Showing papers by "Swiss Federal Institute of Aquatic Science and Technology published in 2017"

Environmental DNA metabarcoding: transforming how we survey animal and plant communities

Abstract: The genomic revolution has fundamentally changed how we survey biodiversity on earth. High-throughput sequencing ("HTS") platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed "environmental DNA" or "eDNA"). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called "eDNA metabarcoding" and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education.

Ancient hybridization fuels rapid cichlid fish adaptive radiations.

Abstract: Understanding why some evolutionary lineages generate exceptionally high species diversity is an important goal in evolutionary biology. Haplochromine cichlid fishes of Africa’s Lake Victoria region encompass >700 diverse species that all evolved in the last 150,000 years. How this ‘Lake Victoria Region Superflock’ could evolve on such rapid timescales is an enduring question. Here, we demonstrate that hybridization between two divergent lineages facilitated this process by providing genetic variation that subsequently became recombined and sorted into many new species. Notably, the hybridization event generated exceptional allelic variation at an opsin gene known to be involved in adaptation and speciation. More generally, differentiation between new species is accentuated around variants that were fixed differences between the parental lineages, and that now appear in many new combinations in the radiation species. We conclude that hybridization between divergent lineages, when coincident with ecological opportunity, may facilitate rapid and extensive adaptive radiation. Cichlids underwent a rapid diversification in the Lake Victoria region, expanding to more than 700 species within 150,000 years. Here, Meier and colleagues show that an ancient hybridization between two divergent cichlid lineages generated high genetic diversity that facilitated the rapid radiation.

Water scarcity assessments in the past, present, and future

Abstract: Water scarcity has become a major constraint to socio-economic development and a threat to livelihood in increasing parts of the world. Since the late 1980s, water scarcity research has attracted much political and public attention. We here review a variety of indicators that have been developed to capture different characteristics of water scarcity. Population, water availability and water use are the key elements of these indicators. Most of the progress made in the last few decades has been on the quantification of water availability and use by applying spatially explicit models. However, challenges remain on appropriate incorporation of green water (soil moisture), water quality, environmental flow requirements, globalization and virtual water trade in water scarcity assessment. Meanwhile, inter- and intra- annual variability of water availability and use also calls for assessing the temporal dimension of water scarcity. It requires concerted efforts of hydrologists, economists, social scientists, and environmental scientists to develop integrated approaches to capture the multi-faceted nature of water scarcity.

Nontarget Screening with High Resolution Mass Spectrometry in the Environment: Ready to Go?

Abstract: The vast, diverse universe of organic pollutants is a formidable challenge for environmental sciences, engineering, and regulation. Nontarget screening (NTS) based on high resolution mass spectrometry (HRMS) has enormous potential to help characterize this universe, but is it ready to go for real world applications? In this Feature article we argue that development of mass spectrometers with increasingly high resolution and novel couplings to both liquid and gas chromatography, combined with the integration of high performance computing, have significantly widened our analytical window and have enabled increasingly sophisticated data processing strategies, indicating a bright future for NTS. NTS has great potential for treatment assessment and pollutant prioritization within regulatory applications, as highlighted here by the case of real-time pollutant monitoring on the River Rhine. We discuss challenges for the future, including the transition from research toward solution-centered and robust, harmonize...

Polyester Textiles as a Source of Microplastics from Households: A Mechanistic Study to Understand Microfiber Release During Washing

Abstract: Microplastic fibers make up a large proportion of microplastics found in the environment, especially in urban areas. There is good reason to consider synthetic textiles a major source of microplastic fibers, and it will not diminish since the use of synthetic fabrics, especially polyester, continues to increase. In this study we provide quantitative data regarding the size and mass of microplastic fibers released from synthetic (polyester) textiles during simulated home washing under controlled laboratory conditions. Consideration of fabric structure and washing conditions (use of detergents, temperature, wash duration, and sequential washings) allowed us to study the propensity of fiber shedding in a mechanistic way. Thousands of individual fibers were measured (number, length) from each wash solution to provide a robust data set on which to draw conclusions. Among all the variables tested, the use of detergent appeared to affect the total mass of fibers released the most, yet the detergent composition (...

