Sequence Divergence Research Articles

Genetic differentiation in the MAT-proximal region is not sufficient for suppressing recombination in Podospora anserina.

Recombination is advantageous over the long-term, as it allows efficient selection and purging deleterious mutations. Nevertheless, recombination suppression has repeatedly evolved in sex and mating-type chromosomes. The evolutionary causes for recombination suppression and the proximal mechanisms preventing crossing overs are poorly understood. Several hypotheses have recently been suggested based on theoretical models, and in particular that divergence could accumulate neutrally around a sex-determining region and reduce recombination rates, a self-reinforcing process that could foster progressive extension of recombination suppression. We used the ascomycete fungus Podospora anserina for investigating these questions: a 0.8 Mbp region around its mating-type locus is non-recombining, despite being collinear between the two mating types. This fungus is mostly selfing, resulting in highly homozygous individuals, except in the non-recombining region around the mating-type locus that displays differentiation between mating types. Here, we test the hypothesis that sequence divergence alone is responsible for recombination cessation. We replaced the mat- idiomorph by the sequence of the mat+ idiomorph, to obtain a strain that is sexually compatible with the mat- reference strain and isogenic to this strain in the MAT-proximal region. Crosses showed that recombination was still suppressed in the MAT-proximal region in the mutant strains, indicating that other proximal mechanisms than inversions or mere sequence divergence are responsible for recombination suppression in this fungus. This finding suggests that selective mechanisms likely acted for suppressing recombination, or the spread of epigenetic marks, as the neutral model based on mere nucleotide divergence does not seem to hold in P. anserina.

Drosophila telomeric protein Verrocchio is an ortholog of STN1.

In most Eukaryota, telomeres are protected by the CST complex, composed of CTC1, STN1 and TEN1. In Drosophila, instead, another complex is present, composed of Modigliani, Tea and Verrocchio. We performed a search for STN1 orthologs in Arthropoda, in order to verify if Verrocchio can be considered as such. We found that STN1 in Arthropoda is shorter than in other Metazoa and shares the same architecture with Verrocchio. Despite high sequence divergence between human and Drosophila, we have discovered that Verrocchio is an ortholog of STN1.

Molecular phylogeny and comparative chloroplast genome analysis of the type species Crucigenia quadrata

BackgroundThe confused taxonomic classification of Crucigenia is mainly inferred through morphological evidence and few nuclear genes and chloroplast genomic fragments. The phylogenetic status of C. quadrata, as the type species of Crucigenia, remains considerably controversial. Additionally, there are currently no reports on the chloroplast genome of Crucigenia.ResultsIn this study, we utilize molecular phylogenetics and comparative genomics to show that C. quadrata belongs to Chlorophyceae rather than Trebouxiophyceae. The Bayesian and maximum likelihood (ML) phylogenetic trees support a monophyletic group of C. quadrata and Scenedesmaceae (Chlorophyceae) species. Our study presents the first complete chloroplast genome of C. quadrata, which is 197,184 bp in length and has a GC content of 31%. It has a typical quadripartite structure, and the chloroplast genome codons exhibit usage bias. Nucleotide diversity analysis highlights six genes (ccsA, psbF, chlN, cemA, rps3, rps18) as hotspots for genetic variation. Coding gene sequence divergence analyses indicate that four genes (cemA, clpP, psaA, rps3) are subject to positive selection.ConclusionsThe determination of the phylogenetic status and the comparative chloroplast genomic analyses of C. quadrata will not only be useful in enhancing our understanding of the intricacy of Crucigenia taxonomy but also provide the important basis for studying the evolution of the incertae sedis taxa within Trebouxiophyceae.

β3 accelerates microtubule plus end maturation through a divergent lateral interface.

