Flower formation has been a primary focus in botanical research, leading to the identification of multiple factors regulating flowering over the past 30 years. The MADS transcription factors SEPALLATA3 (SEP3) and APETALA1 (AP1) are essential for floral meristem development and organ identity. In Arabidopsis, SEP3 functions as a central integrator, combining MADS proteins into a tetrameric complex, with its interaction with AP1 playing a key role in sepal and petal formation. This research explores and , with particular emphasis on the Leu residue in the K1 subfunctional domain of , which is necessary for their interaction. A predicted structural model of AP1 was used, followed by protein docking with SEP3, which indicated that Leu residues at positions 115 and 116 are critical binding sites. Mutations at these position were examined through yeast two-hybrid assays and other techniques, identifying Leu 116 as a significant site. Subsequent purification and EMSA analysis revealed that mutations in the leucine zipper of SEP3 decreased its DNA binding ability. Observations of transgenic plants showed that disruption of and interaction resulted in extended vegetative growth, increased size and number of rosette leaves, and modifications in floral structures. This study offers new insights into the interaction mechanism between AP1 and SEP3 during flowering.

Sugars are important both as an energy source and a signaling cue. In SPINDLY (SPY) is the -fucosylation transferase that links sugar with various plant growth and development processes. Previously, was shown to display a strong salt and drought tolerance phenotype. Herein we confirmed the phenotype and further studied its mechanism. We found that abscisic acid (ABA) elevated expression in guard cells, and SPY is involved in ABA-induced stomatal closure. We show that SPY regulates the rearrangement of the microtubule cytoskeleton in guard cells. Moreover, ABA-induced microtubule reorganization is enhanced in mutants. Mechanistically, SPY interacts with α-tubulin1 (TUA1) in both yeast-two hybrid, bimolecular fluorescence complementation and split luciferase complementation imaging assays, indicating that TUA1 may be -fucosylated by SPY. Our work is in line with the notion that SPY has many substrates involved in diverse processes in plants, and also unearths a key mechanism how glycosylation regulates the stomata movement via the microtubule cytoskeleton.

The need for more sustainable agricultural systems is becoming increasingly apparent. The global demand for agricultural products-food, feed, fuel and fiber-will continue to increase as the global population continues to grow. This challenge is compounded by climate change. Not only does a changing climate make it difficult to maintain stable yields but current agricultural systems are a major source of greenhouse gas emissions and continue to drive the problem further. Therefore, future agricultural systems must not only increase production but also significantly decrease negative environmental impacts. One approach to addressing this is to begin breeding and cultivating new plant species that have fundamental sustainability advantages over our existing crops. The Lemnaceae, commonly known as duckweeds, are one family of plants that have potential to increase output and reduce the negative environmental impacts of agricultural production. Herein we describe the Automated Lab-scale PHenotyping Apparatus, ALPHA, for high-throughput phenotyping of Lemnaceae. ALPHA is being used for selective breeding of one species, , toward the goal of creating a new crop for use in sustainable agricultural systems. ALPHA can be used on many small aquatic plant species to assess growth rates in different environmental conditions. A proof of principle use case is demonstrated where ALPHA is used to determine saltwater tolerance of six different clones of .

Transcriptional master regulators are often targeted to improve plant traits, but antagonistic pleiotropic effects of these regulators can hamper this approach. The Myb-bHLH-WDR (MBW) complex is a broadly conserved transcriptional regulator affecting pigmentation, biotic stress resistance, and abiotic stress tolerance. We investigated the function of sorghum grain pigmentation regulator , the ortholog of Arabidopsis pleiotropic WD40 regulator , to test for conserved pleiotropic regulatory effects and to better understand the evolution of the MBW complex in Poaceae. We characterized genome-wide differential expression of leaf tissue using RNA sequencing in near-isogenic lines (NILs) that contrasted wildtype and loss-of-function alleles, under optimal temperature and chilling stress. Notably, Gene Ontology analyses revealed no pathways with differential expression between and NILs, suggesting that, in contrast to Arabidopsis , has no pleiotropic regulatory role in leaves. Further, NILs had no visible difference in anthocyanin pigmentation, and no genes with known or expected function in flavonoid synthesis were differentially expressed. Genome-wide, only 18 total genes were differentially expressed between NILs, with six of these genes located inside the NIL introgression region, an observation most parsimoniously explained by -regulatory effects unrelated to regulation. Comparing our findings with known function of orthologs in maize, rice, and Arabidopsis, we conclude that pleiotropic regulatory function in leaf tissue was likely lost in panicoid grass evolution before the sorghum-maize split. These findings inform future molecular breeding of MBW regulated traits and highlight the benefit of subfunctionalization to relieve pleiotropic constraints.

