Mind the Outgroup and Bare Branches in Total-Evidence Dating: a Case Study of Pimpliform Darwin Wasps (Hymenoptera, Ichneumonidae).

Taxon sampling is a central aspect of phylogenetic study design, but it has received limited attention in the context of total-evidence dating, a widely used dating approach that directly integrates molecular and morphological information from extant and fossil taxa. We here assess the impact of commonly employed outgroup sampling schemes and missing morphological data in extant taxa on age estimates in a total-evidence dating analysis under the uniform tree prior. Our study group is Pimpliformes, a highly diverse, rapidly radiating group of parasitoid wasps of the family Ichneumonidae. We analyze a data set comprising 201 extant and 79 fossil taxa, including the oldest fossils of the family from the Early Cretaceous and the first unequivocal representatives of extant subfamilies from the mid-Paleogene. Based on newly compiled molecular data from ten nuclear genes and a morphological matrix that includes 222 characters, we show that age estimates become both older and less precise with the inclusion of more distant and more poorly sampled outgroups. These outgroups not only lack morphological and temporal information but also sit on long terminal branches and considerably increase the evolutionary rate heterogeneity. In addition, we discover an artifact that might be detrimental for total-evidence dating: "bare-branch attraction," namely high attachment probabilities of certain fossils to terminal branches for which morphological data are missing. Using computer simulations, we confirm the generality of this phenomenon and show that a large phylogenetic distance to any of the extant taxa, rather than just older age, increases the risk of a fossil being misplaced due to bare-branch attraction. After restricting outgroup sampling and adding morphological data for the previously attracting, bare branches, we recover a Jurassic origin for Pimpliformes and Ichneumonidae. This first age estimate for the group not only suggests an older origin than previously thought but also that diversification of the crown group happened well before the Cretaceous-Paleogene boundary. Our case study demonstrates that in order to obtain robust age estimates, total-evidence dating studies need to be based on a thorough and balanced sampling of both extant and fossil taxa, with the aim of minimizing evolutionary rate heterogeneity and missing morphological information. [Bare-branch attraction; ichneumonids; fossils; morphological matrix; phylogeny; RoguePlots.].

Trophic niche, capture efficiency and venom profiles of six sympatric ant-eating spider species (Araneae: Zodariidae).

The arms race between specialist predators and their prey has resulted in the evolution of a variety of specific adaptations. In venomous predators, this can include venom composition, particularly if predators are specialized on dangerous prey. Here, we performed an integrative study using six species of highly specialized ant-eating spiders of the genus Zodarion to investigate their phylogeny, realized trophic niche, efficacy in the capture of various ant species and venom composition. Data on natural diet obtained by next-generation sequencing and field observations showed that the six Zodarion species exploit different ant species. Their phylogeny, based on mitochondrial and nuclear genes, correlated with the composition of their natural prey, indicating that closely related Zodarion species specialize on similar ant species. Prey-capture parameters differed among Zodarion species suggesting prey-specific efficacy. Similarly, the venom profiles of both low and high molecular compounds differed among species. Only the profiles of low molecular compounds were correlated with capture efficacy parameters, suggesting that the venom of Zodarion spiders contains prey-specific components. Our study suggests that Iberian Zodarion spiders are specialized on particular ant species.

David and Goliath: potent venom of an ant-eating spider (Araneae) enables capture of a giant prey.

It is rare to find a true predator that repeatedly and routinely kills prey larger than itself. A solitary specialised ant-eating spider of the genus Zodarion can capture a relatively giant prey. We studied the trophic niche of this spider species and investigated its adaptations (behavioural and venomic) that are used to capture ants. We found that the spider captures mainly polymorphic Messor arenarius ants. Adult female spiders captured large morphs while tiny juveniles captured smaller morphs, yet in both cases ants were giant in comparison with spider size. All specimens used an effective prey capture strategy that protected them from ant retaliation. Juvenile and adult spiders were able to paralyse their prey using a single bite. The venom glands of adults were more than 50 times larger than those of juvenile spiders, but the paralysis latency of juveniles was 1.5 times longer. This suggests that this spider species possesses very potent venom already at the juvenile stage. Comparison of the venom composition between juvenile and adult spiders did not reveal significant differences. We discovered here that specialised capture combined with very effective venom enables the capture of giant prey.

Host specificity and temporal and seasonal shifts in host preference of a web-spider parasitoid Zatypota percontatoria.

