A novel expression domain of extradenticle underlies the evolutionary developmental origin of the chelicerate patella.

Neofunctionalization of duplicated gene copies is thought to be an important process underlying the origin of evolutionary novelty and provides an elegant mechanism for the origin of new phenotypic traits. One putative case where a new gene copy has been linked to a novel morphological trait is the origin of the arachnid patella, a taxonomically restricted leg segment. In spiders, the origin of this segment has been linked to the origin of the paralog dachshund-2 , suggesting that a new gene facilitated the expression of a new trait. However, various arachnid groups that possess patellae do not have a copy of dachshund-2 , disfavoring the direct link between gene origin and trait origin. We investigated the developmental genetic basis for patellar patterning in the harvestman Phalangium opilio , which lacks dachshund-2 . Here, we show that the harvestman patella is established by a novel expression domain of the transcription factor extradenticle . Leveraging this definition of patellar identity, we surveyed targeted groups across chelicerate phylogeny to assess when this trait evolved. We show that a patellar homolog is present in Pycnogonida (sea spiders) and various arachnid orders, suggesting a single origin of the patella in the ancestor of Chelicerata. A potential loss of the patella is observed in Ixodida. Our results suggest that the modification of an ancient gene, rather than the neofunctionalization of a new gene copy, underlies the origin of the patella. Broadly, this work underscores the value of comparative data and broad taxonomic sampling when testing hypotheses in evolutionary developmental biology.

A plurality of morphological characters need not equate with phylogenetic accuracy: A rare genomic change refutes the placement of Solifugae and Pseudoscorpiones in Haplocnemata.

Recent advances in higher-level invertebrate phylogeny have leveraged shared features of genomic architecture to resolve contentious nodes across the tree of life. Yet, the interordinal relationships within Chelicerata have remained recalcitrant given competing topologies in recent molecular analyses. As such, relationships between topologically unstable orders remain supported primarily by morphological cladistic analyses. Solifugae, one such unstable chelicerate order, has long been thought to be the sister group of Pseudoscorpiones, forming the clade Haplocnemata, on the basis of eight putative morphological synapomorphies. The discovery, however, of a shared whole genome duplication placing Pseudoscorpiones in Arachnopulmonata provides the opportunity for a simple litmus test evaluating the validity of Haplocnemata. Here, we present the first developmental transcriptome of a solifuge (Titanopuga salinarum) and survey copy numbers of the homeobox genes for evidence of systemic duplication. We find that over 70% of the identified homeobox genes in T. salinarum are retained in a single copy, while representatives of the arachnopulmonates retain orthologs of those genes as two or more copies. Our results refute the placement of Solifugae in Haplocnemata. Subsequent reevaluation of putative interordinal morphological synapomorphies among chelicerates reveals a high incidence of homoplasy, reversals, and inaccurate coding within Haplocnemata and other small clades, as well as Arachnida more broadly, suggesting existing morphological character matrices are insufficient to resolve chelicerate phylogeny.

Reproductive diapause influences spermatogenesis and testes' size in the diplochronous wolf spider Allocosa senex (Lycosidae, Araneae) - A case study using a non-experimental approach.

Seasonality considerably impacts on the life of organisms and leads to numerous evolutionary adaptations. Some species face seasonal changes by entering a diapause during different life stages. During adulthood, a diapause in the non-reproductive period can affect male gametogenesis as, for example, it occurs in insects. Spiders are distributed worldwide and show a variety of life cycles. However, data on spiders' life cycles and seasonal adaptations are limited. Here, we explored the effect of reproductive diapause in a seasonal spider for the first time. We used the South American sand-dwelling spider Allocosa senex as a model as this species is diplochronous, meaning that individuals live two reproductive seasons, with juveniles and adults overwintering in burrows. It has been observed that individuals of this species reduce their metabolism during the non-reproductive season, diminishing prey consumption and locomotion to a minimum. This species is also well-known for exhibiting wandering and courting females and sedentary males. We analyzed spermatogenesis throughout the male's life cycle and described the male's reproductive system and spermiogenesis using light and transmission electron microscopy. We found that spermatogenesis in A. senex is asynchronous and continuous. However, when males face the non-reproductive season, the late spermatogenic stages and spermatozoa decline, causing an interruption but not a total arrest of this process. This seasonality is also reflected in smaller testes' size in males from the non-reproductive season than in other periods. The mechanisms and constraints are unknown, but they could be related to the metabolic depression during this life cycle period. Since sex-role reversal apparently sets a low-intensity sperm competition scenario compared with other wolf spiders, surviving two reproductive seasons may balance mating opportunities by distributing them between both periods. Thus, the partial interruption of spermatogenesis during diapause could allow new mating encounters during the second reproductive season.

