Evolutionary Events Research Articles

Evolution of two metabolic genes involved in nucleotide and amino acid metabolism in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen causing various severe infections. Understanding genetic mechanisms of its metabolic versatility aids in developing novel antibacterial drugs and therapeutic strategies to address multidrug-resistant P. aeruginosa infections. The metabolism of nucleotides and amino acids contributes to the cycle of two key biological macromolecules in the genetic central dogma. Guanine deaminase (GuaD) catalyzes the deamination of guanine to produce xanthine to maintain the homeostasis of the nucleotide pool, and transporters specific to BCAAs (termed as BraT) import BCAAs to keep its intracellular availability level. However, little is known about the evolution of GuaD and BraT in P. aeruginosa population. Here, two copies turned out to be widespread in P. aeruginosa population for each of GuaD and BraT. The phylogenic analysis demonstrated that GuaD1 and BraB were inherited from the ancestor of Pseudomonas, while GuaD2 and BraZ were additionally acquired via evolutionary events in the ancestors of P. aeruginosa. The functional divergence of two copies was supported by different distribution patterns of dN/dS ratios, divergent expression levels, differentially co-expressed genes, and their functional enrichment modules with few intersections. Besides, some co-expressed genes with known functions are involved in infecting hosts, forming biofilm and resisting antibiotic treatment. Taken together, functional divergence following copy number increase and differentiation of co-expression networks might confer greater metabolic potential to P. aeruginosa, especially in response to host immune responses and antibiotic treatments in clinical settings.

ZF-HD gene family in rapeseed (Brassica napus L.): genome-wide identification, phylogeny, evolutionary expansion and expression analyses

BackgroundThe zinc finger-homeodomain (ZF-HD) transcription factor family is widely involved in regulating plant growth and fruit filling, as well as responding to various abiotic stress. Rapeseed (Brassica napus L.), the second largest oil-producing crop in the world, is an annual or biennial herb of Brassica in Cruciferae. However, there is currently no systematic study on the evolutionary relationship and stress response of ZF-HD transcription factors in rapeseed.ResultsIn this study, 60 ZF-HD genes in B. napus (BnZHDs) were identified and named based on the chromosomal location. The evolutionary relationships, classifications, gene structures, motif compositions, chromosome localization, and gene replication events in these BnZHD genes were systematically analyzed. These 60 BnZHD members were divided into seven groups. According to the phylogenetic tree and repetitive events, subfamilies MIF, and V may have undergone stronger expansions during the evolutionary process. Interestingly, segmental duplications may have a more important contribution, which distinguishes them from other dicotyledon plants. To further investigate the evolutionary relationship of the ZF-HD family, we constructed eleven comparative genomic maps of homologous genes between rapeseed and different representative monocotyledonous and dicotyledonous plants. Finally, the gene expression pattern of 15 BnZHD genes from different subfamilies under different tissues, fruit developmental stages, and different abiotic stress were analyzed. The expression profile from real-time quantitative PCR analysis showed different expression patterns of BnZHD gene in B. napus. We found that certain BnZHD genes are preferentially expressed in specific tissues of B. napus, while most genes are expressed in multiple tissues. For example, BnZHD37, BnZHD53, and BnZHD55 may be sensitive to different hormones. Under different stresses, the expression of BnZHD3, BnZHD4, BnZHD7, BnZHD38, BnZD45, and BnZHD53 significantly increased in roots, stems, and leaves within 24 h. These genes may play important roles in the growth, development, and environmental adaptation of rapeseed.ConclusionsThese findings provide a basis for a comprehensive understanding of the ZF-HD family in rapeseed, which will provide information for further research on the functional characteristics of the BnZHD genes.