β-tubulin isotypes exhibit similar sequences but different activities, suggesting that limited sequence divergence is functionally important. We investigated this hypothesis for TUBB3/β3, a β-tubulin linked to aggressive cancers and chemoresistance in humans. We created mutant yeast strains with β-tubulin alleles that mimic variant residues in β3 and find that residues at the lateral interface are sufficient to alter microtubule dynamics and response to microtubule targeting agents. In HeLa cells, β3 overexpression decreases the lifetime of microtubule growth, and this requires residues at the lateral interface. These microtubules exhibit a shorter region of EB binding at the plus end, suggesting faster lattice maturation, and resist stabilization by paclitaxel. Resistance requires the H1-S2 and H2-S3 regions at the lateral interface of β3. Our results identify the mechanistic origins of the unique activity of β3 tubulin and suggest that tubulin isotype expression may tune the rate of lattice maturation at growing microtubule plus ends in cells. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

A New Species of Anthocotyle (Polyopisthocotyla: Discocotylidae) from the Gills of the European Hake Merluccius merluccius (Teleostei, Merlucciidae) with a Revision of the Composition of the Genus

This study revisits the taxonomy of Anthocotyle merluccii, originally described from the European hake Merluccius merluccius in the northeast Atlantic, addressing discrepancies in clamp morphology across populations. The original description from Belgium noted near-equal anterior clamp sizes, contrasting with populations from Plymouth (Atlantic) and the Mediterranean, which show marked size differences, questioning their conspecificity. We describe A. radkeaminorum n. sp. from M. merluccius in the western Mediterranean (off Algeria), distinguished from A. merluccii (Belgium) by differing anterior clamp size, genital atrium spine number, and overall anterior clamp dimensions. Populations from Plymouth, previously attributed to A. merluccii, are herein assigned to A. aff. merluccii based on differences in morphometrical traits pending further investigations. Additionally, A. radkeaminorum n. sp. differs from A. americanus in body and clamp size, atrial spine count, and hosts. Based on analysis of morphological and molecular data, we refute the synonymy of A. merluccii and A. americanus, and we reinstate the latter as a valid species. The distinction between A. merluccii and A. americanus was further supported by divergence in cox1 gene sequences analyzed from GenBank (10–11%). Finally, inconsistencies in terminal lappet hook morphology are discussed, cautioning against its use in species delineation. This work highlights the need for continued research to resolve species relationships within this genus.

Biomathematical enzyme kinetics model of prebiotic autocatalytic RNA networks: degenerating parasite-specific hyperparasite catalysts confer parasite resistance and herald the birth of molecular immunity.

Catalysis and specifically autocatalysis are the quintessential building blocks of life. Yet, although autocatalytic networks are necessary, they are not sufficient for the emergence of life-like properties, such as replication and adaptation. The ultimate and potentially fatal threat faced by molecular replicators is parasitism; if the polymerase error rate exceeds a critical threshold, even the fittest molecular species will disappear. Here we have developed an autocatalytic RNA early life mathematical network model based on enzyme kinetics, specifically the steady-state approximation. We confirm previous models showing that these second-order autocatalytic cycles are sustainable, provided there is a sufficient nucleotide pool. However, molecular parasites become untenable unless they sequentially degenerate to hyperparasites (i.e. parasites of parasites). Parasite resistance-a parasite-specific host response decreasing parasite fitness-is acquired gradually, and eventually involves an increased binding affinity of hyperparasites for parasites. Our model is supported at three levels; firstly, ribozyme polymerases display Michaelis-Menten saturation kinetics and comply with the steady-state approximation. Secondly, ribozyme polymerases are capable of sustainable auto-amplification and of surmounting the fatal error threshold. Thirdly, with growing sequence divergence of host and parasite catalysts, the probability of self-binding is expected to increase and the trend towards cross-reactivity to diminish. Our model predicts that primordial host-RNA populations evolved via an arms race towards a host-parasite-hyperparasite catalyst trio that conferred parasite resistance within an RNA replicator niche. While molecular parasites have traditionally been viewed as a nuisance, our model argues for their integration into the host habitat rather than their separation. It adds another mechanism-with biochemical precision-by which parasitism can be tamed and offers an attractive explanation for the universal coexistence of catalyst trios within prokaryotes and the virosphere, heralding the birth of a primitive molecular immunity.

