BMC Ecology and Evolution

Conspicuous coloration in animals is generally thought to evolve and be maintained through inter- or intraspecific interactions such as mate choice, but this might not always be the case. The sight-line hypothesis proposes that conspicuous light-dark contrast in front of the eyes (hereafter, eyeline) evolves and is maintained due to viability selection, enhancing an individual visual acuity and thus evolutionarily associated with a particular foraging behavior that requires accurate aiming. However, empirical evidence that supports the sight-line hypothesis is virtually absent, with no studies demonstrating the key prediction that the direction of eyelines matters. Here, I tested the sight-line hypothesis using macroevolutionary analyses in terns and allies, which are a suitable study system, because they have variation in facial color patterns, including presence/absence and, if any, various angles of eyelines. They also have a large variation in foraging behavior, including picking, plunge diving, and skimming. As predicted by the sight-line hypothesis, tern lineages that require accurate aiming at foraging (e.g., plunge diving) are more likely to have eyelines. In addition, the evolutionary transition to the state with eyelines and these foraging behaviors was more likely to occur than the reverse transition. Furthermore, as expected by the fact that the direction of travel is upwardly deviated from the direction of the bills during skimming, the eyeline angle from bills was evolutionarily positively associated with the occurrence of skimming behavior. To my knowledge, the current study is the first to demonstrate that the direction of the eyeline matters, thereby strongly supporting the sight-line hypothesis.

High and low tides occur twice a day (every [~]12.4 hours), with the largest tidal ranges during spring tides around new and full moons (every [~]14.765 days). While these lunar cycles are known to influence many animal phenotypes, particularly the reproduction of coastal animals, the genetic basis of lunar-related rhythms remains unclear. Since phenotypic variation is a valuable resource for elucidating such mechanisms, we examined geographic variation in the lunar-regulated mass spawning of the grass puffer (Takifugu alboplumbeus) along the Japanese coast. We found that western populations spawn during the first half of the spring tides, whereas eastern populations spawn during the second half. Furthermore, although spawning typically occurs a few hours before high tide, this timing is restricted to a specific time window that is earlier in the western populations than in the eastern ones. Behavioral analysis of larvae also revealed a shorter free-running circadian period ({tau}) in the western population than in the eastern ones. As differences in {tau} affect individual variation in the timing of physiological functions and behaviors, we hypothesized that differences in {tau} could account for the different time windows and consequently the observed difference in spawning days. Population genomics analysis identified proline-rich transmembrane protein 1-like (prrt1l) as a candidate gene. Expression of prrt1l was observed in the circadian pacemaker suprachiasmatic nucleus, and triple CRISPR F0 knockout of prrt1l shortened the free-running period in larvae. These findings suggest a potential mechanism underlying the geographic variation in lunar-synchronized spawning behavior. HighlightsO_LIThe geographic variation exists in the lunar-regulated spawning of the grass puffer, with differences in spawning dates and times between western and eastern Japan. C_LIO_LIThe free-running period of western populations is shorter than that of eastern populations, which is consistent with their earlier spawning timing. C_LIO_LIPopulation genomics analysis identified prrt1l as a candidate gene harboring population-specific missense mutations, the knockout of which shortens the free-running period. C_LI

The evolution of body shape reflects the interplay between functional constraints and habitat structure. In fishes, cave environments are well known for promoting regressive traits such as eye and pigment loss, yet their influence on overall body form remains poorly understood. Here, we examine patterns of body shape variation in cave- and surface-dwelling trichomycterid catfishes from northeastern Colombia to assess whether consistent associations exist between habitat type and morphology. Using geometric morphometric analyses, we quantified differences in body shape among species inhabiting subterranean and surface environments. Our results reveal significant habitat-associated differentiation in body shape along the main axes of morphological variation. Cave-dwelling species exhibit more elongated and fusiform body shapes, whereas surface-dwelling species tend to show deeper and more robust morphologies. In a functional context, these contrasting body patterns suggest associations with differing locomotor demands imposed by subterranean versus surface habitats. Although we do not explicitly test convergence or performance, the recurrence of similar body shapes among species from different clades occupying comparable habitats is consistent with repeated morphological responses to shared ecological constraints. Research HighligthsO_LIMultivariate shape analyses reveal significant habitat-associated variation in trichomycterid fishes. Recurrent morphological patterns suggest repeated responses potentially mediated by habitat constraints. C_LIO_LIBody shape differs consistently between cave- and surface-dwelling trichomycterids. Cave species exhibit more elongated and fusiform forms, whereas surface species display deeper body configurations. C_LI

