Taking it with a grain of salt: tolerance to increasing salinization in Culex pipiens (Diptera: Culicidae) across a low-lying delta.

BACKGROUND: Salinity, exacerbated by rising sea levels, is a critical environmental cue affecting freshwater ecosystems. Predicting ecosystem structure in response to such changes and their implications for the geographical distribution of arthropod disease vectors requires further insights into the plasticity and adaptability of lower trophic level species in freshwater systems. Our study investigated whether populations of the mosquito Culex pipiens, typically considered sensitive to salt, have adapted due to gradual exposure. METHODS: Mesocosm experiments were conducted to evaluate responses in life history traits to increasing levels of salinity in three populations along a gradient perpendicular to the North Sea coast. Salt concentrations up to the brackish-marine transition zone (8 g/l chloride) were used, upon which no survival was expected. To determine how this process affects oviposition, a colonization experiment was performed by exposing the coastal population to the same concentrations. RESULTS: While concentrations up to the currently described median lethal dose (LD50) (4 g/l) were surprisingly favored during egg laying, even the treatment with the highest salt concentration was incidentally colonized. Differences in development rates among populations were observed, but the influence of salinity was evident only at 4 g/l and higher, resulting in only a 1-day delay. Mortality rates were lower than expected, reaching only 20% for coastal and inland populations and 41% for the intermediate population at the highest salinity. Sex ratios remained unaffected across the tested range. CONCLUSIONS: The high tolerance to salinity for all key life history parameters across populations suggests that Cx. pipiens is unlikely to shift its distribution in the foreseeable future, with potential implications for the disease risk of associated pathogens.

Trade-offs in soil microbial functions and soil health in agroecosystems.

Soil microbial communities play pivotal roles in maintaining soil health in agroecosystems. However, how the delivery of multiple microbial functions in agroecosystems is maintained remains poorly understood. This may put us at risk of incurring unexpected trade-offs between soil functions. We elucidate how interactions between soil microbes can lead to trade-offs in the functioning of agricultural soils. Interactions within soil microbial communities can result in not only positive but also neutral and negative relationships among soil functions. Altering soil conditions through soil health-improving agricultural management can alleviate these functional trade-offs by promoting the diversity and interrelationships of soil microbes, which can help to achieve more productive and sustainable agroecosystems.

Fungal communities are passengers in community development of dune ecosystems, while bacteria are not.

An increasing number of studies of above-belowground interactions provide a fundamental basis for our understanding of the coexistence between plant and soil communities. However, we lack empirical evidence to understand the directionality of drivers of plant and soil communities under natural conditions: 'Are soil microorganisms driving plant community functioning or do they adapt to the plant community?' In a field experiment in an early successional dune ecosystem, we manipulated soil communities by adding living (i.e., natural microbial communities) and sterile soil inocula, originating from natural ecosystems, and examined the annual responses of soil and plant communities. The experimental manipulations had a persistent effect on the soil microbial community with divergent impacts for living and sterile soil inocula. The plant community was also affected by soil inoculation, but there was no difference between the impacts of living and sterile inocula. We also observed an increasing convergence of plant and soil microbial composition over time. Our results show that alterations in soil abiotic and biotic conditions have long-term effects on the composition of both plant and soil microbial communities. Importantly, our study provides direct evidence that soil microorganisms are not "drivers" of plant community dynamics. We found that soil fungi and bacteria manifest different community assemblies in response to treatments. Soil fungi act as "passengers," that is, soil microorganisms reflect plant community dynamics but do not alter it, whereas soil bacteria are neither "drivers" nor "passengers" of plant community dynamics in early successional ecosystems. These results are critical for understanding the community assembly of plant and soil microbial communities under natural conditions and are directly relevant for ecosystem management and restoration.

Landscape level associations between birds, mosquitoes and microclimates: possible consequences for disease transmission?