In and many other tree species, photoassimilate sucrose diffuses down a concentration gradient via symplastically connected mesophyll cells to minor vein phloem for long-distance transport. There is no evidence for apoplastic phloem-loading in . However, plasma membrane sucrose transporters (SUT1 and SUT3) orthologous to those associated with apoplastic phloem loading are expressed in vascular tissues of poplar. While SUT3 functions in sucrose import into developing xylem, the role of SUT1 remains unclear. Here, we overexpressed in x to examine the effects on sucrose partitioning in transgenic plants. Overall leaf sucrose levels were similar between wild type and transgenic lines. Stem sucrose levels were not changed in bark but were significantly reduced in the adjacent xylem, suggesting hindered intercellular sucrose trafficking from the phloem to the developing xylem. Fully expanded leaves of transgenic plants deteriorated prematurely with declining photosynthesis prior to severe necrotic spotting. Necrotic spotting advanced most rapidly in the distal portion of mature leaves and was accompanied by sharp hexose increases and sharp sucrose decreases there. Leaf transcriptome profiling and network inference revealed the down-regulation of copper proteins and elevated expression of copper microRNAs prior to noticeable leaf injury. Our results suggest ectopic expression of altered sucrose partitioning in stems with systemic effects on leaf health and copper homeostasis mediated in part by sucrose-sensitive copper miRNAs.

Chorismate is an important branchpoint metabolite in the biosynthesis of lignin and a wide array of metabolites in plants. Chorismate mutase (CM), the enzyme responsible for transforming chorismate into prephenate, is a key regulator of metabolic flux towards the synthesis of aromatic amino acids and onwards to lignin. We examined three CM genes in hybrid poplar ( × , abbreviated as ) and used RNA interference (RNAi) to suppress the expression of ×CM1, the most highly expressed isoform found in xylem tissue. Although this strategy was successful in disrupting CM1 transcripts, there was also an unanticipated increase in lignin content, a shift towards guaiacyl lignin units, and more xylem vessels with smaller lumen areas, at least in the most severely affected transgenic line. This was accompanied by compensatory expression of the other two CM isoforms, CM2 and CM3, as well as widespread changes in gene expression and metabolism. This study investigates potential redundancy within the CM gene family in the developing xylem of poplar and highlights the pivotal role of chorismate in plant metabolism, development, and physiology.

Tropical milkweed () serves as a host plant for monarch butterflies () and other insect herbivores that can tolerate the abundant cardiac glycosides that are characteristic of this species. Cardiac glycosides, along with additional specialized metabolites, also contribute to the ethnobotanical uses of . To facilitate further research on milkweed metabolism, we assembled the 197-Mbp genome of a fifth-generation inbred line of into 619 contigs, with an N50 of 10 Mbp. Scaffolding resulted in 98% of the assembly being anchored to 11 chromosomes, which are mostly colinear with the previously assembled common milkweed () genome. Assembly completeness evaluations showed that 98% of the BUSCO gene set is present in the genome assembly. The transcriptomes of six tissue types (young leaves, mature leaves, stems, flowers, buds, and roots), with and without defense elicitation by methyl jasmonate treatment, showed both tissue-specific gene expression and induced expression of genes that may be involved in cardiac glycoside biosynthesis. Expression of a gene, the predicted first gene in the cardiac glycoside biosynthesis pathway, was observed only in the stems and roots and was induced by methyl jasmonate. Together, this genome sequence and transcriptome analysis provide important resources for further investigation of the ecological and medicinal uses of .

Accurate and nondestructive estimation of plant biomass is crucial for optimizing plant productivity, but existing methods are often expensive and require complex experimental setups. To address this challenge, we developed an automated system for estimating plant root and shoot biomass over the plant's lifecycle in hydroponic systems. This system employs a robotic arm and turntable to capture 40 images at equidistant angles around a hydroponically grown lettuce plant. These images are then processed into silhouettes and used in voxel-based volumetric 3D reconstruction to produce detailed 3D models. We utilize a space carving method along with a raytracing-based optical correction technique to create high-accuracy reconstructions. Analysis of these models demonstrates that our system accurately reconstructs the plant root structure and provides precise measurements of root volume, which can be calibrated to indicate biomass.

