Geographic Cline Research Articles

Temporal trends in geographic clines of chum salmon reproductive traits associated with global warming and hatchery programmes

Abstract Geographic clines in life‐history traits are often recognized as adaptations to the associated transitional environments. As life‐history traits evolve in response to anthropogenic processes, these geographic clines can change over time. The geographic and temporal trends of reproductive traits in Japanese chum salmon Oncorhynchus keta were analysed. Data were collected from 23 rivers located between 36° and 45° north latitude and 136° and 146° east longitude from 1994 to 2010. We confirmed the geographic clines of reproductive traits: relative gonad weight increased in more northeasterly locations, and females had fewer, but larger, eggs in more northeasterly locations after standardization by body size. The geographic clines changed over the years. The northeastward geographic trend of increasing gonad weight became more pronounced over time. Temporal trends towards smaller but more numerous eggs were evident, especially in northeasterly locations. Under natural and sexual selection, gonadal investment should be constrained by the energetic demands of the cost of migration, particularly in southwesterly locations (which are farthest from the feeding grounds), and by breeding competition during natural reproduction. In addition, females should have fewer but larger eggs owing to a constraint on growth opportunities for their offspring in more northeasterly regions of Japan, which are colder and have less available food. However, global warming may mitigate this constraint on growth opportunities in northeastern Japan by increasing river water temperatures. Moreover, we consider that relaxation of the effects of natural and sexual selection on intense breeding competition and early growth conditions has occurred through domestication selection by hatchery programmes. These may have caused temporal shifts in geographic clines. We should consider several co‐occurring anthropogenic impacts on natural and sexual selection when evaluating the life‐history traits of organisms. For the sustainable use of biological resources, maintaining geographically adapted life‐history traits during adaptation to climate change is essential. Therefore, the conservation of wild salmon populations formed by natural selection is preferable to the stocking of hatchery‐reared fry.

Ecogeography of Plumage Pigmentation in Great Horned Owls

ABSTRACT Plumage pigmentation is fundamental to a bird's phenotype, with pigment deposition causing relative crypsis or conspicuousness, depending on the environmental context. Geographic variation in plumage melanin tends to be predictable, suggesting that aspects of climate cause local matching of plumage to environment via genetic adaptation. Ecogeographic rules describe this predictability: Gloger's rule predicts that populations in wetter and warmer environments will be more pigmented; Bogert's Rule predicts more pigmentation in cold environments. The Great Horned Owl (Bubo virginianus) exhibits extensive geographic variation in the degree of melanin-based pigmentation. We examined fine-scale spatial variation in owl plumage melanism along environmental gradients in southwestern North America. We tested whether variation is explained by either of two non-mutually exclusive hypotheses: (1) a history of allopatric divergence between subspecies or (2) in situ local adaptation consistent with ecogeographic rules. The allopatric divergence hypothesis predicts a bimodal distribution of plumage melanism, with a geographic cline across a zone of secondary contact, whereas the local adaptation hypothesis predicts that climate explains variation independently of geography. Using a colorimeter, we measured coloration in 101 museum specimens of breeding-season Great Horned Owls that had been obtained from variable environments and elevations. Specimens previously identified as separate subspecies were distinguishable by colorimetry. Plumage lightness, however, was continuously distributed, rather than bimodal. While accounting for males having reduced pigmentation relative to females, linear models revealed that lighter plumage was associated with low latitude, low elevation, high temperature, and low precipitation. These findings suggest that variation in Great Horned Owl plumage pigmentation is best understood as continuous ecogeographic variation, consistent with ecogeographic predictions, and currently maintained in situ along multiple environmental gradients that characterize the “sky island” topography of the southwestern USA.

Distinguishing genomic homogenization from parapatric speciation in an elevationally replacing pair of Ramphocelus tanagers.

