Hybrid Species Research Articles

The Hybrid History of Zebrafish.

Since the description of zebrafish (Danio rerio) in 1822, the identity of its closest living relative has been unclear. To address this problem, we sequenced the exomes of ten species in genus Danio, using the closely related Devario aequipinnatus as outgroup, to infer relationships across the 25 chromosomes of the zebrafish genome. The majority of relationships within Danio were remarkably consistent across all chromosomes. Relationships of chromosome segments, however, depended systematically upon their genomic location within zebrafish chromosomes. Regions near chromosome centers identified D. kyathit and/or D. aesculapii as the closest relative of zebrafish, while segments near chromosome ends supported only D. aesculapii as the zebrafish sister species. Genome-wide comparisons of derived character states revealed that danio relationships are inconsistent with a simple bifurcating species history but support an ancient hybrid origin of the D. rerio lineage by homoploid hybrid speciation. We also found evidence of more recent gene flow limited to the high recombination ends of chromosomes and several megabases of chromosome 20 with a history distinct from the rest of the genome. Additional insights gained from incorporating genome structure into a phylogenomic study demonstrate the utility of such an approach for future studies in other taxa. The multiple genomic histories of species in the genus Danio have important implications for comparative studies in these morphologically varied and beautiful species and for our understanding of the hybrid evolutionary history of zebrafish.

Ancient gene introgression promotes biodiversity of Nymphaea nouchali in Hainan under environmental changes

AbstractHybridization is common in the plant kingdom. Under the influence of global climate change, an increasing number of hybrid species have experienced significant shifts from the geographical distribution of their parents, making it more difficult to detect ancient gene flows. Nymphaea nouchali is a rare aquatic flower native to Hainan Province. Owing to habitat destruction and human interference, the natural range of N. nouchali has gradually reduced to the point of extinction. To explore how hybrid populations form using RAD‐seq with population genetic analysis, it is shown that there are heterozygous deletions and inbreeding in N. nouchali and a number of ancestral components in Nymphaea lotus, low of genetic differentiation, and little gene flow between N. nouchali and N. lotus, and ancient introgression between ancestors may have been more frequent than gene exchange after the origin of the species. Accordingly, the ancient N. nouchali genotypes were gradually infiltrated by N. lotus genes and their forms changed in Hainan. Potential distribution pattern analysis also predicted the possible distribution of N. lotus in Hainan. This lays a foundation for exploring the natural genetic mechanisms of Nymphaea species formation and evolution.

Breaking the Hybrid Myth of Paphiopedilum wenshanense: Double Bifurcated Divergence Followed by Adaptive Introgression Formed a Morphological Intermediate.

Species with intermediate phenotypes are usually hypothesised to have originated from hybrid speciation. However, other mechanisms, such as double bifurcated speciation with introgression may create the same phenotypes. Clarifying the underlying process generating the observed intermediate morphology is important for conservation efforts, as it provides insights into the species' evolutionary background and adaptability. Here, we utilised genome-wide single nucleotide polymorphism data to test evolutionary history models for Paphiopedilum wenshanense Z. J. Liu & J. Yong Zhang, an orchid species that is widely considered to be a hybrid with intermediate morphology of other two closely related species, P. concolor Pfitzer and P. bellatulum Pfitzer, distributed in Indochina. Our results rejected the hybrid speciation hypothesis and ascribed the intermediate floral phenotypes of P. wenshanese to introgression from P. concolor after it diverged from P. bellatulum during the last glacial maximum. Excluding the geographic covariate, the current genetic mixing rate of P. wenshanense from P. concolor is stronger in the south part of its range and is associated with precipitation in the early summer, indicating that population divergence in P. wenshanense is being driven by adaptive introgression. These genetic patterns also correspond to the observed floral variation across P. wenshaense populations. The introgression from P. concolor, along with the intermediate and varying floral morphologies, may expand the ecological opportunities for P. wenshanense, providing an explanation for why high genetic diversity has been maintained despite a high level of inbreeding and small census population size. Our study provides a framework for future research to investigate the characteristics and underlying mechanisms of intermediate morphologies in general.

First report of Diaporthe caulivora causing Phomopsis stem canker on sunflower (Helianthus annuus L.) in Minnesota.

