0.6 0.4 0.2

Probability of capture

0.8

1.0

In the laboratory, S. reticulata captured invertebrate species belonging to 12 different orders, with the highest capture frequency for heteropterans, lepidopterans, cockroaches and dipterans. They ignored collembolans. The capture success for ants was high (76%). Once the prey was attacked it was also consumed in 92.5% (N = 331) of cases. The capture frequency varied for all orders (GEE-b, 29 = 55.1, P < 0.0001, Table 2). The capture frequency for spiders, woodlice and diplopods was lower than for other prey. The capture probability decreased with the body size ratio of prey (GEE-b, 21 = 12.1, P = 0.0006). 50% efficiency was estimated at the 1.25 size ratio, i.e., when the prey body (pooled for all prey species) was only slightly larger than the spider body (Fig. 1). The majority of prey (86%, N = 220) were captured using the grasp-and-hold tactic, in which the spider grasped the prey by the forelegs formed into a basket, bit it, and then held it in its chelicera

0

20

40

60

80

100

Time [Days] Fig. 2. Comparison of the survival of juvenile Selamia reticulata individuals reared on two different diets over a period of 100 days. “+” identifies censored cases.

until immobilisation. However, ants were captured using two different tactics: in 56% (N = 45) of cases, ants were captured using the grasp-and-hold tactic in which the spider bit into the thorax and held the ant in its chelicera until immobilisation with its forelegs stretched away from it. In the remaining cases (44%), the spider used the bite-and-release tactic in which it bit the ant, retreated and stayed away until immobilisation. 3.3. Performance The survival of juveniles did not differ significantly between diets (Cox proportional hazard, 21 = 0.1, P = 0.73, Fig. 2). Neither the developmental rate (Cox proportional hazard, 21 = 0.3, P = 0.6) nor the duration of the first and the second instar phases (Welch test, t45.9 < 1.7, P > 0.1) differed significantly between diets. The duration of the 3rd instar was significantly longer on the ant diet (Welch test, t17.5 < 2.2, P = 0.045), whereas the duration of the 4th instar was longer on the mixed diet (Welch test, t6.6 < 4.4, P = 0.003). Overall, there was no significant difference in the entire duration of the four instars (Welch test, t49.6 < 0.3, P = 0.8, Table 3). Spiders on the mixed diet increased their weight at a significantly higher rate than those on the ant diet (GLS, F1,465 = 8.8, P = 0.003). At the end of the experiment, spiders on the mixed diet weighed two times more than those on the ant diet (Fig. 3). Adult females on the mixed diet increased their weight significantly more than females on the ant diet (ANOVA, F1,51 = 7.8, P = 0.007); thus, females on the mixed diet were significantly heavier before oviposition (ANOVA, F1,51 = 6.3, P = 0.015, Fig. 4A). Females on the mixed diet had a significantly greater clutch size (4 eggs more on average) than those on the ant diet (GLM-p, 21 = 19.5, P = 0.002, Fig. 4B). The size of the female prosoma did not influence

0.0

Table 3 Comparison of the duration (days; means ± s.e.) of four juvenile instars (1st to 4th) of Selamia reticulata reared on two different diets. Instar

0.5

1.0

1.5 2.0 Size ratio

2.5

Mixed

3.0

Fig. 1. Relationship between the probability of successful capture and body size ratio (prey body to Selamia reticulata body) with the estimated logit model.

Diet

1st 2nd 3rd 4th

33.1 41.1 49.2 78.8

± ± ± ±

Ants 1.8 3.7 2.9 5.9

38.6 42.1 63.8 47.0

± ± ± ±

2.6 2.8 5.9 2.5

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the clutch size significantly (ANCOVA, F1,25 = 1.1, P = 0.3). Females on the mixed diet oviposited significantly earlier (by 5 days) than those on the ant diet (GLM-g, F1,48 = 4.4, P = 0.04, Fig. 4C). There was no significant difference in incubation period (GLM-g, F1,29 = 1.1, P = 0.26), hatching success (GLM-b, 21 = 0.1, P = 0.77), size of offspring (GLS, F1,219 = 0.7, P = 0.41) or clutch laying frequency between the two treatment groups (proportion test, 21 = 0.01, P = 0.91).

