Biotoxicity of mercury as influenced by mercury(II) speciation.

Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolated to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s; mercuric chloride, on the other hand, always produced lower IC50s. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1% glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)2(2+)], chloride (HgCl+, HgCl2(0), HgClOH0, and HgCl3-), phosphate (HgHPO4(0), ammonia (HgNH3(2+), glycine [Hg(GLY)+], alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl2(0), and HgClOH0 were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)2(2-) was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.     

Biotoxicity of mercury as influenced by mercury(II) speciation

Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolated to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s; mercuric chloride, on the other hand, always produced lower IC50s. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1% glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)2(2+)], chloride (HgCl+, HgCl2(0), HgClOH0, and HgCl3-), phosphate (HgHPO4(0), ammonia (HgNH3(2+), glycine [Hg(GLY)+], alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl2(0), and HgClOH0 were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)2(2-) was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.     

Effects of chemical speciation in growth media on the toxicity of mercury(II).

The toxicity of metals, including mercury, is expressed differently in different media, and the addition of soluble organics to the growth medium can have a significant impact on bioassay results. Although the effect of medium composition on metal toxicity is generally attributed to its effect on metal speciation (i.e., the chemical form in which the metal occurs), the importance of individual metal-ligand species remains largely unclear. Here, we report the results of a study that investigated, both experimentally and from a modeling perspective, the effects of complex soluble organic supplements on the acute toxicity (i.e., 50% inhibitory concentration [IC50]) of mercury to a Pseudomonas fluorescens isolate in chemically well-defined synthetic growth media (M-IIX). The media consisted of a basal inorganic salts medium supplemented with glycerol (0.1%, vol/vol) and a variety of common protein hydrolysates (0.1%, vol/vol), i.e., Difco beef extract (X = B), Casamino Acids (X = C), peptone (X = P), soytone (X = S), tryptone (X = T), and yeast extract (X = Y). These were analyzed to obtain cation, anion, and amino acid profiles and the results were used to compute the aqueous speciation of Hg(II) in the media. Respirometric bioassays were performed and IC50s were calculated. Medium components varied significantly in their effects on the acute toxicity of Hg(II) to the P. fluorescens isolate. IC50s ranged from 1.48 to 14.54 micrograms of Hg ml-1, and the acute toxicity of Hg(II) in the different media decreased in the order M-IIC >> M-IIP > M-IIB >> M-IIT > M-IIS >>> M-IIY.(ABSTRACT TRUNCATED AT 250 WORDS)     

Computer simulation of the distribution of aluminum speciation in soil solutions in equilibrium with the mineral phase imogolite.

The speciation of aluminum (Al) is a critical issue when evaluating the environmental and biological significance of elevated Al concentrations in soil solutions caused by acidic precipitation. Numerous studies have revealed that, with increased concentrations of silica acid in soil, the activity of Al species in soil solutions is greatly modified by SiO(4)(2-). However, thus far there has been little thorough theoretical modeling of this subject. This paper reports a computer simulation of the distribution of Al speciation in soil solutions in equilibrium with the mineral phase imogolite based on a chemical equilibrium calculation. The unique characteristic associated with imogolite reported by previous researchers can be explained theoretically by the proposed model. The dissolved silica has a remarkable influence on Al speciation: increasing concentrations of silica acid may effectively inhibit the formation of polymeric alumino-hydroxo species, and, furthermore, detoxify Al toxicity to plants.     

Speciation of trace element compounds in samples of biota from marine ecosystems

Abstract

The toxicity, mobility, bioavailability and bioaccumulation of metals are dependent on the particular physico-chemical form in which the element occurs in the environment. Special attention has been paid to metals which are essential for the proper functioning of organisms if present in appropriate amounts but are toxic if in excess (i.e. Se, Cr), and also to non-essential elements (i.e. Hg, Pb, Cd, Sn and As). To assess the potential hazard to the health of marine organisms, qualitative and quantitative analyses of metal species accumulating along the food chain needs to be carried out. This paper reviews the available information on the speciation of trace elements in the food chain in marine ecosystems and the analytical tools used for acquiring reliable information in this field. Advantages and limitations of commonly used techniques indicate that all metal species in different samples need diverse extraction, separation and detection conditions. Although not recommending which procedure is the most suitable to determine a given compound, speciation analysis has the potential to be a powerful tool for the identification of trace element species in biological samples.     