Cultivated sunflower (Helianthus annuus L.) is a globally important oilseed crop that is grown primarily in the Northern Great Plains region of the United States. In September 2018, sunflower stems exhibiting brown stem lesions centered on the leaf axils and accompanied by pith degradation, consistent with symptoms of Phomopsis stem canker (PSC) disease, were sampled from a commercial field of approximately 520 hectares in Polk County, MN (47°50'24" N, 96.34'13" W). Incidence of PSC in this field was approximately 44%. Ten diseased stem samples were collected from plants spaced at least ten meters apart. Pieces of diseased stems 15 mm in length and 10 mm in width were excised, surface sterilized in 10% sodium hypochlorite and plated onto potato dextrose agar (PDA) containing 50 µg/ml streptomycin. Plates were incubated at 22°C for 3-5 days and hyphal tips of emerging mycelia were transferred twice to new PDA plates. Genomic DNA was extracted from each isolate and polymerase chain reaction (PCR) was carried out using primers specific to either Diaporthe helianthi or D. gulyae, the most common species causing PSC on sunflower (Elverson et al., 2020). Eight isolates were identified as D. helianthi by PCR, while the remaining two isolates, designated H48 and H49, exhibited morphologies on PDA plates distinct from those of D. helianthi or D. gulyae. The ITS region, along with portions of the EF-1α and β-tubulin (TUB) genes were amplified and sequenced, and the sequences were deposited in Genbank (H48: ITS-OP429627.1, EF-1α-OP429589.1, TUB-OP429587.1; H49: ITS-OP429628.1, EF-1α-OP429590.1, TUB-OP429588.1). BLAST analyses using the H48 and H49 ITS sequences revealed 100% identity with D. caulivora isolates including the type specimen CBS 127268 (Genbank accessions AF000567.2 and type specimen MH864501.1). Additionally, 100% identity of EF-1α and TUB sequences from H48 and H49 with those of type specimen CBS 127268 (Genbank accessions KC343771.1 and KC344013.1) were also observed. A multi-locus phylogenetic tree was constructed aligning the ITS, EF-1α, and TUB sequences of H48 and H49 with those of twenty Diaporthe species. Isolates H48 and H49 formed a clade with three other D. caulivora isolates, including the type specimen. Pathogenicity of isolate H49 on sunflower was confirmed by inoculating susceptible sunflower inbred line HA 410 using a stem wound method (Mathew et al., 2018; Thompson et al., 2011; Underwood & Misar, 2024). Plants were inoculated by creating a wound approximately 5 mm deep and 8 mm long using a scalpel and affixing a PDA plug carrying mycelium of isolate H49, D. helianthi isolate H45 known to be pathogenic, or mock inoculum of PDA with no mycelium over the wound using laboratory film. Three independent experimental runs were conducted in which two 6-week-old plants grown in 1-gallon pots under greenhouse conditions at 22 ± 3°C were inoculated with each treatment, resulting in inoculation of six total HA 410 plants per treatment. Isolate H49 formed lesions with mean length of 61.27 mm on HA 410 at 14 days-post-inoculation compared to mean lesion length of 173.05 mm for D. helianthi isolate H45 and no disease symptoms were observed upon mock inoculation. D. caulivora was re-isolated from inoculated stems and confirmed by observing morphological features on PDA plates as well as sequencing the ITS region, fulfilling Koch's postulates. D. caulivora is typically associated with stem canker of soybean and has been previously reported to cause PSC on sunflower in Argentina (Zambelli et al., 2021). Additionally, this species was identified as an endophyte on asymptomatic sunflowers in the U.S., though pathogenicity was not confirmed in this study (Dangel, 2022). To our knowledge, this is the first report of D. caulivora causing Phomopsis stem canker on sunflower in the U.S. It will likely be prudent to monitor this species as a potential causal agent of PSC on sunflower and potentially to determine if sunflower lines resistant to other Diaporthe species are also resistant to D. caulivora should a need arise to deploy resistance to this species in commercial sunflower hybrids.

Detecting hybridization in Chilean species of the genus Baccharis L.