Biotin carboxyl carrier protein (BCCP) is a subunit of the heteromeric acetyl-CoA carboxylase (htACCase), and it chemically links the two half-reactions that constitute the formation of malonyl-CoA from acetyl-CoA, a critical reaction in fatty acid biosynthesis. Because plants are a major source of edible fats and oils, it is important to understand the structural organization of the plant htACCase, relative to its potential to regulate fatty acid biosynthesis in plant plastids. Moreover, unique to the plant htACCase, noncatalytic subunits called biotin attachment domain-containing (BADC) proteins are important in the assembly of the holoenzyme, and they specifically interact with the bcCP and the biotin carboxylase (BC) subunits. We report herein NMR structural studies of the Arabidopsis BCCP isozymes (bcCP1 and BCCP2). We calculated the structure of C-terminal domain of BCCP1 (K-P) and explored structural changes in the BCCP1 protein upon its interactions with bc and BADC. The chemical shift perturbation experiments identified potential surface residues on the BCCP1 protein that may facilitate physical interactions between BC and BADC proteins. These studies indicate that the BADC protein interacts with a "thumb"-like protrusion, which is a common structural feature of the bacterial and plant bcCPs, and thereby acts as a potential "cap" to facilitate the assembly of a BC-BCCP-BADC complex.

Halophytes, plants that thrive in high-salinity environments, host unique microbial communities, including fungal endophytes, which contribute to plant growth and pathogen resistance. This study aimed to isolate, identify, and evaluate the antagonistic potential of fungal endophytes from the halophytic plant , collected from both inland and coastal habitats. Fungal endophytes were isolated, identified via molecular techniques, and tested for antagonistic activity against phytopathogenic fungi using dual-culture assays. The results showed a diverse range of fungal endophytes, with and being the dominant genera. A total of 152 endophytic fungi were isolated from both locations, with 95 isolates coming from coastal plants and 57 from inland species. The isolates exhibited varying degrees of antagonistic activity against phytopathogens, highlighting their potential role in plant protection. Further research is needed to clarify these interactions' mechanisms and investigate their practical applications in agriculture. An endophytic isolate of strain ((AL10) lim10qu) (ON210104.1) exhibited potent in vitro antifungal activity against , a pathogenic fungus affecting tomato plants. Greenhouse experiments demonstrated that the fungus significantly increased both the length of tomato seedlings and the overall plant biomass. Both laboratory-based (in vitro) and field-based (in vivo) evaluations of the strain ((AL10) lim10qu) () against suggest the promising role of endophytes as effective biological control agents. Analysis using Gas Chromatography-Mass Spectrometry of the fungal extract detected around 100 compounds (secondary metabolites). In addition to gradually reducing the need for chemical fungicides, bio-products can also contribute to sustainable agriculture.

The Dectes stem borer, LeConte (Coleoptera: Cerambycidae), can significantly reduce yields by causing significant lodging in soybean. While this stem borer has not been considered a major pest of soybean, damage from it is increasing in the United States Midwest region with no current elite cultivars found resistant. Our objective was to map quantitative trait loci (QTL) that reduce girdled stems caused by Dectes stem borer infection and infestation of Dectes stem borer. A genome-wide association study (GWAS) using 50,000 single nucleotide polymorphisms was used to analyze data from a population of maturity group (MG) V to VII soybean accessions grown in North Carolina, which had been scored for Dectes stem borer larvae infestation and girdled stems caused by Dectes stem borer infestation. The GWAS identified 3 QTL with reduced larvae infestation and 4 QTL for reduced girdled stems. Allele effects ranged from 1% to 9% reduced larvae infestation or girdled stems. The QTL identified and germplasm containing the beneficial alleles can be used for improving resistance to the damage caused by the Dectes stem borer in elite soybean cultivars.

