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New World livebearing fishes (family Poeciliidae) have repeatedly colonised toxic, hydrogen sulphide-rich waters across their natural distribution. Physiological considerations and life-history theory predict that these adverse conditions should favour the evolution of larger offspring. Here, we examined nine poeciliid species that independently colonised toxic environments, and show that these fishes have indeed repeatedly evolved much larger offspring size at birth in sulphidic waters, thus uncovering a widespread pattern of predictable evolution. However, a second pattern, only indirectly predicted by theory, proved additionally common: a reduction in the number of offspring carried per clutch (i.e. lower fecundity). Our analyses reveal that this secondary pattern represents a mere consequence of a classic life-history trade-off combined with strong selection on offspring size alone. With such strong natural selection in extreme environments, extremophile organisms may commonly exhibit multivariate phenotypic shifts even though not all diverging traits necessarily represent adaptations to the extreme conditions., (© 2013 John Wiley & Sons Ltd/CNRS.)

The geological history of the Ponto-Caspian region, with alternating cycles of isolation and reconnection among the three main basins (Black and Azov Seas, and the more distant Caspian Sea) as well as between them and the Mediterranean Sea, profoundly affected the diversification of its aquatic fauna, leading to a high degree of endemism. Two alternative hypotheses on the origin of this amazing biodiversity have been proposed, corresponding to phases of allopatric separation of aquatic fauna among sea basins: a Late Miocene origin (10-6 MYA) vs. a more recent Pleistocene ancestry (<2 MYA). Both hypotheses support a vicariant origin of (1) Black+Azov Sea lineages on the one hand, and (2) Caspian Sea lineages on the other. Here, we present a study on the Ponto-Caspian endemic amphipod Pontogammarus maeoticus. We assessed patterns of divergence based on (a) two mitochondrial and one nuclear gene, and (b) a morphometric analysis of 23 morphological traits in 16 populations from South and West Caspian Sea, South Azov Sea and North-West Black Sea. Genetic data indicate a long and independent evolutionary history, dating back from the late Miocene to early Pleistocene (6.6-1.6 MYA), for an unexpected, major split between (i) a Black Sea clade and (ii) a well-supported clade grouping individuals from the Caspian and Azov Seas. Absence of shared haplotypes argues against either recent or human-mediated exchanges between Caspian and Azov Seas. A mismatch distribution analysis supports more stable population demography in the Caspian than in the Black Sea populations. Morphological divergence largely followed patterns of genetic divergence: our analyses grouped samples according to the basin of origin and corroborated the close phylogenetic affinity between Caspian and Azov Sea lineages. Altogether, our results highlight the necessity of careful (group-specific) evaluation of evolutionary trajectories in marine taxa that should certainly not be inferred from the current geographical proximity of sea basins alone., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We investigated mechanisms of reproductive isolation in livebearing fishes (genus Poecilia) inhabiting sulfidic and nonsulfidic habitats in three replicate river drainages. Although sulfide spring fish convergently evolved divergent phenotypes, it was unclear if mechanisms of reproductive isolation also evolved convergently. Using microsatellites, we found strongly reduced gene flow between adjacent populations from different habitat types, suggesting that local adaptation to sulfidic habitats repeatedly caused the emergence of reproductive isolation. Reciprocal translocation experiments indicate strong selection against immigrants into sulfidic waters, but also variation among drainages in the strength of selection against immigrants into nonsulfidic waters. Mate choice experiments revealed the evolution of assortative mating preferences in females from nonsulfidic but not from sulfidic habitats. The inferred strength of sexual selection against immigrants (RI(s)) was negatively correlated with the strength of natural selection (RI(m)), a pattern that could be attributed to reinforcement, whereby natural selection strengthens behavioral isolation due to reduced hybrid fitness. Overall, reproductive isolation and genetic differentiation appear to be replicated and direct consequences of local adaptation to sulfide spring environments, but the relative contributions of different mechanisms of reproductive isolation vary across these evolutionarily independent replicates, highlighting both convergent and nonconvergent evolutionary trajectories of populations in each drainage., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

The process of ecological speciation drives the evolution of locally adapted and reproductively isolated populations in response to divergent natural selection. In Southern Mexico, several lineages of the freshwater fish species of the genus Poecilia have independently colonized toxic, hydrogen sulfide-rich springs. Even though ecological speciation processes are increasingly well understood in this system, aligning the taxonomy of these fish with evolutionary processes has lagged behind. While some sulfide spring populations are classified as ecotypes of Poecilia mexicana, others, like P. sulphuraria, have been described as highly endemic species. Our study particularly focused on elucidating the taxonomy of the long described sulfide spring endemic, Poecilia thermalis Steindachner 1863, and investigates if similar evolutionary patterns of phenotypic trait divergence and reproductive isolation are present as observed in other sulfidic species of Poecilia. We applied a geometric morphometric approach to assess body shape similarity to other sulfidic and non-sulfidic fish of the genus Poecilia. We also conducted phylogenetic and population genetic analyses to establish the phylogenetic relationships of P. thermalis and used a population genetic approach to determine levels of gene flow among Poecilia from sulfidic and non-sulfidic sites. Our results indicate that P. thermalis' body shape has evolved in convergence with other sulfide spring populations in the genus. Phylogenetic analyses placed P. thermalis as most closely related to one population of P. sulphuraria, and population genetic analyses demonstrated that P. thermalis is genetically isolated from both P. mexicana ecotypes and P. sulphuraria. Based on these findings, we make taxonomic recommendations for P. thermalis. Overall, our study verifies the role of hydrogen sulfide as a main factor shaping convergent, phenotypic evolution and the emergence of reproductive isolation between Poecilia populations residing in adjacent sulfidic and non-sulfidic environments.