20

3.4. Capture efficiency and venom profiles

40

Mixed Ants

35 30

Weight [mg]

5

15 10 5 0 0

20

40

60

80

100

Time [days] Fig. 3. Comparison of weight change in juvenile Selamia reticulata individuals reared on two different diets over a period of100 days. Points are means, whiskers represent SE.

A

The paralysis latency was significantly different for the three kinds of prey (GEE-g, 22 = 96.9, P < 0.0001). It was significantly shorter for Tribolium larvae than for Messor ants (contrast, P = 0.0004) and Reticulitermes termites (contrasts, P < 0.0001, Fig. 5). MALDI-TOF mass spectrometric analysis of venom samples revealed approximately 163 peptides/proteins with masses between 2 and 9 kDa in the profile of S. reticulata (Fig. 6A) and 190 peptides/proteins in the profile of Z. styliferum (Fig. 6B). The spectra of S. reticulata and Z. styliferum did not differ in the diversity of peaks (Simpson index = 3.3 vs. 4.2, Wilcoxon test, W = 7, P = 0.31) but differed markedly in the pattern (Fig. 6A and B): the spectrum of S. reticulata had peaks which were significantly more spread out than those of Z. styliferum (CD = 59 vs. 156, Wilcoxon test, W = 0, P = 0.008). This was because the peaks in the spectrum of Z. styliferum were cumulated between 4 and 6 kDa, whereas those of S. reticulata occurred along the entire mass range.

B

0.12

18 16 14

Clutch size

Weight [g]

0.1 0.08 0.06

12 10 8 6

0.04

4 0.02

2

0

0 Ants

Mixed

Ants

Diet

Days to oviposition

C

Mixed

Diet

30 25 20 15 10 5 0 Ants

Mixed

Diet Fig. 4. Comparison of (A) the final weight, (B) clutch size, and (C) days to oviposition in female Selamia reticulata individuals reared on two different diets. Bars are means, whiskers represent SE.

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Paralysis latency [s]

6

250

number of apomorphic states in the selected traits increases with the proportion of ants in the diet (GEE-b, F1 = 21.8, P = 0.005, Fig. 8).

200

4. Discussion

150

100

50

0

Messor

Reticulitermes

Tribolium

Fig. 5. Comparison of the paralysis latency for female Selamia reticulata individuals towards three prey species: Messor ants, Reticulitermes termites and Tribolium beetle larvae. Bars are medians and whiskers represent 95% confidence intervals of medians.

3.5. Morphological traits By mapping the characters on the phylogeny, it can be seen that the position of the apomorphic states of the six traits related to myrmecophagy has accumulated along the phylogeny (Fig. 7). The

We found that S. reticulata is euryphagous as it has adaptations for the capture of a wide array of prey. The diversity index (estimated prey breadth) was typically high, as in other euryphagous spiders (Pekár et al., 2012b). Although S. reticulata is a euryphagous predator, the diversity of the potential prey was still higher than that of the actual prey. However, the analysis of actual prey probably underestimated the true diversity due to the fast disintegration of soft body structures of some invertebrates (e.g., termites). The spiders did not feed on springtails and very little on millipedes. In the field, S. reticulata captured mainly beetles. In the laboratory, it was able to capture prey of a wide size range but the capture efficiency decreased for prey that was larger or much smaller than its body size. This is consistent with observations for other mainly euryphagous, but not stenophagous spiders (Nentwig and Wissel, 1986). The wide trophic niche found for S. reticulata is probably enhanced by generalised or versatile trophic adaptations. In the following, we will compare the levels of specificity in each class – cognitive, morphological, behavioural, venomic and physiological adaptations – found in S. reticulata with those found in the stenophagous ant-eating Z. styliferum spiders. Ants were detected, identified and attacked by S. reticulata, as well as the majority of offered prey items, so the innate cognitive capacity of S. reticulata seems to be wide. This is in contrast to the

Fig. 6. Comparison of two samples of MALDI-TOF mass spectra (2–8 kDa) of venoms from adult females of (A) Selamia reticulata and (B) Zodarion styliferum.