Genome-wide analyses provide insights into genetic variation, phylo- and co-phylogenetic relationships, and biogeography of the entomopathogenic nematode genus Heterorhabditis.

Multigene, genus-wide phylogenetic studies have uncovered the limited taxonomic resolution power of commonly used gene markers, particularly of rRNA genes, to discriminate closely related species of the nematode genus Heterorhabditis. In addition, conflicting tree topologies are often obtained using the different gene markers, which limits our understanding of the phylo- and co-phylogenetic relationships and biogeography of the entomopathogenic nematode genus Heterorhabditis. Here we carried out phylogenomic reconstructions using whole nuclear and mitochondrial genomes, and whole ribosomal operon sequences, as well as multiple phylogenetic reconstructions using various single nuclear and mitochondrial genes. Using the inferred phylogenies, we then investigated co-phylogenetic relationships between Heterorhabditis and their Photorhabdus bacterial symbionts and biogeographical patterns. Robust, well-resolved, and highly congruent phylogenetic relationships were reconstructed using both whole nuclear and mitochondrial genomes. Similarly, whole ribosomal operon sequences proved valuable for phylogenomic reconstructions, though they have limited value to discriminate closely related species. In addition, two mitochondrial genes, the cytochrome c oxidase subunit I (cox-1) and the NADH dehydrogenase subunit 4 (nad-4), and two housekeeping genes, the fanconi-associated nuclease 1 (fan-1) and the serine/threonine-protein phosphatase 4 regulatory subunit 1 (ppfr-1), provided the most robust phylogenetic reconstructions compared to other individual genes. According to our findings, whole nuclear and/or mitochondrial genomes are strongly recommended for reconstructing phylogenetic relationships of the genus Heterorhabditis. If whole nuclear and/or mitochondrial genomes are unavailable, a combination of nuclear and mitochondrial genes can be used as an alternative. Under these circumstances, sequences of multiple conspecific isolates in a genus-wide phylogenetic context should be analyzed to avoid artefactual species over-splitting driven by the high intraspecific sequence divergence of mitochondrial genes and to avoid artefactual species lumping driven by the low interspecific sequence divergence of some nuclear genes. On the other hand, we observed that the genera Heterorhabditis and Photorhabdus exhibit diverse biogeographic patterns, ranging from cosmopolitan species to potentially endemic species, and show high phylogenetic congruence, although host switches have also occurred. Our study contributes to a better understanding of the biodiversity and phylo- and co-phylogenetic relationships of an important group of biological control agents and advances our efforts to develop more tools that are compatible with sustainable and eco-friendly agricultural practices.

Reassessing leatherback turtle lineages and unveiling the first evidence of nuclear mitochondrial DNA in sea turtles

A recent study proposed a new genetic lineage of leatherback turtles (Dermochelys coriacea) based on genetic analysis, environmental history, and local ecological knowledge (LEK), suggesting the existence of two possible species or subspecies on the beaches of Oaxaca, diverging ~ 13.5 Mya. However, this hypothesis may be influenced by nuclear mitochondrial DNA segments (NUMTs), which could have been misamplified as true mtDNA. NUMTs are sequences that have migrated from the mitochondrial genome to the nuclear genome and can co-amplify with mtDNA, potentially leading to erroneous phylogenetic interpretations. We re-examined the evidence for this proposed lineage by reviewing taxonomic literature and additional genetic data. Our analysis indicates that the divergent sequences, previously associated with a new lineage of D. coriacea, are NUMTs rather than true mitochondrial sequences. This is the first evidence of NUMTs in sea turtles. We also proposed a more specific primer for the mitochondrial control region (D-loop) for leatherback turtles to avoid amplifying nuclear copies. Our findings highlight the importance of rigorous genetic validation in conservation genetics, where misinterpretations can significantly impact species management. Finally, we developed a general protocol for detecting NUMTs applicable to any species.