Mate choice in many species is sensitive to social cues from neighboring individuals; for example, animals can copy mate choice decisions. If males copy other males' choices, sperm of two or more males can compete for fertilization of the female's ova. In the internally fertilizing fish Poecilia mexicana, males respond to the presence of rivals with reduced expression of mating preferences (audience effect), thereby lowering the risk of by-standing rivals copying their mate choice. Also, males interact initially more with a non-preferred female when observed by a rival, which has been interpreted in previous studies as a strategy to mislead rivals, again reducing sperm competition risk (SCR). Using a comparative approach, we tested the hypothesis that SCR is indeed a driving force explaining the occurrence of audience-induced changes in poeciliid male mate choice behavior. If this were true, then males of species with higher overall sexual activity - and, thus, higher potential for multiple mating - should show stronger audience effects. We investigated ten poeciliid species (in two cases including multiple populations) and found support for our hypothesis as mean sexual activity correlated positively with the occurrence of potentially deceptive behavior. An alternative explanation for audience effects would be that males attempt to avoid aggressive encounters, which would predict stronger audience effects in more aggressive species, and so we also characterized the examined species for aggressiveness using staged contests of size-matched males. We demonstrate a positive correlation between mean aggressiveness and sexual activity (suggesting a hormonal link as a mechanistic explanation), but we detected no correlation between aggressiveness and audience effects. Suites of correlated behavioral tendencies are termed behavioral syndromes, and our present study provides correlational evidence for the evolutionary significance of SCR in shaping a behavioral syndrome at the species level across poeciliid taxa.

Extreme habitats are often characterized by reduced predation pressures, thus representing refuges for the inhabiting species. The present study was designed to investigate predator avoidance of extremophile populations of Poecilia mexicana and P. sulphuraria that either live in hydrogen sulfide-rich (sulfidic) springs or cave habitats, both of which are known to have impoverished piscine predator regimes. Focal fishes that inhabited sulfidic springs showed slightly weaker avoidance reactions when presented with several naturally occurring predatory cichlids, but strongest differences to populations from non-sulfidic habitats were found in a decreased shoaling tendency with non-predatory swordtail (Xiphophorus hellerii) females. When comparing avoidance reactions between P. mexicana from a sulfidic cave (Cueva del Azufre) and the adjacent sulfidic surface creek (El Azufre), we found only slight differences in predator avoidance, but surface fish reacted much more strongly to the non-predatory cichlid Vieja bifasciata. Our third experiment was designed to disentangle learned from innate effects of predator recognition. We compared laboratory-reared (i.e., predator-naïve) and wild-caught (i.e., predator-experienced) individuals of P. mexicana from a non-sulfidic river and found no differences in their reaction towards the presented predators. Overall, our results indicate (1) that predator avoidance is still functional in extremophile Poecilia spp. and (2) that predator recognition and avoidance reactions have a strong genetic basis.

This study asked whether reductive traits in cave organisms evolve at a slower pace (suggesting neutral evolution under relaxed selection) than constructive changes, which are likely to evolve under directional selection. We investigated 11 subterranean and seven surface populations of Sundathelphusa freshwater crabs on Bohol Island, Philippines, and examined constructive traits associated with improved food finding in darkness (increased leg and setae length) and reductive traits (reduced cornea size and eyestalk length). All changes occurred rapidly, given that the age of the most recent common ancestor was estimated to be 722-271 ka based on three mitochondrial markers. In order to quantify the speed of character change, we correlated the degree of morphological change with genetic distances between surface and subterranean individuals. The temporal pattern of character change following the transition to subterranean life was indistinguishable for constructive and reductive traits, characterized by an immediate onset and rapid evolutionary change. We propose that the evolution of these reductive traits-just like constructive traits-is most likely driven by strong directional selection.

Ecological speciation assumes reproductive isolation to be the product of ecologically based divergent selection. Beside natural selection, sexual selection via phenotype-assortative mating is thought to promote reproductive isolation. Using the neotropical fish Poecilia mexicana from a system that has been described to undergo incipient ecological speciation in adjacent, but ecologically divergent habitats characterized by the presence or absence of toxic H2S and darkness in cave habitats, we demonstrate a gradual change in male body colouration along the gradient of light/darkness, including a reduction of ornaments that are under both inter- and intrasexual selection in surface populations. In dichotomous choice tests using video-animated stimuli, we found surface females to prefer males from their own population over the cave phenotype. However, female cave fish, observed on site via infrared techniques, preferred to associate with surface males rather than size-matched cave males, likely reflecting the female preference for better-nourished (in this case: surface) males. Hence, divergent selection on body colouration indeed translates into phenotype-assortative mating in the surface ecotype, by selecting against potential migrant males. Female cave fish, by contrast, do not have a preference for the resident male phenotype, identifying natural selection against migrants imposed by the cave environment as the major driver of the observed reproductive isolation.

Male homosexual behaviour-although found in most extant clades across the Animal Kingdom-remains a conundrum, as same-sex mating should decrease male reproductive fitness. In most species, however, males that engage in same-sex sexual behaviour also mate with females, and in theory, same-sex mating could even increase male reproductive fitness if males improve their chances of future heterosexual mating. Females regularly use social information to choose a mate; e.g. male attractiveness increases after a male has interacted sexually with a female (mate choice copying). Here, we demonstrate that males of the tropical freshwater fish Poecilia mexicana increase their attractiveness to females not only by opposite-sex, but likewise, through same-sex interactions. Hence, direct benefits for males of exhibiting homosexual behaviour may help explain its occurrence and persistence in species in which females rely on mate choice copying as one component of mate quality assessment.