Geographically connected species pairs with weakly differentiated genomes could either represent cases of genomic homogenization in progress or of incipient parapatric speciation. Discriminating between these processes is difficult because intermediate stages of either may produce weakly differentiated genomes that diverge at few locations. We used coalescent modelling applied to a genome-wide sample of SNPs to discriminate between speciation with gene flow and genomic homogenization in two phenotypically distinct but genomically weakly diverged species of elevationally replacing Ramphocelus tanagers, forming a hybrid zone in the Andean foothills. We found overwhelming support for a model of genomic homogenization following secondary contact. Simulating under this model suggested that our species pair was differentiated (FST = 0.30) at secondary contact but that most of the genome has rapidly homogenized during 254Ky of high gene flow towards the present (FST = 0.02). Despite extensive genome-wide homogenization, plumage remains distinctive with a narrower than expected geographic cline width, indicating divergent selection on colour. We found two SNPs significantly associated with plumage colour, which retain moderately high FST . We conclude that the majority of the genome has fused, but that divergent selection on select loci probably maintains the geographically structured colour differences between these incipient species.

Nuclear phylogenies and genomics of a contact zone establish the species rank of Podarcis lusitanicus (Squamata, Lacertidae)

Unravelling when divergent lineages constitute distinct species can be challenging, particularly in complex scenarios combining cryptic diversity and phylogenetic discordances between different types of molecular markers. Combining a phylogenetic approach with the study of contact zones can help to overcome such difficulties. The Podarcis hispanicus species complex has proven to be prosperous in independent evolutionary units, sometimes associated with cryptic diversity. Previous studies have revealed that one of the species of this complex, P. guadarramae, comprises two deeply divergent yet morphologically indistinguishable evolutionary units, currently regarded as subspecies (P. g. guadarramae and P. g. lusitanicus). In this study we used molecular data to address the systematics of the two lineages of Podarcis guadarramae and the closely related P. bocagei. Firstly, we reconstructed the species tree of these three and two additional taxa based on 30 nuclear loci using the multispecies coalescent with and without gene flow. Secondly, we used SNPs obtained from RADseq data to analyze the population structure across the distribution limits P. g. lusitanicus and P. g. guadarramae, and for comparison, a contact zone between P. bocagei and P. g. lusitanicus. Nuclear phylogenetic relationships between these three taxa are clearly difficult to determine due to the influence of gene flow, but our results give little support to the monophyly of P. guadarramae, potentially due to a nearly simultaneous divergence between them. Genetic structure and geographic cline analysis revealed that the two lineages of P. guadarramae replace each other abruptly across the sampled region and that gene flow is geographically restricted, implying the existence of strong reproductive isolation. Podarcis bocagei and P. g. lusitanicus show a similar degree of genetic differentiation and reproductive isolation, with very low levels of admixture in syntopy. These results support that all three forms are equally differentiated and reproductively isolated. In consequence, we conclude that the two former subspecies of Podarcis guadarramae constitute valid, yet cryptic species, that should be referred to as P. lusitanicus and P. guadarramae.

A star is torn-molecular analysis divides the Mediterranean population of Poli's stellate barnacle, Chthamalus stellatus (Cirripedia, Chtamalidae).

Poli’s stellate barnacle, Chthamalus stellatus Poli, populates the Mediterranean Sea, the North-Eastern Atlantic coasts, and the offshore Eastern Atlantic islands. Previous studies have found apparent genetic differences between the Atlantic and the Mediterranean populations of C. stellatus, suggesting possible geological and oceanographic explanations for these differences. We have studied the genetic diversity of 14 populations spanning from the Eastern Atlantic to the Eastern Mediterranean, using two nuclear genes sequences revealing a total of 63 polymorphic sites. Both genotype-based, haplotype-based and the novel SNP distribution population-based methods have found that these populations represent a geographic cline along the west to east localities. The differences in SNP distribution among populations further separates a major western cluster into two smaller clusters, the Eastern Atlantic and the Western Mediterranean. It also separates the major eastern cluster into two smaller clusters, the Mid-Mediterranean and Eastern Mediterranean. We suggested here environmental conditions like surface currents, water salinity and temperature as probable factors that have formed the population structure. We demonstrate that C. stellatus is a suitable model organism for studying how geological events and hydrographic conditions shape the fauna in the Mediterranean Sea.

