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The work was supported by a grant from the Spanish Ministerio de Ciencia e Innovación, PID2020-119163 GB-I00, and SG benefited from a Ramón y Cajal contract (RYC-2014–16608), both funded by MCIN/AEI/10.13039/501100011033., Introduction Materials and methods Results and discussion Data availability References Acknowledgements Funding Author information Ethics declarations Additional information Rights and permissions About this article

Summary While Sherwin J. Carlquist (1930–2021) did not originate the concept of long-distance dispersal and its role in evolution — a major pillar in Darwin’s theory (1859) — he almost single-handedly turned research on dispersal to insular habitats into an empirical and experimental research area. This contribution explains how and why this occurred based on Carlquist’s own papers and personal account, and provides a brief assessment of the historical context of his research on long-distance dispersal. I end on a personal note; in 1981, when I was a graduate student, Carlquist participated in a symposium on ‘Dispersal and Distribution’ in Hamburg, and the paper he gave there on intercontinental dispersal greatly influenced my own work.

A special‐purpose Committee on DNA Sequences as Types was established at the XIX International Botanical Congress (IBC) in Shenzhen, China, in 2017, with a mandate to report to the XX IBC in Madrid in 2024 with recommendations on a preferred course of action with respect to potential amendments of the International Code of Nomenclature for algae, fungi, and plants to allow DNA sequences as types. This is the first in an expected series of papers from the Special‐purpose Committee on this issue. We set out the background to the establishment of the Committee, explore key issues around typification that are pertinent to the question of DNA sequences as types, enumerate pros and cons of allowing DNA sequences as types, and foreshadow options for future discussion and potential recommendations.

Microscopically dimorphic sex chromosomes in plants are rare, reducing our ability to study them. One difficulty has been the paucity of cultivatable species pairs for cytogenetic, genomic and experimental work. Here, we study the newly recognized sisters Coccinia grandis and Coccinia schimperi, both with large Y chromosomes as we here show for Co. schimperi. We built genetic maps for male and female Co. grandis using a full-sibling family, inferred gene sex-linkage, and, with Co. schimperi transcriptome data, tested whether X- and Y-alleles group by species or by sex. Most sex-linked genes for which we could include outgroups grouped the X- and Y-alleles by species, but some 10% instead grouped the two species' X-alleles. There was no relationship between XY synonymous-site divergences in these genes and gene position on the non-recombining part of the X, suggesting recombination arrest shortly before or after species divergence, here dated to about 3.6 Ma. Coccinia grandis and Co. schimperi are the species pair with the most heteromorphic sex chromosomes in vascular plants (the condition in their sister remains unknown), and future work could use them to study mechanisms of Y chromosome enlargement and parallel degeneration, or to test Haldane's rule about lower hybrid fitness in the heterogametic sex. This article is part of the theme issue ‘Sex determination and sex chromosome evolution in land plants’.

Iconographic evidence from Egypt suggests that watermelon pulp was consumed there as a dessert by 4,360 BP. Earlier archaeobotanical evidence comes from seeds from Neolithic settlements in Libya, but whether these were watermelons with sweet pulp or other forms is unknown. We generated genome sequences from 6,000- and 3,300-year-old seeds from Libya and Sudan, and from worldwide herbarium collections made between 1824 and 2019, and analyzed these data together with resequenced genomes from important germplasm collections for a total of 131 accessions. Phylogenomic and population-genomic analyses reveal that (1) much of the nuclear genome of both ancient seeds is traceable to West African seed-use “egusi-type” watermelon (Citrullus mucosospermus) rather than domesticated pulp-use watermelon (Citrullus lanatus ssp. vulgaris); (2) the 6,000-year-old watermelon likely had bitter pulp and greenish-white flesh as today found in C. mucosospermus, given alleles in the bitterness regulators ClBT and in the red color marker LYCB; and (3) both ancient genomes showed admixture from C. mucosospermus, C. lanatus ssp. cordophanus, C. lanatus ssp. vulgaris, and even South African Citrullus amarus, and evident introgression between the Libyan seed (UMB-6) and populations of C. lanatus. An unexpected new insight is that Citrullus appears to have initially been collected or cultivated for its seeds, not its flesh, consistent with seed damage patterns induced by human teeth in the oldest Libyan material.