Heterospecific neighbors may reduce damage to a focal plant by lowering specialist herbivore loads (associational resistance hypothesis), or enhance damage by increasing generalist herbivore loads (associational susceptibility hypothesis). We tested the associational effects of tree diversity on herbivory patterns of the tropical focal tree Tabebuia rosea in an experimental plantation setup, which contained tree monocultures and mixed stands. We found higher herbivore damage to T. rosea at higher tree diversity, indicating that T. rosea did not benefit from associational resistance but rather experienced associational susceptibility. The specific consideration of the two dominant insect herbivore species of T. rosea, the specialist chrysomelid Walterianella inscripta and the specialist pyralid Eulepte gastralis, facilitated understanding of the detected damage patterns. Tree diversity exerted opposite effects on tree infestation by the two herbivores. These findings point to resource concentration effects for the chrysomelid beetle (favored by tree monoculture) and to resource dilution effects for the pyralid caterpillar (favored by tree mixture) as underlying mechanisms of herbivore distribution. A strong contribution of the pyralid to overall damage patterns in diversified stands suggests that associational susceptibility may not necessarily be related to higher abundances of generalist herbivores but may also result from specialized herbivores affected by resource dilution effects. Thus, the identity and biology of herbivore species has to be taken into account when attempting to predict damage patterns in forest ecosystems.

Reproductive isolation among locally adapted populations may arise when immigrants from foreign habitats are selected against via natural or (inter-)sexual selection (female mate choice). We asked whether also intrasexual selection through male-male competition could promote reproductive isolation among populations of poeciliid fishes that are locally adapted to extreme environmental conditions [i.e., darkness in caves and/or toxic hydrogen sulphide (H(2)S)]. We found strongly reduced aggressiveness in extremophile P. oecilia mexicana, and darkness was the best predictor for the evolutionary reduction of aggressiveness, especially when combined with presence of H(2)S. We demonstrate that reduced aggression directly translates into migrant males being inferior when paired with males from non-sulphidic surface habitats. By contrast, the phylogenetically old sulphur endemic P. sulphuraria from another sulphide spring area showed no overall reduced aggressiveness, possibly indicating evolved mechanisms to better cope with H(2)S.

Divergent selection through biotic factors like predation or parasitism can promote reproductive isolation even in the absence of geographical barriers. On the other hand, evidence for a role of adaptation to abiotic factors during ecological speciation in animals is scant. In particular, the role played by perpetual darkness in establishing reproductive isolation in cave animals (troglobites) remains elusive. We focused on two reproductively isolated ecotypes (surface- and cave-dwelling) of the widespread livebearer Poecilia mexicana, and raised offspring of wild-caught females to sexual maturity in a 12-month common-garden experiment. Fish were reared in light or darkness combined with high- or low-food conditions. Females, but not males, of the surface ecotype suffered from almost complete reproductive failure in darkness, especially in the low-food treatment. Furthermore, surface fish suffered from a significantly higher rate of spontaneous, stress-related infection with bacterial columnaris disease. This experimental evidence for strong selection by permanent darkness on non-adapted surface-dwelling animals adds depth to our understanding of the selective forces establishing and maintaining reproductive isolation in cave faunas.

Our study was designed to evaluate if, and to what extent, restrictive environmental conditions affect otolith morphology. As a model, we chose two extremophile livebearing fishes: (i) Poecilia mexicana, a widespread species in various Mexican freshwater habitats, with locally adapted populations thriving in habitats characterized by the presence of one (or both) of the natural stressors hydrogen sulphide and darkness, and (ii) the closely related Poecilia sulphuraria living in a highly sulphidic habitat (Baños del Azufre). All three otolith types (lapilli, sagittae, and asterisci) of P. mexicana showed a decrease in size ranging from the non-sulphidic cave habitat (Cueva Luna Azufre), to non-sulphidic surface habitats, to the sulphidic cave (Cueva del Azufre), to sulphidic surface habitats (El Azufre), to P. sulphuraria. Although we found a distinct differentiation between ecotypes with respect to their otolith morphology, no clear-cut pattern of trait evolution along the two ecological gradients was discernible. Otoliths from extremophiles captured in the wild revealed only slight similarities to aberrant otoliths found in captive-bred fish. We therefore hypothesize that extremophile fishes have developed coping mechanisms enabling them to avoid aberrant otolith growth - an otherwise common phenomenon in fishes reared under stressful conditions., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

We investigated replicated ecological speciation in the livebearing fish Poecilia mexicana and P. sulphuraria (Poeciliidae), which inhabit freshwater habitats and have also colonized multiple sulfidic springs in southern Mexico. These springs exhibit extreme hypoxia and high concentrations of hydrogen sulfide, which is lethal to most metazoans. We used phylogenetic analyses to test whether springs were independently colonized, performed phenotypic assessments of body and gill morphology variation to identify convergent patterns of trait differentiation, and conducted an eco-toxicological experiment to detect differences in sulfide tolerances among ecotypes. Our results indicate that sulfidic springs were colonized by three different lineages, two within P. mexicana and one representing P. sulphuraria. Colonization occurred earlier in P. sulphuraria, whereas invasion of sulfidic springs in P. mexicana was more recent, such that each population is more closely related to neighboring populations from adjacent nonsulfidic habitats. Sulfide spring fish also show divergence from nonsulfidic phenotypes and a phenotypic convergence toward larger heads, larger gills, and increased tolerance to H(2) S. Together with previous studies that indicated significant reproductive isolation between fish from sulfidic and nonsulfidic habitats, this study provides evidence for repeated ecological speciation in the independent sulfide spring populations of P. mexicana and P. sulphuraria., (© 2011 The Author(s). Evolution© 2011 The Society for the Study of Evolution.)

