Large-scale environmental signals in seagrass blue carbon stocks are hidden by high variability at local scales.

Increasing focus on nature-based climate change mitigation and adaptation strategies has led to the recognition of seagrasses as globally significant organic carbon (Corg) stocks. However, estimates of carbon stocks have been generally confined to a few regions, with few African studies represented in global datasets. In addition, the extent to which biogeographical and environmental variation shape carbon stocks in marine vegetated environments remains uncertain. For South Africa, Zostera capensis is the dominant seagrass species with limited mapping and quantification of its Corg stocks. Here, we measured Z. capensis Corg stocks at six South African estuaries spanning ∼1800 km of the cool-temperate to subtropical marine environmental gradient. Targeting the intertidal zone of the upper and lower estuary reaches, we collected Z. capensis sediments to a depth of 50 cm and measured the Corg, with the median Corg stock estimated at 24.11 Mg C ha-1 (40.4 ± 53.02; mean ± SD). While this is lower than the global average, these data demonstrate that Z. capensis ecosystems are important contributors to blue carbon stocks in the region. Measured Corg stocks showed significant differences between sampling sites for estuaries; however, we did not detect significant differences between estuaries due to high intra-estuarine Corg variability. Examination of biogeographical regions, terrestrial and marine environmental variables as drivers of Corg variability revealed that annual mean sea surface temperature may explain variation in Corg stocks. Furthermore, we found evidence of signals of biogeographical regions and precipitation driving some of the variability in Corg stocks; however, this requires further investigation. Overall, our estimates for Z. capensis add to ongoing national and global efforts to quantify seagrass Corg stocks across environmental and biogeographic gradients to better determine their contributions as nature-based solutions to climate change.

Using transplantation to restore seagrass meadows in a protected South African lagoon.

Background: Seagrass meadows provide valuable ecosystem services but are threatened by global change pressures, and there is growing concern that the functions seagrasses perform within an ecosystem will be reduced or lost without intervention. Restoration has become an integral part of coastal management in response to major seagrass declines, but is often context dependent, requiring an assessment of methods to maximise restoration success. Here we investigate the use of different restoration strategies for the endangered Zostera capensis in South Africa. Methods: We assessed restoration feasibility by establishing seagrass transplant plots based on different transplant source materials (diameter (ø) 10 cm cores and anchored individual shoots), planting patterns (line, dense, bullseye) and planting site (upper, upper-mid and mid-intertidal zones). Monitoring of area cover, shoot length, and macrofaunal diversity was conducted over 18 months. Results: Mixed model analysis showed distinct effects of transplant material used, planting pattern and site on transplant survival and area cover. Significant declines in seagrass cover across all treatments was recorded post-transplantation (2 months), followed by a period of recovery. Of the transplants that persisted after 18 months of monitoring (~58% plots survived across all treatments), seagrass area cover increased (~112%) and in some cases expanded by over >400% cover, depending on type of transplant material, planting arrangement and site. Higher bioturbator pressure from sandprawns (Kraussillichirus kraussi) significantly reduced transplant survival and area cover. Transplant plots were colonised by invertebrates, including seagrass specialists, such as South Africa's most endangered marine invertebrate, the false-eelgrass limpet (Siphonaria compressa). For future seagrass restoration projects, transplanting cores was deemed the best method, showing higher long-term persistence and cover, however this approach is also resource intensive with potentially negative impacts on donor meadows at larger scales. There is a clear need for further research to address Z. capensis restoration scalability and improve long-term transplant persistence.

Management and conservation implications of cryptic population substructure for two commercially exploited fishes (Merluccius spp.) in southern Africa.

Genomic information can aid in the establishment of sustainable management plans for commercially exploited marine fishes, aiding in the long-term conservation of these resources. The southern African hakes (Merluccius capensis and M. paradoxus) are commercially valuable demersal fishes with similar distribution ranges but exhibiting contrasting life histories. Using a comparative framework based on Pool-Seq genome-wide SNP data, we investigated whether the evolutionary processes that shaped extant patterns of diversity and divergence are shared among these two congeneric fishes, or unique to each one. Our findings revealed that M. capensis and M. paradoxus show similar levels of genome-wide diversity, despite different census sizes and life-history features. In addition, M. capensis shows three highly structured geographic populations across the Benguela Current region (one in the northern Benguela and two in the southern Benguela), with no consistent genome-environment associations detected. In contrast, although population structure and outlier analyses suggested panmixia for M. paradoxus, reconstruction of its demographic history suggested the presence of an Atlantic-Indian Ocean subtle substructuring pattern. Therefore, it appears that M. paradoxus might be composed by two highly connected populations, one in the Atlantic and one in the southwest Indian Ocean. The reported similar low levels of genomic diversity, as well as newly discovered genetically distinct populations in both hake species can thus assist in informing and improving conservation and management plans for the commercially important southern African Merluccius.