Challenging monogamy in a spider with nontraditional sexual behavior.

Each species and sex can develop different reproductive strategies to optimize their fitness while assigning reproductive effort. Allocosa senex is a sex-role reversed spider whose males construct long burrows in the sand. They wait for wandering females to approach, assess their sexual partners and donate their constructions to females after copulation. Females stay in the burrow and lay their egg-sac. When offspring are ready for dispersion, females leave the burrow and gain access to new mating opportunities. Males are choosy during mate courtship, preferring to mate with virgin females over copulated ones, which can even be cannibalized if males reject them. This situation turns new mating opportunities dangerous for copulated females. We wondered whether a copulated female inside the previous mate's burrow responds to courtship from a new male and if this new male can copulate, avoiding burrow construction costs. We also explored whether courtship and copulation behaviors during the first sexual encounter affected the probability of occurrence of a second copulation. For that purposes we exposed copulated females inside male burrows to new males (non-donor males). Males could locate and court females inside the previous male's burrow, and females accepted a second copulation. Hence, A. senex females are not monogamous as was expected but increase their reproductive success by copulating with non-donor males. Also, males can develop opportunistic tactics, suggesting a more dynamic mating system for this sex-role reversed spider than assumed.

Low temperatures induce physiological changes in lipids, fatty acids and hydrocarbons, in two rare winter scorpions of genus Urophonius (Scorpiones, Bothriuridae).

Different organisms (mainly poikilotherms) are subject to environmental fluctuations that could affect their normal physiological functioning (e.g., by destabilization of biomembranes and rupture of biomolecules). As a result, animals regulate their body temperature and adapt to different environmental conditions through various physiological strategies. These adaptations are crucial in all organisms, although they are more relevant in those that have reached a great adaptive diversity such as scorpions. Within scorpions, the genus Urophonius presents species with winter activity, being this a peculiarity within the Order and an opportunity to study the strategies deployed by these organisms when facing different temperatures. Here, we explore three basic issues of lipid remodeling under high and low temperatures, using adults and juveniles of Urophonius achalensis and U. brachycentrus. First, as an indicator of metabolic state, we analyzed the lipidic changes in different tissues observing that low temperatures generate higher quantities of triacylglycerols and fewer amount of structural lipids and sphyngomielin. Furthermore, we studied the participation of fatty acids in adaptive homeoviscosity, showing that there are changes in the quantity of saturated and unsaturated fatty acids at low temperature (mainly 16:0, 18:0, 18:1 and 18:2). Finally, we observe that there are quantitative and qualitative variations in the cuticular hydrocarbons (with possible water barrier and chemical recognition function). These fluctuations are in some cases species-specific, metabolic-specific, tissue-specific and in others depend on the ontogenetic state.

The morphology of mating plugs and its formation in scorpions: Implications for intersexual participation.

Mating plugs have been proposed as a mechanism that has evolved to avoid sperm competition. Their structure and composition vary across taxa and are related to the effectiveness of its function. This effectiveness could be related to different evolutionary interests of the sexes. Urophonius brachycentrus and Urophonius achalensis (Scorpiones, Bothriuridae) are highly suitable species to study mating plugs because both are monandrous species with specific morphological and physiological responses in the female's genitalia. Here, we analyze (a) the morphology and fine structure of the mating plugs of both species, (b) the site of production in males and the formation process of the mating plug, and (c) the changes that it undergoes over time in the female's reproductive tract. In both species, a complex mating plug obliterates the female's genital aperture and fills the genital atrium. We observed considerable interspecific variation in the mating plug morphology. A mating hemi-plug was found surrounding the capsular lobes of the hemispermatophore, which could have a mixed composition (involving portions of the hemispermatophore and glandular products). The glandular portion was transferred in a semi-solid state filling the female's genital atrium and then hardening. Changes that the plug undergoes in the female's genitalia (darkening and increase of the "distal" area of the plug) indicate a participation of the female to the formation of this type of plug. Our study provides new insights into the plugging phenomenon in scorpions, and we discussed the adaptive significance as a post-copulatory mechanism to avoid sperm competition.