The genus Baccharis in Chile is an extraordinary example of admixture, previously described only morphologically and chemically. In Chile, the genus forms a homoploid complex with at least 16 species and 21 hybrids. Genotyping-by-Sequencing (GBS) was used to clarify the hybrid character of Baccharis × intermedia, which originated from the species B. macraei and B. linearis. Additionally, B. vernalis, another species with a morphological resemblance to B. macraei, was subjected to analysis to ascertain its role in the hybridization process. A total of 11,006 SNPs and 72 individuals were analysed using Treemix, D- and f-statistics, which revealed genetic evidence of hybridization between B. macraei and B. linearis. Furthermore, other genetic indicators, such as a high level of heterozygosity, also provided evidence of the hybrid nature of Baccharis × intermedia. Additionally, one individual exhibited strong genetic proportions derived from B. vernalis, B. macraei, and B. linearis. Distinct individuals were clustered using sparse Non-Negative Matrix Factorization (sNMF) into five distinct groups, representing the described species and the hybrid. B. macraei exhibited division into a northern and a southern subpopulation. The morphological and chemical evidence of the hybrid character of Baccharis × intermedia is corroborated by genetic data. Further, the most likely evolutionary scenario is a hybrid swarm. Genetic differentiation between B. linearis and B. macraei indicates separation prior to secondary contact. The close relationship of B. macraei and B. vernalis was confirmed, suggesting that it may emerge as a vicariant species on different soil types.

Full-length transcriptome reveals alternative splicing regulation pattern of skin color variant in red tilapia (Oreochromis spp.)

The hybrid species red tilapia (Oreochromis spp.) is favored in aquaculture for its bright color, rapid growth, and high market value. However, color variation hinders market development, which is influenced by both genetic and environmental factors. We aim to identify candidate genes and pathways related to pigmentation, uncovering the molecular basis of these color variations in red tilapia by employing third-generation full-length transcriptome sequencing combined with second-generation transcriptome analysis. Our analysis focused on three distinct phenotypic classes: whole pink, pink with black blotch, and whole black. Pigment cell and pigment composition analysis highlighted that the pigment pattern in red tilapia is primarily determined by the distribution and interaction of various pigment cells, including melanophores, xanthophores, erythrophores, and iridophores. The whole pink individuals were characterized by an absence of melanophores and a high concentration of carotenoids, while the whole black individuals exhibited active melanogenesis. Transcriptomic data indicated that genes associated with melanin synthesis (wnt, fzd7, lef, tcf7, tyrp1, oca2, pmel, bmp2, cbs) were up-regulated in whole black individuals, while genes related to carotenoid uptake and deposition (scarb1, ldlr, ttc39b, stard5, stard10, plin2, fitm2, bscl2), as well as pteridine synthesis (gch1, ptps), were more actively expressed in whole pink individuals. Retinol metabolism was identified as a mediator of the interaction between melanin synthesis and carotenoid deposition. Further, alternative splicing (AS) act as a crucial post-transcriptional mechanism regulating body coloration, beyond transcriptional differences. Analysis of AS events identifies multiple splice variants of pigment-related genes (scarb1, pax7, ctnnb1, rxrb, bscl2, gjb10, bmp4, frzb) that exhibit differential expression and splicing patterns across different color phenotypes. Notably, AS events of the scarb1 gene, related to carotenoid deposition, and the polr2d gene, associated with the transcription machinery, may significantly influence pigment deposition patterns in red tilapia. Our work not only deepens the genetic understanding of pigmentation in red tilapia but also establishes a solid foundation for improving color stability through selective breeding and genetic improvement programs across various hybrid species in aquaculture, which is crucial for marketability and consumer attraction.

Screening of Psidium Species and Interspecific Hybrid Progenies for Salinity Stress Tolerance

Screening of Psidium Species and Interspecific Hybrid Progenies for Salinity Stress Tolerance

A newborn F-box gene blocks gene flow by selectively degrading phosphoglucomutase in species hybrids