BACKGROUND: Mosquito-borne diseases are on the rise. While climatic factors have been linked to disease occurrences, they do not explain the non-random spatial distribution in disease outbreaks. Landscape-related factors, such as vegetation structure, likely play a crucial but hitherto unquantified role. METHODS: We explored how three critically important factors that are associated with mosquito-borne disease outbreaks: microclimate, mosquito abundance and bird communities, vary at the landscape scale. We compared the co-occurrence of these three factors in two contrasting habitat types (forest versus grassland) across five rural locations in the central part of the Netherlands between June and September 2021. RESULTS: Our results show that forest patches provide a more sheltered microclimate, and a higher overall abundance of birds. When accounting for differences in landscape characteristics, we also observed that the number of mosquitoes was higher in isolated forest patches. CONCLUSIONS: Our findings indicate that, at the landscape scale, variation in tree cover coincides with suitable microclimate and high Culex pipiens and bird abundance. Overall, these factors can help understand the non-random spatial distribution of mosquito-borne disease outbreaks.

Assessing nutrient fate from terrestrial to freshwater systems using a semi-distributed model for the Fuxian Lake Basin, China.

The growing and increasingly intensified agricultural sector exerts major pressures on the environment. Specifically, nitrogen (N) and phosphorus (P) runoff can induce eutrophication in freshwater ecosystems. To formulate environmental strategies for controlling eutrophication, decision-makers commonly consider the importance of pollutant contributors before developing sector-specific environmental policies. These types of science-based decisions benefit from nutrient models that quantify nutrient transport and fate. However, due to a lack of fertilizer application data, distributed models are generally not suitable for most rural regions with extensive agriculture, while lumped models cannot properly characterize the spatial variation of nutrient fate in these regions. To assess the nutrient contributions from different emission sources to freshwater, we developed a localized semi-distributed model to simulate total nitrogen (TN) and total phosphorus (TP) in 52 inflow rivers of Fuxian Lake Basin in China. The results show that diffuse sources contributed 82 % TN and 92 % TP loading to the inflow rivers. The highest eutrophication potentials (i.e., loading per area) is from the built environment, which is more than 10 times that of forests, but the contribution of the built environment to total diffuse loading is only the second-highest as it occupies 8.7 % of the surface area. Farmland is the main contributor, generating 49 % of diffuse TN and 57 % TP, respectively. Our results show that promoting a 10 % increase in nutrient use efficiency would reduce 5 % of N and 30 % of P diffuse loadings to the rivers. Through examining the impact of nutrient use efficiency, we emphasize the potential trade-offs between food productivity and environmental effects. This analysis workflow can be applied to other agricultural regions.

Distribution of Culex pipiens life stages across urban green and grey spaces in Leiden, The Netherlands.

BACKGROUND: There is an urgent need for cities to become more climate resilient; one of the key strategies is to include more green spaces in the urban environment. Currently, there is a worry that increasing green spaces might increase mosquito nuisance. As such, this study explores a comprehensive understanding of how mosquitoes utilise contrasting grey and green habitats at different life stages and which environmental factors could drive these distributions. METHODS: We used a setup of six paired locations, park (green) vs. residential (grey) areas in a single model city (Leiden, The Netherlands), where we sampled the abundances of different mosquito life stages (eggs, larvae, adults) and the local microclimatic conditions. In this study, we focused on Culex pipiens s.l., which is the most common and abundant mosquito species in The Netherlands. RESULTS: Our results show that while Cx. pipiens ovipositioning rates (number of egg rafts) and larval life stages were far more abundant in residential areas, adults were more abundant in parks. These results coincide with differences in the number of suitable larval habitats (higher in residential areas) and differences in microclimatic conditions (more amenable in parks). CONCLUSIONS: These findings suggest that Cx. pipiens dispersal may be considerably more important than previously thought, where adult Cx. pipiens seek out the most suitable habitat for survival and breeding success. Our findings can inform more targeted and efficient strategies to mitigate and reduce mosquito nuisance while urban green spaces are increased, which make cities more climate resilient.

Global regionalized characterization factors for phosphorus and nitrogen impacts on freshwater fish biodiversity.

Inefficient global nutrient (i.e., phosphorus (P) and nitrogen (N)) management leads to an increase in nutrient delivery to freshwater and coastal ecosystems and induces eutrophication in these aquatic environments. This process threatens the various species inhabiting these ecosystems. In this study, we developed regionalized characterization factors (CFs) for freshwater eutrophication at 0.5 × 0.5-degree resolution, considering different fates for direct emissions to freshwater, diffuse emissions, and increased erosion due to agricultural land use. The CFs were provided for global and regional species loss of freshwater fish. CFs for global species loss were quantified by integrating global extinction probabilities. Results showed that the CFs for P and N impacts on freshwater fish are higher in densely populated regions that encompass either large lakes or the headwaters of large rivers. Focusing on nutrient-limited areas increases country-level CFs in 51.9 % of the countries for P and 49.5 % of the countries for N compared to not considering nutrient limitation. This study highlights the relevance of considering freshwater eutrophication impacts via both P and N emissions and identifying the limiting nutrient when performing life cycle impact assessments.

