Salamandra Salamandra Research Articles

Larval development and poor trophic resource availability: Local adaptations and plasticity in a widespread amphibian species.

Theory predicts that, in organisms with complex life cycles, if the earlier-stage limiting factor induces weak later-stage phenotypes, the development of the later-stage trait should evolve to reduce carry-over effects. Local adaptations could thus favour decoupling of later stages. However, decoupling is not always possible. In this study, we used a widespread amphibian, the European fire salamander (Salamandra salamandra), to assess the role of local adaptations to environmental stressful conditions experienced at the larval stage. We exposed 150 larvae from different altitudes to two conditions: rich food and poor food condition. Conditions in early life stages can affect an individual's traits, either as a direct effect or mediated through outcomes in successive life stages. To distinguish between effects of rearing conditions and local adaptation, we searched for a causal model. The causal model detected effects of both food treatment and population origin (altitude) on all life stages. Larvae reared under rich food condition metamorphosed earlier, had higher growth rates and reached smaller size at metamorphosis. Significant differences occurred between larvae of different origin: low-altitude individuals performed poorly under the poor food treatment. Moreover, larvae from higher altitudes were slower with rich food and faster with poor food compared to those from lower altitudes. Our results underline that environmental conditions and local adaptation can interplay in determining the plasticity of larval stages, still adaptations can maximize the growth efficiency of early stages in oligotrophic environments, leading to divergent pathways across populations and environmental conditions.

Behavioural responses to chemical cues of predators differ between fire salamander larvae from two different habitats

AbstractPrey organisms need to detect predators and respond with adequate antipredator behaviours. To recognize predators, chemosensory cues play a pivotal role, particularly in aquatic ecosystems, where sight may be restricted. However, it is less known whether the ability to use these cues, enabling appropriate antipredator behaviour, varies between subpopulations occupying different habitats with different predation risks. We examined antipredator behaviour of wild‐caught larval fire salamanders (Salamandra salamandra) from two different habitats, ponds and streams. Ponds and streams are inhabited by habitat‐specific predators, such as alpine newts (Ichthyosaura alpestris), which only occur in ponds. We confronted larvae from both habitats with either tap water (control) or tap water that had contained alpine newts (newt treatment) and investigated potential behavioural differences in activity and shelter emergence. In the activity test, pond larvae, but not stream larvae, were significantly less active when faced with chemical cues (potentially kairomones) from newts than those faced with a control stimulus, but stream larvae generally exhibited significantly lower activity than pond larvae. In the shelter‐emergence test, larvae from both habitats spent significantly less time outside a shelter in the newt treatment than in control water. This suggests that larval fire salamanders, independent of their habitat, recognize chemical cues from potential predators and alter their behaviour accordingly. Larvae of both habitats were able to detect and use chemical cues released from potential predators, but they differed slightly in their response to these chemical cues. This finding reflects varying antipredator strategies in response to the habitat‐specific differences.

Bedrock may dictate the distribution of the fire salamander in the southern border of its global range

Abstract Understanding the factors that determine the spatial distribution of species is crucial for conservation planning. In this short communication, we review previous distribution models of the fire salamander (Salamandra infraimmaculata) in northern Israel, produced by the group of the late Prof. Leon Blaustein, while suggesting a biologically-informed reinterpretation of their main predictions. We argue for the prime importance of bedrock, specifically hard limestone, because it is tightly associated with the availability of karstic formations that are key to adult survival throughout the summer. Furthermore, we suggest that the spatial distribution of limestone bedrock also determines large-scale inter-population connectivity, and may explain the observed genetic differentiation among populations, as well as the southernmost limit of the species’ global distribution.

Larval development and habitat usage of stream-breeding Fire salamanders in an urban environment

Urbanisation adversely affects the abiotic and biotic characteristics of watercourses, including freshwater streams that support the development of stream-breeding salamanders. We conducted a study over four years on an isolated fire salamander population inhabiting a stream valley northwest of Budapest, Hungary. Our aim was to understand aspects of larval development and habitat usage within this population. The maximum number of larvae was observed in April and the first weeks of May. Due to drifting caused by heavy rainfall, there was a mean decrease of 63.3% in the number of larvae. The abundance of larval salamanders within 16 stream segments showed strong temporal and spatial variation, and there was a strong relationship between larval abundance and the % cover of fine gravel substrate. Some of the larvae could escape drift by entering pools with slower water flow and shelter. Larvae were predominantly solitary in smaller pools but occasionally aggregated in high numbers in some segments. The first larvae with yellow spots (indicative of metamorphosis) appeared in June, and by early September, all larvae were metamorphosing. Our results show that in this urbanised environment, larval development through to metamorphosis is occurring, but increasing urbanisation and alterations to stream flow threaten the persistence of the local population.