The establishment of reproductive barriers such as postzygotic hybrid incompatibility (HI) remains the key to speciation. Gene duplication followed by differential functionalization has long been proposed as a major model underlying HI, but little supporting evidence exists. Here, we demonstrate that a newborn F-box gene, Cni-neib-1, of the nematode Caenorhabditis nigoni specifically inactivates an essential phosphoglucomutase encoded by Cbr-shls-1 in its sister species Caenorhabditis briggsae and their hybrids. Zygotic expression of Cni-neib-1 specifically depletes Cbr-SHLS-1, but not Cni-SHLS-1, in approximately 40 min starting from gastrulation, causing embryonic death. Cni-neib-1 is one of thirty-three paralogues emerging from a recent surge in F-box gene duplication events within C. nigoni, all of which are evolving under positive selection. Cni-neib-1 undergoes turnover even among C. nigoni populations. Differential expansion of F-box genes between the two species could reflect their distinctive innate immune responses. Collectively, we demonstrate how recent duplication of genes involved in protein degradation can cause incidental destruction of targets in hybrids that leads to HI, providing an invaluable insight into mechanisms of speciation.

Experimental Insights into the Formation, Reactivity, and Crosstalk of Thionitrite (SNO−) and Perthionitrite (SSNO−)

AbstractHydrogen sulfide (H2S) and nitric oxide (NO) are important gaseous biological signaling molecules that are involved in complex cellular pathways. A number of physiological processes require both H2S and NO, which has led to the proposal that different H2S/NO⋅ crosstalk species, including thionitrite (SNO−) and perthionitrite (SSNO−), are responsible for this observed codependence. Despite the importance of these S/N hybrid species, the reported properties and characterization, as well as the fundamental pathways of formation and subsequent reactivity, remain poorly understood. Herein we report new experimental insights into the fundamental reaction chemistry of pathways to form SNO− and SSNO−, including mechanisms for proton‐mediated interconversion. In addition, we demonstrate new modes of reactivity with other sulfur‐containing potential crosstalk species, including carbonyl sulfide (COS).

Experimental Insights into the Formation, Reactivity, and Crosstalk of Thionitrite (SNO-) and Perthionitrite (SSNO-).

Hydrogen sulfide (H2S) and nitric oxide (NO) are important gaseous biological signaling molecules that are involved in complex cellular pathways. A number of physiological processes require both H2S and NO, which has led to the proposal that different H2S/NO⋅ crosstalk species, including thionitrite (SNO-) and perthionitrite (SSNO-), are responsible for this observed codependence. Despite the importance of these S/N hybrid species, the reported properties and characterization, as well as the fundamental pathways of formation and subsequent reactivity, remain poorly understood. Herein we report new experimental insights into the fundamental reaction chemistry of pathways to form SNO- and SSNO-, including mechanisms for proton-mediated interconversion. In addition, we demonstrate new modes of reactivity with other sulfur-containing potential crosstalk species, including carbonyl sulfide (COS).

Intersectional Hybrids of the Relict Species, Box Huckleberry (Gaylussacia brachycera), with Dwarf Huckleberry (Gaylussacia dumosa)

Gaylussacia brachycera (box huckleberry) is a unique relict species and is the monospecific member of Gaylussacia sect. Vitis-idaea. As part of investigations to understand interspecific crossability in Gaylussacia, pollinations were made between evergreen 2x G. brachycera and deciduous 2x G. dumosa (dwarf huckleberry). The primary pollinations succeeded at a low level and produced four viable hybrids. Three of these hybrids had box huckleberry as the female, and one of them, dwarf huckleberry, as the female. Morphologically, these hybrids were distinct from the parents and were generally intermediate. The fertility of the hybrids was low, but one hybrid flowered sufficiently to allow collection of pollen and to backcross it as a male to box huckleberry. Four BC1 hybrids were produced from this cross. The F1 and BC1 hybrids were all verified using simple sequence repeat markers developed for the closely related genus Vaccinium. These hybrids are notable for being the first recorded hybrids of this rare species, G. brachycera, with any other Gaylussacia species, and these hybrids are expected to lead to a better understanding of species relationships both within Gaylussacia and between Gaylussacia and Vaccinium.