Background: In many species males face a higher predation risk than females because males display elaborate traits that evolved under sexual selection, which may attract not only females but also predators. Females are, therefore, predicted to avoid such conspicuous males under predation risk. The present study was designed to investigate predator-induced changes of female mating preferences in Atlantic mollies (Poecilia mexicana). Males of this species show a pronounced polymorphism in body size and coloration, and females prefer large, colorful males in the absence of predators., Results: In dichotomous choice tests predator-naïve (lab-reared) females altered their initial preference for larger males in the presence of the cichlid Cichlasoma salvini, a natural predator of P. mexicana, and preferred small males instead. This effect was considerably weaker when females were confronted visually with the non-piscivorous cichlid Vieja bifasciata or the introduced non-piscivorous Nile tilapia (Oreochromis niloticus). In contrast, predator experienced (wild-caught) females did not respond to the same extent to the presence of a predator, most likely due to a learned ability to evaluate their predators' motivation to prey., Conclusions: Our study highlights that (a) predatory fish can have a profound influence on the expression of mating preferences of their prey (thus potentially affecting the strength of sexual selection), and females may alter their mate choice behavior strategically to reduce their own exposure to predators. (b) Prey species can evolve visual predator recognition mechanisms and alter their mate choice only when a natural predator is present. (c) Finally, experiential effects can play an important role, and prey species may learn to evaluate the motivational state of their predators.

Mate choice as one element of sexual selection can be sensitive to public information from neighbouring individuals. Here, we demonstrate that males of the livebearing fish Poecilia mexicana gather complex social information when given a chance to familiarize themselves with rivals prior to mate choice. Focal males ceased to show mating preferences when being observed by a rival (which prevents rivals from copying mating decisions), but this effect was only seen when focal males have perceived rivals as sexually active. In addition, focal males that were observed by a familiar, sexually active rival showed a stronger behavioural response when rivals were larger and thus, more attractive to females. Our study illustrates an unparalleled adjustment in the expression of mating preferences based on social cues, and suggests that male fish are able to remember and strategically exploit information about rivals when performing mate choice.

Mate choice can be sensitive to social cues from neighboring individuals, e.g., animals can copy mate choice decisions. Males that are at risk of being copied by others may respond to this with reduced preference expression ("audience effects"). We review the various pathways by which sperm competition risk affects (1) male mate copying behavior and (2) audience effects. For example, a recent study suggests that males gather complex social information on rivals' sexual competitiveness (sexual activity and attractiveness to females) and respond with reduced expression of mating preferences only "when it matters," i.e., when a sexually competitive rival is present.

Background: Fishes show an amazing diversity in hearing abilities, inner ear structures, and otolith morphology. Inner ear morphology, however, has not yet been investigated in detail in any member of the diverse order Cyprinodontiformes. We, therefore, studied the inner ear of the cyprinodontiform freshwater fish Poecilia mexicana by analyzing the position of otoliths in situ, investigating the 3D structure of sensory epithelia, and examining the orientation patterns of ciliary bundles of the sensory hair cells, while combining μ-CT analyses, scanning electron microscopy, and immunocytochemical methods. P. mexicana occurs in different ecotypes, enabling us to study the intra-specific variability (on a qualitative basis) of fish from regular surface streams, and the Cueva del Azufre, a sulfidic cave in southern Mexico., Results: The inner ear of Poecilia mexicana displays a combination of several remarkable features. The utricle is connected rostrally instead of dorso-rostrally to the saccule, and the macula sacculi, therefore, is very close to the utricle. Moreover, the macula sacculi possesses dorsal and ventral bulges. The two studied ecotypes of P. mexicana showed variation mainly in the shape and curvature of the macula lagenae, in the curvature of the macula sacculi, and in the thickness of the otolithic membrane., Conclusions: Our study for the first time provides detailed insights into the auditory periphery of a cyprinodontiform inner ear and thus serves a basis--especially with regard to the application of 3D techniques--for further research on structure-function relationships of inner ears within the species-rich order Cyprinodontiformes. We suggest that other poeciliid taxa, or even other non-poeciliid cyprinodontiforms, may display similar inner ear morphologies as described here.

Background: Closely related lineages of livebearing fishes have independently adapted to two extreme environmental factors: toxic hydrogen sulphide (H(2)S) and perpetual darkness. Previous work has demonstrated in adult specimens that fish from these extreme habitats convergently evolved drastically increased head and offspring size, while cave fish are further characterized by reduced pigmentation and eye size. Here, we traced the development of these (and other) divergent traits in embryos of Poecilia mexicana from benign surface habitats ("surface mollies") and a sulphidic cave ("cave mollies"), as well as in embryos of the sister taxon, Poecilia sulphuraria from a sulphidic surface spring ("sulphur mollies"). We asked at which points during development changes in the timing of the involved processes (i.e., heterochrony) would be detectible., Methods and Results: Data were extracted from digital photographs taken of representative embryos for each stage of development and each type of molly. Embryo mass decreased in convergent fashion, but we found patterns of embryonic fat content and ovum/embryo diameter to be divergent among all three types of mollies. The intensity of yellow colouration of the yolk (a proxy for carotenoid content) was significantly lower in cave mollies throughout development. Moreover, while relative head size decreased through development in surface mollies, it increased in both types of extremophile mollies, and eye growth was arrested in mid-stage embryos of cave mollies but not in surface or sulphur mollies., Conclusion: Our results clearly demonstrate that even among sister taxa convergence in phenotypic traits is not always achieved by the same processes during embryo development. Furthermore, teleost development is crucially dependent on sufficient carotenoid stores in the yolk, and so we discuss how the apparent ability of cave mollies to overcome this carotenoid-dependency may represent another potential mechanism explaining the lack of gene flow between surface and cave mollies.