Improving trajectories of amphibians in wildlife passages

Linear transport infrastructure can alter the viability of populations and wildlife passages are used to mitigate their impacts. The assessment of their outcomes is often limited to recording the use of the tunnels by a focal species. For amphibians, the effectiveness of tunnels is poorly evaluated with little information about whether certain features encourage individuals that may be reluctant to pass through tunnels. One study showed that acoustic enrichment with anuran calls can increase the crossing of tunnels by newts. This study recorded the behavior of three European amphibian species in three tunnels, tracking them with PIT tags and detection with four RFID antennas installed on the floor of the tunnels. We tested (1) the effectiveness of the antennas in detecting the species, (2) the effect of the length of the tunnels, and (3) the effect of acoustic enrichment. Using a multi-state capture–recapture model, we evaluated the probability of an individual advancing between the tunnel sections. The effectiveness of the antennas varied according to species, higher for Urodela species than for Anuran species. Several types of paths were detected (constant and varying speeds, halt, and back-and-forth movements). The fire salamander and the great crested newt individuals exhibited a similar variety of movements in the tunnels (21 and 40 m length). Triturus cristatus made similar movements in the tunnels with and without acoustic enrichment. In water frogs, all the individuals (n = 16) made a complete crossing in the tunnel with enrichment vs. 75% (n = 71) in the tunnel without enrichment. In T. cristatus, the probability of going forward at the entrance of the tunnel was 18% higher with enrichment in one tunnel. No significant effect of acoustic enrichment was observed in two others tunnels for this species. In Pelophylax esculentus, this probability was 78% higher in the tunnel with enrichment. This multi-antenna RFID system was able to provide valuable information on the behavior of these small animals when traversing the tunnels, as well as to test the effectiveness of tunnel features. The findings indicate that acoustic enrichment to attract animals to specific locations holds promise as a new conservation tool.

Factors determining the dorsal coloration pattern of aposematic salamanders

Aposematic bright colors have a key role for animal defense and can be expressed through metabolic production or by acquiring pigments from diet. Aposematic coloration can be related to both local adaptations and availability of trophic resources. The European fire salamander (Salamandra salamandra) shows significant color variability and occurs across a broad range of habitats. Here we combined field observations with common rearing experiments to disentangle the role of environmental conditions and local adaptations in determining aposematic coloration of salamander populations. We assessed color variation and measured habitat features and food availability in adults from 25 populations. Furthermore, we reared newborn larvae from 10 populations under different food availability and analyzed color of metamorphs. To assess color pattern, we measured the percentage of yellow covering the body, and the Hue, Saturation and Value of yellow coloration. Adult showed strong variation of color pattern; variation was strongly related to the individual's size, to habitat productivity and to food availability. Under common garden conditions, differences between populations were not anymore evident, and coloration was only affected by resource availability during larval development. Our results suggest that environmental conditions and food availability are more important than local adaptations in determining differences in aposematic color pattern.

Predicted current and future distribution of the fire salamander, Salamandra infraimmaculata in Turkey

Predicted current and future distribution of the fire salamander, Salamandra infraimmaculata in Turkey

Host Identity Matters-Up to a Point: The Community Context of Batrachochytrium dendrobatidis Transmission.