Luminescent bacteria toxicity assay in the study of mercury speciation

The toxicities of solutions of 10 mercury compounds to luminescent bacteria were measured using the Microtox Toxicity Bioassay. The aim of this study was to assess the influence that the counter-ions have on the aquatic toxicity of mercury salts. The toxicities of these mercury compounds were very similar, except for mercurous tannate and mercuric salicylate. This can be attributed to differences in the ionization and speciation patterns of these compounds relative to the other compounds tested. In general, the toxicity of the solutions at pH 5 was not significantly different from the toxicity of these solutions at pH 6, but a clear reduction in toxicity was observed when the pH of the solution was adjusted to pH 9. Significant differences were found between the toxicity of Hg(I) and Hg(II) salts of the same anion at pH 9. When cysteine was added to a mercuric nitrate solution (at pH 6), a reduction in the toxicity was observed. This can be explained in terms of the strong binding of mercury to cysteine, thus reducing the concentration of mercury species available to cause an observable toxic effect to the bioluminescent bacteria.     

Impacts of Mercury on Freshwater Fish-Eating Wildlife and Humans

This paper reviews the current state of knowledge of the toxic effects of mercury on fish-eating birds, mammals, and humans associated with freshwater ecosystems, including new information on the relative risk of elevated methyl Hg exposure for fish-eating birds inhabiting aquatic ecosystems impacted by mining/smelting activities and areas characterized by high geological sources of Hg. The influence of various environmental conditions such as lake pH, DOC, and chemical speciation of Hg, on fish-Hg concentrations and Hg exposure in fish-eating wildlife, are discussed. Although a continuing global effort to decrease the release of this nonessential metal into the environment is warranted, Hg methylation and biomagnification may be limited in some environments due to chemical speciation of mercury in soils and sediments (e.g., HgS) and water quality conditions (e.g., high alkalinity and pH) that do not facilitate high methylation rates. We have shown such limitations for a lake where historic Hg mining greatly increased sediment-Hg loadings, yet Hg increases in small fish of various species are currently lower than expected, and top predators (bald eagles), despite having elevated concentrations of Hg in their blood compared with individuals from nearby lakes, exhibit no Hg-related reproductive impairment or other signs of MeHg intoxication. Recent epidemiological studies have shown that fish-eating human populations may be exposed to Hg sufficient to cause significant developmental effects. However, for humans, we conclude that the current USEPA reference dose for MeHg may be too restrictive, particularly for the less sensitive adult. The health status of indigenous peoples relying on the subsistence harvest of wild foods may be negatively affected by such restrictions.     

Effect of anionic micelles on speciation of L-glutamine in presence/absence of metals

Abstract

Protonation equilibria of L-glutamine and speciation of its complexes with Co(II), Ni(II) and Cu(II) have been studied in 0–2.5% w/v SLS–water media using pH-metric method. The protonation constants and binary stability constants have been calculated with the computer program MINIQUAD75. Selection of the best fit chemical models is based on standard deviation in stability constants and residual analysis using crystallographic R-factor and sum of squares of residuals in all mass-balance equations. The trend in the variation of stability constants of the complexes with mole fraction of the medium is attributed to the compartmentalisation of complexation equilibria. Distribution of species and effect of influential parameters on chemical speciation have also been presented.     

Specific interactions of mercury chloride with membranes and other ligands as revealed by mercury-NMR.

High resolution mercury nuclear magnetic resonance (199Hg-NMR) experiments have been performed in order to monitor mercury chemical speciation when HgCl2 is added to water solutions and follow mercury binding properties towards biomembranes or other ligands. Variations of 199Hg chemical shifts by several hundred ppm depending upon pH and/or pCl changes or upon ligand or membrane addition afforded to determine the thermodynamic parameters which describe the equilibria between the various species in solution. By comparison to an external reference, the decrease in concentration of mercury species in solution allowed to estimate the amount as well as the thermodynamic parameters of unlabile mercury-ligand or mercury-membrane complexes. Hence, some buffer molecules can be classified in a scale of increasing complexing power towards Hg(II): EGTA greater than Tris greater than HEPES. In contrast, MOPS, Borax, phosphates and acetates show little complexation properties for mercury, in our experimental conditions. Evidence for complexation with phosphatidylethanolamine (PE), phosphatidylserine (PS) and human erythrocyte membranes has been found. Hg(II) does not form complexes with egg phosphatidylcholine membranes. Interaction with PE and PS model membranes can be described by the presence of two mercury sites, one labile, the other unlabile, in the NMR time scale. In the labile site Hg(PE) and Hg(PS)2 would be formed whereas in the unlabile site Hg(II) would establish bridges between three PE or PS molecules. Calculated thermodynamic data clearly indicate that PE is a better complexing agent than PS. Evidence is also found that complexation with lipids uses at first the HgCl2 species. Interestingly, mercury complexation with ligands or membranes can be completely reversed by addition of decimolar NaCl solutions. Minute mechanisms for mercury complexation with the primary amine of PE or PS membrane head groups are discussed.     