Interactive effects of climate, land use and soil type on Culex pipiens/torrentium abundance.

The incidence and risk of mosquito-borne disease outbreaks in Northwestern Europe has increased over the last few decades. Understanding the underlying environmental drivers of mosquito population dynamics helps to adequately assess mosquito-borne disease risk. While previous studies have focussed primarily on the effects of climatic conditions (i.e., temperature and precipitation) and/or local environmental conditions individually, it remains unclear how climatic conditions interact with local environmental factors such as land use and soil type, and how these subsequently affect mosquito abundance. Here, we set out to study the interactive effects of land use, soil type and climatic conditions on the abundance of Culex pipiens/torrentium, highly abundant vectors of West Nile virus and Usutu virus. Mosquitoes were sampled at 14 sites throughout the Netherlands. At each site, weekly mosquito collections were carried out between early July and mid-October 2020 and 2021. To assess the effect of the aforementioned environmental factors, we performed a series of generalized linear mixed models and non-parametric statistical tests. Our results show that mosquito abundance and species richness consistently differ among land use- and soil types, with peri-urban areas with peat/clay soils having the highest Cx. pipiens/torrentium abundance and sandy rural areas having the lowest. Furthermore, we observed differences in precipitation-mediated effects on Cx. pipiens/torrentium abundance between (peri-)urban and other land uses and soil types. In contrast, effects of temperature on Cx. pipiens/torrentium abundance remain similar between different land use and soil types. Our study highlights the importance of both land use and soil type in conjunction with climatic conditions for understanding mosquito abundances. Particularly in relation to rainfall events, land use and soil type has a marked effect on mosquito abundance. These findings underscore the importance of local environmental parameters for studies focusing on predicting or mitigating disease risk.

Leaf-level coordination principles propagate to the ecosystem scale.

Fundamental axes of variation in plant traits result from trade-offs between costs and benefits of resource-use strategies at the leaf scale. However, it is unclear whether similar trade-offs propagate to the ecosystem level. Here, we test whether trait correlation patterns predicted by three well-known leaf- and plant-level coordination theories - the leaf economics spectrum, the global spectrum of plant form and function, and the least-cost hypothesis - are also observed between community mean traits and ecosystem processes. We combined ecosystem functional properties from FLUXNET sites, vegetation properties, and community mean plant traits into three corresponding principal component analyses. We find that the leaf economics spectrum (90 sites), the global spectrum of plant form and function (89 sites), and the least-cost hypothesis (82 sites) all propagate at the ecosystem level. However, we also find evidence of additional scale-emergent properties. Evaluating the coordination of ecosystem functional properties may aid the development of more realistic global dynamic vegetation models with critical empirical data, reducing the uncertainty of climate change projections.

DNA-based seed intake quantification for enhanced ecological risk assessment of small mammals.

To prevent the non-acceptable effects of agrochemicals on arable fields, Environmental Risk Assessment (ERA) aims to assess and protect against a wide range of risks due to stressors to non-target species. While exposure to stress is a key factor in ERA models, exposure values are difficult to obtain and rely on laboratory studies with often debatable relevance to field situations. To improve intake estimates, data from realistic field-based scenarios are needed. We developed calibration curves relating known seed numbers of up to 20 onion and carrot seeds consumed by wild-caught wood mice (Apodemus sylvaticus) to the seed DNA content in the feces. Based on these inferred quantitative relationships, a field trial was run to determine seed intake in a natural setting using realistic levels of seed spillage. Onion DNA was detected in the fecal samples of the wood mice caught in the field, which resembled a seed intake of up to 1 onion seed. No intake of carrot seeds was detected. This is the first-ever study to quantify seed intake in a realistic field scenario using a DNA-based analysis, showing that accurate seed intake estimates can be obtained. Our approach can help to improve risk assessment models through its minimally-invasive and accurate assessment of seed intake by ERA representative and non-target species, which would otherwise be undetectable with traditional methods. Our novel approach and its results are highly relevant to studies of food intake and diet composition for basic and applied research alike.