Interindividual plasticity in metabolic and thermal tolerance traits from populations subjected to recent anthropogenic heating.

To better understand temperature's role in the interaction between local evolutionary adaptation and physiological plasticity, we investigated acclimation effects on metabolic performance and thermal tolerance among natural Fundulus heteroclitus (small estuarine fish) populations from different thermal environments. Fundulus heteroclitus populations experience large daily and seasonal temperature variations, as well as local mean temperature differences across their large geographical cline. In this study, we use three populations: one locally heated (32°C) by thermal effluence (TE) from the Oyster Creek Nuclear Generating Station, NJ, and two nearby reference populations that do not experience local heating (28°C). After acclimation to 12 or 28°C, we quantified whole-animal metabolic (WAM) rate, critical thermal maximum (CTmax) and substrate-specific cardiac metabolic rate (CaM, substrates: glucose, fatty acids, lactate plus ketones plus ethanol, and endogenous (i.e. no added substrates)) in approximately 160 individuals from these three populations. Populations showed few significant differences due to large interindividual variation within populations. In general, for WAM and CTmax, the interindividual variation in acclimation response (log2 ratio 28/12°C) was a function of performance at 12°C and order of acclimation (12–28°C versus 28–12°C). CTmax and WAM were greater at 28°C than 12°C, although WAM had a small change (2.32-fold) compared with the expectation for a 16°C increase in temperature (expect 3- to 4.4-fold). By contrast, for CaM, the rates when acclimatized and assayed at 12 or 28°C were nearly identical. The small differences in CaM between 12 and 28°C temperature were partially explained by cardiac remodeling where individuals acclimatized to 12°C had larger hearts than individuals acclimatized to 28°C. Correlation among physiological traits was dependent on acclimation temperature. For example, WAM was negatively correlated with CTmax at 12°C but positively correlated at 28°C. Additionally, glucose substrate supported higher CaM than fatty acid, and fatty acid supported higher CaM than lactate, ketones and alcohol (LKA) or endogenous. However, these responses were highly variable with some individuals using much more FA than glucose. These findings suggest interindividual variation in physiological responses to temperature acclimation and indicate that additional research investigating interindividual may be relevant for global climate change responses in many species.

Incomplete reproductive isolation and low genetic differentiation despite floral divergence across varying geographic scales in Castilleja.

Divergence depends on the strength of selection and frequency of gene flow between taxa, while reproductive isolation relies on mating barriers and geographic distance. Less is known about how these processes interact at early stages of speciation. Here, we compared population-level differentiation in floral phenotype and genetic sequence variation among recently diverged Castilleja to explore patterns of diversification under different scenarios of reproductive isolation. Using target enrichment enabled by the Angiosperms353 probe set, we assessed genetic distance among 50 populations of four Castilleja species. We investigated whether patterns of genetic divergence are explained by floral trait variation or geographic distance in two focal groups: the widespread C. sessiliflora and the more restricted C. purpurea species complex. We document that C. sessiliflora and the C. purpurea complex are characterized by high diversity in floral color across varying geographic scales. Despite phenotypic divergence, groups were not well supported in phylogenetic analyses, and little genetic differentiation was found across targeted Angiosperms353 loci. Nonetheless, a principal coordinate analysis of single nucleotide polymorphisms revealed differentiation within C. sessiliflora across floral morphs and geography and less differentiation among species of the C. purpurea complex. Patterns of genetic distance in C. sessiliflora suggest species cohesion maintained over long distances despite variation in floral traits. In the C. purpurea complex, divergence in floral color across narrow geographic clines may be driven by recent selection on floral color. These contrasting patterns of floral and genetic differentiation reveal that divergence can arise via multiple eco-evolutionary paths.

