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At least 199 records · Page 11

A defined medium based on R2A for cultivation and exometabolite profiling of soil bacteria

SummaryExometabolomics is an approach to assess how microorganisms alter their environments through the depletion and secretion of chemical compounds. Comparisons of inoculated with uninoculated media can be used to provide direct biochemical observations on depleted and secreted metabolites which can be used to predict resource competition, cross-feeding and secondary metabolite production in microbial isolates and communities. This approach is most powerful when used with defined media that enable tracking of all depleted metabolites. However, microbial growth media have traditionally been developed for the isolation and growth of microorganisms but not metabolite utilization profiling through LC-MS/MS. Here, we describe the construction of a defined medium, the Northen Lab Defined Medium (NLDM), that not only supports the growth of diverse bacteria but is defined and therefore suited for exometabolomic experiments. Metabolites included in NLDM were selected based on their presence in R2A medium and soil, elemental stoichiometry requirements, as well as knowledge of metabolite usage by different bacteria. We found that NLDM supported the growth of 53 phylogenetically diverse soil bacterial isolates and all of its metabolites were trackable through LC–MS/MS analysis. These results demonstrate the viability and utility of the constructed NLDM medium for cultivating and characterizing diverse microbial isolates and communities.Originality-Significance StatementWe build a defined medium based on the metabolite composition of R2A medium and soil, elemental stoichiometry requirements, and knowledge of metabolite usage by different bacteria. The newly formulated defined medium was evaluated on its ability to support the growth of soil isolates and its application for metabolite utilization profiling. We found that of 53 phylogenetically diverse soil bacterial isolates grew on the defined medium and all of its metabolites were trackable through LC–MS/MS analysis. This demonstrates the viability and utility of the constructed defined medium for cultivating and characterizing diverse microbial isolates and communities.

de Raad, Markus↗

An archaeal genomic signature

Comparisons of complete genome sequences allow the most objective and comprehensive descriptions possible of a lineage's evolution. This communication uses the completed genomes from four major euryarchaeal taxa to define a genomic signature for the Euryarchaeota and, by extension, the Archaea as a whole. The signature is defined in terms of the set of protein-encoding genes found in at least two diverse members of the euryarchaeal taxa that function uniquely within the Archaea; most signature proteins have no recognizable bacterial or eukaryal homologs. By this definition, 351 clusters of signature proteins have been identified. Functions of most proteins in this signature set are currently unknown. At least 70% of the clusters that contain proteins from all the euryarchaeal genomes also have crenarchaeal homologs. This conservative set, which appears refractory to horizontal gene transfer to the Bacteria or the Eukarya, would seem to reflect the significant innovations that were unique and fundamental to the archaeal "design fabric." Genomic protein signature analysis methods may be extended to characterize the evolution of any phylogenetically defined lineage. The complete set of protein clusters for the archaeal genomic signature is presented as supplementary material (see the PNAS web site, www.pnas.org).

Non-NASA Center↗

Application of prophage sequence analysis to investigate a disease outbreak involving Salmonella Adjame, a rare serovar and implications for the population structure

Introduction Outbreak investigation of foodborne salmonellosis is hindered when the food source is contaminated by multiple strains of Salmonella , creating difficulties matching an incriminated organism recovered from patients with the specific strain in the suspect food. An outbreak of the rare Salmonella Adjame was caused by multiple strains of the organism as revealed by single-nucleotide polymorphism (SNP) variation. The use of highly discriminatory prophage analysis to characterize strains of Salmonella should enable a more precise strain characterization and aid the investigation of foodborne salmonellosis. Methods We have carried out genomic analysis of S. Adjame strains recovered during the course of a recent outbreak and compared them with other strains of the organism ( n = 38 strains), using SNPs to evaluate strain differences present in the core genome, and prophage sequence typing (PST) to evaluate the accessory genome. Phylogenetic analyses were performed using both total prophage content and conserved prophages. Results The PST analysis of the S. Adjame isolates showed a high degree of strain heterogeneity. We observed small clusters made up of 2-6 isolates ( n = 27) and singletons ( n = 11) in stark contrast with the three clusters observed by SNP analysis. In total, we detected 24 prophages of which only four were highly prevalent, namely: Entero_p88 (36/38 strains), Salmon_SEN34 (35/38 strains), Burkho_phiE255 (33/38 strains) and Edward_GF (28/38 strains). Despite the marked strain diversity seen with prophage analysis, the distribution of the four most common prophages matched the clustering observed using core genome. Discussion Mutations in the core and accessory genomes of S. Adjame have shed light on the evolutionary relationships among the Adjame strains and demonstrated a convergence of the variations observed in both fractions of the genome. We conclude that core and accessory genomes analyses should be adopted in foodborne bacteria outbreak investigations to provide a more accurate strain description and facilitate reliable matching of isolates from patients and incriminated food sources. The outcomes should translate to a better understanding of the microbial population structure and an 46 improved source attribution in foodborne illnesses.

