Hybrid Species Research Articles

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).

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.

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).

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.

Making Noah's Ark Work for Fishing Cat Conservation: A Blueprint for Connecting Populations across an Interactive Wild Ex Situ Spectrum.

The One Plan Approach advocates for a hybrid species management framework, wherein captive-bred populations are considered metapopulations nested within a broader network of zoos and wild populations Additionally, the Opportunities to Thrive framework aims to enhance animal welfare by addressing the physiological, psychological, and emotional needs of captive individuals, thereby improving conservation outcomes. Here, we present an integrated framework for the conservation of a globally threatened wetland wild cat species, the fishing cat, by synthesizing optimal ex situ management practices and in situ conservation strategies. Further, we examined the genetic constitution of the founder population in a fishing cat captive breeding program that was recently initiated by the West Bengal Zoo Authority, India and conducted a population viability analysis to suggest how best to maintain the genetic diversity of the population. We found that the present genetic diversity of 56% and maximum carrying capacity of the captive population (30 individuals) can be maintained for more than 100 years with a combination of supplementation and harvesting. Keeping stochastic events in mind, the introduction of two adult males and females to the existing population each year will seamlessly allow the harvesting of two adult males and two adult females every alternate year to supplement wild populations. Further, we adopted the proposed integrated framework to delineate recommendations for the supplementation of wild populations in West Bengal. We used environmental criteria known to influence fishing cat occurrence to identify 21 potential reintroduction zones in the Sundarbans landscape and Terai region in northern West Bengal with habitable areas for the fishing cat that are larger than the maximum known species' home range. Our study is timely and insightful because it provides a holistic blueprint for implementing the One Plan Approach in safeguarding a threatened species.

Species limits and hybridization in Andean leaf-eared mice (Phyllotis).

Leaf-eared mice (genus Phyllotis) are among the most widespread and abundant small mammals in the Andean Altiplano, but species boundaries and distributional limits are often poorly delineated due to sparse survey data from remote mountains and high-elevation deserts. Here we report a combined analysis of mitochondrial DNA variation and whole-genome sequence (WGS) variation in Phyllotis mice to delimit species boundaries, to assess the timescale of diversification of the group, and to examine evidence for interspecific hybridization. Estimates of divergence dates suggest that most diversification of Phyllotis occurred during the past 3 million years. Consistent with the Pleistocene Aridification hypothesis, our results suggest that diversification of Phyllotis largely coincided with climatically induced environmental changes in the mid- to late Pleistocene. Contrary to the Montane Uplift hypothesis, most diversification in the group occurred well after the major phase of uplift of the Central Andean Plateau. Species delimitation analyses revealed surprising patterns of cryptic diversity within several nominal forms, suggesting the presence of much undescribed alpha diversity in the genus. Results of genomic analyses revealed evidence of ongoing hybridization between the sister species Phyllotis limatus and P. vaccarum and suggest that the contemporary zone of range overlap between the two species represents an active hybrid zone.

Chromosome-level subgenome-aware de novo assembly provides insight into Saccharomyces bayanus genome divergence after hybridization.

Interspecies hybridization is prevalent in various eukaryotic lineages and plays important roles in phenotypic diversification, adaptation, and speciation. To better understand the changes that occurred in the different subgenomes of a hybrid species and how they facilitate adaptation, we have completed chromosome-level de novo assemblies of all chromosomes for a recently formed hybrid yeast, Saccharomyces bayanus strain CBS380, using Oxford Nanopore Technologies' MinION long-read sequencing. We characterize the S. bayanus genome and compare it with its parent species, Saccharomyces uvarum and Saccharomyces eubayanus, and other S. bayanus genomes to better understand genome evolution after a relatively recent hybridization event. We observe multiple recombination events between the subgenomes in each chromosome, followed by loss of heterozygosity (LOH) in nine chromosome pairs. In addition to maintaining nearly all gene content and synteny from its parental genomes, S. bayanus has acquired many genes from other yeast species, primarily through the introgression of Saccharomyces cerevisiae, such as those involved in the maltose metabolism. Finally, the patterns of recombination and LOH suggest an allotetraploid origin of S. bayanus The gene acquisition and rapid LOH in the hybrid genome probably facilitated its adaptation to maltose brewing environments and mitigated the maladaptive effect of hybridization. This paper describes the first in-depth study using long-read sequencing technology of an S. bayanus hybrid genome, which may serve as an excellent reference for future studies of this important yeast and other yeast strains.

Impact of Cucurbita and Cucumis rootstocks on the performance and quality of Piel de Sapo melon

Melon grafting represent an increasingly important strategy against biotic and abiotic stresses. Cucurbita hybrid rootstocks have been extensively developed for this purpose, but it is necessary to find new alternatives. In this work, several experimental rootstocks of cultivated and wild resources from the Cucurbita and Cucumis genera have been evaluated using Piel de Sapo melon as scion. Cucurbita rootstocks tended to increase fruit size and seed cavity and offered more rounded shapes at the early stages of ripening, but this effect was later minimised. Cucumis rootstocks tended to show less vigour than those of Cucurbita. C. metuliferus hybrid rootstock caused plant collapse at specific environmental conditions, probably due to differences in the growth of rootstock and scion. One of the Cucurbita maxima x C. moschata and the Cucurbita pepo experimental hybrids were also less vigorous, delaying flowering and fruit set, and compromising yield. The effects of Cucurbita and Cucumis rootstocks on sugar and acid accumulation of Piel de Sapo were almost negligible, but they altered the volatile profile. This last impact depended on the specific scion x rootstock interaction and the environmental conditions. Cucumis rootstocks tended to minimise it, especially the hybrid Cucumis melo subsp. melo x C. melo subsp. agrestis Pat 81, which offered a volatile profile highly resembling the non-grafted control. Among Cucurbita alternatives, the commercial hybrid Cucurbita maxima x C moschata had the lowest impact, while other experimental hybrids C. moschata x C. moschata and C. maxima x C. moschata increased levels of alcohols. Among Cucumis rootstocks, the hybrids of wild species Cucumis ficifolius x C. anguria and C. ficifolius x C. myriocarpus, and C. metuliferus had a higher impact on the volatile profile.