Adaptation to ecologically heterogeneous environments can drive speciation. But what mechanisms maintain reproductive isolation among locally adapted populations? Using poeciliid fishes in a system with naturally occurring toxic hydrogen sulfide, we show that (a) fish from non-sulfidic sites (Poecilia mexicana) show high mortality (95 %) after 24 h when exposed to the toxicant, while locally adapted fish from sulfidic sites (Poecilia sulphuraria) experience low mortality (13 %) when transferred to non-sulfidic water. (b) Mate choice tests revealed that P. mexicana females exhibit a preference for conspecific males in non-sulfidic water, but not in sulfidic water, whereas P. sulphuraria females never showed a preference. Increased costs of mate choice in sulfidic, hypoxic water, and the lack of selection for reinforcement due to the low survival of P. mexicana may explain the absence of a preference in P. sulphuraria females. Taken together, our study may be the first to demonstrate independent-but complementary-effects of natural and sexual selection against immigrants maintaining differentiation between locally adapted fish populations.

Cave fish have rarely been investigated with regard to their inner ear morphology, hearing abilities, and acoustic communication. Based on a previous study that revealed morphological differences in the saccular otolith between a cave and two surface populations of Poecilia mexicana, we checked for additional differences in utricular and lagenar otoliths and tested whether different populations have similar hearing sensitivities. We found pronounced differences in the shape of all three otoliths. Otoliths of the saccule and lagena from cave fish differed from those of surface fish in the features of the face oriented towards the sensory epithelium. In addition, otoliths of the utricle and lagena were significantly heavier in cave fish. Auditory sensitivities were measured between 100 and 1500Hz, utilizing the auditory evoked potential recording technique. We found similar hearing abilities in cave and surface fish, with greatest sensitivity between 200 and 300Hz. An acoustic survey revealed that neither ecotype produced species-specific sounds. Our data indicate that cave dwelling altered the otolith morphology in Atlantic mollies, probably due to metabolic differences. Different otolith morphology, however, did not affect general auditory sensitivity or acoustic behavior., (Copyright 2010 Elsevier B.V. All rights reserved.)

Life-history traits are very sensitive to extreme environmental conditions, because resources that need to be invested in somatic maintenance cannot be invested in reproduction. Here we examined female life-history traits in the Mexican livebearing fish Poecilia mexicana from a variety of benign surface habitats, a creek with naturally occurring toxic hydrogen sulfide (H2S), a sulfidic cave, and a non-sulfidic cave. Previous studies revealed pronounced genetic and morphological divergence over very small geographic scales in this system despite the absence of physical barriers, suggesting that local adaptation to different combinations of two selection factors, toxicity (H2S) and darkness, is accompanied by very low rates of gene flow. Hence, we investigated life-history divergence between these populations in response to the selective pressures of darkness and/or toxicity. Our main results show that toxicity and darkness both select for (or impose constraints on) the same female trait dynamics: reduced fecundity and increased offspring size. Since reduced fecundity in the sulfur cave population was previously shown to be heritable, we discuss how divergent life-history evolution may promote further ecological divergence: for example, reduced fecundity and increased offspring autonomy are clearly beneficial in extreme environments, but fish with these traits are outcompeted in benign habitats.

The majority of studies on ecological speciation in animals have investigated the divergence caused by biotic factors like divergent food sources or predatory regimes. Here, we examined a system where ecological speciation can clearly be ascribed to abiotic environmental gradients of naturally occurring toxic hydrogen sulfide (H(2)S). In southern Mexico, two genera of livebearing fishes (Poeciliidae: Poecilia and Gambusia) thrive in various watercourses with different concentrations of H(2)S. Previous studies have revealed pronounced genetic differentiation between different locally adapted populations in one species (Poecilia mexicana), pointing towards incipient speciation. In the present study, we examined female reproductive life-history traits in two species pairs: Gambusia sexradiata (from a nonsulfidic and a sulfidic habitat) and Gambusia eurystoma (sulfide-endemic), as well as P. mexicana (nonsulfidic and sulfidic) and Poecilia sulphuraria (sulfide endemic). We found convergent divergence of life-history traits in response to sulfide; most prominently, extremophile poeciliids exhibit drastically increased offspring size coupled with reduced fecundity. Furthermore, within each genus, this trend increased with increasing sulfide concentrations and was most pronounced in the two endemic sulfur-adapted species. We discuss the adaptive significance of large offspring size in toxic environments and propose that divergent life-history evolution may promote further ecological divergence through isolation by adaptation.