Glacier shrinkage driving global changes in downstream systems

Abstract: Glaciers cover ∼10% of the Earth’s land surface, but they are shrinking rapidly across most parts of the world, leading to cascading impacts on downstream systems. Glaciers impart unique footprints on river flow at times when other water sources are low. Changes in river hydrology and morphology caused by climate-induced glacier loss are projected to be the greatest of any hydrological system, with major implications for riverine and near-shore marine environments. Here, we synthesize current evidence of how glacier shrinkage will alter hydrological regimes, sediment transport, and biogeochemical and contaminant fluxes from rivers to oceans. This will profoundly influence the natural environment, including many facets of biodiversity, and the ecosystem services that glacier-fed rivers provide to humans, particularly provision of water for agriculture, hydropower, and consumption. We conclude that human society must plan adaptation and mitigation measures for the full breadth of impacts in all affected regions caused by glacier shrinkage.

Towards a theory of regional diversification : Combining insights from Evolutionary Economic Geography and Transition Studies

Abstract: Towards a theory of regional diversification: combining insights from Evolutionary Economic Geography and Transition Studies. Regional Studies. This paper develops a theoretical framework of regional diversification by combining insights from Evolutionary Economic Geography and Transition Studies. It argues that a theory of regional diversification should not only build on the current understanding of related diversification but also account for processes of unrelated diversification by looking at the role of agency in processes of institutional entrepreneurship, and at enabling and constraining factors at various spatial scales. This paper proposes a typology of four regional diversification trajectories by cross-tabulating related versus unrelated diversification with niche creation versus regime adoption, and it develops a number of propositions.

Solar forcing for CMIP6 (v3.2)

Abstract: This paper describes the recommended solar forcing dataset for CMIP6 and highlights changes with respect to CMIP5. The solar forcing is provided for radiative properties, namely total solar irradiance (TSI), solar spectral irradiance (SSI), and the F10.7 index as well as particle forcing, including geomagnetic indices Ap and Kp, and ionization rates to account for effects of solar protons, electrons, and galactic cosmic rays. This is the first time that a recommendation for solar-driven particle forcing has been provided for a CMIP exercise. The solar forcing datasets are provided at daily and monthly resolution separately for the CMIP6 preindustrial control, historical (1850–2014), and future (2015–2300) simulations. For the preindustrial control simulation, both constant and time-varying solar forcing components are provided, with the latter including variability on 11-year and shorter timescales but no long-term changes. For the future, we provide a realistic scenario of what solar behavior could be, as well as an additional extreme Maunder-minimum-like sensitivity scenario. This paper describes the forcing datasets and also provides detailed recommendations as to their implementation in current climate models. For the historical simulations, the TSI and SSI time series are defined as the average of two solar irradiance models that are adapted to CMIP6 needs: an empirical one (NRLTSI2–NRLSSI2) and a semi-empirical one (SATIRE). A new and lower TSI value is recommended: the contemporary solar-cycle average is now 1361.0 W m−2. The slight negative trend in TSI over the three most recent solar cycles in the CMIP6 dataset leads to only a small global radiative forcing of −0.04 W m−2. In the 200–400 nm wavelength range, which is important for ozone photochemistry, the CMIP6 solar forcing dataset shows a larger solar-cycle variability contribution to TSI than in CMIP5 (50 % compared to 35 %). We compare the climatic effects of the CMIP6 solar forcing dataset to its CMIP5 predecessor by using time-slice experiments of two chemistry–climate models and a reference radiative transfer model. The differences in the long-term mean SSI in the CMIP6 dataset, compared to CMIP5, impact on climatological stratospheric conditions (lower shortwave heating rates of −0.35 K day−1 at the stratopause), cooler stratospheric temperatures (−1.5 K in the upper stratosphere), lower ozone abundances in the lower stratosphere (−3 %), and higher ozone abundances (+1.5 % in the upper stratosphere and lower mesosphere). Between the maximum and minimum phases of the 11-year solar cycle, there is an increase in shortwave heating rates (+0.2 K day−1 at the stratopause), temperatures ( ∼ 1 K at the stratopause), and ozone (+2.5 % in the upper stratosphere) in the tropical upper stratosphere using the CMIP6 forcing dataset. This solar-cycle response is slightly larger, but not statistically significantly different from that for the CMIP5 forcing dataset. CMIP6 models with a well-resolved shortwave radiation scheme are encouraged to prescribe SSI changes and include solar-induced stratospheric ozone variations, in order to better represent solar climate variability compared to models that only prescribe TSI and/or exclude the solar-ozone response. We show that monthly-mean solar-induced ozone variations are implicitly included in the SPARC/CCMI CMIP6 Ozone Database for historical simulations, which is derived from transient chemistry–climate model simulations and has been developed for climate models that do not calculate ozone interactively. CMIP6 models without chemistry that perform a preindustrial control simulation with time-varying solar forcing will need to use a modified version of the SPARC/CCMI Ozone Database that includes solar variability. CMIP6 models with interactive chemistry are also encouraged to use the particle forcing datasets, which will allow the potential long-term effects of particles to be addressed for the first time. The consideration of particle forcing has been shown to significantly improve the representation of reactive nitrogen and ozone variability in the polar middle atmosphere, eventually resulting in further improvements in the representation of solar climate variability in global models.