PREMISE State‐sponsored weather stations became ubiquitous by the 1880s, yet many old climate data and phenological observations still need to be digitized and made accessible. METHODS We here make available flowering times for 450 species of herbs and shrubs gathered in 1844 by Carl Friedrich Philipp von Martius (1794–1868), director of the Munich Botanical Garden. The data formed part of the world’s third‐oldest phenological monitoring network as we explain in a brief overview of the history of such networks. Using data from one of the world’s oldest continuously functioning weather stations, Hohenpeißenberg, we relate temperature to flowering in three species with short flowering times and herbarium collections made since 1844 within the city’s perimeter, namely, Anemone patens, A. pulsatilla, and Arum maculatum. RESULTS Mean advances in flowering dates were 1.3–2.1 days/decade or 3.2–4.2 days/1°C warming. These advances are in keeping with similar advances in other European herbs during more recent periods. CONCLUSIONS Future studies might use the 1844 flowering data made available here as a source of information on the availability of particular flowers for specialized pollinators including insects looking for oviposition sites, such as the Psychoda flies that become trapped in Arum inflorescences. Another use of Martius’s 1844 data would be their incorporation into larger‐scale analyses of flowering in southern‐central Europe., American Journal of Botany, 108 (4), ISSN:1914-2016, ISSN:0002-9122

Hundreds of land plant lineages have independently evolved separate sexes in either gametophytes (dioicy) or sporophytes (dioecy), but 43% of all dioecious angiosperms are found in just 34 entirely dioecious clades, suggesting that their mode of sex determination evolved a long time ago. Here, we review recent insights on the molecular mechanisms that underlie the evolutionary change from individuals that each produce male and female gametes to individuals specializing in the production of just one type of gamete. The canonical model of sex chromosome evolution in plants predicts that two sex-determining genes will become linked in a sex-determining region (SDR), followed by expanding recombination suppression, chromosome differentiation and, ultimately, degeneration. Experimental work, however, is showing that single genes function as master regulators in model systems, such as the liverwort Marchantia and the angiosperms Diospyros and Populus. In Populus, this type of regulatory function has been demonstrated by genome editing. In other systems, including Actinidia, Asparagus and Vitis, two coinherited factors appear to independently regulate female and male function, yet sex chromosome differentiation has remained low. We discuss the best-understood systems and evolutionary pathways to dioecy, and present a meta-analysis of the sizes and ages of SDRs. We propose that limited sexual conflict explains why most SDRs are small and sex chromosomes remain homomorphic. It appears that models of increasing recombination suppression with age do not apply because selection favours mechanisms in which sex determination depends on minimal differences, keeping it surgically precise.

The increase in managed honeybees (Apis mellifera) in many European cities has unknown effects on the densities of wild bees through competition. To investigate this, we monitored honeybees and non-honeybees from 01 April to 31 July 2019 and 2020 at 29 species of plants representing diverse taxonomic and floral-functional types in a large urban garden in the city of Munich in which the same plant species were cultivated in both years. No bee hives were present in the focal garden, and all bee hives in the adjacent area were closely monitored by interviewing the relevant bee keepers in both 2019 and 2020. Honeybee numbers were similar in April of both years, but increased from May to July 2020 compared to 2019. The higher densities correlated with a significant increase in shifts from wild bee to honeybee visits in May/June/July, while visitor spectra in April 2019 and 2020 remained the same. Most of the species that experienced a shift to honeybee visits in 2020 were visited mostly or exclusively for their nectar. There were no shifts towards increased wild bee visits in any species. These results from a flower-rich garden have implications for the discussion of whether urban bee keeping might negatively impact wild bees. We found clear support that high honeybee densities result in exploitative competition at numerous types of flowers.