Haplotype-Based Approach Represents Locus Specificity in the Genomic Diversification Process in Humans (Homo sapiens)

Background/Objectives: Recent progress in evolutionary genomics on human (Homo sapiens) populations has revealed complex demographic events and genomic changes. These include population expansion with complicated migration, substantial population structure, and ancient introgression from other hominins, as well as human characteristics selections. Nevertheless, the genomic regions in which such evolutionary events took place have remained unclear. Methods: Here, we focused on eight loci containing the haplotypes that were previously presented as atypical for the mutation pattern in sequence and/or geographic distribution pattern with the model of recent African origin, which constitute two major clusters: African only, and global. This was the consensus model before information regarding introgression from Neanderthal (Homo neanderthalensis) was available. We compared diversity in identical datasets of the modern human population genome, with the 1000 Genomes project among them. Results/Conclusions: This study identified representative genomic regions that show traces of various demographic events and genomic changes that modern humans have undergone by categorizing the relationships in sequence similarity and in worldwide geographic distribution among haplotypes.

Pred-ID: Future event prediction based on event type schema mining by graph induction and deduction

In the field of information management, effective event intelligence management is crucial for its development. With the continuous evolution of events, predicting future events has become a key task in information management. Event Prediction aims to predict upcoming events based on given contextual information. This requires modeling events and their relationships in the context to infer the structure of future events. However, the existing event prediction methods ignore that the event graph schema based on core events can provide more knowledge about history and future for event prediction through induction and deduction, so as to achieve accurate event prediction. In addressing this issue, we directed our focus towards Event Schema Induction. Inspired by it, we propose the Pred-ID model, designed to build event evolutionary pattern through Inductive Event Graph Generation, Deductive Event Graph Expansion, and Graph Fusion for Event Prediction. Specifically, in the Inductive Event Graph Generation phase, Pred-ID extracts the event core subgraph and event developmental trends from the instance event graph, learning the global structure and uncovering the main processes of event development. Then, in the Deductive Event Graph Expansion phase, by expanding future event node and stretching the main processes of event development into future directions, Pred-ID obtains deductive results, so as to construct the event evolutionary pattern. Finally, in the Graph Fusion for Event Prediction phase, aligning and merging the event evolutionary pattern with the instance event graph enables collaborative prediction of future events. The experimental results indicate that our proposed Pred-ID achieves optimal performance in event evolutionary pattern generation and event prediction tasks.

Recombination across distant coronavirid species and genera is a rare event with distinct genomic features.

Understanding the evolutionary events that led to SARS-CoV-2 emergence, spillover, and spread is crucial to prevent, or at least be prepared for, the same type of occurrence in the future. Given that SARS-CoV-2 has some characteristics not found in other closely related viruses, we aimed to systematically assess how likely these unique features may have been acquired through recombination. We found that, although recombination is a frequent phenomenon among betacoronaviruses, it is mostly limited to closely related members of the same species. Therefore, we conclude that the most likely scenario involved feature acquisition from recombination with a closely related virus that was circulating in a geographically overlapping area or through a different biological process, but not recombination from a virus of a different species, genus, or subgenus.

Origins of xyloglucan-degrading enzymes in fungi.

The origin story of land plants - the pivotal evolutionary event that paved the way for terrestrial ecosystems of today to flourish - lies within their closest living relatives: the streptophyte algae. Streptophyte cell wall composition has evolved such that profiles of cell wall polysaccharides can be used as taxonomic markers. Since xyloglucan is restricted to the streptophyte lineage, we hypothesized that fungal enzymes evolved in response to xyloglucan availability in streptophyte algal or land plant cell walls. The record of the origins of these enzymes is embedded in fungal genomes, and comparing genomes of fungi that share an ancient common ancestor can provide insights into fungal interactions with early plants. This Viewpoint contributes a review of evidence underlying current assumptions about the distribution of xyloglucan in plant and algal cell walls. We evaluate evolutionary scenarios that may have given rise to the observed distribution of putative xyloglucanases in fungi and discuss possible biological contexts in which these enzymes could have evolved. Our findings suggest that fungal xyloglucanase evolution was more likely driven by land plant diversification and biomass accumulation than by the first origins of xyloglucan in streptophyte algal cell walls.

The Evolution of Complex Multicellularity in Land Plants.