The role of silk in courtship and communication in mygalomorph spiders: Do males regulate their courtship in response to female mating status?

In spiders, pheromones are known to be responsible for attracting the opposite sex, eliciting male searching and courtship behaviors, as well as for synchronizing potential mates in space and time. Most spiders are cannibalistic and aggressive. Thus, early recognition of a female as a possible mate is essential for males, who may suffer high energetic or reproductive costs to the extreme of losing all fitness opportunities. In Acanthogonatus centralis Goloboff 1995, a mygalomorph spider, what female signs might be triggering male courtship behavior remain unknown, as well as whether males can discriminate between females. The aims of the present work were (1) establishing whether males can detect the presence of females using airborne and silk-borne signals and (2) determining whether males can discriminate the reproductive status and body condition of females. We found no evidence that airborne pheromones play a role in the sexual communication of A. centralis, but silk-bound contact signals function as a female advertisement. Also, this is the first study that demonstrates that male mygalomorph spiders can discriminate between different signals on silk through direct contact, showing a preference for unmated females.

Female secondary sexual traits in spiders: Adaptive interpretations of the sternum projection in the pholcid Holocnemus pluchei.

Sexually dimorphic traits are the outcome of a network of selective pressures acting upon each sex. While male secondary sexual traits are frequently restricted to intrasexual competition, female ornaments or weapons are mostly associated to social interactions for the access to limited resources. Here we investigate a sexually dimorphic trait expressed within the female's sternum of the spider species Holocnemus pluchei (Pholcidae), and its adaptive maintenance under the three leading hypotheses: sexual receptivity signaling, mate choice and sexual conflict hypothesis. We provide fine-scaled behavioral descriptions of the copulatory behavior of Holocnemus pluchei, corroborating that mating interactions are mediated by the female's sternum projection. The single moment of contact between the female projection and male body occurs during sperm transfer, always followed by a reduction on the intensity of the male genital movement. Additionally, biometrical properties of the female sternum projection corroborate our functional interpretation for the trait as restricting the intensity of males' copulatory movements. We claim that female sternum projection is a sexually selected trait mediating sexual conflict, with several adaptive consequences upon the sexes.

Specificity of the female's local cellular immune response in genital plug producing scorpion species.

Immune defense is a key feature in the life history of organisms, expensive to maintain, highly regulated by individuals and exposed to physiological and evolutionary trade-offs. In chelicerates, relatively scarce are the studies that relate postcopulatory mechanisms and immune response parameters. This work makes an approximation to the female's immunological consequences produced after the placement of a foreign body in the genitalia of three scorpions species, two species that normally receive genital plugs during mating (Urophonius brachycentrus and U. achalensis) and one that does not (Zabius fuscus). Here we performed the first morphological description of the natural plugs of the two Urophonius species. We described complex three zoned structure anchored to the female genital atrium and based on this information we placed implants in the genitalia (for eliciting the local immune response) of virgin females of the three species and measured the immune encapsulation response to this foreign body. We found a greater and heterogeneous response in different zones of the implants in the plug producing species. To corroborate the specificity of this immune response, we compared the local genital reaction with the triggered response at a systemic level by inserting implants into the female body cavity of U. brachycentrus and Zabius fuscus. We found that the systemic response did not differ between species and that only in the plug producing species the local response in the genitalia was higher than the systemic one. We also compared the total hemocyte load before and after the genital implantation to see if this parameter was compromised by the immunological challenge. We confirmed that in Urophonius species the presence of a strange body in the genitalia caused a decrease in the hemocyte load. Besides, we find correlations between the body weight and the immunological parameters, as well as between different immunological parameters with each other. Complementarily, we characterized the hemocytes of the three scorpion species for the first time. This comparative study can help to provide a wider framework of the immunological characteristics of the species, their differences and their relationship with the particular postcopulatory mechanism such as the genital plugs.

Differences in mating behavior between two allopatric populations of a Neotropical scorpion.