Honeypot ants represent an example of convergent evolution, where a group of workers specialized in storing liquid food in their crops (i.e., stomach) has independently evolved multiple times across different ant genera. While seasonal resource scarcity and arid conditions are thought to drive the evolution of repletism, the role of environmental variables in this process has not been tested. With this is mind, species ensemble models were computed to assess suitability and richness areas, and the importance of predictors. Predictor importance was compared between genera and groups occupying a similar geographical area. Niche overlap and similarity between honeypot ant species were also evaluated to determine whether they occupy similar environmental spaces. Similarity was mainly found within genera, and Leptomyrmex and Myrmecocystus showed striking niche differences. Overall, Leptomyrmex distribution was mainly influenced by atmospheric bioclimatic variables like precipitation and temperature, while Myrmecocystus had soil bioclimatic variables as the most important predictors for their current distribution. Our results indicate that honeypot ants species currently do not occupy the same environmental space, and are not experiencing the same contemporary environmental stressors. While our results suggest that contemporary environmental factors cannot explain the convergence of honeypot ants, future research will examine past climatic conditions along with investigations into the ant genomes to understand more about the causes and consequences of the convergence.

The diversity of body shapes is one of the most prominent features of phenotypic variation in mammals. Yet, mammalian body shapes are poorly quantified and the underlying components contributing to its diversity as well as its relationship to other components of the skeleton are rarely tested. Here, we use lagomorphs (hares, rabbits and pikas) as a model system to (1) investigate which components of the skeleton contributed the most to body shape diversity, (2) examine the relationships between body shape and relative limb lengths, and (3) test how body size, ecotype, burrowing behavior, and locomotor mode influenced variation in lagomorph body shape and appendicular morphology. We quantified the body shape and functional proxies of the appendicular skeleton in 40 lagomorph species from osteological specimens held at museum collections. Using phylogenetic comparative methods, we found the relative length of the ribs and elongation or shortening of the thoracic and lumbar regions contributed the most to body shape evolution across lagomorphs. Second, we found that only leporids (hares and rabbits) exhibited a significant relationship between limb length and body shape, where more elongate species exhibit relatively shorter forelimbs and hindlimbs. Lastly, we found that models incorporating body size were the best predictors of lagomorph body shape and the majority of the appendicular traits, whereas models incorporating burrowing behavior and locomotor mode were largely poor fits. Broadly, these results indicate that larger lagomorphs tend to exhibit more robust body shapes with longer, more gracile forelimbs, whereas smaller lagomorphs tend to exhibit more elongate body shapes with shorter, more robust forelimbs. Overall, this work contributes to the growing understanding of mammalian body shape evolution and demonstrates the importance of not omitting body size in ecomorphological analyses.

Genetically identical cells grown in the same environment show variation in gene expression known as expression noise. Expression noise can be heritable and impact fitness, making it subject to natural selection. Increasing expression noise for the Saccharomyces cerevisiae TDH3 gene was shown to be beneficial in glucose-based media when mean TDH3 expression was far from the fitness optimum but deleterious when it was close to this optimum. Here, we show that growth on different carbon sources alters the effects of new mutations on TDH3 expression noise and examine the fitness effects of changing expression noise. In galactose-based media, we observed the same relationship between expression noise and fitness seen in glucose-based media, but in glycerol- and ethanol-based media, we observed the opposite relationship or no significant relationship, respectively. Using simulations of single-cell organisms, we found that these differences were most likely explained by environment-specific relationships between gene expression and fitness. We also found that, far from the optimum, the fitness effects of noise were greatest when expression was highly heritable between mother and daughter cells. The empirical observations and simulations reported in this study show how environments influence both the production of expression noise and its impacts on fitness.