The presence of a selection marker in transgenic plants has raised public concerns regarding health safety. We have developed a CRISPR/Cas9-based DNA delivery system termed transgenic selection-associated fragment elimination (T-SAFE). The T-SAFE system comprises four cassettes: the selection marker, CRISPR/Cas9, spacer-plus-protospacer adjacent motif (SP), and the cargo. The first two cassettes, the selection marker and CRISPR/Cas9, are collectively referred to as SCC. The SCC is flanked by two identical SPs derived from the fruit fly gene, which efficiently facilitate the SCC cleavage and subsequently lead to self-elimination of the selection marker upon integration of exogenous DNA into the plant genome. To inhibit the production of a functional Cas9 protein in bacteria, the IV2 intron of the potato ST-LS1 gene has been incorporated into the Cas9 gene. Additionally, the Cas9 gene is driven by a reproductive cell-specific or inducible promoter to avoid SCC cleavage in nonreproductive plant cells. These innovative features allow the T-SAFE system to achieve an elimination efficiency of the selection marker ranging from 10%-30% in and 5%-8% in rice, with a DNA delivery capacity of approximately 10 kb. This approach offers a safe means for genetically modifying plants.

The health and productivity of plants, particularly those in agricultural and horticultural industries, are significantly affected by timely and accurate disease detection. Traditional manual inspection methods are labor-intensive, subjective, and often inaccurate, failing to meet the precision required by modern agricultural practices. This research introduces an innovative deep transfer learning method utilizing an advanced version of the Xception architecture, specifically designed for identifying plant diseases in roses, mangoes, and tomatoes. The proposed model introduces additional convolutional layers following the base Xception architecture, combined with multiple trainable dense layers, incorporating advanced regularization and dropout techniques to optimize feature extraction and classification. This architectural enhancement enables the model to capture complex, subtle patterns within plant leaf images, contributing to more robust disease identification. A comprehensive dataset comprising 5491 images across four distinct disease categories was employed for the training, validation, and testing of the model. The experimental results showcased outstanding performance, achieving 98% accuracy, 99% precision, 98% recall, and a 98% F1-score. The model outperformed traditional techniques as well as other deep learning-based methods. These results emphasize the potential of this advanced deep learning framework as a scalable, efficient, and highly accurate solution for early plant disease detection, providing substantial benefits for plant health management and supporting sustainable agricultural practices.

Stomatal aperture is driven by changes in turgor of the guard cells that surround the stomatal pore. Epidermal cells immediately surrounding the guard cells are thought to contribute to the kinetics of aperture changes through changes in their turgor that opposes the guard cells and thought their putative roles in solute storage for use by the guard cells. Nonetheless, our knowledge remains fragmentary of surrounding cell mechanics, in large part because the tools and strategies needed to target the surrounding cells independent of the guard cells are limited. Here, we have analyzed sets of promoters for , , and barley that will allow physiological studies of the roles of epidermal cells and also surrounding cells in the case of barley in stomatal behavior. These tissue-specific promoters offer distinct advantages over widely used, constitutive promoters by enabling precise and targeted gene expression within guard cells and the adjacent epidermal cells. As genetic tools, the promoters will have applications in strategies centered on physiological analyses and differential comparisons following expression targeted between the guard cells and the foliar epidermis as a whole. As such, they are well suited to questions around the mechanics of solute and water flux that will advance an understanding of the stomatal complex in these model species.

Light is one of the most critical ecological cues controlling plant growth and development. Plants have evolved complex mechanisms to cope with fluctuating light signals. In Arabidopsis, bHLH transcription factors MYC2, MYC3, and MYC4 have been shown to play a vital role in protecting plants against herbivory and necrotrophic pathogens. While the role of MYC2 in light-mediated seedling development has been studied in some detail, the role of MYC3 and MYC4 still needs to be discovered. Here, we show that MYC4 negatively regulates seedling photomorphogenesis, while the MYC3 function seems redundant. However, the genetic analysis reveals that MYC3/MYC4 together act as positive regulators of seedling photomorphogenic growth as the double mutants showed exaggerated hypocotyl growth compared to the and single mutants and Col-0. Intriguingly, the loss of function in the double mutant background () resulted in further enhancement in the hypocotyl growth than double mutants in WL, BL and FRL, suggesting that MYC2/3/4 together play an essential and positive role in meditating optimal seedling photomorphogenesis. Besides, MYC3/MYC4 genetically and physically interact with HY5 to partially inhibit its function in controlling hypocotyl and photo-pigment accumulation. Moreover, our results suggest that COP1 physically interacts and degrades MYC3 and MYC4 through the 26S proteasomal pathway and controls their response to dark and light for fine-tuning HY5 function and seedling photomorphogenesis.

