Revision of goblin spiders (Araneae: Oonopidae) in the Nansei Islands, Southwest Japan, with description of a new species.

We revised the fauna of goblin spiders (Oonopidae) in the Nansei Islands, southwest Japan, and recognized 12 species of seven genera. Eleven of them were described species, namely, Gamasomorpha cataphracta Karsch, 1881, Heteroonops spinimanus (Simon, 1892), Ischnothyreus narutomii (Nakatsudi, 1942), Ischnothyreus peltifer (Simon, 1891), Ischnothyreus velox Jackson, 1908, Opopaea cornuta Yin & Wang, 1984, Opopaea deserticola Simon, 1892, Opopaea syarakui (Komatsu, 1967), Orchestina flava Ono, 2005, Xestaspis parmata (Thorell, 1890), and Xyphinus karschi (Bsenberg & Strand, 1906). A new species is described under the name of Ischnothyreus ogatai sp. nov. This paper provides photographs of each oonopid species, notes on habitat and distribution, and keys to the species. A detailed description of I. ogatai sp. nov. (for both sexes), morphological data regarding three species newly added to the Japanese fauna (X. parmata, I. velox, and O. cornata), and additional data on the genital morphology of G. cataphracta are also presented.

Population genetic structure of the globally introduced big-headed ant in Taiwan.

Global commerce and transportation facilitate the spread of invasive species. The African big-headed ant, Pheidole megacephala (Fabricius), has achieved worldwide distribution through globalization. Since the late 19th century, Taiwan has served as a major seaport because of its strategic location. The population genetic structure of P. megacephala in Taiwan is likely to be shaped by international trade and migration between neighboring islands. In this study, we investigated the population genetics of P. megacephala colonies sampled from four geographical regions in Taiwan and elucidated the population genetic structures of P. megacephala sampled from Taiwan, Okinawa, and Hawaii. We observed a low genetic diversity of P. megacephala across regions in Taiwan. Moreover, we noted low regional genetic differentiation and did not observe isolation by distance, implying that long-distance jump dispersal might have played a crucial role in the spread of P. megacephala. We sequenced the partial cytochrome oxidase I gene and observed three mitochondrial haplotypes (TW1-TW3). TW1 and TW3 most likely originated from populations within the species' known invasive range, suggesting that secondary introduction is the predominant mode of introduction for this invasive ant. TW2 represents a novel haplotype that was previously unreported in other regions. P. megacephala populations from Taiwan, Okinawa, and Hawaii exhibited remarkable genetic similarity, which may reflect their relative geographic proximity and the historical connectedness of the Asia-Pacific region.

Two new species and a new genus of ray spiders (Araneae, Theridiosomatidae) from the Ryukyu Islands, southwest Japan, with notes on their natural history.

This paper provides descriptions of two new theridiosomatid species, Theridiosomanigrivirgatum sp. nov. and Sennintanikawai gen. nov., sp. nov. from the Ryukyu Islands, southwest Japan, with photographs and illustrations of both sexes. Sennin gen. nov. is a troglophilic genus composed of two species, S.tanikawai sp. nov. (Iriomote Island, Japan) and S.coddingtoni (Zhu, Zhang & Chen, 2001), comb. nov. (southern China). Zomadibaiyin Miller, Griswold & Yin, 2009, which recently joined the Japanese fauna, was morphologically reexamined based on specimens from the Ryukyus, and taxonomic features of Zoma males were reassessed. A distributional map of theridiosomatid spiders in the Ryukyus is also provided, including T.dissimulatum Suzuki, Serita & Hiramatsu, 2020, and T.alboannulatum Suzuki, Serita & Hiramatsu, 2020 with their habitat types, web morphology, and web-building behavior in detail.

Alternative reproductive tactics in male freshwater fish influence the accuracy of species recognition.

Sexual conflict can result in coercive mating. Because males bear low costs of heterospecific mating, coercive males may engage in misdirected mating attempts toward heterospecific females. In contrast, sexual selection through consensual mate choice can cause mate recognition cues among species to diverge, leading to more accurate species recognition. Some species show both coercive mating and mate choice-associated courtship behaviors as male alternative reproductive tactics. We hypothesized that if the selection pressures on each tactic differ, then the accuracy of species recognition would also change depending on the mating tactic adopted. We tested this hypothesis in the guppy (Poecilia reticulata) and mosquitofish (Gambusia affinis) by a series of choice experiments. Poecilia reticulata and G. affinis males both showed imperfect species recognition and directed all components of mating behavior toward heterospecific females. They tended to direct courtship displays more frequently toward conspecific than heterospecific females. With male P. reticulata, however, accurate species recognition disappeared when they attempted coercive copulation: they directed coercions more frequently toward heterospecific females. We also found that heterospecific sexual interaction had little effect on the fecundity of gravid females, which suggests that prepregnancy interactions likely underpin the exclusion of G. affinis by P. reticulata in our region.