Optimizing Saccharum species hybrid plantlets growth in vitro by using different photon flux density and wavelengths in photoautotrophic system (sugar-free medium)

Optimizing Saccharum species hybrid plantlets growth in vitro by using different photon flux density and wavelengths in photoautotrophic system (sugar-free medium)

Regulatory options for cultivars and hybrids of invasive plant species – the South African experience

Abstract Invasive plant taxa are generally regulated at the species level, without considering infra- or inter-specific variation. However, cultivars or hybrids can be of a lower level of risk, e.g., due to sterility. We evaluate six general approaches to regulating cultivars and hybrids -1) “Globally guilty by association”; 2) “Nationally guilty by association”; 3) “Guilty until proven innocent”; 4) “Negotiated guilt”; 5) “Claimed to be innocent”; and 6) “Innocent until proven guilty”. We discuss these approaches in the context of South Africa (which has a typified “Negotiated guilt” approach). Following negotiations since 2001 between the South African horticultural industry/green industry and legislators, an unofficial consensus list of “presumed sterile” cultivars and hybrids was produced in 2014 containing 187 entities from 34 taxa. In 2020 this was reduced to 157 entities from 16 taxa. But the evidence supporting the original lists and the subsequent revisions were not published. To address this issue, we developed a generic pro-forma for reporting sterility based on observations and/or experiments on: flowering, fruiting, pollen, and seeds; the potential for vegetation propagation; and the potential for genetic changes (including hybridisation and reversion to fertility). We recommend that such information should be incorporated into risk analyses conducted specifically for infra- and inter- specific entities, and only if the risk of a harmful invasion is demonstrated to be acceptably low or can be easily mitigated should such entities be exempted from regulation. This will be time-consuming, but by setting out the evidence clearly, the approach is transparent and provides a clear route for stakeholders to seek exemptions for entities of importance. In summary, although we suspect the simplicity of the “Negotiated guilt” approach is desirable to many stakeholders; in general, and specifically for South Africa, we recommend a shift towards the “Guilty until proven innocent” approach.

Complex Hybridization in a Clade of Polytypic Salamanders (Plethodontidae: Desmognathus) Uncovered by Estimating Higher-Level Phylogenetic Networks.

Reticulation between incipient lineages is a common feature of diversification. We examine these phenomena in the Pisgah clade of Desmognathus salamanders from the southern Appalachian Mountains of the eastern United States. The group contains four to seven species exhibiting two discrete phenotypes, aquatic "shovel-nosed" and semi-aquatic "black-bellied" forms. These ecomorphologies are ancient and have apparently been transmitted repeatedly between lineages through introgression. Geographically proximate populations of both phenotypes exhibit admixture, and at least two black-bellied lineages have been produced via reticulations between shovel-nosed parentals, suggesting potential hybrid speciation dynamics. However, computational constraints currently limit our ability to reconstruct network radiations from gene-tree data. Available methods are limited to level-1 networks wherein reticulations do not share edges, and higher-level networks may be non-identifiable in many cases. We present a heuristic approach to recover information from higher-level networks across a range of potentially identifiable empirical scenarios, supported by theory and simulation. When extrinsic information indicates the location and direction of reticulations, our method can successfully estimate a reduced possible set of non-level-1 networks. Phylogenomic data support a single backbone topology with up to five overlapping hybrid edges in the Pisgah clade. These results suggest an unusual mechanism of ecomorphological hybrid speciation, wherein a binary threshold trait causes some hybrid populations to shift between microhabitat niches, promoting ecological divergence between sympatric hybrids and parentals. This contrasts with other well-known systems in which hybrids exhibit intermediate, novel, or transgressive phenotypes. The genetic basis of these phenotypes is unclear and further data are needed to clarify the evolutionary basis of morphological changes with ecological consequences.

Morphometric Tomographic Analysis of the Head of the Tridactyl Humanoid Specimen from Nasca-Peru

Objective: To report the tomographic and morphometric analysis of the head of the tridactyl humanoid specimen from Nazca, Peru. Method: A qualitative case study approach. RadiAnt DICOM Viewer software version 4.2.1 was used to analyze lengths, diameters, and volume in tomographic images of the head. The morphological biometric analysis applied anthropometric methods using some craniometric points and cephalometric angles. Results and Discussion: As a result, it is reported that there are atypical, strange, and exceptional morphometric imaging features in the cephalic segment of specimen M01. The cephalometric analysis based on the ANB angle revealed a class II skeletal pattern, and according to the Bjorl-Jarabak analysis, it has a convex facial profile with hypodivergent growth (brachyfacial). The patient also has maxillary biprotrusion, severe protrusion of upper and lower incisors, loss of teeth, presence of root remnants, severe dental abrasion, non-assessable canine class, absence of three third molars and ocular protrusion. Based on craniometric points, the SNA angle (Sella, Nasion and Point A) and the SNB angle (Sella, Nasion and Point B) maxillary protrusion and mandibular protrusion were diagnosed. Implications of the research: It is concluded that the combination of uncommon morpho-anatomical features in the maxillofacial and cranial massif do not correspond to a human craniofacial biotype, but could be considered as suggestive findings of morpho-anatomical features typical of a hominid species similar to humans. Originality/Value: The expansion of biometric and morphoanatomical data of the head reinforces the hypothesis that morphologically it would be a non-human hybrid species.