Handbook of protocols for standardized measurement of terrestrial invertebrate functional traits

Abstract: Summary 1. Trait-based approaches are increasingly being used to test mechanisms underlying species assemblages and biotic interactions across a wide range of organisms including terrestrial arthropods and to investigate consequences for ecosystem processes. Such an approach relies on the standardized measurement of functional traits that can be applied across taxa and regions. Currently, however, unified methods of trait measurements are lacking for terrestrial arthropods and related macroinvertebrates (terrestrial invertebrates hereafter). 2. Here, we present a comprehensive review and detailed protocol for a set of 29 traits known to be sensitive to global stressors and to affect ecosystem processes and services. We give rec- ommendations how to measure these traits under standardized conditions across various ter- restrial invertebrate taxonomic groups. 3. We provide considerations and approaches that apply to almost all traits described, such as the selection of species and individuals needed for the measurements, the importance of intraspecific trait variability, how many populations or communities to sample and over which spatial scales. 4. The approaches outlined here provide a means to improve the reliability and predictive power of functional traits to explain community assembly, species diversity patterns and ecosystem processes and services within and across taxa and trophic levels, allowing compar- ison of studies and running meta-analyses across regions and ecosystems. 5. This handbook is a crucial first step towards standardizing trait methodology across the most studied terrestrial invertebrate groups, and the protocols are aimed to balance general applicability and requirements for special cases or particular taxa. Therefore, we envision this handbook as a common platform to which researchers can further provide methodological input for additional special cases.

Variation in recombination frequency and distribution across eukaryotes: patterns and processes

Abstract: Recombination, the exchange of DNA between maternal and paternal chromosomes during meiosis, is an essential feature of sexual reproduction in nearly all multicellular organisms. While the role of recombination in the evolution of sex has received theoretical and empirical attention, less is known about how recombination rate itself evolves and what influence this has on evolutionary processes within sexually reproducing organisms. Here, we explore the patterns of, and processes governing recombination in eukaryotes. We summarize patterns of variation, integrating current knowledge with an analysis of linkage map data in 353 organisms. We then discuss proximate and ultimate processes governing recombination rate variation and consider how these influence evolutionary processes. Genome-wide recombination rates (cM/Mb) can vary more than tenfold across eukaryotes, and there is large variation in the distribution of recombination events across closely related taxa, populations and individuals. We discuss how variation in rate and distribution relates to genome architecture, genetic and epigenetic mechanisms, sex, environmental perturbations and variable selective pressures. There has been great progress in determining the molecular mechanisms governing recombination, and with the continued development of new modelling and empirical approaches, there is now also great opportunity to further our understanding of how and why recombination rate varies. This article is part of the themed issue ‘Evolutionary causes and consequences of recombination rate variation in sexual organisms’.