Courtship and mating behavior generally evolve rapidly in diverging populations and species. The adaptation to different environments may cause behavioral divergence in characteristics involved in mate choice. Our objective in this study was to compare the sexual behavior of two distant populations of the scorpion Bothriurus bonariensis. This species has a broad distribution in South America, inhabiting Central Argentina, Uruguay and south-eastern Brazil. It is known that in this species there is a divergence in morphological patterns (body size, coloration, allometry and fluctuating asymmetry indexes) among distant populations. Considering the differences in environmental conditions between localities, we compare the sexual behavior in intra-population and inter-population matings from Central Argentina and southern Uruguay populations. We found significant differences in mating patterns, including differences in the frequency and duration of important stimulatory courtship behaviors. In addition, most inter-population matings were unsuccessful. In this framework, the differences in reproductive behavior could indicate reproductive isolation between these populations, which coincides with their already known morphological differences. This is the first study comparing the sexual behavior of allopatric populations of scorpions; it provides new data about the degree of intraspecific geographical divergence in the sexual behavior of B. bonariensis.

No pain, no gain: Male plasticity in burrow digging according to female rejection in a sand-dwelling wolf spider.

Behavioral plasticity allows individuals to reversibly respond to short-term variations in their ecological and social environment in order to maximize their fitness. Allocosa senex is a burrow-digging spider that inhabits the sandy coasts of South America. This species shows a reversal in typical sex roles expected in spiders: females are wanderers that visit males at their burrows and initiate courtship. They prefer males with long burrows for mating, and males prefer virgin over mated females. We tested whether female sexual rejection induced males to enlarge their burrows and if female reproductive status affected males' responses. We exposed males who had constructed burrows to: a) virgin females or b) mated females, (n=16 for each category). If female rejection occurred, we repeated the trial 48h later with the same female. As control, we maintained a group of males without female exposure (unexposed group, n=32). Rejected males enlarged their burrows more frequently and burrows were longer compared to unexposed males. However, frequency and length of enlargement did not differ according to female reproductive status. Males of A. senex showed plasticity in digging behavior in response to the availability of females, as a way to maximize the possibilities of future mating.

Geographical variation in sexual behavior and body traits in a sex role reversed wolf spider.

Mating partners need to recognize, assess each other, and exchange information through behavioral events that occur before, during, and after mating. Sexual signals, as well as life history traits, are influenced by selective pressures and environmental factors that can vary across distant geographical areas. Allocosa senex is a sand-dwelling wolf spider which constructs burrows along the sandy coasts of Argentina, Brazil, and Uruguay. Females are the mobile sex that searches for males and initiates courtship. They prefer males which construct longer burrows, and males prefer virgin females in good body condition. The objective of this study was to compare sexual behavior patterns, as well as body characteristics and burrow dimensions, between two geographically distant locations of A. senex, one in Uruguay (Uruguayan location) and the other from central Argentina (Argentinean location). We found differences in the number of male abdominal vibrations, male and female touches during mating, and number of erections of male leg spines, which all were higher in matings of Argentinean pairs. On the other hand, male body mass and female body condition were higher in Uruguayan individuals. The wide distribution of A. senex could be determining variations in the biotic and abiotic features that affect the species, generating differences in the strength of selective forces acting on individuals from the two studied locations.

Worthless and Nutritive Nuptial Gifts: Mating Duration, Sperm Stored and Potential Female Decisions in Spiders.

In nuptial gift-giving species females sometimes select their potential mates based on the presence and size of the gift. But in some species, such as the Neotropical polyandrous spider Paratrechalea ornate male gifts vary in quality, from nutritive to worthless, and this male strategy can be in conflict with female nutritional benefits. In this species, males without gifts experience a reduction in mating success and duration, while males that offer worthless or genuine nutritive gifts mate with similar frequencies and durations. The female apparently controls the duration of copulation. Thus, there is scope for females to favour males offering gifts and further if these are nutritious, via post-copulatory processes. We first tested whether females differentially store sperm from males that offer the highest nutritional benefits by experimentally presenting females with males that offer either nutritive or worthless gifts (uninterrupted matings). Second, we carried out another set of experiments to examine whether females can select sperm based only on gift presence. This time we interrupted matings after the first pedipalp insertion, thus matching number of insertions and mating duration for males that: offered and did not offer gift. Our results showed that the amount of sperm stored is positive related to mating duration in all groups, except in matings with worthless gifts. Gift presence itself did not affect the sperm stored by females, while they store similar number of sperm in matings with males offering either nutritive or worthless gifts. We discuss whether females prefer males with gifts regardless, if content, because it represents an attractive and/or reliable signal. Or alternatively, they prefer nutritive nuptial gifts, as they are an important source of food supply and/or signal of male donor ability.