Inference of Phylogenetic Networks from Sequence Data using Composite Likelihood.

While phylogenies have been essential in understanding how species evolve, they do not adequately describe some evolutionary processes. For instance, hybridization, a common phenomenon where interbreeding between two species leads to formation of a new species, must be depicted by a phylogenetic network, a structure that modifies a phylogenetic tree by allowing two branches to merge into one, resulting in reticulation. However, existing methods for estimating networks become computationally expensive as the dataset size and/or topological complexity increase. The lack of methods for scalable inference hampers phylogenetic networks from being widely used in practice, despite accumulating evidence that hybridization occurs frequently in nature. Here, we propose a novel method, PhyNEST (Phylogenetic Network Estimation using SiTe patterns), that estimates binary, level-1 phylogenetic networks with a fixed, user-specified number of reticulations directly from sequence data. By using the composite likelihood as the basis for inference, PhyNEST is able to use the full genomic data in a computationally tractable manner, eliminating the need to summarize the data as a set of gene trees prior to network estimation. To search network space, PhyNEST implements both hill climbing and simulated annealing algorithms. PhyNEST assumes that the data are composed of coalescent independent sites that evolve according to the Jukes-Cantor substitution model and that the network has a constant effective population size. Simulation studies demonstrate that PhyNEST is often more accurate than two existing composite likelihood summary methods (SNaQ and PhyloNet) and that it is robust to at least one form of model misspecification (assuming a less complex nucleotide substitution model than the true generating model). We applied PhyNEST to reconstruct the evolutionary relationships among Heliconius butterflies and Papionini primates, characterized by hybrid speciation and widespread introgression, respectively. PhyNEST is implemented in an open-source Julia package and is publicly available at https://github.com/sungsik-kong/PhyNEST.jl.

A Dominance Hypothesis Argument for Historical Genetic Gains and the Fixation of Heterosis in Octoploid Strawberry.

Heterosis was the catalyst for the domestication of cultivated strawberry (Fragaria × ananassa), an interspecific hybrid species that originated in the 1700s. The hybrid origin was discovered because the phenotypes of spontaneous hybrids transgressed those of their parent species. The transgressions included fruit yield increases and other genetic gains in the twentieth century that sparked the global expansion of strawberry production. The importance of heterosis to the agricultural success of the hybrid species, however, has remained a mystery. Here we show that heterosis has disappeared (become fixed) among improved hybrids within a population (the California population) that has been under long-term selection for increased fruit yield, weight, and firmness. We found that the highest yielding hybrids are among the most highly inbred (59-79%), which seems counterintuitive for a highly heterozygous, outbreeder carrying heavy genetic loads. Although faint remnants of heterosis were discovered, the between-parent allele frequency differences and dispersed favorable dominant alleles necessary for heterosis have decreased nearly genome-wide within the California population. Conversely, heterosis was prevalent and significant among wide hybrids, especially for fruit count, a significant driver of genetic gains for fruit yield. We attributed the disappearance (fixation) of heterosis within the California population to increased homozygosity of favorable dominant alleles and inbreeding associated with selection, random genetic drift, and selective sweeps. Despite historical inbreeding, the highest yielding hybrids reported to-date are estimated to be heterozygous for 20,370-44,280 of 97,000-108,000 genes in the octoploid genome, the equivalent of an entire diploid genome or more.