The evolution of complex multicellularity in land plants represents a pivotal event in the history of life on Earth, characterized by significant increases in biological complexity. This transition, classified as a Major Evolutionary Transition (MET), is best understood through the framework of Evolutionary Transitions in Individuality (ETIs), which focuses on formerly independent entities forming higher-level units that lose their reproductive autonomy. While much of the ETI literature has concentrated on the early stages of multicellularity, such as the formation and maintenance stages, this paper seeks to address the less explored transformation stage. To do so, we apply an approach that we call Transitions in Structural Complexity (TSCs), which focuses on the emergence of new units of organization via the three key evolutionary processes of modularization, subfunctionalization, and integration to the evolution of land plants. To lay the groundwork, we first explore the relationships between sex, individuality, and units of selection to highlight a sexual life cycle-based perspective on ETIs by examining the early stages of the transition to multicellularity (formation) in the sexual life cycle of the unicellular common ancestor of land plants, emphasizing the differences between the transition to multicellularity in eumetazoans and land plants. We then directly apply the TSC approach in this group, identifying key evolutionary events such as the distinct evolutionary innovations like shoot, root, vascular systems, and specialized reproductive structures, arguing that bringing these under the broader rubric of TSCs affords a degree of explanatory unification. By examining these evolutionary processes, this paper provides a new perspective on the evolution of multicellularity in land plants, highlighting both parallels and distinctions with the animal kingdom.

Pangenome Reveals Gene Content Variations and Structural Variants Contributing to Pig Characteristics.

Pigs are among the most essential sources of high-quality protein in human diets. Structural variants (SVs) are a major source of genetic variants associated with diverse traits and evolutionary events. However, the current linear reference genome of pigs limits the presentation of position information for SVs. In this study, we generated a pangenome of pigs and a genome variation map of 599 deep-sequenced genomes across Eurasia. Moreover, a section-wide gene repertoire was constructed, which indicated that core genes were more evolutionarily conserved than variable genes. Subsequently, we identified 546,137 SVs, their enrichment regions, and relationships with genomic features and found significant divergence across Eurasian pigs. More importantly, the pangenome-detected SVs could complement heritability estimates and genome-wide association studies based only on single nucleotide polymorphisms. Among the SVs shaped by selection, we identified an insertion in the promoter region of the TBX19 gene, which may be related to the development, growth, and timidity traits of Asian pigs and may affect the gene expression. Our constructed pig pangenome and the identified SVs provide rich resources for future functional genomic research on pigs.

Extreme hydroclimatic events and response of vegetation in the eastern QTP since 10 ka

Abstract Climate variations during the Holocene significantly impacted vegetation dynamics in the eastern Qinghai-Tibet Plateau (QTP). However, vegetation evolution in response to regional climatic trends and events during this interval remains controversial. Here, we present well-dated decadal-resolution loss on ignition (LOI) and grain size records from the Xing Co Lake on the eastern QTP. The records show an overall drying trend since 10 thousand years ago (ka), with multiple extreme precipitation events observed during 10 to 7 ka. An extreme drought event occurred at around 5.5 ka, after which the climate was drier and unstable with several drought events. In comparison with the hydroclimate, insolation, and El Niño Southern Oscillation records, our data show a close correspondence with the summer insolation differential between 30°N and 30°S and El Niño events on orbital-millennium timescales. This suggested that the increased rainfall during the early Holocene on the eastern QTP can be attributed to the high insolation differential between 30°N and 30°S and low El Niño events. Conversely, the drying trend in the late Holocene appears to correlate with a low insolation differential and high El Niño events. Whenever ice-rafted debris events occurred in the North Atlantic, there was a corresponding occurrence of drying events in the late Holocene in the Zoige Basin. This suggested that teleconnection between the precipitation on the eastern QTP and the North Atlantic climate exists in the Holocene. When compared to independent hydroclimatic and arboreal pollen (AP%) records on the eastern QTP, the evolutionary trends and events of AP% align closely with local hydroclimate changes. This suggested that arboreal coverage could rapidly respond to climate change during the Holocene, but further studies are needed.