Morphological trait datasets and phylogenies are routinely paired to investigate macroevolutionary patterns during disparity analyses. However, incomplete fossil sampling can distort disparity estimates, obscuring true evolutionary signals. Ancestral state estimation can be used for both continuous and discrete traits to extend these analyses beyond incomplete fossil data, such as investigations into disparity through time. However, when ancestral state estimation occurs in the disparity pipeline, and the inevitable uncertainty in these estimates, complicate their integration. Determining the most robust workflow for integrating ancestral state estimation in disparity analyses remains a critical methodological challenge. Using simulations to attain a ground-truth disparity value, we evaluated different approaches to performing ancestral state estimation and incorporating uncertainty across varying continuous and discrete trait models, fossil sampling densities and disparity metrics. Ancestral state estimation generally improved recovery of true disparity relative to tip-only analyses, though the optimal approach depended on the interaction between trait model and fossil sampling density. For continuous traits, probabilistic approaches were most accurate, but were sensitive to model misspecification under low fossil sampling density. For discrete traits, pre-ordination methods were most reliable and probabilistic approaches outperformed point estimates under low sampling, while point estimates became increasingly accurate as sampling density increased. Fossil sampling density was a stronger predictor of disparity accuracy than estimation method choice, underscoring that methodologies are only as powerful as the data provided. Our findings offer a practical decision framework for selecting the most appropriate workflow given the sampling density and trait characteristics of a dataset.

Diatoms exhibit remarkable diversity in valve morphology, with the raphe system being a fundamental feature in classification of raphid pennate diatoms. The repeated loss of one of the two raphes during evolution has led to multiple independent origins of monoraphid diatoms. The phylogenetic affinities of the monoraphid genus Crenotia A. Z. Wojtal, erected from Achnanthidium thermale Rabenhorst, have not yet been clarified with molecular data. In this study, natural populations of Crenotia and monoclonal strains derived from them were examined using morphological observations and multilocus phylogenetic analyses based on nuclear and plastidial molecular markers. Three species of the genus Crenotia form a well-supported clade placed within a subgroup of monoraphid genera, which are closely related to Cymbellales D.G. Mann and other biraphid diatoms. This study establishes the first molecular framework for representatives of the genus Crenotia, demonstrating their monophyly and congruent interspecific relationships recovered with multiple molecular markers. The low intraspecific sequence variability and substantial interspecific divergence, together with clear morphological and ecological differentiation, support the recognition of the three investigated species.

Silk glands have been found in two groups of amphipods: the Corophiida and the Ampeliscidae. The silk glands in Ampeliscidae, however, have yet to be examined in detail. Here we report, for the first time, the morphology and distribution of pereopodal glands in the Ampeliscidae, in non-thread producing Synopiidae, and in the Paragammaropsidae. In the Ampeliscidae we found two gland types distributed throughout all pereopods which have the ability to create threads. Pereopods three and four have additional silk extrusion morphology at the tip of the dactylus in which silk is transformed into semi-cylindrical threads used for building domiciles. Synopiid outgroup species have one of the gland types but lack silk extrusion morphology. Using ancestral state reconstruction analysis, we find that glands in the Synopiidae are likely ancestral and hypothesize that silk glands in Ampeliscidae are derived from these ancestral glands. Silk-spinning pereopods in the Paragammaropsidae had similarities with both Corophiida and Ampeliscidae but had distinctions. Ampeliscidae silk-spinning systems bear surprising resemblance to the Corophiida which presents one to reconsider the taxonomic placement of Ampeliscidae and the origins of silk-spinning in amphipods. This is the first comprehensive study on the glandular systems of Ampeliscidae, Synopiidae, and Paragammaropsidae using advanced microscopy, providing pertinent morphological data to the study of arthropod silk gland evolution and complex traits.