Rac-2-(2′-Ferrocenyl-2′-hydroxy-n-propyl)-1,3-benzothiazole

The synthesis and characterisation (UV-Vis, IR, HRESI-MS, 1H and 13C NMR spectroscopies, electrochemistry) is reported of the novel title material (1: alternatively named rac-1-(2′-benzothiazolyl)-2-ferrocenyl-2-propanol): a rare example of a ferrocenyl-benzothiazole hybrid species. Compound 1 is produced by the low temperature reaction of acetylferrocene (3) with a solution of the methyl anion derived via the deprotonation of 2-methyl-1,3-benzothiazole. The yield of 1 is moderate (34%) after purification and is an air and thermally stable solid under ambient conditions. Attempts to sublime 1, however, result in decomposition with one of the products being identified (NMR) as 3. The spectroscopic features of 1 are presented. Attempts to obtain suitable crystalline material of 1 for a single crystal X-ray diffraction study were unfortunately unsuccessful. Compound 1 also does not form stable coordination complexes with various metal salts (e.g., Ni[2+], Co[2+], etc.) under the conditions tested.

Optimal conditions for cryopreservation by vitrification of largemouth bass (Micropterus salmoides) embryos

The largemouth bass (Micropterus salmoides) is one of the important freshwater aquaculture species in the world. However, due to limitations on introduction scale, high-density farming, inbreeding, and species hybridization, the germplasm resources of largemouth bass face threats such as degradation and susceptibility to diseases. Therefore, it is urgent to conduct research on the conservation of its original and good germplasm resources. We optimized the conditions of cryopreservation to vitrify and revive largemouth bass embryos, including the mixing ratio of cryoprotectants, embryo stage, equilibration step and temperature, and washing regent. The results showed that the least toxic single, binary, and ternary mixed permeating cryoprotectants were PG, PM (PG: MeOH = 2:1), and PMD (PM: DMSO = 3:1), respectively. The least toxic non-permeating cryoprotectant was 5 % glucose. The optimal vitrification solution selected was PMDG (30 % PMD + 5 % glucose) with an 80.67 % survival rate of embryos. Embryos at the heartbeat stage exhibited strong tolerance to the PMDG solution, which is the optimal embryo stage for cryopreservation. During the equilibration process, either the five-step equilibration method or pre-cooling the cryoprotectant to 4°C could reduce its toxicity. During the washing process, a 0.125 mol·L−1 sucrose solution yielded the best results. Based on the optimized conditions, 650 embryos at the heartbeat stage were subjected to cryopreservation by vitrification, resulting in a total of 350 intact transparent eggs, two of which hatched successfully. The results provide a reference for further improving the efficiency of cryopreservation by vitrification of largemouth bass and other fish species.

Fluctuating selection in a monkeyflower hybrid zone

Abstract While hybridization was viewed as a hindrance to adaptation and speciation by early evolutionary biologists, recent studies have demonstrated the importance of hybridization in facilitating evolutionary processes. However, it is still not well-known what role spatial and temporal variation in natural selection play in the maintenance of naturally occurring hybrid zones. To identify whether hybridization is adaptive between two closely related monkeyflower species, Mimulus guttatus and Mimulus laciniatus, we performed repeated reciprocal transplants between natural hybrid and pure species’ populations. We planted parental genotypes along with multiple experimental hybrid generations in a dry (2021) and extremely wet (2023) year in the Sierra Nevada, CA. By taking fine-scale environmental measurements, we found that the environment of the hybrid zone is more similar to M. laciniatus’s seasonally dry rocky outcrop habitat than M. guttatus’s moist meadows. In our transplants hybridization does not appear to be maintained by a consistent fitness advantage of hybrids over parental species in hybrid zones, but rather a lack of strong selection against hybrids. We also found higher fitness of the drought-adapted species, M. laciniatus, than M. guttatus in both species’ habitats, as well as phenotypic selection for M. laciniatus-like traits in the hybrid habitat in the dry year of our experiment. These findings suggest that in this system, hybridization might function to introduce drought-adapted traits and genes from M. laciniatus into M. guttatus, specifically in years with limited soil moisture. However, we also find evidence of genetic incompatibilities in second generation hybrids in the wetter year, which may balance a selective advantage of M. laciniatus introgression. Therefore, we find that hybridization in this system is both potentially adaptive and costly, and that the interaction of positive and negative selection likely determines patterns of gene flow between these Mimulus species.