AbstractThe level of detail on host communities needed to understand multihost parasite invasions is an unresolved issue in disease ecology. Coarse community metrics that ignore functional differences between hosts, such as host species richness, can be good predictors of invasion outcomes. Yet if host species vary in the extent to which they maintain and transmit infections, then explicitly accounting for those differences may be important. Through controlled mesocosm experiments and modeling, we show that interspecific differences between host species are important for community-wide infection dynamics of the multihost fungal parasite of amphibians (Batrachochytrium dendrobatidis [Bd]), but only up to a point. The most abundant host species in our system, fire salamander larvae (Salamandra salamandra), did not maintain or transmit infections. Rather, two less abundant "auxiliary" host species, Iberian tree frog (Hyla molleri) and spiny toad (Bufo spinosus) larvae, maintained and transmitted Bd. Frogs had the highest mean rates of Bd shedding, giving them the highest contributions to the basic reproduction number, R0. Toad contributions to R0 were substantial, however, and when examining community-level patterns of infection and transmission, the effects of frogs and toads were similar. Specifying more than just host species richness to distinguish salamanders from auxiliary host species was critical for predicting community-level Bd prevalence and transmission. Distinguishing frogs from toads, however, did not improve predictions. These findings demonstrate limitations to the importance of host species identities in multihost infection dynamics. Host species that exhibit different functional traits, such as susceptibility and infectiousness, may play similar epidemiological roles in the broader community.

The Trophic Niche of Two Sympatric Species of Salamanders (Plethodontidae and Salamandridae) from Italy.

Simple SummaryStudies on species’ trophic niches are essential to understand the characteristics of species’ ecology and life traits, as well as to improve conservation strategies. In the absence of competitors, species realize their trophic niche including in their diet the most profitable food resources. In the presence of competitors, species modify their preferences to reduce competition and maintain the highest benefits at the same time. In this study, we assessed the trophic niche of two species of salamanders coexisting in a forested area of Italy and evaluated which might be the mechanisms that these two species adopted to reduce competition. We found that the Italian cave salamander (Speleomantes italicus) mostly consumed flying prey with a hard cuticle, while the fire salamander (Salamandra salamandra) preferred worm-like and soft-bodied prey. In conclusion, we hypothesize that in our case, the two species of salamanders did not have to change their prey preference in order to avoid competition, but divergences in metabolism and behavioral traits likely worked as natural deterrent.The trophic niche of a species is one of the fundamental traits of species biology. The ideal trophic niche of a species is realized in the absence of interspecific competition, targeting the most profitable and easy-to-handle food resources. However, when a competitor is present, species adopt different strategies to reduce competition and promote coexistence. In this study, we assessed the potential mechanisms that allow the coexistence of two generalist salamanders: the Italian cave salamander (Speleomantes italicus) and the fire salamander (Salamandra salamandra). We surveyed, in April 2021, a forested area of Emilia-Romagna (Italy) during rainy nights. Analyzing the stomach contents of the captured individuals, we obtained information on the trophic niche of these two sympatric populations. Comparing our results with those of previous studies, we found that the two species did not modify their trophic niche, but that alternative mechanisms allowed their coexistence. Specifically, different prey preferences and predator metabolisms were likely the major factors allowing reduced competition between these two generalist predators.

In vivo and ex vivo range of motion in the fire salamander Salamandra salamandra.

Joint range of motion (RoM) analyses are fundamental to our understanding of how an animal moves throughout its ecosystem. Recent technological advances allow for more detailed quantification of this RoM (e.g. including interaction of degrees of freedom) both in ex vivo joints and in vivo experiments. Both types of data have been used to draw comparisons with fossils to reconstruct locomotion. Salamanders are often used as analogues for early tetrapod locomotion; testing such hypotheses requires an in‐depth analysis of salamander joint RoM. Here, we provide a detailed dataset of the ex vivo ligamentous rotational joint RoM in the hindlimb of the fire salamander Salamandra salamandra, using a new method for collecting and visualising joint RoM. We also characterise in vivo joint RoM used during walking, via scientific rotoscoping and compare the in vivo and ex vivo data. In summary, we provide (1) a new method for joint RoM data experiments and (2) a detailed analysis of both in vivo and ex vivo data of salamander hindlimbs, which can be used for comparative studies.

A Protocol for Prolonged Surgical Anaesthesia with Recovery in Fire Salamanders Using Tricaine Mesylate (MS-222): A Case Series