IDENTIFICATION AND GENETIC DIVERSITY OF PINEAPPLE LOCAL ACCESSIONS FROM KEDIRI AND CLOSELY RELATED SPECIES (BROMELIACEAE) BASED ON matK GENE

Ananas comosus L. belongs to the Bromelliaceae family and has around 8 subfamilies. Accessions of local pineapple have spread widely in several areas, one of which is in Kediri. However, some accessions and closely related species (Bromeliaceae) likely remain poorly understood. The highly diverse morphological characters and low levels of sequence divergence in Bromeliaceae have been problematic in resolving the reconstruction of phylogenetic relationships. This study aimed to evaluate the efficiency of the matK gene as a DNA barcode to determine genetic diversity and reconstruct a phylogenetic tree in local pineapple and closely related species (Bromeliaceae). A total of 15 specimens were used in this research, of which 7 were local pineapple accessions from Kediri and 8 were closely related species from the Bromeliaceae family. Whole genome DNA was isolated using a Tiangen kit and amplified using the polymerase chain reaction (PCR) technique using a specific primer. The results showed that matK was easily amplified with a DNA fragment length of 750-800 bp. The average composition value of each base was 36.9% T (U) bases, 30.1% in A bases, 15.9% in G bases, and 17.1% in C. BLAST analysis of the sequences showed that all samples were confirmed and identified according to the species names spread across local pineapple production areas (with query cover of 96-98%). However, the species names registered in the PBG database were confirmed to be different species but still in the same genus, i.e., samples B9-B14. The phylogenetic relationship analysis distinguished each accession into 2 large clades according to sub-families, i.e., Bromelioideae and Pitcairnioideae. This research shows that the matK gene is suitable and recommended as a DNA barcode for local pineapple and closely related species.

Fixation of Expression Divergences by Natural Selection in Arabidopsis Coding Genes.

Functional divergences of coding genes can be caused by divergences in their coding sequences and expression. However, whether and how expression divergences and coding sequence divergences coevolve is not clear. Gene expression divergences in differentiated cells and tissues recapitulate developmental models within a species, while gene expression divergences between analogous cells and tissues resemble traditional phylogenies in different species, suggesting that gene expression divergences are molecular traits that can be used for evolutionary studies. Using transcriptomes and evolutionary proxies to study gene expression divergences among differentiated cells and tissues in Arabidopsis, expression divergences of coding genes are shown to be strongly anti-correlated with phylostrata (gene ages), indicators of selective constraint Ka/Ks (nonsynonymous replacement rate/synonymous substitution rate) and indicators of positive selection (frequency of loci with Ka/Ks > 1), but only weakly or not correlated with indicators of neutral selection (Ks). Our results thus suggest that expression divergences largely coevolve with coding sequence divergences, suggesting that expression divergences of coding genes are selectively fixed by natural selection but not neutral selection, which provides a molecular framework for trait diversification, functional adaptation and speciation. Our findings therefore support that positive selection rather than negative or neutral selection is a major driver for the origin and evolution of Arabidopsis genes, supporting the Darwinian theory at molecular levels.

Comparative Analysis of Drosophila Bam and Bgcn Sequences and Predicted Protein Structural Evolution.

The protein encoded by the Drosophila melanogaster gene bag of marbles (bam) plays an essential role in early gametogenesis by complexing with the gene product of benign gonial cell neoplasm (bgcn) to promote germline stem cell daughter differentiation in males and females. Here, we compared the AlphaFold2 and AlphaFold Multimer predicted structures of Bam protein and the Bam:Bgcn protein complex between D. melanogaster, D. simulans, and D. yakuba, where bam is necessary in gametogenesis to that in D. teissieri, where it is not. Despite significant sequence divergence, we find very little evidence of significant structural differences in high confidence regions of the structures across the four species. This suggests that Bam structure is unlikely to be a direct cause of its functional differences between species and that Bam may simply not be integrated in an essential manner for GSC differentiation in D. teissieri. Patterns of positive selection and significant amino acid diversification across species is consistent with the Selection, Pleiotropy, and Compensation (SPC) model, where detected selection at bam is consistent with adaptive change in one major trait followed by positively selected compensatory changes for pleiotropic effects (in this case perhaps preserving structure). In the case of bam, we suggest that the major trait could be genetic interaction with the endosymbiotic bacteria Wolbachia pipientis. Following up on detected signals of positive selection and comparative structural analysis could provide insight into the distribution of a primary adaptive change versus compensatory changes following a primary change.