Environmental and biochemical trace-metal speciation studies by radiotracers and neutron activation analysis

Although knowledge of the total concentrations of trace metals in the environment and living organisms is still the essential starting point for any toxicological evaluation, it is, however, not sufficient to explain the mechanisms responsible for retention and toxic effect of trace metals. Differentiation between their chemical and biochemical forms is necessary. The need to resolve the total concentration of trace metals into single chemical species poses great experimental difficulties and imposes the use of very sensitive analytical techniques for trace metal determinations after specific preseparation procedures of the different chemical species. Thus, once the chemical or biochemical metal species have been selectively isolated, the speciation becomes merely an analytical problem, which requires high sensitivity and accuracy, as well as the evaluation of matrix effects, blanks, contamination, loss, and sampling. This paper describes the use of nuclear and radiochemical techniques related to chemical and biochemical speciation problems at the Joint Research Centre, Ispra, with carrier-free radiotracers and radiochemical neutron activation analysis.     

The chemical forms of mercury and selenium in whale skeletal muscle

Human exposure to potentially neurotoxic methylmercury species is a public-health concern for many populations worldwide. Both fish and whale are known to contain varying amounts of methylmercury species. However studies of populations that consume large quantities of fish or whale have provided no clear consensus as to the extent of the risk. The toxicological profile of an element depends strongly on its chemical form. We have used X-ray absorption spectroscopy to investigate the comparative chemical forms of mercury and selenium in fish and whale skeletal muscle. The predominant chemical form of mercury in whale is found to closely resemble that found in fish. In the samples of skeletal muscle studied, no involvement of selenium in coordination of mercury is indicated in either whale or fish, with no significant inorganic HgSe or HgS type phases being detected. The selenium speciation in fish and whale shows that similar chemical types are present in each, but in significantly different proportions. Our results suggest that for equal amounts of Hg in skeletal muscle, the direct detrimental effects arising from the mercury content from consuming skeletal muscle from whale and fish should be similar if the effects of interactions with other components in the meat are not considered.     

Chemical ecology and pollinator-driven speciation in sexually deceptive orchids

Sexually deceptive orchids mimic females of their pollinator species to attract male insects for pollination. Pollination by sexual deception has independently evolved in European, Australian, South African, and South American orchid taxa. Reproductive isolation is mainly based on pre-mating isolation barriers, the specific attraction of males of a single pollinator species, mostly bees, by mimicking the female species-specific sex-pheromone. However, in rare cases post-mating barriers have been found. Sexually deceptive orchids are ideal candidates for studies of sympatric speciation, because key adaptive traits such as the pollinator-attracting scent are associated with their reproductive success and with pre-mating isolation.During the last two decades several investigations studied processes of ecological speciation in sexually deceptive orchids of Europe and Australia. Using various methods like behavioural experiments, chemical, electrophysiological, and population-genetic analyses it was shown that minor changes in floral odour bouquets might be the driving force for pollinator shifts and speciation events. New pollinators act as an isolation barrier towards other sympatrically occurring species. Hybridization occurs because of similar odour bouquets of species and the overlap of flowering periods. Hybrid speciation can also lead to the displacement of species by the hybrid population, if its reproductive success is higher than that in the parental species.     

Effect of pH on Intracellular Accumulation of Trace Concentrations of Hg(II) in Escherichia coli under Anaerobic Conditions, as Measured Using a mer-lux Bioreporter