Multiple Speciation and Extinction Rate Shifts Shaped the Macro-Evolutionary History of the Genus Lycium Towards a Rather Gradual Accumulation of Species Within the Genus

The Neotropics are the most species-rich region on Earth, and spectacular diversification rates in plants are reported in plants, mostly occurring in oceanic archipelagos, making Neotropical and island plant lineages a model for macro-evolutionary studies. The genus Lycium in the Solanaceae family, originating from the Neotropics and exhibiting a unique disjunct geography across several islands, is therefore expected to experience exceptional diversification events. In this study, we aimed to quantify the diversification trajectories of the genus Lycium to elucidate the diversification events within the genus. We compiled a DNA matrix of six markers on 75% of all the species in the genus to reconstruct a dated phylogeny. Based on this phylogeny, we first revisited the historical biogeography of the genus. Then, we fitted a Compound Poisson Process on Mass Extinction Time model to investigate the following key evolutionary events: speciation rate, extinction rate, as well as mass extinction events. Our analysis confirmed that South America is the origin of the genus, which may have undergone a suite of successive long-distance dispersals. Also, we found that most species arose as recently as 5 million years ago, and that the diversification rate found is among the slowest rates in the plant kingdom. This is likely shaped by the multiple speciation and extinction rate shifts that we also detected throughout the evolutionary history of the genus, including one mass extinction at the early stage of its evolutionary history. However, both speciation and extinction rates remain roughly constant over time, leading to a gradual species accumulation over time.

Chromosome-level assembly for the complex genome of land hermit crab Coenobita brevimanus

Land hermit crabs are a group of shell-carrying crabs that have evolved remarkable terrestrial adaptations in behavior, morphology, physiology, and biochemistry. However, the genetic mechanisms underlying these adaptations remain unclear. In addition, usually it is very difficult to get good genome assemblies for crustaceans. In this study, we managed to assemble the first chromosome-level genome for a land hermit crab (Coenobita brevimanus) with careful manual curation. The final assembly spans 4.74 Gb, with the contig N50 of 1.75 Mb and scaffold N50 of 42.95 Mb, encompassing 117 chromosomes that account for 96.54% of the genome. The evaluations including genome BUSCO (95.26%), Merqury qv (35.88) and the mapping ratio of pair-end short reads (99.48%) showed the high-continuity of C. brevimanus genome assembly, making it the genome with the highest quality in crustaceans with genome size bigger than 3 Gb. The availability of this chromosome-scale genome of crustaceans represents a valuable resource for the land hermit crab, which represents an independent water-to-land transition evolutionary event in the animal kingdom.

Combining GWAS and RNA-Seq approaches identifies the FtADH1 gene for drought resistance in Tartary buckwheat

Drought is one of the major environmental constraints that significantly affects seedling emergence, yield, and quality of Tartary buckwheat, thereby hindering the development of its industry. However, the molecular mechanisms underlying drought tolerance genes in Tartary buckwheat remain largely unexplored. Alcohol dehydrogenase (ADH), one of the essential plant proteins, plays a crucial role in growth, development, and stress responses, but its specific role in drought resistance is still unclear. In this study, we identified an ADH gene FtADH1, using a membership function value of drought tolerance (MFVD) combined with a genome-wide association study (GWAS) and transcriptomic profiles that confers drought tolerance in Tartary buckwheat. Our findings demonstrated that the overexpression of FtADH1 in Arabidopsis and Tartary buckwheat hairy roots enhances drought tolerance by promoting root elongation and mitigating elevated levels of reactive oxygen species (ROS). Our findings demonstrated that FtADH1 can enhanced tolerance to drought stresses in both Tartary buckwheat and Arabidopsis. This study identifies the FtADH1 as a new player in affecting ROS level and the stress response of Tartary buckwheat by regulating protective enzyme activities at a high level to scavenge ROS and modulating root growth under drought stress. Further, we identified proteins interacting with FtADH1 through a prokaryotic expression pull-down assay combined with mass spectrometry, revealing that FtADH1 specifically interacts with the S-adenosyl-L-methionine (SAM) synthetase protein, FtSAMS1. Overexpression of FtSAMS1 was found to enhance ADH enzymatic activity, leading to increased SAM content in overexpressing Tartary buckwheat hairy roots under water-deficit conditions. Additionally, FtSAMS1 overexpression induced a drought-resistant phenotype in Arabidopsis and Tartary buckwheat hairy roots under drought stress, revealing the biological function of FtADH1. Evolutionary analysis indicates that ADH1 in Fagopyrum species has undergone significant evolutionary events, including duplication and purifying selection, which may contribute to functional diversification and adaptive advantages such as drought resistance in cultivated buckwheat. In summary, this study proposes that FtADH1 is a key contributor to drought tolerance, and its interaction with FtSAMS1 holds potential for the development of drought-resistant varieties in Tartary buckwheat and its relative species.