Body size variation in polyplacophoran molluscs: Geographical clines and community structure along the south‐eastern Pacific

AbstractAimTo evaluate the latitudinal pattern of body size within and among chiton species employing phylogenetically structured analyses and examine the role ofgeographical variation in temperature, productivity and oxygen availability as potential environmental drivers.LocationCoastal habitats of the south‐eastern Pacific along a latitudinal range of nearly 6,000 km, from the equator to Patagonia (c. 2° to 56° S).Time periodPresent (2011–2017).Major taxa studiedThirty‐one species of polyplacophoran molluscs.MethodsWe measured the body length of 6,162 individuals collected in 62 sites, and reconstructed the phylogeny of this group based on two mitochondrial and one nuclear gene regions. We combined this information with data of sea surface temperature, chlorophyll‐a concentration—as a proxy of primary productivity—and dissolved oxygen, and assessed which variables best explain the variation in size both within and among species employing phylogenetic generalized least squares (PGLS) and a model comparison approach.Main conclusionsOur analyses show that body size increases consistently with latitude, both within and among species, following Bergmann’s rule. Variation in sea surface temperature along the latitudinal gradient provided a substantially better fit than chlorophyll‐a and dissolved oxygen. Our results support the temperature–size rule for this lineage and suggest that similar processes could underlie the emergence of intra‐ and interspecific gradients in body size of polyplacophorans. At the community level, chiton species richness was higher at intermediate latitudes and positively correlated with body size variation, suggesting that heterogeneity in size may reduce interspecific competition and contribute to species coexistence in this group. Overall, our study demonstrates that historical events, macroecological adaptive trends and local processes at the community level contribute to the distribution and size variation of polyplacophoran species throughout the south‐eastern Pacific.

Haplotype tagging reveals parallel formation of hybrid races in two butterfly species

Genetic variation segregates as linked sets of variants or haplotypes. Haplotypes and linkage are central to genetics and underpin virtually all genetic and selection analysis. Yet, genomic data often omit haplotype information due to constraints in sequencing technologies. Here, we present "haplotagging," a simple, low-cost linked-read sequencing technique that allows sequencing of hundreds of individuals while retaining linkage information. We apply haplotagging to construct megabase-size haplotypes for over 600 individual butterflies (Heliconius erato and H. melpomene), which form overlapping hybrid zones across an elevational gradient in Ecuador. Haplotagging identifies loci controlling distinctive high- and lowland wing color patterns. Divergent haplotypes are found at the same major loci in both species, while chromosome rearrangements show no parallelism. Remarkably, in both species, the geographic clines for the major wing-pattern loci are displaced by 18 km, leading to the rise of a novel hybrid morph in the center of the hybrid zone. We propose that shared warning signaling (Müllerian mimicry) may couple the cline shifts seen in both species and facilitate the parallel coemergence of a novel hybrid morph in both comimetic species. Our results show the power of efficient haplotyping methods when combined with large-scale sequencing data from natural populations.

Multivariate analysis of geographic variation in Darevskia clarkorum (Darevsky amp; Vedmederja, 1977), correlation with geographic and climatic parameters, and true status of Darevskia dryada (Darevsky amp; Tuniyev, 1997).