The effects of pH on the uptake and accumulation of Hg(II) by Escherichia coli were determined at trace, environmentally relevant, concentrations of Hg and under anaerobic conditions. Hg(II) accumulation was measured using inducible light production from E. coli HMS174 harboring a mer-lux bioreporter plasmid (pRB28). The effect of pH on the toxicity of higher concentrations of Hg(II) was measured using a constitutive lux plasmid (pRB27) in the same bacterial host. In this study, intracellular accumulation and toxicity of Hg(II) under anaerobic conditions were both significantly enhanced with decreasing pH over the pH range of 8 to 5. The pH effect on Hg(II) accumulation was most pronounced at pHs of <6, which substantially enhanced the Hg(II)-dependent light response. This enhanced response did not appear to be due to pH stress, as similar results were obtained whether cells were grown at the same pH as the assay or at a different pH. The enhanced accumulation of Hg(II) was also not related to differences in the chemical speciation of Hg(II) in the external medium resulting from the changes in pH. Experiments with Cd(II), also detectable by the mer-lux bioreporter system, showed that Cd(II) accumulation responded differently to pH changes than the net accumulation of Hg(II). Potential implications of these findings for our understanding of bacterial accumulation of Hg(II) under anaerobic conditions and for bacteria-mediated cycling of Hg(II) in aquatic ecosystems are discussed. Arguments are provided suggesting that this differential accumulation is due to changes in uptake of mercury.     

Mercury–Selenium Species Ratio in Representative Fish Samples and Their Bioaccessibility by an In Vitro Digestion Method

The potential toxicity of mercury (Hg) content in fish has been widely evaluated by the scientific community, with Methylmercury (MeHg) being the only legislated species (1 mg kg⁻¹, maximum concentration allowed in predatory fish). On the other hand, selenium (Se) is recognized to decrease its toxicity when both elements are simultaneously administrated. In the present paper, the total content of Se and Hg and their species in fish of high consumption, such as tuna, swordfish, and sardine, have been evaluated. The percentage of MeHg is higher than 90% of total Hg content. The results show that, for all of them, the Se/Hg ratio is significantly higher than one, being the maximum ratio for sardine. As only studying the bioaccessible fraction the extent of a toxic effect caused by an element can be predicted, the bioaccessibility of both analytes through an in vitro digestion method has been carried out. The results show that MeHg in all fishes is very low bioaccessible in both gastric and intestinal digestion. Because the MeHg bioaccessible fraction might be correlated to the Se content, the potential toxicity cannot be only related to the total Hg content but also to Se/Hg ratio.     

Acclimation of aquatic microbial communities to Hg(II) and CH3Hg+ in polluted freshwater ponds

The relationship of mercury resistance to the concentration and chemical speciation of mercurial compounds was evaluated for microbial communities of mercury-polluted and control waters. Methodologies based on the direct viable counting (DVC) method were adapted to enumerate mercury-resistant communities. Elevated tolerance to Hg(II) was observed for the microbial community of one mercury-polluted pond as compared to the community of control waters. These results suggest an in situ acclimation to Hg(II). The results of the methylmercury resistance-DVC assay suggested that minimal acclimation to CH₃Hg⁺ occurred since similar concentrations of CH₃HgCl inhibited growth of 50% of organisms in both the control and polluted communities. Analyses of different mercury species in pond waters suggested that total mercury, but not CH₃Hg⁺ concentrations, approached toxic levels in the polluted ponds. Thus, microbial acclimation was specific to the chemical species of mercury present in the water at concentrations high enough to cause toxic effects to nonacclimated bacterial communities.     

A multidimensional concept for mercury neuronal and sensory toxicity in fish - From toxicokinetics and biochemistry to morphometry and behavior

BackgroundNeuronal and sensory toxicity of mercury (Hg) compounds has been largely investigated in humans/mammals with a focus on public health, while research in fish is less prolific and dispersed by different species. Well-established premises for mammals have been governing fish research, but some contradictory findings suggest that knowledge translation between these animal groups needs prudence [e.g. the relative higher neurotoxicity of methylmercury (MeHg) vs. inorganic Hg (iHg)]. Biochemical/physiological differences between the groups (e.g. higher brain regeneration in fish) may determine distinct patterns. This review undertakes the challenge of identifying sensitive cellular targets, Hg-driven biochemical/physiological vulnerabilities in fish, while discriminating specificities for Hg forms.Scope of reviewA functional neuroanatomical perspective was conceived, comprising: (i) Hg occurrence in the aquatic environment; (ii) toxicokinetics on central nervous system (CNS)/sensory organs; (iii) effects on neurotransmission; (iv) biochemical/physiological effects on CNS/sensory organs; (v) morpho-structural changes on CNS/sensory organs; (vi) behavioral effects. The literature was also analyzed to generate a multidimensional conceptualization translated into a Rubik’s Cube where key factors/processes were proposed.Major conclusionsHg neurosensory toxicity was unequivocally demonstrated. Some correspondence with toxicity mechanisms described for mammals (mainly at biochemical level) was identified. Although the research has been dispersed by numerous fish species, 29 key factors/processes were pinpointed.General significanceFuture trends were identified and translated into 25 factors/processes to be addressed. Unveiling the neurosensory toxicity of Hg in fish has a major motivation of protecting ichtyopopulations and ecosystems, but can also provide fundamental knowledge to the field of human neurodevelopment.     