Communication during copulation in the sex-role reversed wolf spider Allocosa brasiliensis: Female shakes for soliciting new ejaculations?

Traditional studies on sexual communication have focused on the exchange of signals during courtship. However, communication between the sexes can also occur during or after copulation. Allocosa brasiliensis is a wolf spider that shows a reversal in typical sex roles and of the usual sexual size dimorphism expected for spiders. Females are smaller than males and they are the roving sex that initiates courtship. Occasional previous observations suggested that females performed body shaking behaviors during copulation. Our objective was to analyze if female body shaking is associated with male copulatory behavior in A. brasiliensis, and determine if this female behavior has a communicatory function in this species. For that purpose, we performed fine-scaled analysis of fifteen copulations under laboratory conditions. We video-recorded all the trials and looked for associations between female and male copulatory behaviors. The significant difference between the time before and after female shaking, in favor of the subsequent ejaculation is analyzed. We discuss if shaking could be acting as a signal to accelerate and motivate palpal insertion and ejaculation, and/or inhibiting male cannibalistic tendencies in this species.

Testes mass, but not sperm length, increases with higher levels of polyandry in an ancient sex model.

There is strong evidence that polyandrous taxa have evolved relatively larger testes than monogamous relatives. Sperm size may either increase or decrease across species with the risk or intensity of sperm competition. Scorpions represent an ancient direct mode with spermatophore-mediated sperm transfer and are particularly well suited for studies in sperm competition. This work aims to analyze for the first time the variables affecting testes mass, ejaculate volume and sperm length, according with their levels of polyandry, in species belonging to the Neotropical family Bothriuridae. Variables influencing testes mass and sperm length were obtained by model selection analysis using corrected Akaike Information Criterion. Testes mass varied greatly among the seven species analyzed, ranging from 1.6 ± 1.1 mg in Timogenes dorbignyi to 16.3 ± 4.5 mg in Brachistosternus pentheri with an average of 8.4 ± 5.0 mg in all the species. The relationship between testes mass and body mass was not significant. Body allocation in testes mass, taken as Gonadosomatic Index, was high in Bothriurus cordubensis and Brachistosternus ferrugineus and low in Timogenes species. The best-fitting model for testes mass considered only polyandry as predictor with a positive influence. Model selection showed that body mass influenced sperm length negatively but after correcting for body mass, none of the variables analyzed explained sperm length. Both body mass and testes mass influenced spermatophore volume positively. There was a strong phylogenetic effect on the model containing testes mass. As predicted by the sperm competition theory and according to what happens in other arthropods, testes mass increased in species with higher levels of sperm competition, and influenced positively spermatophore volume, but data was not conclusive for sperm length.

Copulatory behavior in a pholcid spider: males use specialized genitalic movements for sperm removal and copulatory courtship.

Sexual selection may operate on pre-copulatory, copulatory, and post-copulatory traits. An example of a copulatory target of sexual selection is the genitalic movements a male performs during copulation. These movements may function either to prevent sperm competition or to influence a female's fertilization decision. Here we investigated how copulation duration, pedipalp movements, and abdominal movements that males of the pholcid spider Holocnemus pluchei produce during copulation influence sperm removal and/or patterns of successful sperm transfer. We compared mating events with virgin and mated females for differences in copulatory and post-copulatory behavior. We expected longer copulation duration, longer pedipalp movement duration, and more complex and frequent pedipalp and abdominal movements when males mated with mated females compared to virgin females. Except for abdominal movements, our results corroborated these predictions. Furthermore, when we investigated mating events with mated females, we observed sperm mass ejection from the female gonopore and physical removal of sperm by males' procursi. Females with interrupted second mating events showed a significant reduction of stored sperm masses compared to females with completed mating events. We suggest that males use alternating pedipalp movements to remove most of the rival sperm stored by mated females prior to sperm transfer. Copulation duration and pedipalp movements can be further used to transfer sperm and/or as a form of genitalic copulatory courtship.

