Change and stability in a steep morph‐frequency cline in the snail Cepaea nemoralis (L.) over 43 years

Populations of the polymorphic land snail Cepaea nemoralis (L.) from Deepdale, Derbyshire, UK, sampled in 1965–67, showed a pattern of area effects, with steep clines among groups of populations differing in shell colour and banding morph frequencies. In 2010, most of these populations were resampled. In particular, a continuous transect made in 1967 of 42 quadrats (18.34 × 18.34 m) across a steep cline in several morph frequencies was completely resampled. In the dale as a whole, yellow shells had increased in frequency. In the transect, the frequencies of banding morphs showed no significant changes, although colour morphs showed some changes. Pink shells had increased in frequency in a section in which scrub had developed, and brown shells had increased in frequency in the area in which they had originally been at the highest frequency. In each case, the selection coefficients were of the order of 4%. Yellow had increased elsewhere. Nevertheless, both in the dale as a whole and in the transect, the pattern of geographical change in morph frequencies had remained essentially the same. Estimates of migration based on previous studies of marked snails and on modelling of the effect of drift and migration suggest that, regardless of whether the cline is a product of differential selection or of the gradual merging of previously separate founding populations, it has been in existence for a long time, and that migration occurs over greater distances than estimated from direct observation on marked snails. Although we can demonstrate that selection has occurred, the origin and maintenance of the cline and others similar to it remain in doubt; the development and maintenance of polymorphism in this species may require consideration of several processes operating on different time scales. © 2012 The Linnean Society of London     

Variable evolutionary response to regional climate change in a polymorphic species

The evolutionary response to regional and global climate change may vary in widespread polymorphic species, so predicting future genetic responses will require careful tracking of genetic variability in local populations. We surveyed chromosomal inversion polymorphisms in 25 populations of Drosophila robusta, many of which have been sampled repeatedly starting in the 1940s, 50s, and 60s up until 2007, across its range in the USA. Frequencies of some northerly, or cold‐adapted, gene arrangements have declined in the face of increasing temperatures, whereas frequencies of several southern, or warm‐adapted, gene arrangements were positively correlated with increasing temperature changes. Over a finer geographic scale, populations from the west‐central part of the species range from the Ozark Plateau, Ouachita mountains, and eastern Oklahoma showed genetic differentiation between south‐central Ozark and western Ozark/Ouachita regions that has persisted in the face of recent shifts in gene arrangement frequencies. Overall, populations of D. robusta exhibited dynamic genetic changes over time, with some populations shifting chromosome frequencies in just 10–15 years. Some temporal genetic shifts were widespread and significantly correlated with temperature increases, but regions of the genome marked by different gene arrangements have responded in different sections of the species range. In some parts of the species range, chromosome frequencies shifted but were not associated with changing temperatures, showed little or no temporal change, or temporal shifts stopped for temperature sensitive gene arrangements near fixation. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 702–718.     

Genetic diversity,sexual condition,and microhabitat preference determine mating patterns in Sphagnum (Sphagnaceae) peat‐mosses

In bryophytes, the possibility of intragametophytic selfing creates complex mating patterns that are not possible in seed plants, although relatively little is known about patterns of inbreeding in natural populations. In the peat‐moss genus Sphagnum, taxa are generally bisexual (gametophytes produce both sperm and egg) or unisexual (gametes produced by separate male and female plants). We sampled populations of 14 species, aiming to assess inbreeding variation and inbreeding depression in sporophytes, and to evaluate correlations between sexual expression, mating systems, and microhabitat preferences. We sampled maternal gametophytes and their attached sporophytes at 12–19 microsatellite loci. Bisexual species exhibited higher levels of inbreeding than unisexual species but did generally engage in some outcrossing. Inbreeding depression did not appear to be common in either unisexual or bisexual species. Genetic diversity was higher in populations of unisexual species compared to populations of bisexual species. We found a significant association between species microhabitat preference and population genetic diversity: species preferring hummocks (high above water table) had populations with lower diversity than species inhabiting hollows (at the water table). We also found a significant interaction between sexual condition, microhabitat preference, and inbreeding coefficients, suggesting a vital role for species ecology in determining mating patterns in Sphagnum populations. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 115 , 96–113.     