Population genetic structure and evolution of Batesian mimicry in Papilio polytes from the Ryukyu Islands, Japan, analyzed by genotyping-by-sequencing.

Batesian mimicry is a striking example of Darwinian evolution, in which a mimetic species resembles toxic or unpalatable model species, thereby receiving protection from predators. In some species exhibiting Batesian mimicry, nonmimetic individuals coexist as polymorphism in the same population despite the benefits of mimicry. In a previous study, we proposed that the abundance of mimics is limited by that of the models, leading to polymorphic Batesian mimicry in the swallowtail butterfly, Papilio polytes, on the Ryukyu Islands in Japan. We found that their mimic ratios (MRs), which varied among the Islands, were explained by the model abundance of each habitat, rather than isolation by distance or phylogenetic constraint based on the mitochondrial DNA (mtDNA) analysis. In the present study, this possibility was reexamined based on hundreds of nuclear single nucleotide polymorphisms (SNPs) of 93 P. polytes individuals from five Islands of the Ryukyus. We found that the population genetic and phylogenetic structures of P. polytes largely corresponded to the geographic arrangement of the habitat Islands, and the genetic distances among island populations show significant correlation with the geographic distances, which was not evident by the mtDNA-based analysis. A partial Mantel test controlling for the present SNP-based genetic distances revealed that the MRs of P. polytes were strongly correlated with the model abundance of each island, implying that negative frequency-dependent selection interacting with model species shaped and maintained the mimetic polymorphism. Taken together, our results support the possibility that predation pressure, not isolation by distance or other neutral factors, is a major driving force of evolution of the Batesian mimicry in P. polytes from the Ryukyus.

Mimicry genes reduce pre-adult survival rate in Papilio polytes: A possible new mechanism for maintaining female-limited polymorphism in Batesian mimicry.

Batesian mimicry, in which harmless organisms resemble unpalatable or harmful species, is a well-studied adaptation for predation avoidance. The females of some Batesian mimic species comprise mimetic and nonmimetic individuals. Mimetic females of such polymorphic species clearly have a selective advantage due to decreased predation pressure, but the selective forces that maintain nonmimetic females in a population remain unclear. In the swallowtail butterfly, Papilio polytes, female polymorphism is controlled by the H (mimetic) and h (nonmimetic) alleles at a single autosomal locus. Here, we examined whether the dominant H allele has a deleterious effect on the pre-adult survival rate (egg-to-adult emergence rate). We repeated an assortative mating-like treatment-that is breeding of males and females whose mothers had the same phenotype (mimetic or nonmimetic)-for three consecutive generations, while avoiding inbreeding. Results showed that pre-adult survival rate decreased over generations only in lines derived from mothers with the mimetic phenotype (hereafter, mimetic-assorted lines). This lowered survival was due to an increased mortality at the final instar larval stage and the pupal stages. Interestingly, the pre-adult mortality in the mimetic-assorted lines seemed to be associated with a male-biased sex ratio at adult emergence. These results suggest that the dominant H allele displays a mildly deleterious effect that is expressed more strongly in females and homozygous individuals than in heterozygous individuals. We propose that this cost of mimicry in larval and pupal stages contributes to the maintenance of female-limited polymorphism in P. polytes.

Cytogenetic markers reveal a reinforcement of variation in the tension zone between chromosome races in the brachypterous grasshopper Podisma sapporensis Shir. on Hokkaido Island.

The cytogenetic characteristics of the grasshopper Podisma sapporensis (two races 2n = 23♂ X0/XX and 2n = 22♂ neo-XY/neo-XX) were analysed through fluorescence in situ hybridization with rDNA and telomeric DNA probes, C-banding, fluorochrome and silver staining. For the first time, samples from the neighbourhood of a hybrid population (i.e., Mikuni Pass population) were studied. Our results indicated a significant degree of chromosomal differentiation between P. sapporensis races when comparing the number and position of the rDNA sites, as well as the heterochromatin composition and distribution obtained by C-banding and DAPI/CMA3 staining. Telomeric signals were usually detected at the distal and/or subdistal position of the autosomes; however, some chromosome ends lacked signals, probably due to a low number of telomeric repeats. On the other hand, telomeric DNA sequences were found as interstitial telomeric repeats in some autosomes, which can trigger a variety of genome instability. B chromosomes were found in specimens belonging to both main races from nine out of 22 localities. Four types of X chromosomes in the X0/XX race were identified. It was concluded that the physical mapping of rDNA sequences and heterochromatin are useful as additional markers for understanding the phylogeographic patterns of cytogenetic differentiation in P. sapporensis populations.