Responses of population structure and genomic diversity to climate change and fishing pressure in a pelagic fish.

The responses of marine species to environmental changes and anthropogenic pressures (e.g., fishing) interact with ecological and evolutionary processes that are not well understood. Knowledge of changes in the distribution range and genetic diversity of species and their populations into the future is essential for the conservation and sustainable management of resources. Almaco jack (Seriola rivoliana) is a pelagic fish with high importance to fisheries and aquaculture in the Pacific Ocean. In this study, we assessed contemporary genomic diversity and structure in loci that are putatively under selection (outlier loci) and determined their potential functions. Using a combination of genotype-environment association, spatial distribution models, and demogenetic simulations, we modeled the effects of climate change (under three different RCP scenarios) and fishing pressure on the species' geographic distribution and genomic diversity and structure to 2050 and 2100. Our results show that most of the outlier loci identified were related to biological and metabolic processes that may be associated with temperature and salinity. The contemporary genomic structure showed three populations-two in the Eastern Pacific (Cabo San Lucas and Eastern Pacific) and one in the Central Pacific (Hawaii). Future projections suggest a loss of suitable habitat and potential range contractions for most scenarios, while fishing pressure decreased population connectivity. Our results suggest that future climate change scenarios and fishing pressure will affect the genomic structure and genotypic composition of S. rivoliana and lead to loss of genomic diversity in populations distributed in the eastern-central Pacific Ocean, which could have profound effects on fisheries that depend on this resource.

Las respuestas de las especies marinas ante los cambios ambientales y presiones antropogénicas (por ejemplo, la sobrepesca) interactúan con procesos ecológicos y evolutivos que no se comprenden bien. El conocimiento del cambio en el rango de distribución y la diversidad genética de las especies y sus poblaciones en el futuro es fundamental para la conservación y gestión sostenible de los recursos. El jurel (Seriola rivoliana) es un pez pelágico de gran importancia para la pesca y la acuicultura en el Océano Pacífico. En este estudio, evaluamos la diversidad y estructura genómica contemporánea en loci que supuestamente están bajo selección (loci atípicos) y determinamos sus funciones potenciales. Se utilizó la combinación de métodos de asociación genotipo-ambiente, modelos de distribución espacial y simulaciones demogenéticas, para modelar los efectos del cambio climático (bajo tres escenarios RCP diferentes) y presión de pesca sobre la distribución geográfica de la especie, la diversidad y estructura genómica para los años 2050 y 2100. Nuestros resultados mostraron que la mayoría de los loci atípicos están relacionados con procesos biológicos y metabólicos que pueden estar asociados con la temperatura y la salinidad. La estructura genómica contemporánea mostró tres poblaciones: dos en el Pacífico oriental (Cabo San Lucas y el Pacífico oriental) y una en el Pacífico central (Hawai). Las proyecciones futuras sugieren una pérdida de hábitat idóneo y posibles contracciones del área de distribución para la mayoría de los escenarios, mientras que la presión de la pesca redujo la conectividad de las poblaciones. Nuestros resultados sugieren que los escenarios de cambio climático y la presión pesquera afectarán la estructura genómica y la composición genotípica de S. rivoliana y conducirán a la pérdida de diversidad genómica en las poblaciones distribuidas en el Océano Pacífico centro-oriental, lo que podría tener efectos en las pesquerías que dependen de este recurso.

The role of seagrass meadows in the accumulation of microplastics: Insights from a South African estuary.