Patterns of genetic variation in isolated Danish populations of the endangered butterfly Euphydryas aurinia

In the present study, we report an investigation on molecular variation in the endangered univoltine butterfly Euphydryas aurinia (Rottemburg, 1775), a species heavily affected by habitat degradation and fragmentation in Denmark. Levels of genetic variation in extant populations were estimated using six variable number tandem repeat loci and were found to be low compared to other butterfly species with low migration rates. An analysis of genetic structure, based on both allele frequencies and genotype distributions, divided the entire sample into four distinct clusters. This was partially concordant with the a priori subdivision based on collection areas. An overall F_ST value of 0.16 (pairwise values ranging from 0.087–0.276) indicated restrictions of gene flow. Especially two populations had higher F_ST values than the others, suggesting their isolation, and showed signs of bottlenecks/founder events. One population deviated significantly from Hardy–Weinberg equilibrium, suggesting a possible Wahlund effect or the presence of null alleles. The results suggest habitat fragmentation, resulting in genetic drift and possibly inbreeding. Future management is therefore recommended to increase gene flow between the remaining populations while habitats are restored in order to increase carrying capacity. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 677–687.     

Body size variation and caste ratios in geographically distinct populations of the invasive big‐headed ant,Pheidole megacephala (Hymenoptera: Formicidae)

Body size is an important life history trait that can evolve rapidly as a result of how species interact with each other and their environment. Invasive species often encounter vastly different ecological conditions throughout their introduced range that can influence relative investment in growth, reproduction and defence among populations. In this study, we quantified variation in worker size, morphology and proportion of majors among five populations of a worldwide invasive species, the big‐headed ant, Pheidole megacephala (Fabricius). The sampled populations differed in ant community composition, allowing us to examine if P. megacephala invests differently in the size and number of majors based on the local ant fauna. We also used genetic data to determine if these populations of P. megacephala represented cryptic species or if morphological differences could be attributed to change following introduction. We found significant variation in worker mass among the populations. Both major and minor workers were largest in Australia, where the ant fauna was most diverse, and minor workers were smallest in Hawaii and Mauritius, where P. megacephala interacted with few to no other ants. We also found differences in major and minor worker morphology among populations. Majors from Mauritius had significantly larger heads (width and length) relative to whole body size than those from Hawaii and Florida. Minors had longer heads and hind tibias in South Africa compared with populations from Australia, Hawaii and Florida. The proportion of majors did not differ among populations, suggesting that these populations may not be subject to trade‐offs in investment in major size versus number. Our molecular data place all samples within the same clade, supporting that these morphologically different populations represent the same species. These results suggest that the variation in shape and morphology of major and minor workers may therefore be the result of rapid adaptation or plastic responses to local conditions. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 423–438.     

Geographic morphological variation in parapatric Western Palearctic tree frogs,Hyla arborea and Hyla savignyi: are related species similarly affected by climatic conditions?

Tree frogs Hyla arborea and Hyla savignyi are similar, closely‐related species distributed in Europe and the Middle East. We investigated geographic variation in body shape within and between these species, and tested its relationships to macroclimatic conditions. We used morphometric distances (based on size corrected external measurements) to construct phenetic trees (unweighted pair‐group method of arithmetical averages, Neighbour‐joining), and to test correlations between morphology, geography, and climate by the partial Mantel test. Regardless of their specific affiliation, the parapatric populations of both species from the eastern Mediterranean, where they occupy comparable habitats, are closer to each other in morphospace than to conspecific populations from distal regions. This local interspecific similarity is probably driven by the common response to environment, expressed here as macroclimatic conditions. In support, the geographically close but ecologically vicariant populations of both species from the Caucasus region differ quite substantially in body shape. We suggest that climate‐provoked phenotypic variation in closely‐related parapatric species should be taken into account as a potential complication to character displacement in morphology. Contrariwise, morphological diversification between related species or their populations could be enhanced by habitat shifts resulting in occupation of different environmental space. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 539–556.     

Temporal morph frequency changes in sand‐dune populations of Cepaea nemoralis (L.)