Gao, Ruimin↗

Full ribosomal operon sequencing of anaerobic gut fungi (phylum Neocallimastigomycota ): insights on its markers and phylogenetic resolution

The phylogenetic affiliations of anaerobic gut fungi (Neocallimastigomycota) are typically evaluated using single-gene markers. However, this approach often fails to resolve relationships between closely related lineages. To address this issue and identify alternative markers, we created a curated database comprising the complete ribosomal operon sequences of 156 isolates, representing 20 of the 22 recognized genera and two new genus-level clades. Using long-read sequencing, we obtained ~9 kbp operon sequences and developed a robust analysis pipeline. Incorporating both coding genes and non-coding regions (excluding IGS1) improved phylogenetic resolution. This phylogenetic approach successfully resolved the Cyllamyces and Caecomyces clades (hard-to-distinguish genetically), as well as seven analysed Piromyces species. We also scanned the operon for markers that are suitable for short-read sequencing platforms, with the aim of enhancing biodiversity and phylogenetic studies. Notably, the ETS1 genetic region also enabled the distinction between these lineages, indicating its phylogenetic value within the ribosomal operon. The resulting database is a valuable resource for expanding and strengthening phylogenetic frameworks.

High-throughput sequencing↗

A meta-evaluation of the quality of reporting and execution in ecological meta-analyses

Quantitatively summarizing results from a collection of primary studies with meta-analysis can help answer ecological questions and identify knowledge gaps. The accuracy of the answers depends on the quality of the meta-analysis. We reviewed the literature assessing the quality of ecological meta-analyses to evaluate current practices and highlight areas that need improvement. From each of the 18 review papers that evaluated the quality of meta-analyses, we calculated the percentage of meta-analyses that met criteria related to specific steps taken in the meta-analysis process (i.e., execution) and the clarity with which those steps were articulated (i.e., reporting). We also re-evaluated all the meta-analyses available from Pappalardo et al. to extract new information on ten additional criteria and to assess how the meta-analyses recognized and addressed non-independence. In general, we observed better performance for criteria related to reporting than for criteria related to execution; however, there was a wide variation among criteria and meta-analyses. Meta-analyses had low compliance with regard to correcting for phylogenetic non-independence, exploring temporal trends in effect sizes, and conducting a multifactorial analysis of moderators (i.e., explanatory variables). In addition, although most meta-analyses included multiple effect sizes per study, only 66% acknowledged some type of non-independence. The types of non-independence reported were most often related to the design of the original experiment (e.g., the use of a shared control) than to other sources (e.g., phylogeny). We suggest that providing specific training and encouraging authors to follow the PRISMA EcoEvo checklist recently developed by O’Dea et al. can improve the quality of ecological meta-analyses.

59 BASIC BIOLOGICAL SCIENCES↗

Microbial communities of Auka hydrothermal sediments shed light on vent biogeography and the evolutionary history of thermophily

Abstract Hydrothermal vents have been key to our understanding of the limits of life, and the metabolic and phylogenetic diversity of thermophilic organisms. Here we used environmental metagenomics combined with analysis of physicochemical data and 16S rRNA gene amplicons to characterize the sediment-hosted microorganisms at the recently discovered Auka vents in the Gulf of California. We recovered 325 metagenome assembled genomes (MAGs) representing 54 phyla, over 30% of those currently known, showing the microbial community in Auka hydrothermal sediments is highly diverse. 16S rRNA gene amplicon screening of 224 sediment samples across the vent field indicates that the MAGs retrieved from a single site are representative of the microbial community in the vent field sediments. Metabolic reconstruction of a vent-specific, deeply branching clade within the Desulfobacterota suggests these organisms metabolize sulfur using novel octaheme cytochrome-c proteins related to hydroxylamine oxidoreductase. Community-wide comparison between Auka MAGs and MAGs from Guaymas Basin revealed a remarkable 20% species-level overlap, suggestive of long-distance species transfer over 400 km and subsequent sediment colonization. Optimal growth temperature prediction on the Auka MAGs, and thousands of reference genomes, shows that thermophily is a trait that has evolved frequently. Taken together, our Auka vent field results offer new perspectives on our understanding of hydrothermal vent microbiology.