Temperature-dependent toxicity of acetaminophen in Japanese medaka larvae.

Because of its analgesic properties, acetaminophen (AAP) is widely used to relieve headache. AAP is generally considered safe for humans, but its effects on aquatic organisms are not well known. Here, we have hypothesis that effects of AAP on aquatic organisms would be environmental temperature dependent, because their physiological function depend on the temperature. To test this hypothesis, we used medaka (Oryzias latipes) as a model, because they can live at a wide range of temperatures (0-40 °C). We exposed medaka larvae to 0 (control), 50, or 150 mg/L of AAP at 15, 25 (optimal temperature), or 30 °C for 4 days. Egg yolk absorption was accelerated with raising temperature at any AAP dose. AAP exposure did not have biologically significant effects on survival ratio and body length of larvae at any tested temperature or dose, but heart rate decreased as the dose of AAP and environmental temperature increased. In addition, as the temperature increased, amount of ATP in individual larvae increased in control group, but decreased in AAP exposed group. Subsequently, exposure to 150 mg/L of AAP at 30 °C decreased the number of red blood cells in the gills; we used 150 mg/L of AAP in subsequent hematological and histological analyses. Hematological analysis showed that rising temperature increased the proportion of morphologically abnormal red blood cells in AAP-exposed larvae, suggesting that AAP induced anemia-like signs in larvae. Histological observation of the kidney, which is a hematopoietic organ in fish, revealed no abnormalities. However, in the liver, which is responsible for drug metabolism, the proportion of vacuoles increased with increasing temperature. Although the exposure concentration we tested was higher than environmentally relevant concentrations, our data indicated that rising temperature enhances the toxicity of AAP to medaka larvae, suggesting an ecological risk of AAP due to global warming.

Evidence for frequency-dependent selection maintaining polymorphism in the Batesian mimic Papilio polytes in multiple islands in the Ryukyus, Japan.

Batesian mimicry is a well-studied adaptation for predation avoidance, in which a mimetic species resembles an unpalatable model species. Batesian mimicry can be under positive selection because of the protection gained against predators, due to resemblance to unpalatable model species. However, in some mimetic species, nonmimetic individuals are present in populations, despite the benefits of mimicry. The mechanism for evolution of such mimetic polymorphism remains an open question. Here, we address the hypothesis that the abundance of mimics is limited by that of the models, leading to mimetic polymorphism. In addition, other forces such as the effects of common ancestry and/or isolation by distance may explain this phenomenon. To investigate this question, we focused on the butterfly, Papilio polytes, that exhibits mimetic polymorphism on multiple islands of the Ryukyus, Japan, and performed field surveys and genetic analysis. We found that the mimic ratio of P. polytes was strongly correlated with the model abundance observed on each of the five islands, suggesting negative frequency-dependent selection is driving the evolution of polymorphism in P. polytes populations. Molecular phylogenetic analysis indicated that the southern island populations are the major source of genetic diversity, and the middle and northern island populations arose by relatively recent migration. This view was also supported by mismatch distribution and Tajima's D analyses, suggesting a recent population expansion on the middle and northern islands, and stable population persistence on the southern islands. The frequency of the mimetic forms within P. polytes populations is thus explained by variations in the model abundance rather than by population structure. Thus, we propose that predation pressure, rather than neutral forces, have shaped the Batesian mimicry polymorphism in P. polytes observed in the Ryukyus.

Latitudinal variation and coevolutionary diversification of sexually dimorphic traits in the false blister beetle Oedemera sexualis.