High-avidity IgA protects the intestine by enchaining growing bacteria

Abstract: Vaccine-induced high-avidity IgA can protect against bacterial enteropathogens by directly neutralizing virulence factors or by poorly defined mechanisms that physically impede bacterial interactions with the gut tissues ('immune exclusion'). IgA-mediated cross-linking clumps bacteria in the gut lumen and is critical for protection against infection by non-typhoidal Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium). However, classical agglutination, which was thought to drive this process, is efficient only at high pathogen densities (≥108 non-motile bacteria per gram). In typical infections, much lower densities (100-107 colony-forming units per gram) of rapidly dividing bacteria are present in the gut lumen. Here we show that a different physical process drives formation of clumps in vivo: IgA-mediated cross-linking enchains daughter cells, preventing their separation after division, and clumping is therefore dependent on growth. Enchained growth is effective at all realistic pathogen densities, and accelerates pathogen clearance from the gut lumen. Furthermore, IgA enchains plasmid-donor and -recipient clones into separate clumps, impeding conjugative plasmid transfer in vivo. Enchained growth is therefore a mechanism by which IgA can disarm and clear potentially invasive species from the intestinal lumen without requiring high pathogen densities, inflammation or bacterial killing. Furthermore, our results reveal an untapped potential for oral vaccines in combating the spread of antimicrobial resistance.

Absolute quantification of microbial taxon abundances.

Abstract: High-throughput amplicon sequencing has become a well-established approach for microbial community profiling. Correlating shifts in the relative abundances of bacterial taxa with environmental gradients is the goal of many microbiome surveys. As the abundances generated by this technology are semi-quantitative by definition, the observed dynamics may not accurately reflect those of the actual taxon densities. We combined the sequencing approach (16S rRNA gene) with robust single-cell enumeration technologies (flow cytometry) to quantify the absolute taxon abundances. A detailed longitudinal analysis of the absolute abundances resulted in distinct abundance profiles that were less ambiguous and expressed in units that can be directly compared across studies. We further provide evidence that the enrichment of taxa (increase in relative abundance) does not necessarily relate to the outgrowth of taxa (increase in absolute abundance). Our results highlight that both relative and absolute abundances should be considered for a comprehensive biological interpretation of microbiome surveys.

Selenium deficiency risk predicted to increase under future climate change

Abstract: Deficiencies of micronutrients, including essential trace elements, affect up to 3 billion people worldwide. The dietary availability of trace elements is determined largely by their soil concentrations. Until now, the mechanisms governing soil concentrations have been evaluated in small-scale studies, which identify soil physicochemical properties as governing variables. However, global concentrations of trace elements and the factors controlling their distributions are virtually unknown. We used 33,241 soil data points to model recent (1980–1999) global distributions of Selenium (Se), an essential trace element that is required for humans. Worldwide, up to one in seven people have been estimated to have low dietary Se intake. Contrary to small-scale studies, soil Se concentrations were dominated by climate–soil interactions. Using moderate climate-change scenarios for 2080–2099, we predicted that changes in climate and soil organic carbon content will lead to overall decreased soil Se concentrations, particularly in agricultural areas; these decreases could increase the prevalence of Se deficiency. The importance of climate–soil interactions to Se distributions suggests that other trace elements with similar retention mechanisms will be similarly affected by climate change.

Toward a Comprehensive Strategy to Mitigate Dissemination of Environmental Sources of Antibiotic Resistance.

Abstract: Antibiotic resistance is a pervasive global health threat. To combat the spread of resistance, it is necessary to consider all possible sources and understand the pathways and mechanisms by which resistance disseminates. Best management practices are urgently needed to provide barriers to the spread of resistance and maximize the lifespan of antibiotics as a precious resource. Herein we advise upon the need for coordinated national and international strategies, highlighting three essential components: (1) Monitoring, (2) Risk Assessment, and (3) Mitigation of antibiotic resistance. Central to all three components is What exactly to monitor, assess, and mitigate? We address this question within an environmental framework, drawing from fundamental microbial ecological processes driving the spread of resistance.