54 ENVIRONMENTAL SCIENCES↗

Isotope analysis reveals dietary overlap among sympatric canids

Abstract When colonizing new regions, invading species might compete strongly with phylogenetically related species native to the regions they are colonizing, eventually leading to coexistence or displacement. In the southeast of the United States, recently established coyotes (Canis latrans) compete with red fox (Vulpes vulpes) and gray fox (Urocyon cinereoargenteus), although it remains unclear if competition is leading to resource partitioning or displacement by species. Using nitrogen and carbon stable isotopes, we tested the hypothesis that coyotes compete with foxes for food resources, with canids partitioning those resources to mitigate competition. We compared diets of canids in the southeast to those in the Plains region of the United States, a region where all three species historically have coexisted. We analyzed 217 hair samples from both regions pre-1960, prior to coyote colonization of the southeast, and post-2000, after coyotes were ubiquitous there, to assess differences in diet among species for both regions (southeast versus Plains and time periods, pre- versus postcolonization by coyotes). Modeling revealed significant dietary overlap among historical and contemporary populations in the southeast. Historically, all species partitioned resources in the Plains. Contemporarily, red fox and coyotes co-occurring in the Plains overlapped in diet; however, gray fox diet did not overlap with those of red fox and coyotes. Absence of partitioning in diet among co-occurring canids in the southeast indicates that interspecific competition could be strong in the region. Competition among canid populations in the southeast could lead to further resource partitioning among species that promotes coexistence or competitive exclusion of smaller fox species where coyote populations are abundant.

Zoology↗

Long-read metagenomics of soil communities reveals phylum-specific secondary metabolite dynamics

Abstract Microbial biosynthetic gene clusters (BGCs) encoding secondary metabolites are thought to impact a plethora of biologically mediated environmental processes, yet their discovery and functional characterization in natural microbiomes remains challenging. Here we describe deep long-read sequencing and assembly of metagenomes from biological soil crusts, a group of soil communities that are rich in BGCs. Taking advantage of the unusually long assemblies produced by this approach, we recovered nearly 3,000 BGCs for analysis, including 712 full-length BGCs. Functional exploration through metatranscriptome analysis of a 3-day wetting experiment uncovered phylum-specific BGC expression upon activation from dormancy, elucidating distinct roles and complex phylogenetic and temporal dynamics in wetting processes. For example, a pronounced increase in BGC transcription occurs at night primarily in cyanobacteria, implicating BGCs in nutrient scavenging roles and niche competition. Taken together, our results demonstrate that long-read metagenomic sequencing combined with metatranscriptomic analysis provides a direct view into the functional dynamics of BGCs in environmental processes and suggests a central role of secondary metabolites in maintaining phylogenetically conserved niches within biocrusts.

59 BASIC BIOLOGICAL SCIENCES↗

Genetic Subtypes and Natural Resistance Mutations in HCV Genotype 4 Infected Saudi Arabian Patients

This study aimed to characterize the HCV genetic subtypes variability and the presence of natural occurring resistance-associated substitutions (RASs) in Saudi Arabia patients. A total of 17 GT patients were analyzed. Sequence analysis of NS3, NS5A, and NS5B regions was performed by direct sequencing, and phylogenetic analyses were used to determine genetic subtypes, RAS, and polymorphisms. Nine patients were infected by GT 4a, two with GT 4o and three with GT 4d. Two patients were infected with apparent recombinant virus (4a/4o/4a in NS3/NS5A/NS5B), and one patient was infected with a previously unknown, unclassifiable, virus of GT 4. Natural RASs were found in six patients (35%), including three infected by GT 4a, two by GT 4a/GT 4o/GT 4a, and one patient infected by an unknown, unclassifiable, virus of GT 4. In particular, NS3-RAS V170I was demonstrated in three patients, while NS5A-RASs (L28M, L30R, L28M + M31L) were detected in the remaining three patients. All patients were treated with sofosbuvir plus daclatasvir; three patients were lost to follow-up, whereas 14 patients completed the treatment. A sustained virological response (SVR) was obtained in all but one patient carrying NS3-RAS V170I who later relapsed. GT 4a is the most common subtype in this small cohort of Saudi Arabia patients infected with hepatitis C infection. Natural RASs were observed in about one-third of patients, but only one of them showed a treatment failure.