Sexual selection arises from individual differences in reproductive success, which can drive the maintenance of genetic polymorphisms in genes subject to balancing selection by the pleiotropic effects that trade-off between survival and reproduction. However, the extent to which sexual selection maintains genetic polymorphisms in wild populations remains unclear. Here, we explored on genomic signatures of balancing selection and selective sweep in the northern medaka, Oryzias sakaizumii in Japan by performing whole-genome resequencing of wild individuals. In addition, we re-evaluated the population genetic structure and admixture of Oryzias latipes and O. sakaizumii across the Japanese archipelago and detected genomic regions affected by introgression. Regions with signatures of selection from multiple statistics were located on eleven chromosomes. In particular, a region spanning 4.25 to 6.80 Mb on chromosome 18 showed high genetic diversity that could not be explained by sex differentiation or introgression from O. latipes in Eastern Japan. This pattern suggests that balancing selection maintains genetic polymorphisms in O. sakaizumii. Specifically, because a previously reported quantitative trait locus associated with female mating behavior overlaps with this region, we infer that sexual selection contributes to the maintenance of genetic polymorphism at this locus.

BackgroundAlpha satellites, a superfamily of AT-rich tandem repeats, are the primary DNA component of centromeres in Platyrrhini and Catarrhini. Analyses of the human genome suggest that centromeres behave like biological ridges, with new alpha satellite families expanding at the centromere core, splitting and displacing older ones towards the pericentromeres. The Cercopithecini tribe, which displays an unusual chromosomal evolution involving multiple chromosomal fissions and centromere formations, represents a promising model to enhance our understanding of alpha satellite DNA evolutionary history. We previously applied targeted sequencing to centromere DNA from two distant species drawn from the Cercopithecini terrestrial and arboreal lineages, and characterized six alpha satellite families exhibiting varying mean sequence identities. MethodsCombining classical and molecular cytogenetics, we mapped the chromosomal distribution of these alpha satellite families across 13 Cercopithecini, one Papionini, and one Colobinae species. A nuclear marker-based phylogeny provided an evolutionary framework for interpretation. ResultsOur phylogeny identifies the terrestrial and arboreal lineages, and a newly designated swamp clade. We observed significant interspecies variations in alpha satellite patterns, including differences in presence/absence and distinct chromosomal distribution patterns (centromeric, pericentromeric, or subtelomeric). Families previously described as heterogeneous (83-87% mean sequence identity) exhibit a centromeric position in the swamp lineage, which is characterized by conserved karyotypes. In contrast, these families show a pericentromeric distribution in the terrestrial and arboreal lineages, replaced at the centromere core by more homogeneous families (95-98% mean sequence identity). In the arboreal clade, which is characterized by highly fissioned karyotypes, putative evolutionary new centromeres show a unique co-occurrence of highly homogeneous and heterogeneous families. Conclusion & ImplicationsWe propose a comprehensive evolutionary scenario for alpha satellite DNA in Cercopithecini, where younger families arise at the centromere core, shift toward the pericentromeres as they age, and eventually face extinction. Our study suggests that alpha satellite DNA and chromosomes evolve in an interdependent manner, with satellite diversification and displacement occurring in parallel with chromosome fissions and centromere repositioning. This comparative cytogenomic approach provides both support for the human-based evolutionary model for alpha satellite DNA and novel temporal insights into its diversification dynamics. Beyond evolutionary genomics, our findings highlight the potential of alpha satellite DNA to complement systematic studies in deciphering complex primate evolutionary histories.