Effects of Nitrogen Emissions on Fish Species Richness across the World's Freshwater Ecoregions.

The increasing application of synthetic fertilizer has tripled nitrogen (N) inputs over the 20th century. N enrichment decreases water quality and threatens aquatic species such as fish through eutrophication and toxicity. However, the impacts of N on freshwater ecosystems are typically neglected in life cycle assessment (LCA). Due to the variety of environmental conditions and species compositions, the response of species to N emissions differs among ecoregions, requiring a regionalized effect assessment. Our study tackled this issue by establishing regionalized species sensitivity distributions (SSDs) of freshwater fish against N concentrations for 367 ecoregions and 48 combinations of realms and major habitat types globally. Subsequently, effect factors (EFs) were derived for LCA to assess the effects of N on fish species richness at a 0.5 degree × 0.5 degree resolution. Results show good SSD fits for all of the ecoregions that contain sufficient data and similar patterns for average and marginal EFs. The SSDs highlight strong effects on species richness due to high N concentrations in the tropical zone and the vulnerability of cold regions. Our study revealed the regional differences in sensitivities of freshwater ecosystems against N content in great spatial detail and can be used to assess more precisely and comprehensively nutrient-induced impacts in LCA.

Climate change threats to the global functional diversity of freshwater fish.

Climate change impacts on freshwater ecosystems and freshwater biodiversity show strong spatial variability, highlighting the importance of a global perspective. While previous studies on biodiversity mostly focused on species richness, functional diversity, which is a better predictor of ecosystem functioning, has received much less attention. This study aims to comprehensively assess climate change threats to the functional diversity of freshwater fish across the world, considering three complementary metrics-functional richness, evenness and divergence. We built on existing spatially explicit projections of geographical ranges for 11,425 riverine fish species as affected by changes in streamflow and water temperature extremes at four warming levels (1.5°C, 2.0°C, 3.2°C and 4.5°C). To estimate functional diversity, we considered the following four continuous, morphological and physiological traits: relative head length, relative body depth, trophic level and relative growth rate. Together, these traits cover five ecological functions. We treated missing trait values in two different ways: we either removed species with missing trait values or imputed them. Depending on the warming level, 6%-25% of the locations globally face a complete loss of functional diversity when assuming no dispersal (6%-17% when assuming maximal dispersal), with hotspots in the Amazon and Paraná River basins. The three facets of functional diversity do not always follow the same pattern. Sometimes, functional richness is not yet affected despite species loss, while functional evenness and divergence are already reducing. Other times, functional richness reduces, while functional evenness and/or divergence increase instead. The contrasting patterns of the three facets of functional diversity show their complementarity among each other and their added value compared to species richness. With increasing climate change, impacts on freshwater communities accelerate, making early mitigation critically important.

Antibiotic Resistance Genes in Interconnected Surface Waters as Affected by Agricultural Activities.

Pastures have become one of the most important sources of antibiotic resistance genes (ARGs) pollution, bringing risks to human health through the environment and the food that is grown there. Another significant source of food production is greenhouse horticulture, which is typically located near pastures. Through waterways, pasture-originated ARGs may transfer to the food in greenhouses. However, how these pasture-originated ARGs spread to nearby waterways and greenhouses has been much less investigated, while this may pose risks to humans through agricultural products. We analyzed 29 ARGs related to the most used antibiotics in livestock in the Netherlands at 16 locations in an agricultural area, representing pastures, greenhouses and lakes. We found that ARGs were prevalent in all surface waters surrounding pastures and greenhouses and showed a similar composition, with sulfonamide ARGs being dominant. This indicates that both pastures and greenhouses cause antibiotic resistance pressures on neighboring waters. However, lower pressures were found in relatively larger and isolated lakes, suggesting that a larger water body or a non-agricultural green buffer zone could help reducing ARG impacts from agricultural areas. We also observed a positive relationship between the concentrations of the class 1 integron (intl1 gene)-used as a proxy for horizontal gene transfer-and ARG concentration and composition. This supports that horizontal gene transfer might play a role in dispersing ARGs through landscapes. In contrast, none of the measured four abiotic factors (phosphate, nitrate, pH and dissolved oxygen) showed any impact on ARG concentrations. ARGs from different classes co-occurred, suggesting simultaneous use of different antibiotics. Our findings help to understand the spatial patterns of ARGs, specifically the impacts of ARGs from pastures and greenhouses on each other and on nearby waterways. In this way, this study guides management aiming at reducing ARGs' risk to human health from agricultural products.