All the Turkish populations studied, both those previously assigned to D. dryada (Subaşı and Yoldere villages, near Hopa) and those attributed to D. clarkorum (the largest sample studied so far, 177 specimens in total), are indistinguishable from each other and therefore must all be ascribed to the natural variability of a monotypic D. clarkorum. The Georgian specimens from the Type Locality of D. dryada (Charnaly river gorge, Chevachauri district) are clearly different, so that taxon cannot be considered a simple synonym for D. clarkorum, but as a valid taxon, although its proper status (more probably as a subspecies of D. clarkorum), is yet to be clarified. It is a highly threatened population, so studies should be done in vivo or with as low intrusiveness as possible. Darevskia dryada is clearly larger (SVL) than any D. clarkorum studied, with strongly longer heads and pilei in adult males (and hence more teeth in dentary bone), and higher dorsalia counts. There also seem to be (but need to be studied in a larger sample) more longitudinal rows of temporal scales between tympanic and parietal plates, a tendency to have more supralabial scales; comparatively smaller values for longitudinal rows of scales on the ventral surface of the thigh between the femoral pores and the outer row of enlarged scales, and higher collaria, and circumanalia scales. Other differences in femoralia and gularia are also reflected in Darevsky Tuniyev's (1997) tables and should also be investigated with more Georgian specimens. Two supposed discriminant characters, the frontonasal index and the presence of developed masseteric, are not valid. The frontonasal index does not discriminate both taxa; D dryada specimens fall inside the variation of D. clarkorum for this character. Also the presence of a developed masseteric plate is supposed to be rare if at all in D. clarkorum but always present in D. dryada; however, it appears in nearly 75% of D. clarkorum studied and in all D. dyada, so is also no longer valid for taxa discrimination. Although very similar, D. clarkorum and D. dryada are morphologically different, and genetic studies (as the unpublished results mentioned by Fu, 1999) do not make the provenance of the specimens clear, and hence the correct identification of the supposed specimens of D. dryada used. There are no geographical clines in D. clarkorum. However, as stated by Schmidtler et al. (2002), there is an inverse relationship between altitude and dorsalia values in D. clarkorum. Both the general differentiation between populations and the scalation (dorsalia) appear statistically correlated with the altitude and also with latitude (being both factors not strictly the same). The correlation seems to be stronger with morphology in general (multiple scalation characters and head biometry) than only with dorsalia. In the case of the general differentiation among samples, it is also significantly correlated with temperatures during the activity period (April-September) and with precipitation during incubation (July-August). As these climatic parameters of temperature and precipitation are not directly correlated with the dorsalia variation, the relation with altitude (and perhaps latitude) must be linked to some other climatic parameter not studied here, perhaps solar radiation or evapotranspiration.

A narrow window for geographic cline analysis using genomic data: Effects of age, drift, and migration on error rates.

The use of genomic and phenotypic data to scan for outliers is a mainstay for studies of hybridization and speciation. Geographic cline analysis of natural hybrid zones is widely used to identify putative signatures of selection by detecting deviations from baseline patterns of introgression. As with other outlier-based approaches, demographic histories can make neutral regions appear to be under selection and vice versa. In this study, we use a forward-time individual-based simulation approach to evaluate the robustness of geographic cline analysis under different evolutionary scenarios. We modelled multiple stepping-stone hybrid zones with distinct age, deme sizes, and migration rates, and evolving under different types of selection. We found that drift distorts cline shapes and increases false positive rates for signatures of selection. This effect increases with hybrid zone age, particularly if migration between demes is low. Drift can also distort the signature of deleterious effects of hybridization, with genetic incompatibilities and particularly underdominance prone to spurious typing as adaptive introgression. Our results suggest that geographic clines are most useful for outlier analysis in young hybrid zones with large populations of hybrid individuals. Current approaches may overestimate adaptive introgression and underestimate selection against maladaptive genotypes.

Broad Concordance in the Spatial Distribution of Adaptive and Neutral Genetic Variation across an Elevational Gradient in Deer Mice.

When species are continuously distributed across environmental gradients, the relative strength of selection and gene flow shape spatial patterns of genetic variation, potentially leading to variable levels of differentiation across loci. Determining whether adaptive genetic variation tends to be structured differently than neutral variation along environmental gradients is an open and important question in evolutionary genetics. We performed exome-wide population genomic analysis on deer mice sampled along an elevational gradient of nearly 4,000 m of vertical relief. Using a combination of selection scans, genotype−environment associations, and geographic cline analyses, we found that a large proportion of the exome has experienced a history of altitude-related selection. Elevational clines for nearly 30% of these putatively adaptive loci were shifted significantly up- or downslope of clines for loci that did not bear similar signatures of selection. Many of these selection targets can be plausibly linked to known phenotypic differences between highland and lowland deer mice, although the vast majority of these candidates have not been reported in other studies of highland taxa. Together, these results suggest new hypotheses about the genetic basis of physiological adaptation to high altitude, and the spatial distribution of adaptive genetic variation along environmental gradients.