Sarita Lake, British Columbia houses a distinctive population of threespine stickleback (Gastrosteus aculeatus L.) with a phenotype characterized by unusually large individuals relative to nearby conspecifics. We tested the hypothesis that members of this population are not isometrically larger but rather exhibit variation in allometric trajectories that reflect changes in developmental timing impacting the developmental-genetic architecture of the phenotype. We used 3D geometric morphometrics to characterize the size and shape of skulls, pectoral girdles and pelvic girdles from a sample of individuals from nearby freshwater and marine populations and compare them to a sample from Sarita Lake. We showed that individuals from the Sarita Lake population are larger in each body region compared to most other populations examined. Further, these individuals have dorsally expanded skulls and relatively robust pelvic armour. We also showed that the relationship between size and shape is differently structured among body regions and is heavily influenced by non-uniform sexually-mediated variation across populations sampled. Our results reflect complex underlying developmental trajectories, and we suggest that the large phenotype observed may be driven by fecundity selection on female size in combination with a limnetic trophic niche and relatively increased predation pressure in Sarita Lake.

Multivariate phylogenetic comparative methods for modelling high-dimensional traits such as 3D shapes or gene expression proBiles have been recently developed. However, these approaches are impractical and almost impossible to use when the number of traits exceeds a few thousands, as they become computationally prohibitive. We overcome these limitations by proposing a new maximum likelihood approach based on the Empirical Bayes framework. This approach takes into account the information of the complete covariances (among species and traits) to infer parameters and compare models of trait evolution for high-dimensional datasets. Through simulations, we demonstrate that the proposed approach can accurately estimate parameters of various trait evolution models, even when the number of traits is ten times larger than the number of lineages; it requires less memory and is at least 10 times faster than currently available approaches. This fast, efBicient framework enabled us to extend the high-dimensional multivariate phylogenetic comparative toolkit by including an Ornstein-Uhlenbeck process with multiple optima to study adaptation to various selective regimes. Applying our approach to the evolution of jaw morphology in relation to dietary adaptation in mammals, we demonstrate morphological convergence in carnivorous and herbivorous lineages. The proposed Empirical Bayes framework, implemented in the R package mvMORPH, enables phylogenetic comparative methods to efBiciently handle high-dimensional datasets and complex models of trait evolution.

We examined the overlap in the genes associated with daily rhythms and with behavioral plasticity in ants. We first investigated the daily rhythms of gene expression in the harvester ant, Pogonomyrmex barbatus, and how the rhythmic genes overlap with others previously shown to be associated with plasticity of foraging behavior. Then, to consider whether the overlap is conserved across ant species, we compared rhythms of gene expression in the diurnal, desert harvester ants with those previously reported for a distantly related nocturnal, subtropical carpenter ant, Camponotus floridanus. First, daily transcriptomes in P. barbatus showed that most genes were expressed in light-dark (LD) and constantly dark (DD) conditions at about the same levels; only 11 genes showed at least a two-fold change in expression. Network analysis identified eleven modules of P. barbatus genes under LD conditions. Of these 11 clusters, modules C1 and C2 seem to be central nodes of the gene expression network, because they are the most highly connected in LD, and show the strongest preservation in DD vs. LD, and contain core clock gene Period. Only one module, C2, showed significant overlap with P. barbatus genes that have 24h-rhythmic expression in both LD and DD. There was significant overlap between modules C1, C2, C10, C11, and P. barbatus genes found previously to be associated with plasticity in the regulation of foraging activity to manage water loss. A comparison of the daily transcriptome of P. barbatus with that of C. floridanus showed significant overlap of 24h-rhythmic genes in LD. Modules C1 and C2 of P. barbatus also overlap with C. floridanus genes previously shown to differ in expression rhythms in nurses and foragers. In combination, these results indicate that genes linking plasticity of the circadian clock and of behavior may be broadly conserved in ants.

Computational workshops are common in evolutionary biology and are used to share discipline-specific tools and skills with researchers. Despite the perceived importance of these workshops, there is no common set of criteria for workshop success, and there are few peer-reviewed studies investigating the efficacy of workshops or assessing the value of particular instructional techniques in this context. Here, we focused on one key element of a successful workshop: its ability to increase participants motivation to use the methods and tools presented during the workshop. We analyzed the goals, perceptions, and future plans of research practitioners engaging in a workshop on phylogenetic methods of historical biogeography using pre- and post-workshop surveys. Overall, the workshop was successful at motivating participants, and survey responses provided insights into participants perceptions of different activities, including "participatory live coding". Apart from this case study, we aim to highlight the importance of developing a common set of workshop goals in collaboration with other workshop stakeholders and the need for specialized, validated tools for assessing the efficacy of computational workshops for researchers.