The pseudoknot structure of a viral RNA reveals a conserved mechanism for programmed exoribonuclease resistance.

Exoribonuclease-resistant RNAs (xrRNAs) are viral RNA structures that block degradation by cellular 5'-3' exoribonucleases to produce subgenomic viral RNAs during infection. Initially discovered in flaviviruses, xrRNAs have since been identified in wide range of RNA viruses, including those that infect plants. High sequence variability among viral xrRNAs raises questions about the shared molecular features that characterize this functional RNA class. Here, we present the first structure of a plant-virus xrRNA in its active exoribonuclease-resistant conformation. The xrRNA forms a 9 base pair pseudoknot that creates a knot-like topology similar to that of flavivirus xrRNAs, despite lacking sequence similarity. Biophysical assays confirm a compact pseudoknot structure in solution, and functional studies validate its relevance both in vitro and during infection. Our study reveals how viral RNAs achieve a common functional outcome through highly divergent sequences and identifies the knot-like topology as a defining feature of xrRNAs.

Molecular and morphological assessment of the Liopholis inornata (Rosén, 1905) species group (Reptilia: Squamata: Scincidae), with descriptions of two new species from northern Western Australia

Molecular genetic and morphological assessments were undertaken on the Liopholis inornata species group of skinks that occur on sandy soils in both mesic and arid regions of Australia. The primary objective was the taxonomic identification of two outlying peripheral populations from the North-West Cape and Purnululu National Park, in northern Western Australia. To provide adequate context, molecular genetic and morphometric variation was assessed across the wide geographic range of L. inornata, a taxon that is strongly phylogeographically structured. It was also necessary to reassess the taxonomic identity of two previously named taxa from central Australia, L. s. slateri (Storr) and L. s. virgata (Storr). Phylogenetic analyses of nuclear SNPs from extant populations confirm that L. inornata, L. s. slateri and the two outlying populations from northern Western Australia are each distinct evolutionary lineages. Prior to analyses of the extent of morphological divergence between the lineages, we assessed the extent of morphometric differences between the sexes using two well-sampled species, L. inornata and L. striata (Sternfeld) from Western Australian populations. We determined that while males have relatively longer, wider and deeper heads throughout life these differences are relatively slight and the data from both sexes could be pooled for subsequent morphological interspecific comparisons. The two outlying populations are distinguished from congeners based on phylogenetic relationships and divergence in nuclear nucleotide sequences and distinctive morphometric and colour attributes and thus represent new species. The new species both occur in biogeographically significant areas that contain several other endemic reptile species. Liopholis s. virgata, for which we currently lack genetic data, is morphometrically and chromatically different from all the other taxa and very likely represents a distinct species that is potentially extinct as it has not been sighted for more than a century.

Meira qingyuanensis sp. nov., an anamorphic yeast species isolated from plant leaves.

Two yeast strains, NYNU 224191 and NYNU 22411, were isolated from plant leaves of Cyclobalanopsis jenseniana and Schisandra sp. harvested from Qingyuan Mountain, Quanzhou City, Fujian Province, southeast China. Molecular phylogenetic analyses demonstrated that these two strains represent a novel species in the genus Meira. They are more closely related to M. marina, M. nashicola, M. plantarum and M. pileae than other Meira species based on phylogenetic analysis of the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit (LSU) rRNA gene. The novel species differed from its closest relatives, M. marina, M. nashicola, M. plantarum and M. pileae, by 1.8-2% sequence divergence (11-12 substitutions and 1-2 gaps) in the D1/D2 domain and over 6.7% sequence divergence (40-60 substitutions and 53-77 gaps) in the ITS region, respectively. We propose the name Meira qingyuanensis sp. nov. (Holotype CICC 33567; Mycobank MB 855328) for the new species, which differs phenotypically from M. marina, M. plantarum and M. pileae in its ability to assimilate methyl-α-d-glucoside and the inability to assimilate salicin, and from M. nashicola in its ability to assimilate nitrate and nitrite and grow at 30 °C.