Current knowledge about polysphinctine parasite wasps' interactions with their spider hosts is very fragmented and incomplete. This study presents the host specificity of Zatypota percontatoria (Müller) (Hymenoptera: Ichneumonidae) and its adaptation to varying host availability. Two years of field observations show that Z. percontatoria is a stenophagous parasitoid that parasitizes only five closely related web-building spiders of the family Theridiidae (Araneae). Within the Theridiidae it attacks only species belonging to a small group of species, here called the "Theridion" group. These hosts have a similar biology, but are available at different levels of abundance and at different sizes over the season. Laboratory experiments showed that this wasp species ignores linyphiid, araneid or dictynid spiders and accepts only theridiid spiders of the "Theridion" group. In the field study, wasp females preferred older juvenile and sub-adult female spider instars with intermediate body size. Only 5% of the parasitized spiders were males. Parasitism in the natural population of theridiid spiders was on average 1.3%. Parasitism was most frequent on two species, Theridion varians Hahn in 2007 and Neottiura bimaculata Linnaeus in 2008. The parasitization rate was positively correlated with spider abundance. The wasp responded adaptively to seasonal changes in host abundance and host body size and shifted host preference according to the availability of suitable hosts during, as well as between, seasons. In spring and summer the highest percentage of parasitism was on T. varians and in autumn it was on N. bimaculata.

Neonicotinoids suppress contact chemoreception in a common farmland spider.

Neonicotinoid insecticides are increasingly recognized for their role as information disruptors by modifying the chemical communication system of insects and therefore decreasing the chances of reproduction in target insects. However, data from spiders are lacking. In the present study, we tested the responses of males of a common agrobiont spider, Pardosa agrestis, to the application of field-realistic concentration of acetamiprid, which was formulated as Mospilan, and trace amounts of thiacloprid, which was formulated as Biscaya. We applied fresh or 24-h-old residues of Mospilan or Biscaya to the males just prior to the experiment or treated only the surface of a tunnel containing female draglines. We evaluated the ability of the males to recognize female cues from female dragline silk in a Y-maze. The field-realistic, sublethal doses of Mospilan altered pheromone-guided behavior. The choice of the tunnel with female draglines by males was hampered by tarsal treatment of the males with 24 h-old residues of Mospilan. The mating dance display was commonly initiated in control males that came into contact with female draglines and was suppressed by the Mospilan treatments in all three experimental settings. Some males only initiated the mating dance but did not manage to complete it; this was particularly true for males that were treated tarsally with fresh Mospilan residues, as none of these males managed to complete the mating dance. All three experimental settings with Mospilan decreased the frequency of males that managed to both select the tunnel with female draglines and complete the mating dance. The responses to the low-dose Biscaya were much milder and the study was not sufficiently powered to confirm the effects of Biscaya; however, the surprisingly observed trends in responses to very low Biscaya concentrations call for further analyses of long-term effects of trace amounts of neonicotinoids on the pheromone-guided behavior of spiders. These are the first conclusive data regarding the effects of commercially available formulations of neonicotinoid insecticides on the intraspecific chemical communication of spiders.

Koinobint life style of the spider wasp Minagenia (Hymenoptera, Pompilidae) and its consequences for host selection and sex allocation.

Spider wasps of the genus Minagenia have evolved koinobiontism as a relatively rare life strategy within the widely diversified hymenopteran family Pompilidae. In this study, we evaluated several aspects of the parasitic strategy of the wasp Minagenia sp. (hereafter, Minagenia) - namely host specificity, ontogeny, and sex determination as a function of host size. We found that Minagenia is highly host specific, being associated only with the genus Lycosa from the family Lycosidae, namely Lycosa u-album (Mello-Leitão, 1938), Lycosa erythrognatha (Lucas, 1836) and Lycosa poliostoma (Koch, 1847) with a parasitism incidence of 18.9%, 15.8% and 12.5%, respectively. Both ecological and taxonomical host traits determine the host selection and sex allocation of Minagenia female wasps. Charnov's host-size model explains Minagenia's host-size-dependent sex ratio in combination with the effect of host development stage, host species, and host foraging strategy. We also found that the final instar larva of Minagenia induces behavioural changes in spider hosts. The manipulated spider builds a protective silk chamber as a shelter for parasitoid pupation. Our results suggest that host manipulation seems to be narrowly connected with koinobiont life style throughout Hymenoptera. This study provides new information about the host-parasitoid koinobiont life strategy among spider wasps, which probably arose convergently in distant taxonomical groups within Pompilidae.

Prey contaminated with neonicotinoids induces feeding deterrent behavior of a common farmland spider.