The objective of this study was to describe prolonged surgical anaesthesia and recovery in fire salamanders (Salamandra salamandra) using tricaine methanesulfonate (MS-222). A total of 14 salamanders were anaesthetised for electromyography wire implantation. Sodium bicarbonate buffered solutions (0.5-4 g l-1) of MS-222 were prepared (adjusted to pH7.0). Anaesthesia was induced by partial immersion in pre-oxygenated 3 g l-1 solution for 20 min. Buprenorphine (0.5 mg kg-1) was administered subcutaneously. During microsurgery, heart rate (HR), solution pH and temperature were recorded. Reflectance pulse oximeter (SpO2) (Masimo Rad-57) was recorded in two salamanders. Anaesthetic plane and MS-222 pH stability (pH7.6) were maintained by renewing administration of oxygenated MS-222 solution (0.5-3 g l-1) onto swabs that partially covered the body. Recovery started at the end of surgery (MS-222 0 g l-1). Postoperatively, salamanders were given oral meloxicam (0.2 mg kg-1). Mean time for loss of righting reflex during induction was 13.7 ± 2.2 min. Duration of anaesthesia and time to recovery were 111 ± 24.2 and 31 ± 10.3 min, respectively. Due to complications, two salamanders did not recover. Baseline HR was 67.4 ± 34.5 beats/min, and it decreased significantly until recovery (p ≤ 0.0001). In two salamanders, baseline SpO2 was 85.5% ± 14.5, SpO2 during surgery was 61% ± 6.4, improving to 80.5% ± 2.1 on recovery.In conclusion, prolonged recovery anaesthesia is achievable with MS-222 dilutions in salamander. Reflectance SpO2 could prove valuable during immersion anaesthesia.

The roles of landscape of fear and light in allowing the exploitation of spring habitats by subterranean amphipods: an experimental and field approach

Border habitats such as interfaces and ecotones promise research targets from an evolutionary and zoological point of view. Springs are typical ecotones that border two strongly distinct environments: surface and underground. They are exploited by both subterranean and surface species for which they may provide specific environmental pressures promoting phenotypic plasticity and local adaptations. The aim of this study is to understand how the landscape of fear (LOF) and physical constraints, like light occurrence, affect springs' exploitation by both a subterranean (Niphargus thuringius) and a surface crustacean amphipod species (Echinogammarus stammeri). From March to May 2021, we surveyed 15 springs, divided into 25 plots according to their distance to the border, and both day and night, we recorded amphipods activity and LOF levels for them. In a subterranean laboratory, we also reared 80 N. thuringius and 80 E. stammeri in safe and risky conditions with constant darkness and diel light variation assessing their activity and survival for 30 days. Risky conditions were represented by meso-predators (four fire salamander larvae) alone or with a top-predator (a dragonfly larva of the species Cordulegaster boltonii). While in field conditions, the activity of N. thuringius seemed negatively affected by the number of active predators, in laboratory experiments, the main role was played by the light treatment; activity was significantly higher in constant darkness conditions. E. stammeri activity in the field was higher in surface plots, while in laboratory conditions was affected by LOF. Predation risk negatively affected the survival of both amphipods. Our findings reveal that while light conditions seem to shape activity patterns of stygobionts strongly, predators have a lower effect on activity, even though predators have negative effects on survival. Moreover, physical constraints, such as light exposure, can affect antipredator responses of subterranean organisms, thus representing selective pressures for the exploitation of surface environments.

Can antioxidant responses be induced by habitat fragmentation process?

Animal populations are increasingly forced to live in small residual natural or semi‐natural areas due to habitat loss and fragmentation. Here, the viability of populations is often compromised by intrinsic threat factors typical of small and isolated populations, such as inbreeding depression, genetic drift and environmental and demographic stochasticity. Under these circumstances, organisms may have low fitness due to inadequate physiological responses needed to face environmental challenges. However, few studies have investigated the relationship between habitat fragmentation and stress defences. In this study, we aimed to test whether an increase in the level of individual inbreeding produced an increase in the antioxidant system response. To this purpose, we genotyped 151 individuals of fire salamander Salamandra salamandra (Amphibia: Urodela) within five sampling populations, located in forest landscapes with different degree of fragmentation in northern Italy. For 113 individuals we also measured the glutathione‐S‐transferase (GST) and catalase (CAT) enzyme activity. Results showed a significant increase in individual GST activity for increasing levels of inbreeding, whereas no relationship was found for CAT. We also measured acetylcholinesterase to test the possible confounding effects of pesticides that might have occurred in fragmented landscapes with forests interspersed with agricultural areas. However, no difference in this enzyme activity was found among sampling populations. We argue that high levels of GST activity may be symptomatic of oxidative stress derived from inbreeding. An increased frequency of homozygous deleterious alleles due to inbreeding may cause homeostatic alterations and trigger the expression of GST for protection against hydrogen peroxide reactive oxygen species. We suggest using GST as a biomarker for environmental stressors with great caution and not to underestimate that the sources of stress deriving from habitat fragmentation could lead to an unbalance in the oxidative status, possibly increasing population susceptibility to infectious diseases and, potentially, spillover events and zoonoses.