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Fig. 7. Topology of 39 zodariid spider species, with the proportion of ants in the diet indicated by the amount of black in pie charts at the tips of the branches. The six morphological traits are mapped on the tree.

innate capacity of Zodarion spiders, in which only ants and termites elicited an attack when the spiders were moderately hungry (Pekár, 2004). Furthermore, Zodarion locates ants using their pheromone plumes (Cárdenas et al., 2012), which is probably not the case in sedentary S. reticulata as it has never been found in close proximity to ant nests. In Zodarion the cognitive capacity is fine-tuned to ants.

Fig. 8. Relationship between the number of apomorphic traits and the proportion of ants in the diet in 12 zodariid genera. The logit model is displayed. Each point represents a genus and the size of points is scaled to the number of species included in the analysis.

With respect to morphological adaptations in comparison with the ant-specialised Zodarion, S. reticulata has stout legs that allow the prey to be grasped and held firmly during the attack. Zodarion, having slender legs instead, never grasps and holds the prey during an attack. Its slender legs enable Zodarion to run fast and avoid counter-attack by ants (Pekár, pers. observ.). The chelicerae of S. reticulata are larger than those of Zodarion and are not fused; thus, they are probably more powerful and can penetrate even the thick cuticle of beetles. In Zodarion the chelicerae are very small and used only to penetrate the rather soft cuticle of ant legs. The fangs are longer in S. reticulata than in Zodarion, presumably to be inserted more easily into the thick body of invertebrates. Thus, all the morphological adaptations appear to be more versatile in S. reticulata than in Zodarion (Pekár, 2004). Similarly, a study on the evolution of morphological characters in carnivorous mammals (Holliday, 2010) revealed that morphological traits related to hypercarnivory show reduced variance. S. reticulata has a repertoire of at least two prey-capture tactics. It did capture a number of different invertebrates using the graspand-hold tactic, which is a general-purpose tactic efficient for a variety of prey types, including crawling and flying invertebrates. However, ants were also captured using a different tactic: bite-andrelease. This tactic seems to be specialised for the capture of ants – a dangerous prey. It is also used by Zodarion (Pekár, 2004; Cushing and Santangelo, 2002), which always remains at a safe distance from the attacked ant. The capture resulted in a counter-attack of S. reticulata in only 7% (N = 45) of cases, suggesting that this tactic is efficient for ant handling. S. reticulata spiders were able to immobilise different prey types. The comparison of capture efficiency with regard to three prey types revealed a lower efficiency against ants than against beetle larvae. Interestingly, Zodarion was unable to immobilise termites,