Microplastics are widespread in marine ecosystems, where they threaten biota and human wellbeing. Seagrasses may act as natural filters of microplastics due to their particle trapping abilities, yet little is known about the extent of microplastics in the sediment of seagrass beds. The aim of this study was to compare microplastic accumulation in the sediments of Zostera capensis meadows with adjacent bare sediments at a small spatial scale (~5 km) in the Knysna estuary in South Africa. No significant difference in total microplastic counts were found between seagrass and unvegetated sediments. However, fibre microplastic counts differed significantly between the two locations at which samples were collected. This may be due to pollution levels and proximity to larger human population densities. Importantly, our study reveals variability in microplastic abundance and type even at small spatial scales, an important consideration for the design of future studies aiming to monitor for microplastics.

Molecular ecology meets systematic conservation planning.

Integrative and proactive conservation approaches are critical to the long-term persistence of biodiversity. Molecular data can provide important information on evolutionary processes necessary for conserving multiple levels of biodiversity (genes, populations, species, and ecosystems). However, molecular data are rarely used to guide spatial conservation decision-making. Here, we bridge the fields of molecular ecology (ME) and systematic conservation planning (SCP) (the 'why') to build a foundation for the inclusion of molecular data into spatial conservation planning tools (the 'how'), and provide a practical guide for implementing this integrative approach for both conservation planners and molecular ecologists. The proposed framework enhances interdisciplinary capacity, which is crucial to achieving the ambitious global conservation goals envisioned for the next decade.

Conservation implications of herbicides on seagrasses: sublethal glyphosate exposure decreases fitness in the endangered Zostera capensis.

Worldwide seagrass populations are in decline, calling for urgent measures in their conservation. Glyphosate is the most widely used herbicide globally, leading to increasing concern about its ecological impact, yet little is known about the prevalence or impact of glyphosate on seagrasses. In this study, we investigated the effect of sublethal glyphosate exposure on the endangered seagrass, Zostera capensis, to identify effects on growth, photosynthetic pigments and leaf morphology as measures of seagrass fitness. Seagrasses were exposed to a single dose of a commercial glyphosate formulation-ranging between 250 to 2,200 µg/L. After three weeks, the median leaf area decreased by up to 27%, with reductions of up to 31% in above ground biomass (p < 0.05). Photosynthetic pigment concentration showed no significant difference between groups. The observed effects on biomass and leaf area were seen at glyphosate levels below the regulatory limits set for surface water by several countries and may negatively affect the long-term resilience of this ecosystem engineer to additional stressors, such as those associated with climate change and anthropogenic pollution. As such, glyphosates and other herbicides that are washed into estuarine and marine ecosystems, pose a significant threat to the persistence of seagrasses and are important factors to consider in seagrass conservation, management and restoration efforts.

De novo assembly and annotation of the transcriptome of the endangered seagrass Zostera capensis: Insights from differential gene expression under thermal stress.

Seagrasses are important marine ecosystem engineers but anthropogenic impacts and climate change have led to numerous population declines globally. In South Africa, Zostera capensis is endangered due to fragmented populations and heavy anthropogenic pressures on estuarine ecosystems that house the core of the populations. Addressing questions of how pressures such as climate change affect foundational species, including Z. capensis are crucial to supporting their conservation and underpin restoration efforts. Here we use ecological transcriptomics to study key functional responses of Z. capensis through quantification of gene expression after thermal stress and present the first reference transcriptome of Z. capensis. Four de novo reference assemblies (Trinity, IDBA-tran, RNAspades, SOAPdenovo) filtered through the EvidentialGene pipeline resulted in 153,755 transcripts with a BUSCO score of 66.1% for completeness. Differential expression analysis between heat stressed (32 °C for three days) and pre-warming plants identified genes involved in photosynthesis, oxidative stress, translation, metabolic and biosynthetic processes in the Z. capensis thermal stress response. This reference transcriptome is a significant contribution to the limited available genomic resources for Z. capensis and represents a vital tool for addressing questions around the species restoration and potential functional responses to warming marine environments.

Distinct interspecific and intraspecific vulnerability of coastal species to global change.