How Trophic Conditions Affect Development of Fire Salamander (Salamandra salamandra) Larvae: Two Extreme Cases

Reduced trophic resources can pose relevant constraints to the development of freshwater animals with complex life cycles. For amphibians, food deprived environments, such as high-altitude ponds and springs and groundwaters are frequently used for breeding. The aim of this study is to outline trophic conditions leading to extreme cases of delayed larval development and increased size at metamorphosis of a European widespread amphibian, the fire salamander (Salamandra salamandra). We collected 150 fire salamander larvae, split them in two groups, one with high and one with low trophic resource availability. We then observed the effects of nutritional conditions on larval development recording time to metamorphosis and average day growth. Moreover, in the field, we surveyed larvae growth and size at metamorphosis in two artificial subterranean sites with low prey availability. Trophic conditions strongly affected larval development and under low food treatment time to metamorphosis reached up to 416 days. In the subterranean environments we observed a similar pattern, with larvae requiring more than one year to attain metamorphosis but reaching unexpected large sizes. Environmental trophic conditions experienced during early stages can induce strong delay in metamorphosis of the fire salamander; this plasticity makes fire salamander larvae optimal models for comparative studies and cross-environment experiments.

Effects of Pine and Eucalypt ashes on bacterial isolates from the skin microbiome of the fire salamander (Salamandra salamandra)

Environmental contamination influences the diversity of the resident skin microbial community of amphibians, ultimately affecting the individual's immune system. Wildfires are expected to impact the skin microbiome, since post-fire runoff typically transports hazardous substances, that can affect terrestrial and aquatic ecosystems. The present study is the first to assess the effects of Eucalypt and Pine wildfire ash on cultivable bacterial isolates from the skin microbiome of amphibians, in particular the fire salamander (Salamandra salamandra), a common species in fire-prone Mediterranean ecosystems. To achieve this goal, samples of skin bacteria of adult individuals of S. salamandra were collected at a site without influence of wildfires. The bacterial isolates were tested against the pathogenic agent Aeromonas salmonicida for assessing their antimicrobial activity, before exposing them to a series of dilutions of aqueous extracts of Eucalypt and Pine ashes (AAEs) from high severity wildfires. From the 80 bacterial isolates collected, 48 (mostly Pseudomonas spp.) showed antimicrobial activity. Exposure of bacteria with antimicrobial activity to the Eucalypt and Pine AAEs at concentrations of 0, 6.25, 12.5, 25, 50, 75, and 100%, revealed that bacterial growth could be significantly inhibited, stimulated or unaffected by ash. Growth inhibition was found for Pine and Eucalypt AAEs at concentrations as low as 6.25% and 12.5%, respectively, but were more expressive at concentrations equal or above 50%. Eucalypt AAEs had a higher negative impact on bacterial growth than Pine AAEs, likely due to differences in metal concentrations between ash types. These findings raise concern about the future of amphibians in fire-prone regions since the foreseen increase in fire frequency and severity owing to climate changes are likely to alter the skin microbiome of amphibians, weaken the immune system and consequently increasing the incidence of infections or diseases, further contributing to the decline of the populations.

Oviparity, viviparity or plasticity in reproductive mode of the olm Proteus anguinus: an epic misunderstanding caused by prey regurgitation?

Abstract Cave animals are biological models of fast evolutionary change induced by transition to extreme subterranean environments. But their concealed lifestyle makes it inherently difficult to study life-history changes. Therefore, currently very little is known on the reproduction of cave species, and even less is known on general patterns and potentially shared reproductive strategies. Theory predicts that the cave environment favours the production of a few well-developed offspring and live birth. For one of the most enigmatic cave animals, the olm (Proteus anguinus), it has been debated fiercely whether they reproduce by live birth (viviparity), egg-laying (oviparity) or facultatively. While successes in captive breeding after the 1950s report oviparity as the single parity mode, some historically older observations claimed viviparity. The controversial neo-Lamarckist Paul Kammerer even claimed to have induced changes in parity mode by altering environmental conditions. Here, we report on the feeding and regurgitation of fire salamander (Salamandra salamandra) larvae by olms. The salamander larvae showed clear teeth marks and other injuries on the head caused by the olm, yet one larva was still alive after regurgitation. We suggest that historical reports of olm viviparity could have been misled by regurgitated salamander larvae. Our data bring additional indications that at least some of Kammerer’s experiments were fraudulent.