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even after repeated attacks (Pekár, 2004). Thus we assume that the venom composition of Zodarion is very specific. This seems to be supported by the profiling of the venoms. In the venom of zodariid spiders small (<6 kDa) and large (>7 kDa) cationic linear peptides seem to predominate (Kuhn-Nentwig et al., 2011). These peptides possess cytolytic activity; in particular, the large ones are superior insectotoxins. In the venom of S. reticulata, several peaks occurred between 2.5 and 8 kDa, which seem to correspond to both small and large peptides. In the venom of Z. styliferum, a group of peaks between 4 and 6 kDa was found, suggesting the presence of short peptides only. Alternatively, the peaks correspond to mini-proteins with a different ion channel inhibition function (Kuhn-Nentwig et al., 2011). Whatever the case, the venom composition of S. reticulata is more varied than that of Z. styliferum. We assume that this corresponds to the wider trophic niche of S. reticulata and indicates generalised venomic adaptations. The performance of juveniles on the two diets was similar in all studied parameters: survival, development and growth. There was basically no marked difference, except for the weight increase. This suggests that S. reticulata can utilise a monotypic ant diet: the body of the ant provides S. reticulata spiders with all essential nutrients required for development. However, the bodies of ants are slim and, therefore, probably less profitable in terms of energetic gain (McNab, 1992) than the bodies of alternative prey, such as beetles. A similar effect was found for females, which achieved lower body size, and as a result females on a mixed diet had higher fecundity than those on ants. When combining survival and fecundity in a Leslie transition matrix, slightly better fitness was achieved on a mixed diet (finite rate of increase:  = 2.6) in comparison with the monotypic ant diet ( = 2.2). This is in agreement with results obtained for other euryphagous predators (e.g., Toft, 1999; Bilde and Toft, 2000). However, Zodarion spiders suffered high mortality and had slow growth rates when reared on prey other than ants (Drosophila flies), although these are nutritionally enriched and thus optimal for euryphagous predators (Pekár and Toft, 2009). For stenophagous predators, alternative prey or even a mixed diet had a negative effect on fitness (Li and Jackson, 1997; Pekár et al., 2008). This study did not reveal any trade-offs in adaptations in S. reticulata. This is in sharp contrast to Zodarion, in which several trade-offs were found in behavioural, venomic and physiological adaptations: Zodarion spiders were not able to attack, paralyse and utilise other prey than ants (Pekár, 2004; Pekár and Toft, 2009). A comparative analysis of spider diets within the Zodariidae (Pekár et al., 2012b) revealed that strict myrmecophagy is a derived condition, which has evolved independently in several spider families, namely Thomisidae, Theridiidae, Corinnidae. The pattern is always similar to that found in Zodariidae: basal genera are euryphagous but able to capture and consume ants, and advanced genera are myrmecophagous specialists. The ability to capture ants seems to be an ancestral trait within Zodariidae. The evolution of myrmecophagy in other families seems to follow a similar pattern as in Zodariidae. There appears to be a gradual change in adaptations, as also observed in myrmecophagous mammals (Reiss, 2001) or lizards (Losos et al., 1994). Myrmecophagy of other true predators, such as Coccinellidae (Giorgi et al., 2009), dendrobatid frogs (Caldwell, 1996) and Lycaenidae (Pierce et al., 2002), was found to be derived, too. It has evolved either from coccidophagy (Coccinellidae), polyphagy (frogs) or phytophagy (Lycaenidae). The obtained data suggest that ants were a profitable prey for the presumed euryphagous generalist ancestor. The conditions that led to myrmecophagy in derived genera are not known, but we presume it could be easy and stable access to ants, either due to interspecific competition, local specialisation or predator avoidance. Under such conditions parallel changes in trophic traits that increased the utilisation of ants were selected. Specifically,