Species Diversity, Host Association, and Evolutionary History of Cronartium: An Important Global Fungal Pathogen to Trees.

Pine stem rust, the most damaging and widespread forest disease occurring in pine trees in the Northern Hemisphere, is primarily caused by Cronartium species (Pucciniales, Melampsorineae). While the phylogenetic relationships of major Cronartium species have been largely elucidated, there is limited understanding of their species diversity and the evolutionary processes shaping their distribution patterns. In this work, we performed broad sampling and sequencing of Cronartium taxa in China together with additional sequence data and other accessions in NCBI to investigate the diversification and to estimate the divergence time of major evolutionary events in this genus. Molecular dating analysis suggested that the divergence of the genus Cronartium probably was around 91.78 Ma during the Upper Cretaceous. It is believed that Cronartium species may have originated in Asia and North America, with intercontinental dispersals occurring primarily during the Middle Eocene, Middle Miocene, and Pliocene. These dispersal events likely took place through the North Atlantic Land Bridge, the De Geer Route, and the Bering Land Bridge, and subsequently diverged through sporadic dispersal and vicariance events. Furthermore, our analysis of host associations revealed that the diversification of Cronartium species was correlated with their telial-hosts, and some species may have experienced host jump events, indicating a complex interplay between host specificity and pathogen-host interaction during Cronartium evolution.

A host shift as the origin of tomato bacterial canker caused by Clavibacter michiganensis.

The Actinomycetota (formerly Actinobacteria) genus Clavibacter includes phytopathogens with devasting effects in several crops. Clavibacter michiganensis, the causal agent of tomato bacterial canker, is the most notorious species of the genus. Yet, its origin and natural reservoirs remain elusive, and its populations show pathogenicity profiles with unpredictable plant disease outcomes. Here, we generate and analyse a decade-long genomic dataset of Clavibacter from wild and commercial tomato cultivars, providing evolutionary insights that directed phenotypic characterization. Our phylogeny situates the last common ancestor of C. michiganensis next to Clavibacter isolates from grasses rather than to the sole strain we could isolate from wild tomatoes. Pathogenicity profiling of C. michiganensis isolates, together with C. phaseoli and C. californiensis as sister taxa and the wild tomato strain, was found to be congruent with the proposed phylogenetic relationships. We then identified gene enrichment after the evolutionary event, leading to the appearance of the C. michiganesis clade, including known pathogenicity factors but also hitherto unnoticed genes with the ability to encode adaptive traits for a pathogenic lifestyle. The holistic perspective provided by our evolutionary analyses hints towards a host shift event as the origin of C. michiganensis as a tomato pathogen and the existence of pathogenic genes that remain to be characterized.