This investigation aimed at compiling all phylogenetic lineages within and around genus Cyanoboletus. The evolutionary inference obtained from the nuclear ribosomal genes internal transcribed spacer region (ITS) suggests that part of the species currently classified in Cyanoboletus belong in lineages separate from the genus, thus suggesting a narrower boundary that includes only the species that develop a strong staining reaction to touch and to air exposure of the context. The separate lineages are the monotypic Cupreoboletus genus and a few species that do not develop such reaction, which are part of a clade together with genera Cacaoporus and Acyanoboletus, thus broadening the concept of Cacaoporus to encompass all of them. The emerging 3C perspective of Cupreoboletus, Cacaoporus and Cyanoboletus offers a remarkably consistent morphological diagnosis, overcoming the problems of a too broad concept for Cyanoboletus. This work reveals that Boletus neotropicus, B. novae-zelandiae and B. sensibilis belong respectively in Cyanoboletus, Cacaoporus and Lanmaoa, and by studying multigene alignment concatenates it identifies lineages that probably represent undescribed species: at least four in Cacaoporus and at least five in Cyanoboletus. Diagnostic tables and dichotomic keys are presented by geographic region. The present work also includes a study of the phylogenetic position of Neoboletus flavosanguineus, a species once classified in Cyanoboletus. The complexity of assigning species epithets in some lineages is addressed, namely for the boundaries between Cacaoporus instabilis and Ca. fagaceophilus as well as the diversity under the names Cyanoboletus sinopulverulentus and Cy. pulverulentus. The overall picture of evolutionary lineages sets a framework for the choice of reference data that can provide, in future phylogenetic studies that involve the 3C, a balanced and efficient coverage. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/724631v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@7f618corg.highwire.dtl.DTLVardef@dd6a14org.highwire.dtl.DTLVardef@5f7399org.highwire.dtl.DTLVardef@9e7443_HPS_FORMAT_FIGEXP M_FIG C_FIG

Text AbstractIn this study, we characterized the genetic structure and reconstructed the demographic history of cactus wrens (Campylorhynchus brunneicapillus), an endemic species of desert regions of North America, that shows a clear phenotypic and genotypic variation. We evaluated the effects of historical climate change on the structure and population dynamics of desert species using genomic data through genotyping by sequencing (GBS) and applied a population structure analysis (FST and ADMIXTURE), revealing two genetically differentiated groups: one continental and another peninsular in Baja California. Subsequently, we implemented the MSMC2 coalescent model on data divided into autosomal regions and the Z sex chromosome to estimate changes in effective population size (Ne) through evolutionary time. Additionally, we developed ecological niche models (ENMs) projected to the Last Glacial Maximum (LGM), Last Interglacial (LIG), Present times, and Future (2060 - 2080). Results indicate that both populations maintained moderated Nes before the LGM, experienced severe bottlenecks (Ne [~] 102-103), followed by a sustained expansion. However, recovery was limited to the Z chromosome of the peninsular population. These findings reveal how glaciations and interglacials shaped the evolutionary history of desert species and provide genomic evidence of the splitting of C. affinis from C. brunneicapillus. Article summaryThis research examines how climate changes shaped genetic diversity of cactus wrens across North American warm deserts. Using coalescent methods, researchers tracked effective population size changes over 100,000 years, using ecological niche modeling they predicted habitat suitability across climate periods. Results showed that continental and peninsular populations experienced bottlenecks during the Last Glacial Maximum, followed by demographic recovery on warm periods. However, the sex chromosome (Z) revealed male-biased demographic patterns in peninsular populations. Future projections indicated habitat suitability reductions for peninsular populations, highlighting conservation concerns. These findings demonstrate that past climate shaped genetic diversity of cactus wrens.