selection was directed towards improving search and capture success through cognitive, behavioural, morphological and venomic adaptations to such an extent that trade-offs evolved in the advanced species. Acknowledgements ˇ We would like to thank S. Henriques and M. Rezᡠc for help in collecting spiders in the field. This study was supported by grant no. MSM0021622416 provided by the Ministry of Education, Youth and Sports of the Czech Republic. MALDI-MS analyses were supported by the European Regional Development Fund under the auspices of the “CEITEC – Central European Institute of Technology” (project CZ.1.05/1.1.00/02.0068). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.zool.2012.05.006. References Bilde, T., Toft, S., 2000. Evaluation of prey for the spider Dicymbium brevisetosum Locket (Araneae: Linyphiidae) in single-species and mixed-species diets. In: Gajdoˇs, P., Pekár, S. (Eds.), Proceedings of the 18th European Colloquium of Arachnology. Ekologia (Bratislava) 19 (Suppl. 3), 9–18. Caldwell, J.P., 1996. The evolution of myrmecophagy and its correlates in poison frogs (Family Dendrobatidae). J. Zool. 240, 75–101. Cárdenas, M., Jiroˇs, P., Pekár, S., 2012. Selective olfactory attention of a specialised predator to intraspecific chemical signals of its prey. Naturwissenschaften 99, 597–605. Cushing, P.E., Santangelo, R.G., 2002. Notes on the natural history and hunting behavior of an ant eating zodariid spider (Arachnida, Araneae) in Colorado. J. Arachnol. 30, 618–621. Gertsch, W.J., 1961. The spider genus Lutica. Senckenb. Biol. 42, 365–374. Giorgi, J.A., Vandenberg, N.J., McHugh, J.V., Forrester, J.A., Slipinski, S.A., Miller, K.B., Shapiro, L.R., Whiting, M.F., 2009. The evolution of food preferences in Coccinellidae. Biol. Control 51, 215–231. Holliday, J.A., 2010. Evolution in Carnivora: identifying a morphological bias. In: Goswami, A., Friscia, A. (Eds.), Carnivoran Evolution. New Views on Phylogeny, Form, and Function. Cambridge University Press, Cambridge, pp. 189–224. Hardin, J.W., Hilbe, J.M., 2003. Generalized Estimating Equations. Chapman & Hall/CRC, Boca Raton. Hölldobler, B., Wilson, E.O., 1990. The Ants. Springer Verlag, Berlin/Heidelberg. Jermy, T., Lábos, E., Molnár, I., 1990. Stenophagy of phytophagous insects – a result of constraints on the evolution of the nervous system. In: Maynard Smith, J., Vida, G. (Eds.), Organizational Constraints on the Dynamics of Evolution. Manchester University Press, Manchester, pp. 157–166. Jocqué, R., 1988. An updating of the genus Leprolochus (Araneae: Zodariidae). Stud. Neotrop. Fauna Environ. 23, 77–87. Jocqué, R., 1991. A generic revision of the spider family Zodariidae (Araneae). Bull. Am. Mus. Nat. Hist. 201, 1–160. Jocqué, R., Bosmans, R., 2001. A revision of the genus Selamia with the description of Amphiledorus gen. n. (Araneae, Zodariidae). Bull. Inst. R. Sci. Nat. Belg. (Ent.) 71, 115–134. Kuhn-Nentwig, L., Stöcklin, R., Nentwig, W., 2011. Venom composition and strategies in spiders: is everything possible? Adv. Insect Physiol. 40, 1–86. Li, D., Jackson, R.R., 1997. Influence of diet on survivorship and growth in Portia fimbriata, an araneophagic jumping spider (Araneae: Salticidae). Can. J. Zool. 75, 1652–1658. Losos, J.B., Irschick, D.J., Schoener, T.W., 1994. Adaptation and constraint in the evolution of specialization of Bahamian Anolis lizards. Evolution 48, 1786–1798. McNab, B.K., 1984. Physiological convergence amongst ant-eating and termite-eating mammals. J. Zool. 203, 485–510. McNab, B.K., 1992. Rate of metabolism in the termite-eating sloth bear (Ursus ursinus). J. Mammal. 731 (1), 168–172. Mead, R., 1988. The Design of Experiments. Cambridge University Press, Cambridge. Nentwig, W., Wissel, C., 1986. A comparison of prey lengths among spiders. Oecologia 68, 595–600. Paradis, E., 2006. Analysis of Phylogenetics and Evolution with R. Springer, New York. Pekár, S., 2004. Predatory behavior of two European ant-eating spiders (Araneae, Zodariidae). J. Arachnol. 32, 31–41. Pekár, S., Brabec, M., 2009. Modern Analysis of Biological Data. 1. Generalised Linear Models in R. Scientia, Praha (in Czech). Pekár, S., Král, J., 2002. Mimicry complex in two central European zodariid spiders (Araneae: Zodariidae): how Zodarion deceives ants. Biol. J. Linn. Soc. 75, 517–532. Pekár, S., Lubin, Y., 2009. Prey and predatory behaviour of two zodariid spiders (Araneae, Zodariidae). J. Arachnol. 37, 118–121.