Genomic changes and stabilization following homoploid hybrid speciation of the Oxford ragwort Senecio squalidus

Oxford ragwort (Senecio squalidus) is one of only two homoploid hybrid species known to have originated very recently, so it is a unique model for determining genomic changes and stabilization following homoploid hybrid speciation. Here, we provide a chromosome-level genome assembly of S.squalidus with 95% of the assembly contained in the 10 longest scaffolds, corresponding to its haploid chromosome number. We annotated30,249 protein-coding genes and estimated that ∼62% of the genome consists of repetitive elements. We then characterized genome-wide patterns of linkage disequilibrium, polymorphism, and divergence in S.squalidus and its two parental species, finding that (1) linkage disequilibrium is highly heterogeneous, with a region on chromosome 4 showing increased values across all three species but especially in S.squalidus; (2) regions harboring genetic incompatibilities between the two parental species tend to be large, show reduced recombination, and have lower polymorphism in S.squalidus; (3) the two parental species have an unequal contribution (70:30) to the genome of S.squalidus, with long blocks of parent-specific ancestry supporting a very rapid stabilization of the hybrid lineage after hybrid formation; and (4) genomic regions with major parent ancestry exhibit an overrepresentation of loci with evidence for divergent selection occurring between the two parental species on Mount Etna. Our results show that both genetic incompatibilities and natural selection play a role in determining genome-wide reorganization following hybrid speciation and that patterns associated with homoploid hybrid speciation-typically seen in much older systems-can evolve very quickly following hybridization.

Adaptive radiation of the Callicarpa genus in the Bonin Islands revealed through double-digest restriction site-associated DNA sequencing analysis.

The Bonin Islands, comprised of the Mukojima, Chichijima, and Hahajima Islands, are known for their isolated and distinctive habitats, hosting a diverse array of endemic flora and fauna. In these islands, adaptive radiation has played a remarkable role in speciation, particularly evident in the Callicarpa genus that is represented by three species: Callicarpa parvifolia and C. glabra exclusive to the Chichijima Islands, and Callicarpa subpubescens, distributed across the entire Bonin Islands. Notably, C. subpubescens exhibits multiple ecotypes, differing in leaf hair density, flowering time, and tree size. In this study, we aimed to investigate species and ecotype diversification patterns, estimate divergence times, and explore cryptic species within Callicarpa in the Bonin Islands using phenotypic and genetic data (double-digest restriction site-associated DNA sequencing). Genetic analysis revealed that C. parvifolia and C. glabra both formed single, distinct genetic groups. Conversely, C. subpubescens consisted of six genetic groups corresponding to different ecotypes and regions, and a hybrid group resulting from the hybridization between two of these genetic groups. Population demography analysis focusing on six Chichijima and Hahajima Islands-based species/ecotypes indicated that all species and ecotypes except one ecotype diverged simultaneously around 73-77 kya. The star-shaped neighbor-net tree also suggests the simultaneous divergence of species and ecotypes. The species and ecotypes that simultaneously diverged adapted to dry environments and understory forests, suggesting that aridification may have contributed to this process of adaptive radiation. Moreover, leaf morphology, flowering time, and genetic analyses suggested the presence of two cryptic species and one hybrid species within C. subpubescens.

Botryosphaeriaceae species associated with branch dieback and decline of macadamia trees in South Africa

Botryosphaeriaceae species are important latent pathogens causing diseases on trees utilized in forestry and agriculture. In recent years, there has been an increase in the incidence and severity of branch dieback and decline on macadamia trees in South Africa, and species of Botryosphaeriaceae have been considered as a possible cause. Although botryosphaeria dieback has been well-studied in Australia, there is little information regarding these fungi on Macadamia in South Africa. The aims of this study were consequently to (i) identify species of Botryosphaeriaceae from Macadamia branches from main production regions in South Africa, (ii) compare the diversity of species between symptomatic and asymptomatic branches, as well as between different growing regions, (iii) and to consider their relative importance in causing dieback. Eight species and three putative hybrids of the Botryosphaeriaceae were identified based on a phylogenetic comparison of sequence data from the ITS rDNA, tub2, tef-1α and rpb2 loci. These included an unidentified Diplodia sp., and Lasiodiplodia sp., as well as L. gilanensis, L. theobromae, L. pseudotheobromae, Neofusicoccum kwambonambiense, N. luteum, N. parvum and three hybrid species. The unidentified species of Diplodia., Lasiodiplodia sp., L. gilanensis, and N. kwambonambiense are reported for the first time on Macadamia in South Africa. All species showed a potential to cause branch dieback symptoms, with species of Neofusicoccum identified as the most aggressive species. This study revealed a high level of diversity of Botryosphaeriaceae species and illustrates their potential as causal agents of dieback on Macadamia in South Africa.