Abscission is a tightly regulated process in which plants shed unnecessary, infected, damaged, or aging organs, as well as ripe fruits, through predetermined abscission zones in response to developmental, hormonal, and environmental signals. Despite its importance, the underlying mechanisms remain incompletely understood. This study highlights the deleterious effects of abscission on chloroplast ultrastructure in the cells of the tomato flower pedicel abscission zone, revealing spatiotemporal differential gene expression and key transcriptional networks involved in chloroplast vesiculation during abscission. Significant changes in chloroplast structure and vesicle formation were observed 8 and 14 h after abscission induction, coinciding with the differential expression of vesiculation-related genes, particularly with upregulation of () and (). This suggests a possible vesicle transport of chloroplast degrading material for recycling by autophagy-independent senescence-associated vacuoles (SAVs) and CV-containing vesicles (CCVs). Ethylene signaling appears to be involved in the regulation of these processes, as treatment with a competitive inhibitor of ethylene action, 1-methylcyclopropene, delayed vesiculation, reduced the expression of , and increased expression of (). In addition, chloroplast vesiculation during abscission was associated with differential expression of photosynthesis-related genes, particularly those involved in light reactions, underscoring the possible functional impact of the observed structural changes. This work provides new insights into the molecular and ultrastructural mechanisms underlying abscission and offers potential new targets for agricultural or biotechnological applications.

L., a medicinal plant renowned for its pharmaceutical alkaloids, has captivated scientific interest due to its rich secondary metabolite profile. This study explores a novel approach to manipulating alkaloid biosynthesis pathways by integrating virus-induced gene silencing (VIGS) with macerozyme enzyme pretreatment. Targeting key genes in the benzylisoquinoline alkaloid (BIA) pathway (, , , ), the research aimed to elucidate the transformative potential of enzymatic preconditioning in somatic embryo cultures. To address the cell wall barrier, a known limitation in genetic manipulation, macerozyme pretreatment was employed, significantly enhancing gene silencing efficacy. Quantitative reverse transcription PCR analyses revealed significant alterations in gene expression profiles with macerozyme pretreatment, whereas no changes were observed in its absence. The T6ODM + DIOX combination was the most effective, reducing , , and expression by 72%, 65%, and 60%, respectively. Conversely, expression increased by up to 107% in the CODM treatment. Notably, expression displayed dual regulatory dynamics, with suppression (47% decrease in T6ODM + DIOX) and enhancement (49% increase in CODM+DIOX) observed under different conditions. These findings underscore the complex interplay of gene regulation in the morphine biosynthesis pathway. This study highlights the critical role of macerozyme enzymatic pretreatment in overcoming cell wall barriers, enabling effective VIGS applications in somatic suspension cultures. The combination of VIGS and enzymatic pretreatment provides a robust platform for targeted metabolic engineering, offering insights into the regulation of morphine biosynthesis and paving the way for advancements in pharmaceutical alkaloid production and functional genomics in medicinal plants.

The superfamily represents a class of transcription factors involved in plant growth, development, and stress responses. ., also known as safflower, is an important plant whose flowers contain carthamin, an expensive aromatic pigment with various medicinal and flavoring properties. This study aimed to elucidate the roles of these transcription factors in plant growth, metabolic regulation, and environmental adaptation in safflower, providing foundational information and theoretical support for genetic improvement and stress resilience research in this crop. In this study, we identified and characterized the family genes in safflower through a comprehensive genomic analysis. A total of 127 genes were identified and clustered into seven groups and 14 subgroups based on phylogenetic analysis. Multiple sequence alignment revealed that the basic region and two helical structures were highly conserved in most AP2/ERF proteins. -acting elements in the promoters of the genes were analyzed, and a degree of safflower specificity was observed among different safflower species. Tissue-specific expression analysis showed that 23, 21, 15, and 9 genes were most abundantly expressed in the roots, leaves, flowers, and buds, respectively, while only eight genes were highly expressed in all tissues examined. These results indicate that the family genes in safflower are diverse and complex, with distinct expression patterns for different genes in different safflower species. The findings provide important fundamental data for in-depth studies of the growth, development, and stress response mechanisms in safflower.