Characterising and predicting species responses to anthropogenic global change is one of the key challenges in contemporary ecology and conservation. The sensitivity of marine species to climate change is increasingly being described with forecasted species distributions, yet these rarely account for population level processes such as genomic variation and local adaptation. This study compares inter- and intraspecific patterns of biological composition to determine how vulnerability to climate change, and its environmental drivers, vary across species and populations. We compare species trajectories for three ecologically important southern African marine invertebrates at two time points in the future, both at the species level, with correlative species distribution models, and at the population level, with gradient forest models. Reported range shifts are species-specific and include both predicted range gains and losses. Forecasted species responses to climate change are strongly influenced by changes in a suite of environmental variables, from sea surface salinity and sea surface temperature, to minimum air temperature. Our results further suggest a mismatch between future habitat suitability (where species can remain in their ecological niche) and genomic vulnerability (where populations retain their genomic composition), highlighting the inter- and intraspecific variability in species' sensitivity to global change. Overall, this study demonstrates the importance of considering species and population level climatic vulnerability when proactively managing coastal marine ecosystems in the Anthropocene.

Multi-model seascape genomics identifies distinct environmental drivers of selection among sympatric marine species.

BACKGROUND: As global change and anthropogenic pressures continue to increase, conservation and management increasingly needs to consider species' potential to adapt to novel environmental conditions. Therefore, it is imperative to characterise the main selective forces acting on ecosystems, and how these may influence the evolutionary potential of populations and species. Using a multi-model seascape genomics approach, we compare putative environmental drivers of selection in three sympatric southern African marine invertebrates with contrasting ecology and life histories: Cape urchin (Parechinus angulosus), Common shore crab (Cyclograpsus punctatus), and Granular limpet (Scutellastra granularis). RESULTS: Using pooled (Pool-seq), restriction-site associated DNA sequencing (RAD-seq), and seven outlier detection methods, we characterise genomic variation between populations along a strong biogeographical gradient. Of the three species, only S. granularis showed significant isolation-by-distance, and isolation-by-environment driven by sea surface temperatures (SST). In contrast, sea surface salinity (SSS) and range in air temperature correlated more strongly with genomic variation in C. punctatus and P. angulosus. Differences were also found in genomic structuring between the three species, with outlier loci contributing to two clusters in the East and West Coasts for S. granularis and P. angulosus, but not for C. punctatus. CONCLUSION: The findings illustrate distinct evolutionary potential across species, suggesting that species-specific habitat requirements and responses to environmental stresses may be better predictors of evolutionary patterns than the strong environmental gradients within the region. We also found large discrepancies between outlier detection methodologies, and thus offer a novel multi-model approach to identifying the principal environmental selection forces acting on species. Overall, this work highlights how adding a comparative approach to seascape genomics (both with multiple models and species) can elucidate the intricate evolutionary responses of ecosystems to global change.

Evolutionary Genomics at the Human-Environment Interface in Africa.

We report on the first meeting of SMBE in Africa. SMBE Malawi was initiated to bring together African and international researchers who use genetics or genomics to study natural systems impacted by human activities. The goals of this conference were 1) to reach a world-class standard of science with a large number of contributions from Africa, 2) to initiate exchange between African and international researchers, and 3) to identify challenges and opportunities for evolutionary genomics research in Africa. As repored, we think that we have achieved these goals and make suggestions on the way forward for African evolutionary genomics research.

Anthropogenic pressures negatively impact genomic diversity of the vulnerable seagrass Zostera capensis.

Zostera capensis is a keystone species providing essential ecosystem services to southern African coastal systems. Like most seagrasses globally, Z. capensis is declining and under threat from anthropogenic pressures, and indicators of seagrass health and resilience may be of interest in preventing further declines. As intraspecific diversity is an important component of resilience, we used a pooled RADseq approach to generate genome-wide measures of variation across the entire South African distribution of Z. capensis. Using nucleotide diversity, heterozygosity and allelic richness we tested for associations with fine-scale anthropogenic pressure data compiled by the South African National Biodiversity Assessment using generalised linear models. Increased fishing effort, habitat loss, sand mining and a change in estuary flow dynamics were found to play an important role in decreasing nucleotide diversity and expected heterozygosity, most likely due to the loss of less resilient genotypes as a result of direct physical damage or indirect consequences. As the building block for adaptation, nucleotide diversity is particularly important for resilience. Because of this, as well as the fact that nucleotide diversity displayed the most distinct difference between the west and east coast, and responded most strongly to anthropogenic pressures, we suggest that this may be a useful measure for monitoring genetic or genomic variation. As genomic diversity influences resilience and resistance to disturbances, the remaining diversity in South African seagrass beds urgently needs to be conserved through restoration efforts and careful management of pressures.