Sexual traits are subject to evolutionary forces that maximize reproductive benefits and minimize survival costs, both of which can depend on environmental conditions. Latitude explains substantial variation in environmental conditions. However, little is known about the relationship between sexual trait variation and latitude, although body size often correlates with latitude. We examined latitudinal variation in male and female sexual traits in 22 populations of the false blister beetle Oedemera sexualis in the Japanese Archipelago. Males possess massive hind legs that function as a female-grasping apparatus, while females possess slender hind legs that are used to dislodge mounting males. Morphometric analyses revealed that male and female body size (elytron length), length and width of the hind femur and tibia, and allometric slopes of these four hind leg dimensions differed significantly among populations. Of these, three traits showed latitudinal variation, namely, male hind femur was stouter; female hind tibia was slenderer, and female body was smaller at lower latitudes than at higher latitudes. Hind leg sizes and shapes, as measured by principal component analysis of these four hind leg dimensions in each sex, covaried significantly between sexes, suggesting coevolutionary diversification in sexual traits. Covariation between sexes was weaker when variation in these traits with latitude was removed. These results suggest that coevolutionary diversification between male and female sexual traits is mediated by environmental conditions that vary with latitude.

When a male perceives a female: the effect of waxy components on the body surface on decision-making in the invasive pest weevil.

Insects use various semiochemicals for sexual communication and mate recognition; these can therefore be used to govern the behaviours of harmful pest species, and several candidate chemicals have been explored for this purpose. For the West Indian sweet potato weevil, Euscepes postfasciatus, which is one of the most serious pests of sweet potato, no effective capture techniques, such as sex pheromone lures, exist. Toward exploring promising procedures for monitoring these weevils, we assessed the effect of secretions on the body surface on the recognition of congeners and on courtship behaviour in the weevils. Our study clearly demonstrated that weevils responded to extracts from the body surface, and the behaviour adopted by the weevils varied significantly depending on the condition of the extracts. Furthermore, we found a significantly prolonged retention time for males on glass beads covered with extracts of females based on survival analysis. These findings are, as far as we are aware, the first to show the effect of lipid components of the body surface on decision-making in these economically important pest weevils.

Ultraviolet exposure has an epigenetic effect on a Batesian mimetic trait in the butterfly Papilio polytes.

Wing polymorphism of butterflies provides a good system in which to study adaptation. The Asian Batesian mimic butterfly Papilio polytes has unmelanized, putative mimetic red spots on its black hind wings. The size of those red spots is non-heritable but it is highly polymorphic, the adaptive significance of which is unknown. We hypothesized that under strong ultraviolet (UV) irradiation, butterflies develop a wider melanized black area to protect the wings from UV damage, and as a result express smaller mimetic red spots. Our field survey on Okinawa Island revealed a negative relationship between the sizes of the red spot and the black area in the wings. The size varied seasonally and was negatively correlated with the intensity of solar UV radiation at the time of capture. Laboratory experiments revealed that the size was reduced by strong UV irradiation not only of the eggs and larvae, but also of their mothers through a putative epigenetic mechanism. The flexible phenotypic expression of the red spots in P. polytes suggests a trade-off between protection against UV damage and predation avoidance, and provides a new insight into the evolution of Batesian mimicry.

A Comparative Chromosome Mapping Study in Japanese Podismini Grasshoppers (Orthoptera: Acrididae: Melanoplinae).

In the present paper, karyotypes of 7 Japanese Podismini species, Anapodisma beybienkoi, Fruhstorferiola okinawaensis, Parapodisma caelestis, P. mikado, P. setouchiensis, P. tenryuensis, and Sinopodisma punctata (2n♂ = 21, all acrocentric), are described and compared on the basis of conventional (C-banding, DAPI/CMA3-staining, Ag-NOR) and molecular (FISH with 18S rDNA and telomeric probes) cytogenetic staining methods. This is the first study to report karyotypes of A. beybienkoi and P. caelestis. Differential staining techniques showed karyotypic diversity in these species. The number of 18S rDNA signals ranged from 2 to 6, and the signals were located on the autosomes or sex chromosomes. In all species, clusters of rDNA coincided with Ag-NORs. Telomeric signals occurred at the chromosome ends at the pachytene stage and seldom at other stages of meiosis. Paracentromeric and some distal and interstitial blocks of constitutive heterochromatin were detected in the chromosomes of Anapodisma, Fruhstorferiola, and Parapodisma species. Staining with DAPI and CMA3 revealed 2 groups of heterochromatin composition. In addition, intraspecific differences in the number of rDNA clusters and C-bands were observed within Parapodisma species. Based on the evidence of cytogenetic characteristics, the monophyly of Tonkinacridina cannot be supported.

Comparative toxicities of silver nitrate, silver nanocolloids, and silver chloro-complexes to Japanese medaka embryos, and later effects on population growth rate.