Two ubiquitous aldo-keto reductases in the genus Papaver support a patchwork model for morphine pathway evolution

The evolution of morphinan alkaloid biosynthesis in plants of the genus Papaver includes permutation of several processes including gene duplication, fusion, neofunctionalization, and deletion resulting in the present chemotaxonomy. A critical gene fusion event resulting in the key bifunctional enzyme reticuline epimerase (REPI), which catalyzes the stereochemical inversion of (S)-reticuline, was suggested to precede neofunctionalization of downstream enzymes leading to morphine biosynthesis in opium poppy (Papaver somniferum). The ancestrally related aldo-keto reductases 1,2-dehydroreticuline reductase (DRR), which occurs in some species as a component of REPI, and codeinone reductase (COR) catalyze the second and penultimate steps, respectively, in the pathway converting (S)-reticuline to morphine. Orthologs for each enzyme isolated from the transcriptomes of 12 Papaver species were shown to catalyze their respective reactions in species that capture states of the metabolic pathway prior to key evolutionary events, including the gene fusion event leading to REPI, thus suggesting a patchwork model for pathway evolution. Analysis of the structure and substrate preferences of DRR orthologs in comparison with COR orthologs revealed structure-function relationships underpinning the functional latency of DRR and COR orthologs in the genus Papaver, thus providing insights into the molecular events leading to the evolution of the pathway.

Genomic and transcriptomic perspectives on the origin and evolution of NUMTs in Orthoptera

Nuclear mitochondrial pseudogenes (NUMTs) result from the transfer of mitochondrial DNA (mtDNA) to the nuclear genome. NUMTs, as “frozen” snapshots of mitochondria, can provide insights into diversification patterns. In this study, we analyzed the origins and insertion frequency of NUMTs using genome assembly data from ten species in Orthoptera. We found divergences between NUMTs and contemporary mtDNA in Orthoptera ranging from 0 % to 23.78 %. The results showed that the number of NUMT insertions was significantly positively correlated with the content of transposable elements in the genome. We found that 39.09 %-68.65 % of the NUMTs flanking regions (2,000 bp) contained retrotransposons, and more NUMTs originated from mitochondrial rDNA regions. Based on the analysis of the mitochondrial transcriptome, we found a potential mechanism of NUMT integration: mitochondrial transcripts are reverse transcribed into double-stranded DNA and then integrated into the genome. The probability of this mechanism occurring accounts for 0.30 %-1.02 % of total mitochondrial nuclear transfer events. Finally, based on the phylogenetic tree constructed using NUMTs and contemporary mtDNA, we provide insights into ancient evolutionary events such as species-specific “autaponumts” and “synaponumts” shared among different species, as well as post-integration duplication events.

Early Hominin Paleoenvironments and Habitat Heterogeneity

Environmental changes are often cited as the main driver of hominin evolutionary events, including major adaptive events such as bipedalism. Thus, researchers are particularly interested in the paleoenvironment of early hominins. The previous prevailing idea that hominins originated in expanding savannas is contradicted by the association of the earliest hominins with more closed and mesic (i.e., moderately wet) habitats. The Pliocene homi-nins that followed lived in a variety of habitats characterized by high levels of heterogeneity and permanent sources of water. This article reviews what we know of Mio-Pliocene hominin paleoenvironments, discusses the nature of the observed habitat heterogeneity associated with early hominins and implications for hominin paleoecology, and considers the challenges we face in showing a causative relationship between environmental change and major evolutionary events.

WRKY Transcription Factors (TFs) as Key Regulators of Plant Resilience to Environmental Stresses: Current Perspective

Plants encounter various stresses in their natural environments and can effectively respond to only one stress at a time. Through a complex gene network, transcription factors (TFs) such as WRKY TFs regulate a diverse array of stress responses. The clarification of the structural characteristics of WRKY proteins, along with recent advancements in molecular dynamics simulations, has shed light on the formation, stability, and interactions of DNA–protein complexes. This has provided a novel viewpoint regarding the control of WRKY TFs. The investigation of superfamilies, encompassing their historical development, diversity, and evolutionary patterns, has become feasible due to the transcriptome approach’s capacity to provide extensive and comprehensive transcripts. The significance of WRKY TFs lies in their pivotal role within several signaling cascades and regulatory networks that influence plant defense responses. The present review summarizes the functional aspects of the high-volume sequence data of WRKY TFs from different species studied to date. Moreover, a comparative analysis approach was utilized to determine the functions of the identified WRKY TFs in response to both abiotic and biotic stresses, as revealed through numerous studies on different plant species. The results of this review will be pivotal in understanding evolutionary events and the significance of WRKY TFs in the context of climate change, incorporating new scientific evidence to propose an innovative viewpoint.