Changes in morph frequency over time in sand‐dune populations of Cepaea nemoralis in the British Isles have been examined using the Evolution Megalab database. Frequencies in colonies on a sand dune at Berrow, Somerset were estimated in 2008–9. This extends a survey started in 1926 by A. E. Boycott and C. Diver and continued in the mid‐20th Century by B. C. Clarke and J. J. Murray. An increase in the frequency of the mid‐banded morph, noted in earlier work, has continued. The apparent decrease in brown was not confirmed but the yellow frequency has increased. At a range of dunes in the British Isles, comparisons between data from the mid‐20th Century and the early 21st Century indicate an increase in yellow and mid‐banded morphs and a decrease in banding and brown morphs. These results differ from the overall trends derived from Europe‐wide comparison of early data with Evolution Megalab data, in which banded and mid‐banded increased in frequency, whereas there was no overall change in yellow. The general pattern of regional variation has been retained, although there is also high heterogeneity between samples, suggesting that a variety of factors are involved in explaining the changes. The mean shifts in frequency are consistent with climatic change over the period. Dunes are probably the most likely habitat in which to detect such a change. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 108 , 315–322.     

Divergence in trophic ecology characterizes colonization of extreme habitats

Extreme habitats are characterized by the presence of physio‐chemical stressors, but also differ in aspects of the biotic environment, such as resource availability or the presence of competitors. The present study quantifies variation in trophic ecology of a small livebearing fish (Poecilia mexicana, Poeciliidae) across four different habitats that included nonsulphidic and sulphidic surface waters, as well as a nonsulphidic and a sulphidic cave. Resource use in different habitat types was investigated using gut content analysis. Populations diverged in resource use from a diet dominated by algae and detritus in nonsulfidic surface habitats to a diet including invertebrate food items in the other habitats. Poecilia mexicana in cave habitats further exhibited a higher dietary niche width than conspecifics from surface habitats. The condition of P. mexicana was analysed using storage lipid extractions. Fish from sulphidic and cave habitats exhibited a very poor condition, suggesting resource limitation and/or high costs of coping with extreme conditions. Finally, divergence in resource use was correlated with variation in viscerocranial morphology. A common garden experiment indicated both a genetic and plastic basis to the morphological variation observed among field populations. It is suggested that the morphological diversification is an adaptation to the differential use of resources among populations. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 517–528.     

Patterns of gene flow and source‐sink dynamics in high altitude populations of the common toad Bufo bufo (Anura: Bufonidae)

Understanding patterns of genetic structure is fundamental for developing successful management programmes for deme‐structured organisms, such as amphibians. We used five microsatellite loci and DNA sequences of the mitochondrial control region to assess the relative influences of landscape (geographic distance, altitude and rivers as corridors for dispersal) and historical factors on patterns of gene flow in populations of the toad Bufo bufo in Central Spain. We sampled 175 individuals from eight populations distributed along two major river drainages and used maximum‐likelihood and Bayesian approaches to infer patterns of gene flow and population structure. The mitochondrial DNA data show closely‐related haplotypes distributed across the Iberian Peninsula with no geographic structuring, suggesting recent differentiation of haplotypes and extensive gene flow between populations. On the other hand, microsatellites provide finer resolution, showing that high altitude populations (> 2000 m) exchange lower numbers of migrants with other populations. The results of Bayesian estimates for recent migration rates in high altitude populations suggest source‐sink dynamics between ponds that are consistent with independent data from monitoring over the past 20 years. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 824–839.     

Twenty years of body shape evolution in a threespine stickleback population adapting to a lake environment

Analysis of contemporary evolution can provide important insights into the pattern and rate of phenotypic evolution. The threespine stickleback population in Loberg Lake was exterminated in 1982, and a new population was founded between 1983 and 1989 by anadromous stickleback. The body shape of the Loberg Lake population resembled that of anadromous populations in 1990, although it had diverged markedly by 1992. Between 1992 and 2009, the population evolved more slowly to resemble typical lake populations in the region, diverging approximately 68% of the distance separating its putative ancestor and the original native population by 2009. Temporal evolution is the main source of variation, although spatial heterogeneity, armour phenotype, and allometry contribute significant variation. There was no significant effect of ancestral phenotypic shape covariance on the evolutionary trajectory of this population. Temporal variation in the Loberg Lake population provides a rare glimpse into the evolutionary response of a complex trait to natural selection after a major habitat shift. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 817–831.     