Evaluation of VP4-VP2 sequencing for molecular typing of human enteroviruses.

Enteroviruses and rhinoviruses are highly diverse, with over 300 identified types. Reverse transcription-polymerase chain reaction (RT-PCR) assays targeting their VP1, VP4, and partial VP2 (VP4-pVP2) genomic regions are used for detection and identification. The VP4-pVP2 region is particularly sensitive to RT-PCR detection, making it efficient for clinical specimen analysis. However, a standard type identification method using this region is lacking. This study aimed to establish such a method by examining the divergence of VP4-pVP2 amino acid sequences between enterovirus and rhinovirus prototypes. Pairwise analysis of 249 types indicated a 95% threshold for enterovirus intra-species identification but not for rhinovirus prototypes. Protein BLAST search analyses of representative enterovirus prototypes, including EV-A71, EV-D68, CVA6, CVA10, CVA16, and polioviruses (PVs), validated the 95% threshold for typing, with a few exceptions such as PV1-PV2 and CVA6-CVA10, as well as some EV-C types. This study proposes a criterion for typing based on VP4-pVP2 amino acids, which can aid in rapid enterovirus diagnosis during routine clinical or environmental surveillance and emergency outbreaks. Our research confirms the reliability of the suggested VP4-pVP2-based threshold for typing and its potential application in laboratory settings.

A review of the genus Pempheris (Teleostei, Pempheridae) found in Japan and Taiwan.

Species of the genus Pempheris Cuvier, 1829 (Pempheridae) from Japan and Taiwan are taxonomically reviewed based on morphology supported by molecular phylogenetic analysis. Ten species are recognized from these countries: Pempherisadusta Bleeker, 1855, Pempherisfamilia Koeda & Motomura, 2017, Pempherisjaponica Döderlein, 1883, Pempherisnyctereutes Jordan & Evermann, 1902, Pempherisoualensis Cuvier, 1831, Pempherissasakii Jordan & Hubbs, 1925, Pempherisschwenkii Bleeker, 1877, Pempherisufuagari Koeda, Yoshino & Tachihara, 2013, Pempherisvanicolensis Cuvier, 1831, Pempherisxanthoptera Tominaga, 1963. Nine of them are distributed in Japan, and five of them in Taiwan. Pempherissasakii and P.xanthoptera, nominal species that have been regarded as invalid are revalidated, redescribed with diagnoses based on examinations of the holotypes and the specimens collected from Japan. Pempherissasakii is morphologically similar to P.nyctereutes and has been thought to be a senior synonym of the latter, but the comparison of the holotypes and non-types of both species revealed that the former species is distinguishable from the latter species in having fewer counts of body scales, also genetically supported with a 3.1% mitochondrial DNA sequence divergence. Pempherisxanthoptera is similar to P.schwenkii, but the coloration of their caudal fins is different, and the genetic analysis supported the difference. The distributions of all species of the genus Pempheris in Japanese waters are also described, based on the specimen localities from literature and new material herein.

Conservation of function without conservation of amino acid sequence in intrinsically disordered transcriptional activation domains.

Protein function is canonically believed to be more conserved than amino acid sequence, but this idea is only well supported in folded domains, where highly diverged sequences can fold into equivalent 3D structures. In contrast, intrinsically disordered protein regions (IDRs) do not fold into a stable 3D structure, thus it remains unknown when and how function is conserved for IDRs that experience rapid amino acid sequence divergence. As a model system for studying the evolution of IDRs, we examined transcriptional activation domains, the regions of transcription factors that bind to coactivator complexes. We systematically identified activation domains on 502 orthologs of the transcriptional activator Gcn4 spanning 600 MY of fungal evolution. We find that the central activation domain shows strong conservation of function without conservation of sequence. This conservation of function without conservation of sequence is facilitated by evolutionary turnover (gain and loss) of key acidic and aromatic residues, the positions most important for function. This high sequence flexibility of functional orthologs mirrors the physical flexibility of the activation domain coactivator interaction interface, suggesting that physical flexibility enables evolutionary plasticity. We propose that turnover of short functional elements, sometimes individual amino acids, is a general mechanism for conservation of function without conservation of sequence during IDR evolution.