Fish embryo toxicology is important because embryos are more susceptible than adults to toxicants. In addition, the aquatic toxicity of chemicals depends on water quality. We examined the toxicities to medaka embryos of three types of silver-AgNO3, silver nanocolloids (SNCs), and silver ions from silver nanoparticle plates (SNPPs)-under three pH conditions (4.0, 7.0, and 9.0) in embryo-rearing medium (ERM) or ultrapure water. Furthermore, we tested the later-life-stage effects of SNCs on medaka and their population growth. "Later-life-stage effects" were defined here as delayed toxic effects that occurred during the adult stage of organisms that had been exposed to toxicant during their early life stage only. AgNO3, SNCs, and silver ions were less toxic in ERM than in ultrapure water. Release of silver ions from the SNPPs was pH dependent: in ERM, silver toxicity was decreased owing to the formation of silver chloro-complexes. SNC toxicity was higher at pH 4.0 than at 7.0 or 9.0. AgNO3 was more toxic than SNCs. To observe later-life effects of SNCs, larvae hatched from embryos exposed to 0.01 mg/L SNCs in ultrapure water were incubated to maturity under clean conditions. The mature medaka were then allowed to reproduce for 21 days. Calculations using survival ratios and reproduction data indicated that the intrinsic population growth rate decreased after exposure of embryos to SNC. SNC exposure reduced the extinction time as a function of the medaka population-carrying capacity.

Rapid evolution of a Batesian mimicry trait in a butterfly responding to arrival of a new model.

Batesian mimicry, a phenomenon in which harmless organisms resemble harmful or unpalatable species, has been extensively studied in evolutionary biology. Model species may differ from population to population of a single mimetic species, so different predation pressures might have driven micro-evolution towards better mimicry among regions. However, there is scant direct evidence of micro-evolutionary change over time in mimicry traits. Papilio polytes shows female-limited Batesian mimicry. On Okinawa, one mimicry model is Pachliopta aristolochiae, which was not present on the island until 1993. In P. polytes, the size of the hind-wing white spot, a mimetic trait, is maternally heritable. Among specimens collected between 1961 and 2016, the average white spot size was unchanged before the model's arrival but has rapidly increased since then. However, white spot size showed greater variance after the model's establishment than before. This suggests that before 1993, white spot size in this population was not selectively neutral but was an adaptive trait for mimicking an unpalatable native, Byasa alcinous, which looks like P. aristolochiae apart from the latter's hind-wing white spot. Thus, some females switched their model to the new one after its arrival.

(Z)-13-Hexadecenyl Acetate: a Novel Moth Sex Pheromone Component from Herpetogramma submarginale (Lepidoptera: Crambidae).

The sex pheromone of Herpetogramma submarginale (Swinhoe) was studied by gas chromatography (GC) with electroantennographic detection and GC coupled with mass spectrometry. Two pheromone candidates detected in the gland extracts of females were identified as (Z)-13-hexadecenyl acetate (Z13-16:OAc) and (E)-13-hexadecenyl acetate (E13-16:OAc) in a ratio of 87:13 by mass spectral analysis of the natural pheromone components and their dimethyldisulfide adducts. In field tests, Z13-16:OAc alone attracted H. submarginale males and caught significantly more males than live virgin females. Addition of E13-16:OAc did not enhance the attractiveness of Z13-16:OAc. Derivatives of Z13-16:OAc also were tested as potential pheromone components. Addition of (Z)-13-hexadecen-1-ol significantly reduced the number of males captured, and (Z)-13-hexadecenal had no effect on the attractiveness of the lure. These results suggest that the female-produced sex pheromone of H. submarginale is Z13-16:OAc. This hexadecenyl acetate is a novel moth sex pheromone component.

Free silver ion as the main cause of acute and chronic toxicity of silver nanoparticles to cladocerans.

We investigated the interspecific variation of silver nanoparticle (SNP) sensitivity in common cladocerans (Daphnia magna, D. galeata, and Bosmina longirostris) and the exact cause of both acute and chronic toxicity focusing on the form of silver (NPs and ions). Materials tested were non-surface-coated silver nanocolloids (SNCs) and AgNO3. The results of the acute toxicity tests support the theory that the effects of SNPs on aquatic organisms is mainly due to Ag(+) released from SNPs. Among the three cladocerans, D. galeata was more sensitive to silver (as Ag(+)) than both D. magna and B. longirostris. Moreover, the chronic toxicity of SNCs was also derived from dissolved silver (especially Ag(+)). SNCs (as total silver concentration) showed far lower chronic compared with acute toxicity to daphnids because the amount of dissolved silver decreased in the presence of prey algae. The chronic end-point values (EC10 values for net reproductive rate and the probability of survival to maturation) did not differ largely from acute ones (48-h EC50 obtained from acute toxicity tests and 48-h LC50 estimated by the biotic ligand model) when the values were calculated based on Ag(+) concentration. The α value (concentration at which intrinsic population growth rate is decreased to zero) estimated by a power function model was a reliable parameter for assessing the chronic toxicity of silver.