A New Disease Caused by an Unidentified Etiological Agent Affects European Salamanders.

Simple SummaryIn the last few years, multiple new infectious diseases have affected amphibians, causing unprecedented declines and extinctions at the global scale. Many of these diseases are caused by pathogens that have been described during recent decades. In this study we report a novel disease, affecting European amphibians. In a protected area of Northern Italy, since the autumn of 2013, we started to find some adult salamanders with cysts at the throat level (subgular region). Following this observation, we ran a regular monitoring of the salamander population and performed multiple morphological and molecular investigations to identify the cause of this undescribed disease. The cysts surround peculiar cells, probably protists, of about 30 µm covered by numerous motile cilia/undulipodia. Despite multiple attempts with a broad spectrum of techniques, the detailed identification remains challenging as we have been unable to match its features with previously described organisms. We provide the results achieved till now to promote a rapid dissemination on this new enigmatic wildlife pathogen and to create a basis for further and deeper studies.New pathologies are causing dramatic declines and extinctions of multiple amphibian species. In 2013, in one fire salamander population of Northern Italy, we found individuals with undescribed cysts at the throat level, a malady whose existence has not previously been reported in amphibians. With the aim of describing this novel disease, we performed repeated field surveys to assess the frequency of affected salamanders from 2014 to 2020, and integrated morphological, histological, and molecular analyses to identify the pathogen. The novel disease affected up to 22% of salamanders of the study population and started spreading to nearby populations. Cysts are formed by mucus surrounding protist-like cells about 30 µm long, characterized by numerous cilia/undulipodia. Morphological and genetic analyses did not yield a clear match with described organisms. The existence of this pathogen calls for the implementation of biosecurity protocols and more studies on the dynamics of transmission and the impact on wild populations.

A near-natural experiment on factors influencing larval drift in Salamandra salamandra

The larval stage of the European fire salamander (Salamandra salamandra) inhabits both lentic and lotic habitats. In the latter, they are constantly exposed to unidirectional water flow, which has been shown to cause downstream drift in a variety of taxa. In this study, a closed artificial creek, which allowed us to keep the water flow constant over time and, at the same time, to simulates with predefined water quantities and durations, was used to examine the individual movement patterns of marked larval fire salamanders exposed to unidirectional flow. Movements were tracked by marking the larvae with VIAlpha tags individually and by using downstream and upstream traps. Most individuals showed stationarity, while downstream drift dominated the overall movement pattern. Upstream movements were rare and occurred only on small distances of about 30 cm; downstream drift distances exceeded 10 m (until next downstream trap). The simulated flood events increased drift rates significantly, even several days after the flood simulation experiments. Drift probability increased with decreasing body size and decreasing nutritional status. Our results support the production hypothesis as an explanation for the movements of European fire salamander larvae within creeks.

A road-killed Fire Salamander (Salamandra salamandra) scavenged by a Round-backed Slug (Arion sp.) in Galicia (Northwestern Spain)

A road-killed Fire Salamander (Salamandra salamandra) scavenged by a Round-backed Slug (Arion sp.) in Galicia (Northwestern Spain)

The Midwife Toad Challenge After (Half) a Century

The evolutionary biologist Paul Kammerer (1880–1926) purportedly demonstrated that environmentally induced character states are carried over to the next generation(s), therewith providing evidence for Lamarckian evolution. Kammerer’s work is generally seen as contentious but has also been valued as daring and insightful, and Kammerer has been heralded as an academic forebear of epigenetics. Most of the more pertinent of Kammerer’s experiments on amphibians and the sea squirt have either been invalidated by replications that failed, or have been dismissed as virtually impossible. An experiment on color pattern change in the fire salamander stands out because it has been confirmed, but only with data for within a generation, so that Kammerer’s claim concerning the inheritance of acquired character states still awaits confirmation. To facilitate and encourage replicate studies, I draw attention to species and populations that would be most practical and promising to work with, for those that are into the challenge.