Processes underlying complex patterns of song trait evolution in a Setophaga hybrid zone.

During secondary contact between two species when hybrids are less fit than parents, mating signals are expected to diverge, while aggressive signals are expected to converge. If a single signal trait is used in both mating and aggression, then the dynamics between these two forces could influence the evolutionary trajectory of that trait. We studied such a situation in an avian hybrid zone between two Setophaga species, where birdsong is used in both mate attraction and territory defense. We hypothesized that song modules of the two species will show separate and distinct geographic patterns due to the influence of selective pressures for effective territorial aggression and for effective mate attraction. We conducted geographic cline analyses and playback experiments across this hybrid zone. We found an unexpected geographic pattern of asymmetric introgression of song rhythm, which may be explained by results of the playback experiments that suggest that differences in song rhythm serve a greater role in mate attraction than in territory defense. In contrast, differences in syllable morphology show little evidence of importance in mate attraction or territorial defense. Song features converge in the hybrid zone, yet patterns of trait change suggest that the song production modules may vary in their modes of development and inheritance. Syringeal motor gesturing, which gives rise to syllable morphology, shows a nonclinal mosaic pattern, suggesting that this trait may be predominantly learned. In contrast, respiratory patterning, which forms song rhythm, shows a clinal geographic transition, suggesting that this trait could be more innate. The results indicate that opposing forces act independently on song via distinct modules of the song production mechanism, driving complex patterns of song trait evolution.

Sex-linked genetic diversity and differentiation in a globally distributed avian species complex.

Sex chromosomes often bear distinct patterns of genetic variation due to unique patterns of inheritance and demography. The processes of mutation, recombination, genetic drift and selection also influence rates of evolution on sex chromosomes differently than autosomes. Measuring such differences provides information about how these processes shape genomic variation and their roles in the origin of species. To test hypotheses and predictions about patterns of autosomal and sex-linked genomic diversity and differentiation, we measured population genetic statistics within and between populations and subspecies of the barn swallow (Hirundo rustica) and performed explicit comparisons between autosomal and Z-linked genomic regions. We first tested for evidence of low Z-linked genetic diversity and high Z-linked population differentiation relative to autosomes, then for evidence that the Z chromosome bears greater ancestry information due to faster lineage sorting. Finally, we investigated geographical clines across hybrid zones for evidence that the Z chromosome is resistant to introgression due to selection against hybrids. We found evidence that the barn swallow mating system, demographic history and linked selection each contribute to low Z-linked diversity and high Z-linked differentiation. While incomplete lineage sorting is rampant across the genome, our results indicate faster sorting of ancestral polymorphism on the Z. Finally, hybrid zone analyses indicate barriers to introgression on the Z chromosome, suggesting that sex-linked traits are important in reproductive isolation, especially in migratory divide regions. Our study highlights how selection, gene flow and demography shape sex-linked genetic diversity and underlines the relevance of the Z chromosome in speciation.

Geometric morphometrics reveal sister species in sympatry and a cline in genital morphology in a ghost spider genus

AbstractMorphological boundaries between species of the maculatipes‐group in the ghost spider genus Sanogasta Mello‐Leitão are ambiguous, and the most widespread species, S. maculatipes, is unusually variable and may represent multiple cryptic species. To resolve these issues, we perform a geometric morphometric analysis on the female genitalia of the group, visualizing and testing for differences in shape between described species, and between putative cryptic species within S. maculatipes. We complement this with a multi‐locus phylogenetic analysis of the group, to place the morphological results in a phylogenetic context. Our study reveals that two species in the group, S. alticola and S. mandibularis, are morphologically and molecularly distinct lineages that can be objectively distinguished. However, we reveal that S. maculatipes actually consists of two widespread sister species which occur in sympatry throughout the grasslands of northern Argentina. We further discover a geographical cline in the shape of the female genitalia of one of these sister species, such that specimens from the east and west of the range display morphological differences comparable to those between species, despite being virtually identical at the COI locus. Our study demonstrates the strength of a geometric morphometric approach for delimiting species in species‐complexes where morphological differences are subtle and confounding factors such as overlapping ranges, allometry and high levels of intraspecific variation are present.