Background: Multidirectional interactions in social networks can have a profound effect on mate choice behavior; e.g., Poecilia mexicana males show weaker expression of mating preferences when being observed by a rival. This may be an adaptation to reduce sperm competition risk, which arises because commonly preferred female phenotypes will receive attention also from surrounding males, and/or because other males can copy the focal male's mate choice. Do P. mexicana males indeed respond to perceived sperm competition risk? We gave males a choice between two females and repeated the tests under one of the following conditions: (1) an empty transparent cylinder was presented (control); (2) another ("audience") male inside the cylinder observed the focal male throughout the 2nd part, or (3) the audience male was presented only before the tests, but could not eavesdrop during the actual choice tests (non-specific sperm competition risk treatments); (4) the focal male could see a rival male interact sexually with the previously preferred, or (5) with the non-preferred female before the 2nd part of the tests (specific sperm competition risk treatments)., Results: The strength of individual male preferences declined slightly also during the control treatment (1). However, this decrease was more than two-fold stronger in audience treatment (2), i.e., with non-specific sperm competition risk including the possibility for visual eavesdropping by the audience male. No audience effect was found in treatments (3) and (5), but a weak effect was also observed when the focal male had seen the previously preferred female sexually interact with a rival male (treatment 4; specific sperm competition risk)., Conclusion: When comparing the two 'non-specific sperm competition risk' treatments, a very strong effect was found only when the audience male could actually observe the focal male during mate choice [treatment (2)]. This suggests that focal males indeed attempt to conceal their mating preferences so as to prevent surrounding males from copying their mate choice. When there is no potential for eavesdropping [treatment (3)], non-specific specific sperm competition risk seems to play a minor or no role. Our results also show that P. mexicana males tend to share their mating effort more equally among females when the resource value of their previously preferred mate decreases after mating with a rival male (perceived specific sperm competition risk), but this effect is comparatively weak.

A unique evolutionary specialization of African weakly electric fish (Mormyridae) is their ability to produce and perceive electric signals. Mormyrids use their electric organs discharge (EOD) for electrolocation and electrocommunication. Here we discuss the adaptive significance of the EOD in foraging (electric prey detection) in light of recent results demonstrating that mormyrid fish mate assortatively according to EOD waveform characteristics (electric mate choice). Therefore the EOD as a single trait pleiotropically combines natural divergent selection and reproductive isolation. Consequently we postulate the EOD as a "magic trait" promoting the diversification of African weakly electric fish.

Mate choice is mediated by a range of sensory cues, and assortative mating based on these cues can drive reproductive isolation among diverging populations. A specific feature of mormyrid fish, the electric organ discharge (EOD), is used for electrolocation and intraspecific communication. We hypothesized that the EOD also facilitates assortative mating and ultimately promotes prezygotic reproductive isolation in African weakly electric fishes. Our behavioural experiments using live males as well as EOD playback demonstrated that female mate recognition is influenced by EOD signals and that females are attracted to EOD characteristics of conspecific males. The dual function of the EOD for both foraging and social communication (including mate recognition leading to assortative mating) underlines the importance of electric signal differentiation for the divergence of African weakly electric fishes. Thus, the EOD provides an intriguing mechanism promoting trophic divergence and reproductive isolation between two closely related Campylomormyrus species occurring in sympatry in the lower Congo rapids.

A fundamental question in sympatric speciation is how trophic divergence is achieved. We used an extremely young (<8,000 years) species flock of pupfish (Cyprinodon spp.) from Laguna Chichancanab in south-eastern Mexico to examine divergent evolution of preferences for different feeding substrates. In a test aquarium, we presented four feeding substrates (sand, gravel, a plastic plant, and blank bottom), but no actual food was offered. The four feeding substrates were chosen to mirror the most common substrate types in Laguna Chichancanab. Previous studies demonstrated that benthic food items prevail in the diet of most Cyprinodon species. C. beltrani preferred sand, whereas C. labiosus preferred gravel. F(1) hybrids of both species showed intermediate preferences. C. maya searched for food equally at all substrates. As the test fish were reared under identical laboratory conditions (i.e., in the absence of feeding substrates), the species-specific preferences appear to be genetically fixed, suggesting rapid divergent evolution of feeding behaviors.

Genes of the major histocompatibility complex (MHC) encode molecules that control immune recognition and are highly polymorphic in most vertebrates. The remarkable polymorphisms at MHC loci may be maintained by selection from parasites, sexual selection, or both. If asexual species show equal (or higher) levels of polymorphisms at MHC loci as sexual ones, this would mean that sexual selection is not necessary to explain the high levels of diversity at MHC loci. In this study, we surveyed the MHC diversity of the asexual amazon molly (Poecilia formosa) and one of its sexual ancestors, the sailfin molly (P. latipinna), which lives in the same habitat. We found that the asexual molly has polymorphic MHC loci despite its clonal reproduction, yet not as polymorphic as the sexual species. Although the nucleotide diversity was similar between the asexual and sexual species, the sexual species exhibited a greater genotypic diversity compared to the asexual one from the same habitats. Within-genome diversity was similar for MHC class I loci, but for class IIB, the sexual species had higher diversity compared to the asexual--despite the hybrid origins and higher levels of heterozygosity at microsatellite loci in the asexual species. The level of positive selection appears to be similar between the two species, which suggests that these polymorphisms are maintained by selection. Thus, our findings do not allow us to rule out the sexual selection hypothesis for the evolution of MHC diversity, and although the sexual fish has higher levels of MHC-diversity compared to the asexual species, this may be due to differences in demography, parasites, or other factors, rather than sexual selection.

Sexual selection by female choice can maintain male traits that are counter selected by natural selection. Alteration of the potential for sexual selection can thus lead to shifts in the expression of male traits. We investigated female mate choice for large male body size in a fish (Poecilia mexicana) that, besides surface streams, also inhabits two caves. All four populations investigated, exhibited an ancestral visual preference for large males. However, only one of the cave populations also expressed this female preference in darkness. Hence, the lack of expression of female preference in darkness in the other cave population leads to relaxation of sexual selection for large male body size. While P. mexicana populations with size-specific female mate choice are characterized by a pronounced male size variation, the absence of female choice in one cave coincides with the absence of large bodied males in that population. Our results suggest that population differences in the potential for sexual selection may affect male trait variation.