Shared genomic outliers across two divergent population clusters of a highly threatened seagrass.

The seagrass, Zostera capensis, occurs across a broad stretch of coastline and wide environmental gradients in estuaries and sheltered bays in southern and eastern Africa. Throughout its distribution, habitats are highly threatened and poorly protected, increasing the urgency of assessing the genomic variability of this keystone species. A pooled genomic approach was employed to obtain SNP data and examine neutral genomic variation and to identify potential outlier loci to assess differentiation across 12 populations across the ∼9,600 km distribution of Z. capensis. Results indicate high clonality and low genomic diversity within meadows, which combined with poor protection throughout its range, increases the vulnerability of this seagrass to further declines or local extinction. Shared variation at outlier loci potentially indicates local adaptation to temperature and precipitation gradients, with Isolation-by-Environment significantly contributing towards shaping spatial variation in Z. capensis. Our results indicate the presence of two population clusters, broadly corresponding to populations on the west and east coasts, with the two lineages shaped only by frequency differences of outlier loci. Notably, ensemble modelling of suitable seagrass habitat provides evidence that the clusters are linked to historical climate refugia around the Last Glacial Maxi-mum. Our work suggests a complex evolutionary history of Z. capensis in southern and eastern Africa that will require more effective protection in order to safeguard this important ecosystem engineer into the future.

Thermal selection as a driver of marine ecological speciation.

Intraspecific genetic structure in widely distributed marine species often mirrors the boundaries between temperature-defined bioregions. This suggests that the same thermal gradients that maintain distinct species assemblages also drive the evolution of new biodiversity. Ecological speciation scenarios are often invoked to explain such patterns, but the fact that adaptation is usually only identified when phylogenetic splits are already evident makes it impossible to rule out the alternative scenario of allopatric speciation with subsequent adaptation. We integrated large-scale genomic and environmental datasets along one of the world's best-defined marine thermal gradients (the South African coastline) to test the hypothesis that incipient ecological speciation is a result of divergence linked to the thermal environment. We identified temperature-associated gene regions in a coastal fish species that is spatially homogeneous throughout several temperature-defined biogeographic regions based on selectively neutral markers. Based on these gene regions, the species is divided into geographically distinct regional populations. Importantly, the ranges of these populations are delimited by the same ecological boundaries that define distinct infraspecific genetic lineages in co-distributed marine species, and biogeographic disjunctions in species assemblages. Our results indicate that temperature-mediated selection represents an early stage of marine ecological speciation in coastal regions that lack physical dispersal barriers.

The ghost of introduction past: Spatial and temporal variability in the genetic diversity of invasive smallmouth bass.

Understanding the demographic history of introduced populations is essential for unravelling their invasive potential and adaptability to a novel environment. To this end, levels of genetic diversity within the native and invasive range of a species are often compared. Most studies, however, focus solely on contemporary samples, relying heavily on the premise that the historic population structure within the native range has been maintained over time. Here, we assess this assumption by conducting a three-way comparison of the genetic diversity of native (historic and contemporary) and invasive (contemporary) smallmouth bass (Micropterus dolomieu) populations. Analyses of a total of 572 M. dolomieu samples, representing the contemporary invasive South African range, contemporary and historical native USA range (dating back to the 1930s when these fish were first introduced into South Africa), revealed that the historical native range had higher genetic diversity levels when compared to both contemporary native and invasive ranges. These results suggest that both contemporary populations experienced a recent genetic bottleneck. Furthermore, the invasive range displayed significant population structure, whereas both historical and contemporary native US populations revealed higher levels of admixture. Comparison of contemporary and historical samples showed both a historic introduction of M. dolomieu and a more recent introduction, thereby demonstrating that undocumented introductions of this species have occurred. Although multiple introductions might have contributed to the high levels of genetic diversity in the invaded range, we discuss alternative factors that may have been responsible for the elevated levels of genetic diversity and highlight the importance of incorporating historic specimens into demographic analyses.

Sleeping with the enemy: introgressive hybridization in two invasive centrarchids.

Introgressive hybridization between Micropterus dolomieu and Micropterus salmoides was assessed in their invaded South African range using nine microsatellite markers and two mtDNA gene regions. Although M. dolomieu and M. salmoides are distantly related, indicated by the large uncorrected pairwise distances observed between the two species, mitochondrial introgression and unidirectional admixture was detected.