Geographical cline and inter‐seaside difference in cone characteristics related to climatic conditions of old planted Pinus thunbergii populations throughout Japan

AbstractAlthough cone morphology has been mainly utilized for taxonomy or forestry purposes, characterization of the environmental factors that influence its variation is still insufficient. Major conifers distributed over diverse climatic conditions like the Japanese archipelago could show cone morphology variation among populations related to large differences in key influential factors such as temperature, snow, irradiance and sunshine. Geographical variation in cone characteristics (cone size, seed production, seed productivity per cone and seed size) was examined across the species distribution in 24 old planted Pinus thunbergii populations (479 trees) along two major seasides in Japan (Pacific Ocean side and Japan Sea side). Variance components of cone characteristics explained by seaside were at similar levels to those for populations nested within seasides. Populations on the Japan Sea side produced cones that had larger size (length and width), higher seed production (number of filled seeds per cone and seed mass per cone) and seed productivity per cone (seed‐to‐ovule ratio). Analysis of covariance showed that significant latitudinal clines observed in cone size and seed productivity per cone were mostly a result of inter‐seaside differences. Linear mixed model analysis detected that geographical variation in cone size, seed production and seed productivity per cone in the populations were affected significantly by low temperature, more snow, less solar irradiance and less sunshine time. Large cone sizes and high seed productivity per cone found in the populations on the Japan Sea side could be explained by a genetic or plastic response to maintain reproductive success.

Selection on a small genomic region underpins differentiation in multiple color traits between two warbler species.

Speciation is one of the most important processes in biology, yet the study of the genomic changes underlying this process is in its infancy. North American warbler species Setophaga townsendi and Setophaga occidentalis hybridize in a stable hybrid zone, following a period of geographic separation. Genomic differentiation accumulated during geographic isolation can be homogenized by introgression at secondary contact, whereas genetic regions that cause low hybrid fitness can be shielded from such introgression. Here, we examined the genomic underpinning of speciation by investigating (1) the genetic basis of divergent pigmentation traits between species, (2) variation in differentiation across the genome, and (3) the evidence for selection maintaining differentiation in the pigmentation genes. Using tens of thousands of single nucleotide polymorphisms (SNPs) genotyped in hundreds of individuals within and near the hybrid zone, genome‐wide association mapping revealed a single SNP associated with cheek, crown, breast coloration, and flank streaking, reflecting pleiotropy (one gene affecting multiple traits) or close physical linkage of different genes affecting different traits. This SNP is within an intron of the RALY gene, hence we refer to it as the RALY SNP. We then examined between‐species genomic differentiation, using both genotyping‐by‐sequencing and whole genome sequencing. We found that the RALY SNP is within one of the highest peaks of differentiation, which contains three genes known to influence pigmentation: ASIP, EIF2S2, and RALY (the ASIP‐RALY gene block). Heterozygotes at this gene block are likely of reduced fitness, as the geographic cline of the RALY SNP has been narrow over two decades. Together, these results reflect at least one barrier to gene flow within this narrow (∼200 kb) genomic region that modulates plumage difference between species. Despite extensive gene flow between species across the genome, this study provides evidence that selection on a phenotype‐associated genomic region maintains a stable species boundary.