60 APPLIED LIFE SCIENCES↗

Transcriptome Sequencing of Agave angustifolia Reveals Conservation and Diversification in the Expression of Cinnamyl Alcohol Dehydrogenase Genes in Agave Species

Agave angustifolia is an important crassulacean acid metabolism plant species, with wide applications in beverage and sisal fiber production. In this study, we carried out a transcriptome analysis of A. angustifolia leaves, generating a total of 58,482,436 clean reads through Illumina paired-end sequencing. De novo transcriptome assembly generated 67,314 unigenes, with about half of them having homologs in four public databases. In the Nr database, Asparagus officinalis was shown to be most closely related to agave, and the early angiosperm Amborella trichopoda was selected as an outgroup species. We further identified five, two, seven, seven, seven, six and six CAD genes in asparagus, amborella, A. deserti, A. tequilana, A. americana, A. hybrid H11648 and A. angustifolia, respectively. The maximum likelihood phylogenetic tree revealed the species-specific expansion of CAD genes in arabidopsis, rice and agave. The expression analysis indicated the conservatively expressed CAD1/2/4/6, providing candidate targets for manipulation to improve lignin traits. The species-specific expression of CAD3/5/7 indicates the existence of different regulatory mechanisms controlling the expression of these genes in agave species. This study presents the first transcriptome dataset of A. angustifolia, facilitating future studies on lignin biosynthesis in agave.

59 BASIC BIOLOGICAL SCIENCES↗

Evolutionary Diversification of Host-Targeted Bartonella Effectors Proteins Derived from a Conserved FicTA Toxin-Antitoxin Module

Proteins containing a FIC domain catalyze AMPylation and other post-translational modifications (PTMs). In bacteria, they are typically part of FicTA toxin-antitoxin modules that control conserved biochemical processes such as topoisomerase activity, but they have also repeatedly diversified into host-targeted virulence factors. Among these, Bartonella effector proteins (Beps) comprise a particularly diverse ensemble of FIC domains that subvert various host cellular functions. However, no comprehensive comparative analysis has been performed to infer molecular mechanisms underlying the biochemical and functional diversification of FIC domains in the vast Bep family. Here, we used X-ray crystallography, structural modelling, and phylogenetic analyses to unravel the expansion and diversification of Bep repertoires that evolved in parallel in three Bartonella lineages from a single ancestral FicTA toxin-antitoxin module. Our analysis is based on 99 non-redundant Bep sequences and nine crystal structures. Inferred from the conservation of the FIC signature motif that comprises the catalytic histidine and residues involved in substrate binding, about half of them represent AMP transferases. A quarter of Beps show a glutamate in a strategic position in the putative substrate binding pocket that would interfere with triphosphate-nucleotide binding but may allow binding of an AMPylated target for deAMPylation or another substrate to catalyze a distinct PTM. The β-hairpin flap that registers the modifiable target segment to the active site exhibits remarkable structural variability. The corresponding sequences form few well-defined groups that may recognize distinct target proteins. The binding of Beps to promiscuous FicA antitoxins is well conserved, indicating a role of the antitoxin to inhibit enzymatic activity or to serve as a chaperone for the FIC domain before translocation of the Bep into host cells. Taken together, our analysis indicates a remarkable functional plasticity of Beps that is mostly brought about by structural changes in the substrate pocket and the target dock. These findings may guide future structure–function analyses of the highly versatile FIC domains.