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G Model ZOOL-25320; No. of Pages 9

ARTICLE IN PRESS S. Pekár et al. / Zoology xxx (2012) xxx–xxx

ˇ J., 2007. Comparative study of the femoral organ in Zodarion Pekár, S., Sobotník, spiders (Araneae: Zodariidae). Arthrop. Struct. Dev. 36, 105–112. Pekár, S., Toft, S., 2009. Can ant-eating Zodarion spiders (Araneae: Zodariidae) develop on a diet optimal for polyphagous predators? Physiol. Entomol. 34, 195–201. Pekár, S., Král, J., Malten, A., Komposch, C., 2005a. Comparison of natural histories and karyotypes of two closely related ant-eating spiders, Zodarion hamatum and Z. italicum (Araneae, Zodariidae). J. Nat. Hist. 39, 1583–1596. Pekár, S., Král, J., Lubin, Y.D., 2005b. Natural history and karyotype of some ant-eating zodariid spiders (Araneae, Zodariidae) from Israel. J. Arachnol. 33, 50–62. Pekár, S., Toft, S., Hruˇsková, M., Mayntz, D., 2008. Dietary and prey-capture adaptations by which Zodarion germanicum, an ant-eating spider (Araneae: Zodariidae), specialises on the Formicinae. Naturwissenschaften 95, 233–239. ˇ ˇ Pekár, S., Smerda, J., Hruˇsková, M., Sedo, O., Muster, C., Cardoso, P., Zdráhal, Z., Korenko, S., Bureˇs, P., Líznarová, E., Sentenská, L., 2012a. Prey-race drives differentiation of biotypes in ant-eating spiders. J. Anim. Ecol. 81, 838–848. Pekár, S., Coddington, J.A., Blackledge, T., 2012b. Evolution of stenophagy in spiders (Araneae): evidence based on the comparative analysis of spider diets. Evolution 66, 776–806. Pierce, N.E., Braby, M.F., Heath, A., Lohman, D.J., Mathew, J., Rand, D.B., Travassos, M.A., 2002. The ecology and evolution of ant association in the Lycaenidae (Lepidoptera). Annu. Rev. Entomol. 47, 733–771. Pigliucci, M., Preston, K., 2004. Phenotypic Integration. Oxford University Press, Oxford.

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Pinheiro, J.C., Bates, D.M., 2000. Mixed-effects Models in S and S-PLUS. Springer, New York. R Development Core Team, 2010. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, Available at http://www.R-project.org Reiss, K.Z., 2001. Using phylogenies to study convergences: the case of the ant-eating mammals. Am. Zool. 41, 507–525. Richardson, P.R.K., 1987. Aardwolf: the most specialised myrmecophagous mammal? S. Afr. J. Sci. 83, 643–646. Sanz, L., Gibbs, H.L., Mackessy, S.P., Calvete, J.J., 2006. Venom proteomes of closely related Sistrurus rattlesnakes with divergent diets. J. Proteome Res. 5, 2098–2112. Singer, M.S., 2008. Evolutionary ecology of polyphagy. In: Tilmon, K.J. (Ed.), Specialization, Speciation, and Radiation: The Evolutionary Biology of Herbivorous Insects. University of California, Berkeley, pp. 29–42. Southwood, T.R.E., Henderson, R., 2000. Ecological Methods. Blackwell Science, Oxford. Tableman, M., Kim, J.S., 2004. Survival Analysis Using S. Analysis of Time-to-Event Data. Chapman & Hall/CRC, Boca Raton. Thompson, J.D., 1994. The Coevolutionary Process. University of Chicago Press, Chicago. Toft, S., 1999. Prey choice and spider fitness. In: Greenstone, M.H., Sunderland, K.D. (Eds.), Spiders in Agroecosystems: Ecological Processes and Biological Control. Proceedings of a Symposium at the XIV International Arachnological Congress. J. Arachnol. 27, 301–307.

Please cite this article in press as: Pekár, S., et al., Phenotypic integration in a series of trophic traits: tracing the evolution of myrmecophagy in spiders (Araneae). Zoology (2012), http://dx.doi.org/10.1016/j.zool.2012.05.006