Geographic clines in Daphnia magna’s circadian clock gene expression: Local adaptation to photoperiod

Nearly all organisms show daily and seasonal physiological and behavioural responses that are necessary for their survival. Often these responses are controlled by the rhythmic activity of an endogenous clock that perceives day length. Day length differs not only between seasons but also along latitudes, with different seasonal day lengths between the north and the south. Both seasonal and latitudinal differences in day length are discussed to be perceived/processed by the endogenous clock. Some species are distributed over a wide range of latitudes; it should be highly adaptive for these species to be able to time physiological responses (e.g. migration behaviour and diapause) according to the organisms’ respective photoperiod, i.e. their respective seasonal and latitudinal day length. The mediator of day length is the indoleamine hormone melatonin which is synthesized by melatonin-producing enzymes (AANAT and HIOMT). These enzymes are in turn controlled by an endogenous clock. The ubiquitous aquatic keystone organism Daphnia possess clock and melatonin synthesis genes that are rhythmically expressed over 24hours. We were able to show that the 24-h rhythm of D. magna’s clock persists in constant darkness and is thus truly circadian. In one particular photoperiod, all D. magna clones produced a similar melatonin concentration due to a fixed AANAT activity. However, we have demonstrated that clones originating from different latitudes are adapted to their respective photoperiod by showing a geographic cline in clock and downstream melatonin synthesis gene expression. These findings hint at the problem locally adapted organisms face when they are forced to leave their respective photoperiod, e.g. because of climate change-driven range-expansion. If such a species is incapable of adjusting its endogenous clock to an unknown photoperiod, it will likely become extinct.

Body size of ectotherms constrains thermal requirements for reproductive activity in seasonal environments

Body size may influence ectotherm behaviour by influencing heating and cooling rates, thereby constraining the time of day that some individuals can be active. The time of day at which turtles nest, for instance, is hypothesized to vary with body size at both inter- and intra-specific levels because large individuals have greater thermal inertia, retaining preferred body temperatures for a longer period of time. We use decades of data on thousands of individual nests from Algonquin Park, Ontario, Canada, to explore how body size is associated with nesting behaviour in Painted Turtles (Chrysemys picta (Schneider, 1783); small bodied) and Snapping Turtles (Chelydra serpentina (Linnaeus, 1758); large bodied). We found that (i) between species, Painted Turtles nest earlier in the evening and at higher mean temperatures than Snapping Turtles, and (ii) within species, relatively large individuals of both species nest at cooler temperatures and that relatively larger Painted Turtles nest later in the evening compared with smaller Painted Turtles. Our data support the thermal inertia hypothesis and may help explain why turtles in general exhibit geographic clines in body size: northern environments experience more daily variation in temperature, and larger size may evolve, in part, for retention of preferred body temperature during terrestrial forays.

Statistical methodology for the evaluation of leukocyte data in wild reptile populations: A case study with the common wall lizard (Podarcis muralis).

The leukocyte profile has the potential to be a reliable method to measure health conditions and stress in wild animals, but limitations occur because current knowledge on reference intervals is largely incomplete, especially because data come from studies on captive animals involving few individuals from single populations. Here we propose a general framework for achieving reliable leukocyte reference intervals, encompassing a set of internal and external factors, potentially affecting the leukogram. To do so, we present a systematic survey of the hematology of the common wall lizard, Podarcis muralis, involving 794 lizards from 54 populations over the whole geographic range of the species in Italy. Reference intervals for white blood cell (WBC) and leukocyte differential count were obtained by using linear mixed models in a Bayesian framework. The application of the procedure clearly showed that both internal (sex and size) and external (latitude and season) factors are a source of variation of leukocyte profile. Furthermore, the leukogram of common wall lizard has a strong variability among populations, which accounts for more than 50% of the whole variation. Consequently, some common assumptions used in studies on captive individuals are no longer supported in wild populations, namely, i) any group of individuals is a representative sample, ii) any population is representative of all others, iii) geographic clines do not occur over the species range, and iv) seasonal variation has limited effects. We encourage researchers aimed at the definition of leukocyte reference intervals for wild populations of reptiles to involve a large number of populations over a wide geographic range in ad hoc statistical models to disentangle local and geographic effects on leukocyte profile variation.