Female sex pheromones of two Japanese saturniid species, Rhodinia fugax and Loepa sakaei: identification, synthesis, and field evaluation.

While 11 species in the family Saturniidae are found in Japan, no sex pheromones of the native species had been investigated previously. We collected larvae of Rhodinia fugax in Nagano and Tottori Prefecture, and of Loepa sakaei in Okinawa Prefecture, and extracted sex pheromones of these two species from virgin female moths. In gas chromatography-electroantennogram detection (GC-EAD) analyses, male antennae of each species responded to one component in the respective pheromone extracts of conspecific females. Chemical analyses of the extracts by GC/mass spectrometry revealed that the EAD-active compounds of R. fugax and L. sakaei were a hexadecadienal and a tetradecadienyl acetate, respectively. The two species belong to the subfamily Saturniinae, and the mass spectra of both were similar to that of the 6,11-hexadecadienyl acetate identified from Antheraea polyphemus, classified in the same subfamily, suggesting the same 6,11-dienyl structure for the C16 aldehyde and a 4,9-dienyl structure for the C14 acetate. Based on this assumption, four geometrical isomers of each dienyl compound were stereoselectively synthesized via acetylene intermediates, compared to the natural products, and tested in the field. Male catches confirmed the pheromone structures of the two Japanese saturniid species as (6E,11Z)-6,11-hexadecadienal for R. fugax and (4E,9Z)-4,9-tetradecadienyl acetate for L. sakaei. The compounds have a characteristic 1,6-dienyl motif common to the pheromones of Saturniinae species.

Retrospective estimation of population-level effect of pollutants based on local adaptation and fitness cost of tolerance.

We present a novel framework for estimating site-specific effects of pollutants on natural populations. Our method is based on fitness optimization and uses observed differences in tolerance (sensitivity) to a particular pollutant between populations at contaminated and uncontaminated sites (i.e., target and reference populations). In addition, the method uses laboratory estimates of the fitness cost of tolerance, that is, the reduction of population growth rate (fitness) of a target population compared to that of a reference population when both are maintained in uncontaminated conditions. As a case study, we applied this framework to analyze observed genetic differentiation in tolerance to the pyrethroid insecticide fenvalerate between Daphnia galeata populations in Lake Kasumigaura and an adjacent agricultural pond. The estimated exposure level at the contaminated site was about 0.015 µg/L, and the population-level risk corresponded to about a 24 % reduction of the intrinsic rate of natural increase.

Habitat association and seasonality in a mosaic and bimodal hybrid zone between Chorthippus brunneus and C. jacobsi (Orthoptera: Acrididae).

Understanding why some hybrid zones are bimodal and others unimodal can aid in identifying barriers to gene exchange following secondary contact. The hybrid zone between the grasshoppers Chorthippus brunneus and C. jacobsi contains a mix of allopatric parental populations and inter-mingled bimodal and unimodal sympatric populations, and provides an ideal system to examine the roles of local selection and gene flow between populations in maintaining bimodality. However, it is first necessary to confirm, over a larger spatial scale, previously identified associations between population composition and season and habitat. Here we use cline-fitting of one morphological and one song trait along two valley transects, and intervening mountains, to confirm previously identified habitat associations (mountain versus valley) and seasonal changes in population composition. As expected from previous findings of studies on a smaller spatial scale, C. jacobsi dominated mountain habitats and mixed populations dominated valleys, and C. brunneus became more prevalent in August. Controlling for habitat and incorporating into the analysis seasonal changes in cline parameters and the standard errors of parental trait values revealed wider clines than previous studies (best estimates of 6.4 to 24.5 km in our study versus 2.8 to 4.7 km in previous studies) and increased percentage of trait variance explained (52.7% and 61.5% for transects 1 and 2 respectively, versus 17.6%). Revealing such strong and consistent patterns within a complex hybrid zone will allow more focused examination of the causes of variation in bimodality in mixed populations, in particular the roles of local selection versus habitat heterogeneity and gene flow between differentiated populations.