Extensive arsenic contamination in high-pH unconfined aquifers in the Indus Valley

Abstract: Arsenic-contaminated aquifers are currently estimated to affect ~150 million people around the world. However, the full extent of the problem remains elusive. This is also the case in Pakistan, where previous studies focused on isolated areas. Using a new data set of nearly 1200 groundwater quality samples throughout Pakistan, we have created state-of-the-art hazard and risk maps of arsenic-contaminated groundwater for thresholds of 10 and 50 μg/liter. Logistic regression analysis was used with 1000 iterations, where surface slope, geology, and soil parameters were major predictor variables. The hazard model indicates that much of the Indus Plain is likely to have elevated arsenic concentrations, although the rest of the country is mostly safe. Unlike other arsenic-contaminated areas of Asia, the arsenic release process in the arid Indus Plain appears to be dominated by elevated-pH dissolution, resulting from alkaline topsoil and extensive irrigation of unconfined aquifers, although pockets of reductive dissolution are also present. We estimate that approximately 50 million to 60 million people use groundwater within the area at risk, with hot spots around Lahore and Hyderabad. This number is alarmingly high and demonstrates the urgent need for verification and testing of all drinking water wells in the Indus Plain, followed by appropriate mitigation measures.

Strong impact of anthropogenic contamination on the co-occurrence patterns of a riverine microbial community.

Abstract: Summary Although the health of rivers is threatened by multiple anthropogenic stressors with increasing frequency, it remains an open question how riverine microbial communities respond to emerging micropollutants. Here, by using 16S rDNA amplicon sequencing of 60 water samples collected during different hydrological seasons, we investigated the spatio-temporal variation and the co-occurrence patterns of microbial communities in the anthropogenically impacted Jiulong River in China. The results indicated that the riverine microbial co-occurrence network had a non-random, modular structure, which was mainly shaped by the taxonomic relatedness of co-occurring species. Fecal indicator bacteria may survive for prolonged periods of time in river water, but they formed an independent module which had fewer interactions with typical freshwater bacteria. Multivariate analysis demonstrated that nutrients and micropollutants (i.e. pharmaceuticals and personal care products, PPCPs) exerted combined effects in shaping α- and β-diversity of riverine microbial communities. Remarkably, we showed that a hitherto unrecognized disruptive effect of PPCPs on the abundance variations of central species and module communities was stronger than the influence of physico-chemical factors, suggesting the key role played by micropollutants for the microbial co-occurrence relationships in lotic ecosystems. Overall, our findings provide novel insights into community assembly in aquatic environments experiencing anthropogenic stresses. This article is protected by copyright. All rights reserved.

Treatment technologies for urban solid biowaste to create value products: a review with focus on low- and middle-income settings

Abstract: Treatment of biowaste, the predominant waste fraction in low- and middle-income settings, offers public health, environmental and economic benefits by converting waste into a hygienic product, diverting it from disposal sites, and providing a source of income. This article presents a comprehensive overview of 13 biowaste treatment technologies, grouped into four categories: (1) direct use (direct land application, direct animal feed, direct combustion), (2) biological treatment (composting, vermicomposting, black soldier fly treatment, anaerobic digestion, fermentation), (3) physico-chemical treatment (transesterification, densification), and (4) thermo-chemical treatment (pyrolysis, liquefaction, gasification). Based on a literature review and expert consultation, the main feedstock requirements, process conditions and treatment products are summarized, and the challenges and trends, particularly regarding the applicability of each technology in the urban low- and middle-income context, are critically discussed. An analysis of the scientific articles published from 2005 to 2015 reveals substantial differences in the amount and type of research published for each technology, a fact that can partly be explained with the development stage of the technologies. Overall, publications from case studies and field research seem disproportionately underrepresented for all technologies. One may argue that this reflects the main task of researchers—to conduct fundamental research for enhanced process understanding—but it may also be a result of the traditional embedding of the waste sector in the discipline of engineering science, where socio-economic and management aspects are seldom object of the research. More unbiased, well-structured and reproducible evidence from case studies at scale could foster the knowledge transfer to practitioners and enhance the exchange between academia, policy and practice.