Divergent natural selection drives evolutionary diversification. It creates phenotypic diversity by favoring developmental plasticity within populations or genetic differentiation and local adaptation among populations. We investigated phenotypic and genetic divergence in the livebearing fish Poecilia mexicana along two abiotic environmental gradients. These fish typically inhabit nonsulfidic surface rivers, but also colonized sulfidic and cave habitats. We assessed phenotypic variation among a factorial combination of habitat types using geometric and traditional morphometrics, and genetic divergence using quantitative and molecular genetic analyses. Fish in caves (sulfidic or not) exhibited reduced eyes and slender bodies. Fish from sulfidic habitats (surface or cave) exhibited larger heads and longer gill filaments. Common-garden rearing suggested that these morphological differences are partly heritable. Population genetic analyses using microsatellites as well as cytochrome b gene sequences indicate high population differentiation over small spatial scale and very low rates of gene flow, especially among different habitat types. This suggests that divergent environmental conditions constitute barriers to gene flow. Strong molecular divergence over short distances as well as phenotypic and quantitative genetic divergence across habitats in directions classic to fish ecomorphology suggest that divergent selection is structuring phenotypic variation in this system.

A fundamental question in animal communication is whether the information provided is honest or deceptive [1, 2]. This problem has received much attention, both in theoretical [1, 3] and experimental [4] work. Here we show that male Atlantic mollies (Poecilia mexicana), when observed during mate choice by another male, reduce their mating activity and no longer prefer mating with one of two females presented, which can be interpreted as an attempt to avoid unintended interception of information by the rival male. Most importantly, focal males directed their first sexual interaction (after they were presented with the rival male) toward the initially nonpreferred female, suggesting that males deceive other males about their mating preferences. Deception by the choosing male may be an adaptation to avoid sperm competition, because surrounding males may use public information and copy the focal male's mate choice.

Male-biased predation has been described from several epigean species, and in many cases, intrinsic differences between the sexes (such as male ornaments) have been suggested as an explanation. Here we report on male-biased predation of a cave fish (Poecilia mexicana) by an aquatic insect (Belostoma sp.) in a Mexican sulfur cave. P. mexicana use aquatic surface respiration (ASR) to survive in their sulfidic, hypoxic habitat. We found that males typically exhibit more ASR activity than females, which leads to increased exposure to the sit-and-wait predator that catches fish near the water surface. Our finding is novel, because male vulnerability to predation is not directly related to male traits involved in courtship, but rather due to other sexual differences in behavior and ultimately, oxygen demands.

In sperm-dependent sexual/asexual mating systems, male mate choice is critical for understanding the mechanisms behind apparent stability observed in natural populations. The gynogenetic Amazon molly (Poecilia formosa) requires sperm from sexual males (e.g. Poecilia latipinna) to trigger embryogenesis, but inheritance is strictly maternal. Consequently, males should try to avoid or reduce the cost of mating with asexuals. We investigated male mate choice by documenting the presence of sperm in natural populations and found that a higher proportion of sexual females had sperm than asexuals. In addition, among those females that had sperm, sexuals had more sperm than asexuals. Our results hint at a role for male mate choice as a stabilizing factor in such systems.

An increasing body of literature considers the question of how mate choice is influenced by the social environment of the choosing individual (non-independent mate choice). For example, individuals may copy the mate choice of others. A very simple form of socially influenced mate choice, however, remained comparatively little investigated: choosing individuals may adjust their mate choice to the mere presence of rivals. Recent studies in our groups1-4 have examined this question. Using live bearing fish (mollies, Poecilia spp.) as a model, we could show that (a) males will copy the mate choice of other males,5 (b) males cease expressing mating preferences in the presence of a conspecific rival male,1,2 whereas (c) females copy each other's mate choice, but are otherwise not affected by an audience.3 (d) Most importantly, males, when presented with an audience (potential rival), first approached the previously non-preferred female, suggesting that males attempt to lead the rival away from their preferred mate, thereby exploiting male mate choice copying behavior.4 We discuss that these effects are best explained as male adaptations to reduce the risk of sperm competition in a highly dynamic mating system with frequent multiple mating.

Extreme habitats challenge animals with highly adverse conditions, like extreme temperatures or toxic substances. In this paper, we report of a fish (Poecilia mexicana) inhabiting a limestone cave in Mexico. Several springs inside the cave are rich in toxic H(2)S. We demonstrate that a behavioural adaptation, aquatic surface respiration (ASR), allows for the survival of P. mexicana in this extreme, sulphidic habitat. Without the possibility to perform ASR, the survival rate of P. mexicana was low even at comparatively low H(2)S concentrations. Furthermore, we show that food limitation affects the survival of P. mexicana pointing to energetically costly physiological adaptations to detoxify H(2)S.

Most eucaryotic organisms classified as living in an extreme habitat are invertebrates. Here we report of a fish living in a Mexican cave (Cueva del Azufre) that is rich in highly toxic H(2)S. We compared the water chemistry and fish communities of the cave and several nearby surface streams. Our study revealed high concentrations of H(2)S in the cave and its outflow (El Azufre). The concentrations of H(2)S reach more than 300 muM inside the cave, which are acutely toxic for most fishes. In both sulfidic habitats, the diversity of fishes was heavily reduced, and Poecilia mexicana was the dominant species indicating that the presence of H(2)S has an all-or-none effect, permitting only few species to survive in sulfidic habitats. Compared to habitats without H(2)S, P. mexicana from the cave and the outflow have a significantly lower body condition. Although there are microhabitats with varying concentrations of H(2)S within the cave, we could not find a higher fish density in areas with lower concentrations of H(2)S. We discuss that P. mexicana is one of the few extremophile vertebrates. Our study supports the idea that extreme habitats lead to an impoverished species diversity.