Neonicotinoids are thought to have negligible repellent or anti-feeding effects. Based on our preliminary observations, we hypothesized that the contamination of spider prey with commonly used neonicotinoids has repellent or feeding deterrent effects on spiders. We tested this hypothesis by providing prey treated or not with field-realistic concentrations of neonicotinoids to the spiders and determining the number of (a) killed only and (b) killed and eaten prey. We exposed adult freshly molted and starved Pardosa agrestis, a common agrobiont lycosid species, to flies treated with neonicotinoids (acetamiprid, imidacloprid, thiacloprid and thiamethoxam) at field-realistic concentrations or with distilled water as a control. There were no effects of the exposure of the prey to neonicotinoids on the number of flies captured. However, the spiders consumed less of the prey treated with neonicotinoids compared to the ratio of control prey consumed, which resulted in increased overkilling (i.e., killing without feeding). In female P. agrestis, the overkilling increased from only 2.6% of control flies to 25-45% of neonicotinoid-treated flies. As the spiders avoided consuming the already captured neonicotinoid-treated prey, the sublethal effects of neonicotinoids extend beyond the simple attractivity/deterrence of the prey itself. The present study demonstrated that prey overkilling serves as a physiological response of spiders to the contact with the prey contaminated with agrochemicals. We speculate that primary contact with neonicotinoids during prey capture may play a role in this unexpected behavior.

Modification of Tetragnatha montana (Araneae, Tetragnathidae) web architecture induced by larva of the parasitoid Acrodactyla quadrisculpta (Hymenoptera, Ichneumonidae, Polysphincta genus-group).

BACKGROUND: Thepolysphinctine wasp, Acrodactylaquadrisculpta,is a koinobiont ecto-parasitoid of spiders and is narrowly associated with the biology of its spider hosts. The larva, attached to the dorsal side of the abdomen, develops while the spider continues foraging. Shortly before pupation, the parasitoid larva manipulates the web-building activity of the host in order to construct a safe shelter against natural elements and predators during parasitoid pupation. RESULTS: A. quadrisculpta was associated exclusively with the orb web weaving spiders Tetragnatha montana, with a parasitism incidence of 19%. The manipulated spider constructed a unique cocoon web that provided strong mechanical support for the parasitoid's pupal cocoon. The cocoon web consisted of one highly reinforced main thread, tensioned in 60% of cases by a reinforced side thread. The wasp cocoon, square in cross-section, was fastened along its length to the main cocoon thread. CONCLUSIONS: The wasp A. quadrisculpta was exclusively associated with an orb-weaving spider T. montana in the family Tetragnathidae. The alteration of the web architecture of T. montana induced by the larva A. quadrisculpta was unique and species specific.

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

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.

Hymenopteran parasitoids of the ant-eating spider Zodarion styliferum (Simon) (Araneae, Zodariidae).

Calymmochilus dispar Boucek & Andriescu (Hymenoptera, Eupelmidae) and Gelis apterus (Pontoppidan) (Hymenoptera, Ichneumonidae) are newly recorded as parasitoids of the ant-eating spider Zodarion styliferum (Simon) (Araneae, Zodariidae). The larvae of both parasitoid species fed on juvenile spiders. The final instar larva and pupa of Calymmochilus dispar and the male of Gelis apterus are described for the first time. Both species represent new distribution records for Portugal. The biology and host associations of the parasitoids are discussed.

A parasitoid wasp induces overwintering behaviour in its spider host.

Parasites and parasitoids control behaviors of their hosts. However, the origin of the behavior evoked by the parasitic organism has been rarely identified. It is also not known whether the manipulation is universal or host-specific. Polysphinctine wasps, koinobiont ectoparasitoids of several spider species that manipulate host web-spinning activity for their own protection during pupation, provide an ideal system to reveal the origin of the evoked behavior. Larva of Zatypota percontatoria performed species-specific manipulation of theridiid spiders, Neottiura bimaculata and Theridion varians, shortly before pupation. Parasitized N. bimaculata produced a dense web, whereas parasitized T. varians built a cupola-like structure. The larva pupated inside of either the dense web or the cupola-like structure. We discovered that unparasitized N. bimaculata produce an analogous dense web around their eggsacs and for themselves during winter, while T. varians construct an analogous 'cupola' only for overwintering. We induced analogous manipulation in unparasitized hosts by altering ambient conditions. We discovered that the behavior evoked by larvae in two hosts was functionally similar. The larva evoked protective behaviors that occur in unparasitized hosts only during specific life-history periods.