Genotyping by sequencing for estimating relative abundances of diatom taxa in mock communities.

BACKGROUND: Diatoms are present in all waters and are highly sensitive to pollution gradients. Therefore, they are ideal bioindicators for water quality assessment. Current indices used in these applications are based on identifying diatom species and counting their abundances using traditional light microscopy. Several molecular techniques have been developed to help automate different steps of this process, but obtaining reliable estimates of diatom community composition and species abundance remains challenging. RESULTS: Here, we evaluated a recently developed quantification method based on Genotyping by Sequencing (GBS) for the first time in diatoms to estimate the relative abundances within a species complex. For this purpose, a reference database comprised of thousands of genomic DNA clusters was generated from cultures of Nitzschia palea. The sequencing reads from calibration and mock samples were mapped against this database for parallel quantification. We sequenced 25 mock diatom communities containing up to five taxa per sample in different abundances. Taxon abundances in these communities were also quantified by a diatom expert using manual counting of cells on light microscopic slides. The relative abundances of strains across mock samples were over- or under-estimated by the manual counting method, and a majority of mock samples had stronger correlations using GBS. Moreover, one previously recognized putative hybrid had the largest number of false positive detections demonstrating the limitation of the manual counting method when morphologically similar and/or phylogenetically close taxa are analyzed. CONCLUSIONS: Our results suggest that GBS is a reliable method to estimate the relative abundances of the N. palea taxa analyzed in this study and outperformed traditional light microscopy in terms of accuracy. GBS provides increased taxonomic resolution compared to currently available quantitative molecular approaches, and it is more scalable in the number of species that can be analyzed in a single run. Hence, this is a significant step forward in developing automated, high-throughput molecular methods specifically designed for the quantification of [diatom] communities for freshwater quality assessments.

Characterization Factors to Assess Land Use Impacts on Pollinator Abundance in Life Cycle Assessment.

While wild pollinators play a key role in global food production, their assessment is currently missing from the most commonly used environmental impact assessment method, Life Cycle Assessment (LCA). This is mainly due to constraints in data availability and compatibility with LCA inventories. To target this gap, relative pollinator abundance estimates were obtained with the use of a Delphi assessment, during which 25 experts, covering 16 nationalities and 45 countries of expertise, provided scores for low, typical, and high expected abundance associated with 24 land use categories. Based on these estimates, this study presents a set of globally generic characterization factors (CFs) that allows translating land use into relative impacts to wild pollinator abundance. The associated uncertainty of the CFs is presented along with an illustrative case to demonstrate the applicability in LCA studies. The CFs based on estimates that reached consensus during the Delphi assessment are recommended as readily applicable and allow key differences among land use types to be distinguished. The resulting CFs are proposed as the first step for incorporating pollinator impacts in LCA studies, exemplifying the use of expert elicitation methods as a useful tool to fill data gaps that constrain the characterization of key environmental impacts.

Fine-resolution global maps of root biomass carbon colonized by arbuscular and ectomycorrhizal fungi.

Despite the recognized importance of mycorrhizal associations in ecosystem functioning, the actual abundance patterns of mycorrhizal fungi belowground are still unknown. This information is key for better quantification of mycorrhizal impacts on ecosystem processes and for incorporating mycorrhizal pathways into global biogeochemical models. Here we present the first high-resolution maps of fine root stocks colonized by arbuscular mycorrhizal (AM) and ectomycorrhizal (EcM) fungi (MgC ha-1). The maps were assembled by combining multiple open-source databases holding information on root biomass carbon, the proportion of AM and EcM tree biomass, plot-level relative abundance of plant species and intensity of AM and EcM root colonization. We calculated root-associated AM and EcM abundance in 881 spatial units, defined as the combination of ecoregions and land cover types across six continents. The highest AM abundances are observed in the (sub-)tropics, while the highest EcM abundances occur in the taiga regions. These maps serve as a basis for future research where continuous spatial estimates of root mycorrhizal stocks are needed.

Environmental DNA methylation of Lymnaea stagnalis varies with age and is hypermethylated compared to tissue DNA.