Selective predation favouring cryptic individuals of marine snails (Littorina)

Many gastropods have inherited conspicuous shell colour polymorphisms. A challenging question is, are colour frequencies under selection or is polymorphism owing to random evolutionary processes? The intertidal species Littorina saxatilis (a rock‐dweller) and L. obtusata (confined to macroalgae) both have genetically determined shell colour variation. In Iceland, Littorina obtusata are mostly cryptic on brown macroalgae by having brown or yellow shells (~95% of the snails), while Littorina saxatilis often appears conspicuous to the background of dark rocks owing to non‐cryptic colours (15–20%). This difference may be due to selective elimination of conspicuously coloured L. obtusata by visual predators, while L. saxatilis, largely living in another habitat, is not under a similarly intense colour selection. To test this hypothesis we increased the frequencies of conspicuous L. saxatilis in experimental populations (from <12 to 55%) and placed these in the seaweed zone, the main habitat of L. obtusata. Fifteen populations were released on isolated spots of seaweed and three of these were covered by net cages to exclude bird predators. One month later, yellow snails had increased in frequency within the patches, and to our surprise the result did not differ between bare and caged patches. This suggests selection favouring a colour that matches the background of fucoid seaweeds by visual predators able to enter the cages. Birds acted as important predators by picking 16% of the experimental snails in the uncaged spots, but were unable to enter the caged spots. However, the bird predation was non‐selective with respect to snail colour. For various reasons the most likely predators able to enter the cages were intertidal fish, these were thus responsible for the selection of non‐cryptic snails. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 76 , 137–144.     

Homalodontus nom. nov., a replacement name for Wapitiodus Mutter,de Blanger and Neuman, 2007 (Homalodontidae nom. nov., ?Hybodontoidea), preoccupied by Wapitiodus Orchard, 2005

The replacement names Homalodontus nom. nov. ( Homalodontidae nom. nov. ) are proposed for the Early Triassic shark Wapitiodus Mutter et al., 2007 (Wapitiodidae Mutter et al., 2007), preoccupied by the Triassic conodont Wapitiodus Orchard, 2005 (Gondolellidea Lindström, 1970 ). © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 154 , 419–420.     

New Streptocarpus species (Gesneriaceae) from South Africa

Two new rosulate species of Streptocarpus are described from the eastern seaboard of South Africa. The first is endemic to Mpumalanga Province. This species has almost actinomorphic corollas with small cylindrical tubes and was previously included within Streptocarpus parviflorus. However, molecular and morphological data and habitat preference do not support this classification. The second species is from the Msikaba River Gorge in the Eastern Cape Province. It adds to the already impressive list of endemic plant species from this region and is allied to other rosulate species of the Eastern Cape. It approaches Streptocarpus rexii in flower size but differs in its much shorter corolla tubes, which lack purple nectar guides. In addition, the corolla floors are marked with yellow bars reminiscent of Streptocarpus cyaneus and also seen in sympatric populations of the small‐flowered Streptocarpus modestus. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 743–748.     

Males are cheaper,or the extreme consequence of size/age‐dependent sex allocation: sexist gender diphasy in Fritillaria montana (Liliaceae)

The bulbous geophyte Fritillaria montana is partially self‐compatible and capable of switching gender. Small flowering plants produce only single male flowers, but larger plants produce hermaphrodite or, rarely, male and hermaphrodite flowers. Eight populations in peninsular Italy were sampled to determine the frequency and size distributions of male and hermaphrodite plants, and to determine the relationship of plant size to male and hermaphrodite flower production. Male plants were significantly smaller than hermaphrodites and made up 14.5–47.8% (100% in one small population) of flowering plants within populations. There were no significant differences in male fitness among female‐sterile and hermaphrodite flowers, as they both possessed full and comparable fertilizing power. Therefore, the gender variation observed in F. montana is likely to depend on resource‐dependent sex allocation. From an evolutionary perspective, we highlight the occurrence of similar mechanisms of gender variation in other representatives of the order Liliales. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 323–333.     