59 BASIC BIOLOGICAL SCIENCES↗

Bioinformatic teaching resources - for educators, by educators - using KBase, a free, user-friendly, open source platform

Over the past year, biology educators and staff at the Department of Energy Systems Biology Knowledgebase (KBase) initiated a collaborative effort to develop a curriculum for bioinformatics education. KBase is a free and easily accessible data science platform that integrates many bioinformatics resources into a graphical user interface built upon reproducible analysis notebooks. KBase held conversations with college and high school instructors to understand how KBase could potentially support their educational goals. These conversations morphed into a working group of biological and data science instructors that adapted the KBase platform to their curriculum needs, specifically around concepts in Genomics, Metagenomics, Pangenomics, and Phylogenetics. The KBase Educators Working Group developed modular, adaptable, and customizable instructional units. Each instructional module contains teaching resources, publicly available data, analysis tools, and markdown capability to tailor instructions and learning goals for each class. The online user interface enables students to conduct hands-on data science research and analyses without requiring programming skills or their own computational resources (these are provided by KBase). Alongside these resources, KBase continues to work with instructors, supporting the development of additional curriculum modules. For anyone new to the platform, KBase, and the growing KBase Educators Organization, provides a community network, accompanied by community-sourced guidelines, instructional templates, and peer support to use KBase within a classroom whether virtual or in-person.

59 BASIC BIOLOGICAL SCIENCES↗

Expanding the genomic encyclopedia of Actinobacteria with 824 isolate reference genomes

The phylum Actinobacteria includes important human pathogens like Mycobacterium tuberculosis and Corynebacterium diphtheriae and renowned producers of secondary metabolites of commercial interest, yet only a small part of its diversity is represented by sequenced genomes. Here, we present 824 actinobacterial isolate genomes in the context of a phylum-wide analysis of 6,700 genomes including public isolates and metagenome-assembled genomes (MAGs). We estimate that only 30%–50% of projected actinobacterial phylogenetic diversity possesses genomic representation via isolates and MAGs. A comparison of gene functions reveals novel determinants of host-microbe interaction as well as environment-specific adaptations such as potential antimicrobial peptides. We identify plasmids and prophages across isolates and uncover extensive prophage diversity structured mainly by host taxonomy. Analysis of >80,000 biosynthetic gene clusters reveals that horizontal gene transfer and gene loss shape secondary metabolite repertoire across taxa. Our observations illustrate the essential role of and need for high-quality isolate genome sequences.

59 BASIC BIOLOGICAL SCIENCES↗

Elucidation of trophic interactions in an unusual single-cell nitrogen-fixing symbiosis using metabolic modeling

Marine nitrogen-fixing microorganisms are an important source of fixed nitrogen in oceanic ecosystems. The colonial cyanobacterium Trichodesmium and diatom symbionts were thought to be the primary contributors to oceanic N 2 fixation until the discovery of the unusual uncultivated symbiotic cyanobacterium UCYN-A ( Candidatus Atelocyanobacterium thalassa ). UCYN-A has atypical metabolic characteristics lacking the oxygen-evolving photosystem II, the tricarboxylic acid cycle, the carbon-fixation enzyme RuBisCo and de novo biosynthetic pathways for a number of amino acids and nucleotides. Therefore, it is obligately symbiotic with its single-celled haptophyte algal host. UCYN-A receives fixed carbon from its host and returns fixed nitrogen, but further insights into this symbiosis are precluded by both UCYN-A and its host being uncultured. In order to investigate how this syntrophy is coordinated, we reconstructed bottom-up genome-scale metabolic models of UCYN-A and its algal partner to explore possible trophic scenarios, focusing on nitrogen fixation and biomass synthesis. Since both partners are uncultivated and only the genome sequence of UCYN-A is available, we used the phylogenetically related Chrysochromulina tobin as a proxy for the host. Through the use of flux balance analysis (FBA), we determined the minimal set of metabolites and biochemical functions that must be shared between the two organisms to ensure viability and growth. We quantitatively investigated the metabolic characteristics that facilitate daytime N 2 fixation in UCYN-A and possible oxygen-scavenging mechanisms needed to create an anaerobic environment to allow nitrogenase to function. This is the first application of an FBA framework to examine the tight metabolic coupling between uncultivated microbes in marine symbiotic communities and provides a roadmap for future efforts focusing on such specialized systems.