Mitochondrial DNA is unsuitable to test for isolation by distance.

Tests for isolation by distance (IBD) are the most commonly used method of assessing spatial genetic structure. Many studies have exclusively used mitochondrial DNA (mtDNA) sequences to test for IBD, but this marker is often in conflict with multilocus markers. Here, we report a review of the literature on IBD, with the aims of determining (a) whether significant IBD is primarily a result of lumping spatially discrete populations, and (b) whether microsatellite datasets are more likely to detect IBD when mtDNA does not. We also provide empirical data from four species in which mtDNA failed to detect IBD by comparing these with microsatellite and SNP data. Our results confirm that IBD is mostly found when distinct regional populations are pooled, and this trend disappears when each is analysed separately. Discrepancies between markers were found in almost half of the studies reviewed, and microsatellites were more likely to detect IBD when mtDNA did not. Our empirical data rejected the lack of IBD in the four species studied, and support for IBD was particularly strong for the SNP data. We conclude that mtDNA sequence data are often not suitable to test for IBD, and can be misleading about species' true dispersal potential. The observed failure of mtDNA to reliably detect IBD, in addition to being a single-locus marker, is likely a result of a selection-driven reduction in genetic diversity obscuring spatial genetic differentiation.

Complex signatures of genomic variation of two non-model marine species in a homogeneous environment.

BACKGROUND: Genomic tools are increasingly being used on non-model organisms to provide insights into population structure and variability, including signals of selection. However, most studies are carried out in regions with distinct environmental gradients or across large geographical areas, in which local adaptation is expected to occur. Therefore, the focus of this study is to characterize genomic variation and selective signals over short geographic areas within a largely homogeneous region. To assess adaptive signals between microhabitats within the rocky shore, we compared genomic variation between the Cape urchin (Parechinus angulosus), which is a low to mid-shore species, and the Granular limpet (Scutellastra granularis), a high shore specialist. RESULTS: Using pooled restriction site associated DNA (RAD) sequencing, we described patterns of genomic variation and identified outlier loci in both species. We found relatively low numbers of outlier SNPs within each species, and identified outlier genes associated with different selective pressures than those previously identified in studies conducted over larger environmental gradients. The number of population-specific outlier loci differed between species, likely owing to differential selective pressures within the intertidal environment. Interestingly, the outlier loci were highly differentiated within the two northernmost populations for both species, suggesting that unique evolutionary forces are acting on marine invertebrates within this region. CONCLUSIONS: Our study provides a background for comparative genomic studies focused on non-model species, as well as a baseline for the adaptive potential of marine invertebrates along the South African west coast. We also discuss the caveats associated with Pool-seq and potential biases of sequencing coverage on downstream genomic metrics. The findings provide evidence of species-specific selective pressures within a homogeneous environment, and suggest that selective forces acting on small scales are just as crucial to acknowledge as those acting on larger scales. As a whole, our findings imply that future population genomic studies should expand from focusing on model organisms and/or studying heterogeneous regions to better understand the evolutionary processes shaping current and future biodiversity patterns, particularly when used in a comparative phylogeographic context.

Multispecies genetic objectives in spatial conservation planning.

Growing threats to biodiversity and global alteration of habitats and species distributions make it increasingly necessary to consider evolutionary patterns in conservation decision making. Yet, there is no clear-cut guidance on how genetic features can be incorporated into conservation-planning processes, despite multiple molecular markers and several genetic metrics for each marker type to choose from. Genetic patterns differ between species, but the potential tradeoffs among genetic objectives for multiple species in conservation planning are currently understudied. We compared spatial conservation prioritizations derived from 2 metrics of genetic diversity (nucleotide and haplotype diversity) and 2 metrics of genetic isolation (private haplotypes and local genetic differentiation) in mitochondrial DNA of 5 marine species. We compared outcomes of conservation plans based only on habitat representation with plans based on genetic data and habitat representation. Fewer priority areas were selected for conservation plans based solely on habitat representation than on plans that included habitat and genetic data. All 4 genetic metrics selected approximately similar conservation-priority areas, which is likely a result of prioritizing genetic patterns across a genetically diverse array of species. Largely, our results suggest that multispecies genetic conservation objectives are vital to creating protected-area networks that appropriately preserve community-level evolutionary patterns.