Sexual dimorphism in immune response, fat reserves and muscle mass in a sex role reversed spider.

In sex role reversed species, females and males adopt behaviors that are not the traditional ones for that animal group. Furthermore, this reversal can translate into physiological differences between the sexes in characteristics such as energetic demands or immune response. Allocosa brasiliensis shows a reversal in the sex roles and sexual size dimorphism that would be expected for spiders. Males are larger than females and are sedentary, while females are the mobile sex that looks for males and initiates courtship. Our objective was to explore the occurrence of sexual dimorphism in immune response, fat content and muscular mass in A. brasiliensis, and relate the results to the reproductive strategies of the species. An encapsulation response was used as an estimate of the immune response. Abdominal fat content and leg muscular mass were quantified and the results were compared between females (N = 19) and males (N = 21). Males showed higher values of the three characteristics as compared to females. Life history divergences between the sexes regarding size, mobility and foraging opportunities could be factors driving these differences in immune response and energy requirements.

Female presence influences sperm velocity in the guppy.

As sperm production is costly, males are expected to strategically allocate resources to sperm production according to mating opportunities. While sperm number adjustments have been reported in several taxa, only a few studies investigated whether sperm quality shows adaptive plasticity as well. We tested this prediction in the guppy, Poecilia reticulata. A total of 46 males were initially stripped of all retrievable sperm before being randomly allocated to one of two treatments simulating different levels of mating opportunities (visual contact with females or female deprived). After 3 days, males were stripped and sperm velocity was assayed using Computer Assisted Sperm Analysis. Males in the presence of females produced significantly faster sperm than their counterparts. Implications for the evolution of this ejaculate plasticity in the light of results of sperm competition studies are discussed.

Chelicerae as male grasping organs in scorpions: sexual dimorphism and associated behaviour.

Specialised structures that enable males to grasp females during sexual interactions are highly susceptible to selection and thus diverge relatively rapidly over evolutionary time. These structures are often used to test hypotheses regarding sexual selection such as sexually antagonistic co-evolution and sexual selection by female choice. In the present study, we determine whether there is a relationship between a novel record of scorpion sexual dimorphism, the sexual dimorphism of chelicerae (CSD), and the presence of the mating behaviour termed "cheliceral grip" (CG). The presence of both traits in the order Scorpiones is also reviewed from a phylogenetic perspective. The results confirm a strong relationship between CSD and the presence of CG. The morphological and behavioural patterns associated with "CSD-CG" are opposed to the predictions postulated by the hypothesis of sexually antagonistic co-evolution. However, if the female shows resistance after the deposition of the spermatophore, the possibility that the male exerts pressure as a "cryptic form" of coercion to prevent the interruption of mating cannot be ruled out completely. Female choice by "mechanical fit" could be another explanation for some aspects of the CG's contact zone. The possibility that the "CG-CSD" complex has evolved under natural selection in order to ensure sperm transfer is also considered.

Patrones de resistencia femenina y respuesta del macho durante el apareamiento en escorpiones bothriuridae y buthidae: qué hipótesis puede explicarlos mejor / Female resistance patterns and male response during the mating in Bothriuridae and Buthidae scorpions: which hypothesis can best explain them?

Las explicaciones para la resistencia femenina que suele aparecer durante el apareamiento son: a) rechazo a coerción masculina (hipótesis de conflicto de intereses) o (b) mecanismo de “screening” para favorecer ciertos machos (hipótesis de elección femenina). Este trabajo examina estas hipótesis según datos obtenidos sobre resistencia femenina en 14 especies de escorpiones. Luego de la resistencia femenina, el macho realizaba sus patrones comportamentales para excitación sexual o apaciguamiento. Si bien nunca el macho efectuaba coerción comportamental directa, podría hacerlo indirectamente mediante el mecanismo de traba de sus pinzas. En Bothriuridae la hembra puede tener dificultad para desprenderse del espermatóforo si intenta interrumpir la inseminación. Los resultados indicarían que en escorpiones la resistencia femenina actuaría como un mecanismo de “screening”. Sin embargo, en algunos Bothriuridae esta posibilidad es compatible con la existencia de un cierto grado de coerción genital(AU)