The connection between female host plant preference and offspring performance is important for understanding how relationships between plants and phytophagous insects have evolved. According to the preference-performance hypothesis, female insects should evolve to oviposit on host plants on which offspring performance is the highest. Here, we examined the preference-performance hypothesis in the northern brown argus (Aricia artaxerxes) butterfly in the province of Uppland, Sweden, by comparing female host plant preference and larval growth between the host plant species wood cranesbill (Geranium sylvaticum) and bloody cranesbill (G. sanguineum). We also investigated if host plant preference in A. artaxerxes was related to the geographic distribution of A. artaxerxes and its host plants in the province Uppland. We found that the A. artaxerxes females, contrary to the preference-performance hypothesis, preferred ovipositing on G. sylvaticum, even though larvae feeding on G. sylvaticum were slightly smaller than those feeding on G. sanguineum. Since G. sylvaticum is more abundant and probably more utilized than G. sanguineum in Uppland, an explanation for this negative preference-performance connection may be that there are advantages associated with utilizing a more common host plant species, even though larvae feeding on this plant show reduced growth rates. Overall, the results show that factors other than offspring performance, such as geographic distribution, may influence female host plant preference in A. artaxerxes.

AO_SCPLOWBSTRACTC_SCPLOWSpecies represent a fundamental unit of biodiversity in evolutionary biology, but the nature of the speciation continuum and inadequate sampling of organisms with broad distributions provide substantial challenges to species delimitation. The Pacific Treefrog complex (Pseudacris regilla sensu lato) is an iconic but systematically poorly understood group of chorus frogs inhabiting a vast portion of western North America. Current studies tentatively recognize three species in this complex (P. hypochondriaca, P. regilla, P. sierra), but disagreement remains among morphological, mitochondrial, and nuclear genetic data. In this study, we used thorough geographic sampling and thousands of nuclear loci to clarify the phylogenetic relationships and divergence history of P. regilla s.l. lineages and recommend a new taxonomic arrangement. We inferred recent divergence with gene flow between P. regilla and P. sierra, topological inconsistencies, and genealogical divergence indices that place P. regilla and P. sierra firmly in the "gray zone" of speciation. Pseudacris hypochondriaca diverged over 0.5 Ma without gene flow until recent secondary contact with the "north" (P. regilla + P. sierra) lineage. Based on inferences from our genomic data and recently published acoustic signal differentiation, we propose a two species taxonomy for this complex, recognizing the "north" lineage as P. regilla. Our study shows how extensive geographic sampling, high-throughput sequencing, and multiple analytical approaches can resolve systematic uncertainties in challenging species complexes.

Understanding how anatomical structures evolve requires disentangling the roles of integration and modularity in shaping morphological variation. The vertebral column, a serially repeated and regionally differentiated structure, provides a powerful system for investigating these processes. Here, we examine how vertebral morphology evolves in relation to whole-body elongation across the adaptive radiation of Lake Malawi cichlid fishes. We tested for evolutionary integration between the precaudal and caudal domains, as well as assessed the contributions of vertebral count, centrum shape, and intervertebral spacing on body elongation. We find strong evolutionary integration between precaudal and caudal vertebral shape, with both vertebral shapes varying along shared axes of multivariate shape change. Despite this, precaudal and caudal vertebral counts evolve independently, indicating a decoupling between the evolution of identity and morphology. Whole-body elongation is significantly associated with coordinated changes in vertebral and rib morphology, including proportional increases in centrum size, posterior displacement of neural and haemal spines, and increased rib curvature. In contrast, centrum elongation and intervertebral spacing do not independently explain body elongation beyond vertebral counts. These results demonstrate that body elongation in cichlids necessitates integrated, multivariate changes in axial morphology. Our findings highlight the importance of morphological integration in facilitating coordinated evolutionary responses in anatomical systems.