Path partitions of phylogenetic networks

In phylogenetics, evolution is traditionally represented in a tree-like manner. However, phylogenetic networks can be more appropriate for representing evolutionary events such as hybridization, horizontal gene transfer, and others. In particular, the class of forest-based networks was recently introduced to represent introgression, in which genes are swapped between species. A network is forest-based if it can be obtained by adding arcs to a collection of trees, so that the endpoints of the new arcs are in different trees. This contrasts with so-called tree-based networks, which are formed by adding arcs within a single tree.We are interested in the computational complexity of recognizing forest-based networks, which was recently left as an open problem by Huber et al. It has been observed that forest-based networks coincide with directed acyclic graphs that can be partitioned into induced paths, each ending at a leaf of the original graph. Several types of path partitions have been studied in the graph theory literature, but to our best knowledge this type of ‘leaf induced path partition’ has not been directly considered before. The study of forest-based networks in terms of these partitions allows us to establish closer relationships between phylogenetics and algorithmic graph theory, and to provide answers to problems in both fields.More specifically, we show that deciding whether a network is forest-based is NP-complete, even on input networks that are tree-based, binary, and have only three leaves. This shows that partitioning a directed acyclic graph into a constant number of induced paths is NP-complete, answering a recent question of Fernau et al. We then show that the problem is polynomial-time solvable on binary networks with two leaves and on the recently introduced class of orchards, which we show to be always forest-based. Finally, for undirected graphs, we introduce unrooted forest-based networks and provide hardness results for this class as well.

Reconstructing the redox environment of the Cambrian Harkless Formation, Nevada, USA: Navigating the effects of iron-rich clay minerals on the iron speciation redox proxy

Understanding the oxygenation of ancient oceans is critical for contextualizing evolutionary events throughout Earth's history. While there are many geochemical proxies available for evaluating paleoredox state, iron speciation is one of the most robust and widely applied recorders of local bottom water redox. The proxy uses a series of sequential extractions which are operationally designed to target redox active iron in different mineral groups, including carbonates, oxides, magnetite, and sulfides. However, clay minerals that contain iron are not specifically targeted but can be important reservoirs of redox active iron. These clay minerals are abundant during many times in Earth's history, so it is critical to understand how they are extracted during iron speciation and their impact on paleoenvironmental interpretations.Here we apply the iron speciation proxy in combination with other geochemical proxies (FeT/Al, Mn enrichments) and mineralogical analyses to determine local bottom water redox conditions during deposition of the early Cambrian Harkless Formation exposed near Gold Point, Nevada, USA. The Harkless Formation contains one of the last occurrences of archaeocyathan reefs before their extinction, thus studying the underlying strata can provide information about the environmental conditions which led to reef development. Iron speciation results indicate oxic to potentially anoxic conditions in the water column. The presence of FeT/Al enrichments, Mn depletion, and the Fe-rich redox sensitive clay minerals chamosite and glauconite suggest intermittent anoxia in the water column and porewaters during some intervals of Harkless deposition. Further, by combining iron speciation extractions with petrographic and x-ray diffraction analyses, we demonstrate that glauconite and chamosite were likely formed at or below the sediment water interface and are also not consistently or fully removed by the standard sequential iron reagents, as noted by previous work.The presence of these authigenic Fe-bearing clay minerals overall results in an underestimation of the proportion of redox active iron during deposition and early sediment diagenesis that may lead to incorrect environmental characterization. We therefore advocate for caution and careful examination of the mineralogy of samples when applying the iron speciation proxy, particularly when authigenic and/or early diagenetic iron-bearing clay minerals are suspected to be present. Based upon these findings, we suggest that the Harkless Formation experienced variable local bottom water redox conditions ranging from oxic to anoxic during deposition. Changes in the relative abundances of chamosite and glauconite may also indicate an increase in oxygenation up section, suggesting that local water column oxygenation played a role in archaeocyathan reef development.