Currently in wheat breeding, genome wide association studies (GWAS) have successfully revealed the genetic basis of complex traits such as nitrogen use efficiency (NUE) and its biological processes. In the GWAS model, thresholding is common strategy to indicate deviation of expected range of -(s), and it can be used to find the distribution of true positive associations under or over of test statistics. Therefore, the threshold plays a critical role to identify reliable and significant associations in wide genome, while the proportion of false positive results is relatively low. The problem of multiple comparisons arises when a statistical analysis involves multiple simultaneous statistical tests, each of them has the potential to be a discovery. There are several ways to address this problem, including the family-wise error rate and false discovery rate (FDR), raw and adjusted -(s), consideration of threshold coherence and consonance, and the properties of proportional hypothesis tests in the threshold definition. We encountered some limitations in the definition of FDR threshold, particularly in the upper bounds of linear and nonlinear approaches. We emphasize that empirical null distributions based on permutation test can be useful when the assumption of linear or parametric FDR approaches do not hold. Nevertheless, we believe that it is necessary to utilize modern statistical optimization techniques to evaluate the stability and performance of our results and to select significant FDR threshold. By incorporating the neural network algorithm, it is possible to improve the reliability of FDR threshold and increase the probability of identifying true genetic associations while minimizing the risk of false positives in GWAS results.

The plant shikimate pathway directs a significant portion of photosynthetically assimilated carbon into the downstream biosynthetic pathways of aromatic amino acids (AAA) and aromatic natural products. 3-Deoxy-d--heptulosonate 7-phosphate (DAHP) synthase (hereafter DHS) catalyzes the first step of the shikimate pathway, playing a critical role in controlling the carbon flux from central carbon metabolism into the AAA biosynthesis. Previous biochemical studies suggested the presence of manganese- and cobalt-dependent DHS enzymes (DHS-Mn and DHS-Co, respectively) in various plant species. Unlike well-studied DHS-Mn, however, the identity of DHS-Co is still unknown. Here, we show that all three DHS isoforms of exhibit both DHS-Mn and DHS-Co activities in vitro. A phylogenetic analysis of various DHS orthologs and related sequences showed that Arabidopsis 3-deoxy-D--octulosonate-8-phosphate synthase (KDOPS) proteins were closely related to microbial Type I DHSs. Despite their sequence similarity, these Arabidopsis KDOPS proteins showed no DHS activity. Meanwhile, optimization of the DHS assay conditions led to the successful detection of DHS-Co activity from Arabidopsis DHS recombinant proteins. Compared with DHS-Mn, DHS-Co activity displayed the same redox dependency but distinct optimal pH and cofactor sensitivity. Our work provides biochemical evidence that the DHS isoforms of Arabidopsis possess DHS-Co activity.

The strong correlation between reproductive life cycle type and chromosome numbers in green plants has been a long-standing mystery in evolutionary biology. Within green plants, the derived condition of heterosporous reproduction has emerged from the ancestral condition of homospory in disparate locations on the phylogenetic tree at least 11 times, of which three lineages are extant. In all green plant lineages where heterospory has emerged, there has been a significant downsizing in chromosome numbers. This dynamic has been investigated without clear answers for many decades. In this study, we combine known ideas from existing literature with novel methods, tools, and data to generate fresh insights into an old question. Using gene family evolution models and selection analyses, we identified gene families that have undergone significant expansion, contraction, or selection in heterosporous lineages. Alongside lineage-specific genomic changes, our results revealed shared genomic changes/trends among heterosporous lineages. We found expansions in gene families related to developmental regulation, signaling pathways, and stress responses across heterosporous groups. Notably, the MATE efflux family showed consistent expansion and evidence of selection in heterosporous lineages, suggesting a potentially conserved role in heterospory evolution. These findings could provide novel avenues to investigate and probe the underlying mechanism that may underpin the association between heterospory and genomic changes. The general importance of chromosome numbers, structure, and sizes in cellular biology notwithstanding, the association between the emergence of heterosporous reproduction and chromosome number reduction/genome downsizing is not fully understood. It remains unclear why there exists an association between aspects of biology at such disparate levels as reproductive life cycles and chromosome numbers/genome size. Exploring and answering this conundrum of evolutionary biology can add to our broader understanding of life sciences and of biology at different levels. Applying the novel tools and methods emerging from ongoing progress in biotechnology and computational sciences presents an opportunity to make new inroads into this long-standing question.