Extending evolutionary forecasts across bacterial species.

Improving evolutionary forecasting requires progressing from studying repeated evolution of a single genotype under identical conditions to formulating broad principles. These principles should enable predictions of how similar species will adapt to similar selective pressures. Evolve-and-resequence experiments with multiple species allow testing forecasts on different biological levels and elucidating the causes for failed predictions. Here, we show that forecasts for adaptation to static culture conditions can be extended to multiple species by testing previous predictions for Pseudomonas syringae and Pseudomonas savastanoi. In addition to sequence divergence, these species differ in their repertoire of biofilm regulatory genes and structural components. Consistent with predictions, both species repeatedly produced biofilm mutants with a wrinkly spreader phenotype. Predominantly, mutations occurred in the wsp operon, with less frequent promoter mutations near uncharacterized diguanylate cyclases. However, mutational patterns differed on the gene level, which was explained by a lack of conservation in relative fitness of mutants between more divergent species. The same mutation was the most frequent for both species suggesting that conserved mutation hotspots can increase parallel evolution. This study shows that evolutionary forecasts can be extended across species, but that differences in the genotype-phenotype-fitness map and mutational biases limit predictability on a detailed molecular level.

Tubulin sequence divergence is associated with the use of distinct microtubule regulators.

Diverse eukaryotic cells assemble microtubule networks that vary in structure and composition. While we understand how cells build microtubule networks with specialized functions, we do not know how microtubule networks diversify across deep evolutionary timescales. This problem has remained unresolved because most organisms use shared pools of tubulins for multiple networks, making it difficult to trace the evolution of any single network. In contrast, the amoeboflagellate Naegleria expresses distinct tubulin genes to build distinct microtubule networks: while Naegleria builds flagella from conserved tubulins during differentiation, it uses divergent tubulins to build its mitotic spindle. This genetic separation makes for an internally controlled system to study independent microtubule networks in a single organismal and genomic context. To explore the evolution of these microtubule networks, we identified conserved microtubule-binding proteins and used transcriptional profiling of mitosis and differentiation to determine which are upregulated during the assembly of each network. Surprisingly, most microtubule-binding proteins are upregulated during only one process, suggesting that Naegleria uses distinct component pools to specialize its microtubule networks. Furthermore, the divergent residues of mitotic tubulins tend to fall within the binding sites of differentiation-specific microtubule regulators, suggesting that interactions between microtubules and their binding proteins constrain tubulin sequence diversification. We therefore propose a model for cytoskeletal evolution in which pools of microtubule network components constrain and guide the diversification of the entire network, so that the evolution of tubulin is inextricably linked to that of its binding partners.

Testing the evolutionary theory of inversion polymorphisms in the yellow monkeyflower (Mimulus guttatus)

Chromosomal inversions have been implicated in a remarkable range of natural phenomena, but it remains unclear how much they contribute to standing genetic variation. Here, we evaluate 64 inversions that segregate within a single natural population of the yellow monkeyflower (Mimulus guttatus). Nucleotide diversity patterns confirm low internal variation for the derived orientation (predicted by recent origin), elevated diversity between orientations (predicted by natural selection), and localized fluctuations (predicted by gene flux). Sequence divergence between orientations varies idiosyncratically by position, not following the suspension bridge pattern predicted if the breakpoints are the targets of selection. Genetic variation in gene expression is not inflated close to inversion breakpoints but is clearly partitioned between orientations. Like sequence variation, the pattern of expression variation suggests that the capture of coadapted alleles is more important than the breakpoints for the fitness effects of inversions. This work confirms several evolutionary predictions for inversion polymorphisms, but clarity emerges only by synthesizing estimates across many loci.