Toxicity and speciation analysis of organotin compounds

Abstract

The use of antifouling paints in shipbuilding has led to a significant concentration of organotin compounds in the marine environment. Antifouling paints have become the main source of tributyltin and triphenyltin derivatives loaded into the sea. The toxicity of organotin compounds has been of great concern. High concentrations of organotin compounds are associated with growth abnormalities in mussels and oysters and have also resulted to the decline in their abundance. High concentration of organotin compounds have also been found in the tissues of marine mammals and its presence has been linked to mass mortalities of marine mammals. It causes imposex and calcification anomalities in mollusks. Seafood is thought to be a possible source of organotin compounds in human. Therefore, to evaluate the environmental distribution and fate of these compounds and to determine the effectives of legal provisions adopted by a number of countries, a variety of analytical methods have been developed for the speciation of organotin compounds in the environment. A detailed review of the toxicity and chemical speciation of organotin compounds is given.     

Biological methods for speciation of heavy metals: different approaches

Heavy metals are found in their different forms in the environment. The distribution, mobility, and toxicity of metals are strongly related to these different forms. This necessitates the exploration of different methods for the remediation and speciation of heavy metals. Some direct and indirect physico-chemical methods such as filtration, chemical precipitation, ion-exchange, electro deposition, and membrane systems have been used for the last four decades. However, it is only in last few years that reliable biological methods have also been used. The biological methods include the use of microorganisms (fungi, algae, bacteria), plants (live or dead) and biopolymers. The use of these methods for the speciation of heavy metals is reviewed here.     

Importance of Clean Techniques and Speciation in Assessing Water Quality for Metals

Trace metals in aquatic and soil systems exist in a number of different soluble and particulate forms that impact the effect of the metals on these ecosystems. Appropriate methods of sampling and analysis are required to accurately determine the low concentrations present. Although assessment of metals in many regulatory programs is based on data for total metal concentrations, such values rarely correlate with effects. Consequently, other means are needed for the prediction of risk. Bioavailability of metals depends on their speciation, whose importance was first established for copper in aquatic systems where the toxicity of metals is related to the activity of the free metal ion. Small concentrations of natural organic matter strongly complex metals ameliorating toxicity. Several electroanalytical techniques are available that allow the assessment of metal species. Recently, a modeling approach, the Biotic Ligand Model (BLM), has been applied to the prediction of acute toxicity. The model accounts for the effects of natural organic matter, pH, and hardness and is able to predict toxicity over several orders of magnitude of soluble metal concentration using only easily determined site parameters. Total metal concentrations in sediment cover several orders of magnitude with no distinction of sediments that cause effects and those that do not except at low total metal concentrations. Relating the metal concentration to the concentrations of sulfide and organic matter binding sites enables the sediments containing higher concentrations of metals to be divided into those that do and those that do not have adverse effects. It is essential that metal speciation be considered to realistically evaluate the potential of metals to pose risk.     

Ring species as bridges between microevolution and speciation

A demonstration of how small changes can lead to species-level differences is provided by ring species, in which two reproductively isolated forms are connected by a chain of intermediate populations. We review proposed cases of ring species and the insights they provide into speciation. Ring species have been viewed both as illustrations of the history of divergence of two species from their common ancestor and as demonstrations that speciation can occur in spite of gene flow between the diverging forms. Theoretical models predict that speciation with gene flow can occur when there is divergent ecological selection, and geographical differentiation increases the likelihood of speciation. Thus ring species are ideal systems for research into the role of both ecological and geographical differentiation in speciation, but few examples have been studied in detail. The Greenish warbler is a ring species in which two northward expansions around the Tibetan plateau have been accompanied by parallel evolution in morphology, ecology, and song length and complexity. However, songs have diverged in structure, resulting in a lack of recognition where the reproductively isolated forms come into contact in Siberia. Our analysis suggests that these differences could have arisen even with gene flow, and that parallel rather than divergent ecological changes have led to divergence in sexually selected traits and subsequent speciation.