Bridging ecology and conservation: from ecological networks to ecosystem function

Abstract: Summary 1. Current approaches to conservation may be inadequate to maintain ecosystem integrity because they are mostly based on rarity status of organisms rather than functional signifi- cance. Alternatively, approaches focusing on the protection of ecological networks lead to more appropriate conservation targets to maintain ecosystem integrity. 2. We propose that a shift in focus from species to interaction networks is necessary to achieve pressing conservation management and restoration ecology goals of conserving biodi- versity, ecosystem processes and ultimately landscape-scale delivery of ecosystem services. 3. Using topical examples from the literature, we discuss historical and conceptual advances, current challenges and ways to move forward. We also propose a road map to ecological net- work conservation, providing a novel ready to use approach to identify clear conservation targets with flexible data requirements. 4. Synthesis and applications. Integration of how environmental and spatial constraints affect the nature and strength of local interaction networks will improve our ability to predict their response to change and to conserve them. This will better protect species, ecosystem pro- cesses, and the resulting ecosystem services we depend on.

The Potential of Knowing More: A Review of Data-Driven Urban Water Management

Abstract: The promise of collecting and utilizing large amounts of data has never been greater in the history of urban water management (UWM). This paper reviews several data-driven approaches which play a key role in bringing forward a sea change. It critically investigates whether data-driven UWM offers a promising foundation for addressing current challenges and supporting fundamental changes in UWM. We discuss the examples of better rain-data management, urban pluvial flood-risk management and forecasting, drinking water and sewer network operation and management, integrated design and management, increasing water productivity, wastewater-based epidemiology and on-site water and wastewater treatment. The accumulated evidence from literature points toward a future UWM that offers significant potential benefits thanks to increased collection and utilization of data. The findings show that data-driven UWM allows us to develop and apply novel methods, to optimize the efficiency of the current network-based approach...

Biased partitioning of the multidrug efflux pump AcrAB-TolC underlies long-lived phenotypic heterogeneity

Abstract: The molecular mechanisms underlying phenotypic variation in isogenic bacterial populations remain poorly understood. We report that AcrAB-TolC, the main multidrug efflux pump of Escherichia coli, exhibits a strong partitioning bias for old cell poles by a segregation mechanism that is mediated by ternary AcrAB-TolC complex formation. Mother cells inheriting old poles are phenotypically distinct and display increased drug efflux activity relative to daughters. Consequently, we find systematic and long-lived growth differences between mother and daughter cells in the presence of subinhibitory drug concentrations. A simple model for biased partitioning predicts a population structure of long-lived and highly heterogeneous phenotypes. This straightforward mechanism of generating sustained growth rate differences at subinhibitory antibiotic concentrations has implications for understanding the emergence of multidrug resistance in bacteria.

Navigating the Range of Statistical Tools for Inferential Network Analysis

Abstract: The last decade has seen substantial advances in statistical techniques for the analysis of network data, as well as a major increase in the frequency with which these tools are used. These techniques are designed to accomplish the same broad goal, statistically valid inference in the presence of highly interdependent relationships, but important differences remain between them. We review three approaches commonly used for inferential network analysis—the quadratic assignment procedure, exponential random graph models, and latent space network models—highlighting the strengths and weaknesses of the techniques relative to one another. An illustrative example using climate change policy network data shows that all three network models outperform standard logit estimates on multiple criteria. This article introduces political scientists to a class of network techniques beyond simple descriptive measures of network structure, and it helps researchers choose which model to use in their own research.

Transitions between phases of genomic differentiation during stick-insect speciation

Abstract: Speciation can involve a transition from a few genetic loci that are resistant to gene flow to genome-wide differentiation. However, only limited data exist concerning this transition and the factors promoting it. Here, we study phases of speciation using data from >100 populations of 11 species of Timema stick insects. Consistent with early phases of genic speciation, adaptive colour-pattern loci reside in localized genetic regions of accentuated differentiation between populations experiencing gene flow. Transitions to genome-wide differentiation are also observed with gene flow, in association with differentiation in polygenic chemical traits affecting mate choice. Thus, intermediate phases of speciation are associated with genome-wide differentiation and mate choice, but not growth of a few genomic islands. We also find a gap in genomic differentiation between sympatric taxa that still exchange genes and those that do not, highlighting the association between differentiation and complete reproductive isolation. Our results suggest that substantial progress towards speciation may involve the alignment of multi-faceted aspects of differentiation.