Extreme,continuous variation in an island snail: local diversification and association of shell form with the current environment

On Rosemary Island, a small continental island (11 km²) in the Dampier Archipelago, Western Australia, snails of the genus Rhagada have extremely diverse morphologies. Their shells vary remarkably in size and shape, with the latter ranging from globose to keeled‐flat, spanning the range of variation in the entire genus. Based primarily on variation in shell morphology, five distinct species are currently recognized. However, a study of 103 populations has revealed continuity of shell form within a very closely‐related group. A phylogenetic analysis of specimens from Rosemary Island, and other islands in the Dampier Archipelago, indicates that much of the morphological variation has evolved on the island, from within a monophyletic group. Within the island, snails with distinct shell morphologies could not be distinguished based on variation in mitochondrial DNA or their reproductive anatomy. The shell variation is geographically structured over a very fine scale, with clines linking the extreme forms over distances less than 200 m. Although there is no evidence that the different forms have evolved in isolation or as a consequence of drift, there is a strong association between keeled‐flat shells and rocky habitats, suggesting that shell shape may be of adaptive significance. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 756–769.     

Effects of grassland intensification on Whinchats Saxicola rubetra and implications for conservation in upland habitats

Modern, intensive grassland management has led to strong declines in ground‐nesting grassland birds, and is now increasingly threatening the last remaining strongholds of the Whinchat Saxicola rubetra in the Central European uplands. In this study, we assess key threats to Whinchat populations in these uplands in order to suggest appropriate conservation measures. We compared the direct threat of early mowing as well as the indirect threat resulting from a deteriorating arthropod food source in an inner‐alpine valley. Five of our seven study sites were mown too early with respect to the chicks' fledging date. Such early mowing was particularly evident on the more intensively farmed, earlier mown valley bottoms than on the valley slopes. Arthropod abundance and biomass did not differ between valley bottoms and slopes. However, valley bottoms had a greater amount of unprofitable prey items such as flies. Breeding bird density was mainly determined by the degree of overlap between the mowing schedule and breeding phenology. These findings suggest that in upland grasslands at an early stage of intensification, early mowing is of greater importance for populations than possible negative effects of a reduced food source. We suggest that mowing is delayed until a sufficient proportion of nestlings are safely fledged.     

Geographic variation in offspring size of a widespread lizard (Takydromus septentrionalis): importance of maternal investment

Geographic variation in offspring size is widespread, but the proximate causes of this variation have not yet been explicitly determined. We compared egg size and egg contents among five populations of a lizard (Takydromus septentrionalis, Günther, 1864) along a latitudinal gradient, and incubated eggs at two temperatures to determine the influence of maternal investment and incubation temperature on offspring size. The mean values for female size and egg size were both greater in the two northern populations (Chuzhou and Anji) than in the three southern populations (Lishui, Dongtou, and Ningde). The larger eggs were entirely attributable to the body size of females in the Anji population, but their increased size also stemmed from further enlargement of egg size relative to female body size in Chuzhou, the northernmost population sampled in this study. Eggs of the Chuzhou population contained more yolk and less water than those of southern populations. Despite the lower lipid content in the yolk, eggs from the Chuzhou population had higher energy contents than those from the two southern populations, owing to the larger egg size and increased volume of yolk. Hatchling size was not affected by incubation temperature, but differed significantly among populations, with hatchlings being larger in the Chuzhou population than in the other populations. Our data provide an inference that oviparous reptiles from cold climates may produce larger offspring, not only by increasing egg size but also by investing more energy into their eggs. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 59–67.     

Parasitic mites influence intra‐ and interpopulational variation in sperm length in a simultaneous hermaphrodite land snail (Gastropoda: Helicidae)