Environmental DNA (eDNA) approaches contributing to species identifications are quickly becoming the new norm in biomonitoring and ecosystem assessments. Yet, information such as age and health state of the population, which is vital to species biomonitoring, has not been accessible from eDNA. DNA methylation has the potential to provide such information on the state of a population. Here, we measured the methylation of eDNA along with tissue DNA (tDNA) of Lymnaea stagnalis at four life stages. We demonstrate that eDNA methylation varies with age and allows distinguishing among age classes. Moreover, eDNA was globally hypermethylated in comparison to tDNA. This difference was age-specific and connected to a limited number of eDNA sites. This differential methylation pattern suggests that eDNA release with age is partially regulated through DNA methylation. Our findings help to understand mechanisms involved in eDNA release and shows the potential of eDNA methylation analysis to assess age classes. Such age class assessments will encourage future eDNA studies to assess fundamental processes of population dynamics and functioning in ecology, biodiversity conservation and impact assessments.

Global patterns of vascular plant alpha diversity.

Global patterns of regional (gamma) plant diversity are relatively well known, but whether these patterns hold for local communities, and the dependence on spatial grain, remain controversial. Using data on 170,272 georeferenced local plant assemblages, we created global maps of alpha diversity (local species richness) for vascular plants at three different spatial grains, for forests and non-forests. We show that alpha diversity is consistently high across grains in some regions (for example, Andean-Amazonian foothills), but regional 'scaling anomalies' (deviations from the positive correlation) exist elsewhere, particularly in Eurasian temperate forests with disproportionally higher fine-grained richness and many African tropical forests with disproportionally higher coarse-grained richness. The influence of different climatic, topographic and biogeographical variables on alpha diversity also varies across grains. Our multi-grain maps return a nuanced understanding of vascular plant biodiversity patterns that complements classic maps of biodiversity hotspots and will improve predictions of global change effects on biodiversity.

A Review on Biotechnological Approaches Applied for Marine Hydrocarbon Spills Remediation.

The increasing demand for petroleum products generates needs for innovative and reliable methods for cleaning up crude oil spills. Annually, several oil spills occur around the world, which brings numerous ecological and environmental disasters on the surface of deep seawaters like oceans. Biological and physico-chemical remediation technologies can be efficient in terms of spill cleanup and microorganisms-mainly bacteria-are the main ones responsible for petroleum hydrocarbons (PHCs) degradation such as crude oil. Currently, biodegradation is considered as one of the most sustainable and efficient techniques for the removal of PHCs. However, environmental factors associated with the functioning and performance of microorganisms involved in hydrocarbon-degradation have remained relatively unclear. This has limited our understanding on how to select and inoculate microorganisms within technologies of cleaning and to optimize physico-chemical remediation and degradation methods. This review article presents the latest discoveries in bioremediation techniques such as biostimulation, bioaugmentation, and biosurfactants as well as immobilization strategies for increasing the efficiency. Besides, environmental affecting factors and microbial strains engaged in bioremediation and biodegradation of PHCs in marines are discussed.

Phylotranscriptomics reveals the reticulate evolutionary history of a widespread diatom species complex.

In contrast to surveys based on a few genes that often provide limited taxonomic resolution, transcriptomes provide a wealth of genomic loci that can resolve relationships among taxonomically challenging lineages. Diatoms are a diverse group of aquatic microalgae that includes important bioindicator species and many such lineages. One example is Nitzschia palea, a widespread species complex with several morphologically defined taxonomic varieties, some of which are critical pollution indicators. Morphological differences among the varieties are subtle and phylogenetic studies based on a few genes fail to resolve their evolutionary relationships. We conducted morphometric and transcriptome analyses of 10 Nitzschia palea strains to resolve the relationships among strains and taxonomic varieties. Nitzschia palea was resolved into three clades, one of which corresponds to a group of strains with narrow linear-lanceolate valves. The other morphological group recovered in the shape outline analysis was not monophyletic and consisted of two clades. Gene-tree concordance analyses and phylogenetic network estimations revealed patterns of incomplete lineage sorting and gene flow between intraspecific lineages. We detected reticulated evolutionary patterns among lineages with different morphologies, resulting in a putative recent hybrid. Our study shows that phylogenomic analyses of unlinked nuclear loci, complemented with morphometrics, can resolve complex evolutionary histories of recently diverged species complexes.