59 BASIC BIOLOGICAL SCIENCES↗

Methanopyrus kandleri: an archaeal methanogen unrelated to all other known methanogens

Analysis of its 16S rRNA sequence shows that the newly discovered hyperthermophilic methanogen, Methanopryus kandleri, is phylogenetically unrelated to any other known methanogen. The organism represents a separate lineage originating near the root of the archaeal tree. Although the 16S rRNA sequence of Mp. kandleri resembles euryarchaeal 16S rRNAs more than it does crenarchaeal, it shows more crenarchaeal signature features than any known euryarchaeal rRNA. Attempts to place it in relation to the root of the archaeal tree show that the Mp. kandleri lineage likely arises from the euryarchaeal branch of the tree. While the existence of so deeply branching a methanogenic lineage brings into question the thesis that methanogenesis evolved from an earlier metabolism similar to that seen in Thermococcus, it at the same time reinforces the notion that the aboriginal [correction of aborginal] archaeon was a thermophile.

NASA Discipline Number 52-30↗

Gene family expansions and transcriptome signatures uncover fungal adaptations to wood decay

Because they comprise some of the most efficient wood-decayers, Polyporales fungi impact carbon cycling in forest environment. Despite continuous discoveries on the enzymatic machinery involved in wood decomposition, the vision on their evolutionary adaptation to wood decay and genome diversity remains incomplete. We combined the genome sequence information from 50 Polyporales species, including 26 newly sequenced genomes and sought for genomic and functional adaptations to wood decay through the analysis of genome composition and transcriptome responses to different carbon sources. The genomes of Polyporales from different phylogenetic clades showed poor conservation in macrosynteny, indicative of genome rearrangements. We observed different gene family expansion/contraction histories for plant cell wall degrading enzymes in core polyporoids and phlebioids and captured expansions for genes involved in signalling and regulation in the lineages of white rotters. Furthermore, we identified conserved cupredoxins, thaumatin-like proteins and lytic polysaccharide monooxygenases with a yet uncharacterized appended module as new candidate players in wood decomposition. Given the current need for enzymatic toolkits dedicated to the transformation of renewable carbon sources, the observed genomic diversity among Polyporales strengthens the relevance of mining Polyporales biodiversity to understand the molecular mechanisms of wood decay.

59 BASIC BIOLOGICAL SCIENCES↗

Phylogenetic relationships of the Fox (Forkhead) gene family in the Bilateria

The Forkhead or Fox gene family encodes putative transcription factors. There are at least four Fox genes in yeast, 16 in Drosophila melanogaster (Dm) and 42 in humans. Recently, vertebrate Fox genes have been classified into 17 groups named FoxA to FoxQ. Here, we extend this analysis to invertebrates, using available sequences from D. melanogaster, Anopheles gambiae (Ag), Caenorhabditis elegans (Ce), the sea squirt Ciona intestinalis (Ci) and amphioxus Branchiostoma floridae (Bf), from which we also cloned several Fox genes. Phylogenetic analyses lend support to the previous overall subclassification of vertebrate genes, but suggest that four subclasses (FoxJ, L, N and Q) could be further subdivided to reflect their relationships to invertebrate genes. We were unable to identify orthologs of Fox subclasses E, H, I, J, M and Q1 in D. melanogaster, A. gambiae or C. elegans, suggesting either considerable loss in ecdysozoans or the evolution of these subclasses in the deuterostome lineage. Our analyses suggest that the common ancestor of protostomes and deuterostomes had a minimum complement of 14 Fox genes.

NASA Program Fundamental Space Biology↗

Taxogenomic analysis of a novel yeast species isolated from soil, Pichia galeolata sp. nov.

A novel budding yeast species was isolated from a soil sample collected in the United States of America. Phylogenetic analyses of multiple loci and phylogenomic analyses conclusively placed the species within the genus Pichia. Strain yHMH446 falls within a clade that includes Pichia norvegensis, Pichia pseudocactophila, Candida inconspicua, and Pichia cactophila. Whole genome sequence data were analyzed for the presence of genes known to be important for carbon and nitrogen metabolism, and the phenotypic data from the novel species were compared to all Pichia species with publicly available genomes. Across the genus, including the novel species candidate, we found that the inability to use many carbon and nitrogen sources correlated with the absence of metabolic genes. Based on these results, Pichia galeolata sp. nov. is proposed to accommodate yHMH446T (=NRRL Y-64187 = CBS 16864). This study shows how integrated taxogenomic analysis can add mechanistic insight to species descriptions.

59 BASIC BIOLOGICAL SCIENCES↗