Sperm morphology can be highly variable among species, but less is known about patterns of population differentiation within species. Sperm morphology is under strong sexual selection, may evolve rapidly, and often co‐varies with other reproductive traits that differ between populations. We investigated variation in sperm morphology in the simultaneous hermaphrodite land snail Arianta arbustorum in relation to parasitic mite infection. Variation in total sperm length and sperm head length was assessed in 23 populations sampled across the distributional range of the species in Central and Northern Europe. We found a pronounced variation in total sperm length among the populations studied, with a difference of 11.0% of total sperm length between the shortest and longest population means. Differences among populations explained 62.9% of the variance in total sperm length, differences among individual snails within population 23.4% and differences within individual snail 13.7%. Mantel tests showed that interpopulation differences in total sperm length increased significantly with geographical distance between populations. A minimal adequate model revealed that parasitic infection had a positive effect and longitude a negative effect on total sperm length. Thus, independent of the population examined, mite‐infected individuals of A. arbustorum produced larger sperm than uninfected snails and total sperm length decreased from west to east. Sperm head length also varied among populations, but it was not influenced by any of the factors examined. In a subsample of 12 populations restricted to the mountains of Switzerland (elevational range 440–2485 m a.s.l.), total sperm length decreased with increasing elevation. Our results suggest that selection pressures acting among populations may differ from those acting within. Stabilizing selection might be a possible mechanism for producing the reduced variation observed in sperm length within a population. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 1036–1046.     

Genetic diversity analysis and conservation implications for the Iberian threatened populations of the irano-turanian relict Krascheninnikovia ceratoides (Chenopodiaceae)

Krascheninnikovia ceratoides (L.) Gueldenst. (Chenopodiaceae) is an irano‐turanian steppe plant with a striking geographical vicariance between the western Mediterranean and the central Asian regions. Its westernmost populations are located in the north‐east part of the Iberian Peninsula, in the narrow middle Ebro and Alfambra valleys, where they are threatened and have been catalogued as ‘vulnerable’. Genetic diversity and structure was studied in 150 individuals from five populations of these two valleys using inter‐simple sequence repeats (ISSR). A total of 121 informative bands allowed the identification of 150 genotypes. Nei's genetic diversity found in all natural populations (h = 0.448) was higher than that of other geographical restricted species. Analysis of molecular variance and spatial correlations analyses showed a strong genetic differentiation among populations (35.88%) and among valleys (22.33%). The unweighted pair‐group method of arithmetical averages and Neighbour‐joining clusterings further demonstrated a substructure of two population cores within the Ebro valley. Our genetic data suggest that the high levels of genetic diversity and the strong genetic structure found among the northern Iberian populations and ranges of K. ceratoides might result from the summed effects of its tetraploidy, its outbreeding nature, and its success in colonizing newly altered areas. However, the ISSR data might also reflect the existence of an ancient wider distribution range of the plant in the Iberian Peninsula coupled with a more recent history of habitat fragmentation. Different management conservation guidelines are recommended for this plant after our genetic analysis: a microreserve of approximately 2 km² is proposed for the highly threatened population of Osera (Ebro valley), whereas seed collection strategies and occasional population reinforcements are suggested for the remaining populations. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 92 , 419–429.     

Species limits and hybridization zones in Icterus cayanensis–chrysocephalus group (Aves: Icteridae)

One of the best examples of differentiation and hybridization among South American passerine birds is exhibited by Icterus cayanensis (Epaulet Oriole) and Icterus chrysocephalus (Moriche Oriole). Icterus chrysocephalus is a monotypic species restricted to northern South America. Icterus cayanensis is a polytypc species that ranges from Suriname and French Guyana to northern Argentina. Five subspecies are recognized to I. cayanensis. Hybrid zones are known between I. cayanensis and I. chrysocephalus as well as between subspecies of I. cayanenis, even though character variation has never been adequately assessed and mapped. Although molecular data support the hypothesis that I. cayanensis and I. chrysocephalus form a monophyletic group, they do not support the species limits currently recognized within this group. We analysed the geographic variation of plumage characters along the range of this group to map the geographic variation of individual plumage characters and identify the populations that have uniform phenotypic character expression and therefore represent genuine phylogenetic species. We also used molecular data to investigate the phylogenetic relationships among these species. Geographic variation of plumage characters, habitat preferences and molecular data identified four species within I. cayanensis–chrysocephalus clade: an Amazonian species group, formed by I. cayanensis and I. chrysocephalus and a Southern species group composed of I. pyrrhopterus and I. tibialis. The Amazonian species are separated by a relatively narrow hybrid zone along the Amazon valley, whereas the Southern species are separated by a hybrid zone that is larger than the ranges of the two species individually. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 583–597.