Atlantic mollies (Poecilia mexicana) inhabit a variety of surface habitats, but they also occur in a sulfur cave in southern Mexico. We examined male mate choice relative to female body size in the cave population and in the most closely related surface-dwelling population from a nearby river. Males from both populations were either light- or dark-reared and could choose between two differently sized females either on the basis of visual cues in light or on the basis of solely nonvisual cues in darkness. Sexual preferences were estimated from the degree of association. Cave molly males always showed a preference for the larger female, both in light and in darkness. Among the surface males, only light-reared males showed a preference in the visual cues test, but not in darkness. In a control experiment, we demonstrated that male association preferences directly translate into actual mating preferences. Apparently, using visual cues for mate choice is the ancestral state in this system, and using nonvisual cues has evolved as a novel trait in the cave population. We discuss the evolution of nonvisual male mate choice in the context of changed environmental conditions, namely the absence of light, hypoxia, and toxic hydrogen sulfide in the cave.

Background: Human pancreatic adenocarcinomas are highly resistant to chemotherapy. The p16 tumor-suppressor protein is inactivated in more than 90% of human pancreatic cancers. The p16 protein transcriptionally inhibits expression of retinoblastoma tumor-suppressor gene pRB. The pRB protein transcriptionally inhibits expression of the p16 gene. Because pRB normally prevents apoptosis, we investigated whether pRB is involved in resistance to chemotherapy-induced apoptosis in pancreatic cancer cells., Methods: pRB expression was examined by immunohistochemistry in 106 human pancreatic tissue specimens. The human pancreatic tumor cell line Capan-1 (pRB+/p16-) was stably transfected with p16 to functionally inactivate pRB. pRB gene expression was examined by western and northern blot analyses, and pRB function was assessed by electrophoretic mobility shift assays and promoter transactivation studies for the transcription factor E2F. Changes in cell sensitivity to chemotherapy were measured by assays for cytotoxicity and apoptosis., Results: pRB was overexpressed in pancreatic ductal adenocarcinomas but was hardly detectable in other pancreatic malignancies, chronic pancreatitis, or nontransformed human pancreatic tissue. Expression of p16 in Capan-1 cells resulted in the loss of pRB gene and protein expression concomitant with increased activity of the transcription factor E2F, which was not detected in wild-type or control-transfected Capan-1 cells. Wild-type and control-transfected Capan-1 cells were resistant to chemotherapy-induced apoptosis, but pRB-depleted (i.e., p16-transfected) Capan-1 cells were highly sensitive. The effect was specific to pRB depletion because two other human pancreatic cancer cell lines that retained high pRB expression after p16 transfection were resistant to chemotherapy-induced apoptosis., Conclusions: Overexpression of pRB is associated with human pancreatic duct-cell cancer and may allow pancreatic cancer cells to evade chemotherapy-induced apoptosis.

The purpose of this study was to examine the changes of the fat compartment and its relations to the corresponding blood chemistry and nutritional parameters of 55 participants of an ultra long distance run of 1000 km which consisted of 20 daily runs of 50 km. During the whole event the weight showed a falling tendency, significantly paralleling the cholesterol levels. In the middle of the run the values did not change any longer. The fat mass and all skinfolds and circumferences showed a continuously falling tendency. Only the thigh-skinfold initially grew and came down from the 4th day on. The serum blood concentrations of cholesterol and triglycerides of both sexes were decreased until the 6th and 8th day. Then a distinct increase was observed, but the initial values were not restored. The average daily energy intake of the men (women) was 4260 (3033) kcal/d, the average nutrient intake being 602.7 (431.5) g carbohydrates/d, 146.5 (103.3) g fat/d, 53.1 (38.4) g saturated fatty acids/d, 43.1 (31.3) g monounsaturated fatty acids/d, 25.3 (17.2) g polyunsaturated fatty acids/d and 382.8 (279.1) mg cholesterol/d. The correlations between the cumulative fat and food-supplies on one hand and the decrease of the anthropometric measurements of fatness on the other are negative, hence body fat mass followed closely total energy and fat intake.

The purpose of the study was to examine the changes of the muscle's fat-free compartment and its relation to the corresponding biochemical and nutritional parameters of 42 men and 13 women, the participants of an ultra long-distance run of 1000 km (20 days of daily running 50 km). The muscle-fractions initially increased, decreased in the middle phase, and remained stable for the rest of the run. Significant changes of the fat-free weight were registered from the 11th day on, the LBM decreasing until the middle of the distance; then the lean body mass enlarged. All the muscle-circumferences were reduced with the exception of the thigh, which grew, paralleling the CK/CKMB-concentrations, this phenomenon being due to the high mechanical stress of the lower extremities. The biochemical parameters exhibit a strain-related reaction of adaptation within the initial 6 days, the hormones and protein-concentration increasing in the beginning and falling from the third day on, uric acid and CK/CKMB-activity decreasing from the 6th day on. The consecutive parallel reduction of both uric acid, urea, and muscle measurements might be seen as a special endurance-related clearance-mechanism of potential toxicants. The negative relationship between the changes of muscle measurements and the cumulative protein intake and the catabolic constellation of the clinical-chemical values might suggest that the absolute protein intake of 1.7 g/kg body mass should be increased in order to diminish the loss of musculature during an ultra-long distance run.