SummaryAnimal-pollinated plants have to get pollen to a conspecific stigma while protecting it from getting eaten. We provide experimental evidence that touch-sensitive stamens function in (i) enhancing pollen export and (ii) reducing pollen loss to thieves. Stamens of Berberis and Mahonia are inserted between paired nectar glands and when touched by an insect’s tongue rapidly snap forward so that their valvate anthers press pollen on the insect’s tongue or face. We immobilized the stamens in otherwise unmodified flowers and studied pollen transfer in the field and under enclosed conditions. On flowers with immobilized stamens, the commonest bee visitor stayed up to 3.6x longer, yet removed 1.3x fewer pollen grains and deposited 2.1x fewer grains on stigmas per visit. Self-pollen from a single stamen hitting the stigma amounted to 6% of the grains received from single bee visits. Bees discarded pollen passively placed on their bodies, likely because of its berberine content; nectar has no berberine. Syrphid flies fed on both nectar and pollen, taking more when stamens were immobilized. Pollen-tracking experiments in two species showed that mobile-stamen-flowers donate pollen to many more recipients. These results demonstrate another mechanism by which plants simultaneously meter out their pollen and reduce pollen theft.HighlightsStamens that snap forward when triggered by a flower visitor may serve to meter out pollen, scare away pollen thieves, or place pollen more accurately.We tested these hypotheses by experimentally immobilizing all six stamens in numerous flowers of Berberis and Mahonia species in the field and under enclosed conditions.In flowers with immobilized stamens, the commonest bee species stayed up to 3.6x longer, yet removed 1.3x fewer pollen grains and deposited 2.1x fewer grains on stigmas per visit. Mobile stamens exported their pollen to significantly more neighboring flowers.Graphic abstractBehaviour and pollen transfer after flower visitors received a beating on the tongue or in the face by the forward-snapping stamens of Berberis. Stamens only snap forward if their filament basis is touched by an insect tongue.

Animal-pollinated plants have to get pollen to a conspecific stigma while protecting it from getting eaten. Touch-sensitive stamens, which are found in hundreds of flowering plants, are thought to function in enhancing pollen export and reducing its loss, but experimental tests are scarce. Stamens of Berberis and Mahonia are inserted between paired nectar glands and when touched by an insect’s tongue rapidly snap forward so that their valvate anthers press pollen on the insect’s tongue or face. We immobilized the stamens in otherwise unmodified flowers and studied pollen transfer in the field and under enclosed conditions. On flowers with immobilized stamens, the most common bee visitor stayed up to 3.6× longer, yet removed 1.3× fewer pollen grains and deposited 2.1× fewer grains on stigmas per visit. Self-pollen from a single stamen hitting the stigma amounted to 6% of the grains received from single bee visits. Bees discarded pollen passively placed on their bodies, likely because of its berberine content; nectar has no berberine. Syrphid flies fed on both nectar and pollen, taking more when stamens were immobilized. Pollen-tracking experiments in two Berberis species showed that mobile-stamen-flowers donate pollen to many more recipients. These results demonstrate another mechanism by which plants simultaneously meter out their pollen and reduce pollen theft.

In 1917, sex chromosomes in plants were discovered in a liverwort with hetermorphic U and V chromosomes. Such heteromorphy is unexpected because, unlike the XY chromosomes in diploid-dominant plants, in haploid-dominant plants the female U and the male V chromosomes experience largely symmetrical potential recombination environments. Here we use molecular cytogenetics and super-resolution microscopy to study Frullania dilatata, a liverwort with one male and two female sex chromosomes. We applied a pipeline to Illumina sequences to detect abundant types of repetitive DNA and developed FISH probes to microscopically distinguish the sex chromosomes. We also determined the phenotypic population sex ratio because biased ratios have been reported from other liverworts with heteromorphic sex chromosomes. Populations had male-biased sex ratios. The sex chromosomes are monocentric, and of 14 probes studied (eight satellites, five transposable elements and one plastid region), four resulted in unique signals that differentiated the sex chromosomes from the autosomes and from each other. One FISH probe selectively marked the centromeres of both U chromosomes, so we could prove that during meiosis each U chromosome associates with one of the opposite telomeres of the V chromosome, resulting in a head-to-head trivalent. The similarity of the two U chromosomes to each other in size and in their centromere FISH signal positions points to their origin via a non-disjunction event (aneuploidy), which would fit with the general picture of sex chromosomes rarely crossing-over and being prone to suffer from non-disjunction.

Previous work has shown that among 428 species of bees occurring in Germany, decline or extinction over the past 40 years have been correlated with late-season emergence and restricted habitats, while other factors, such as pollen specialization, body size, nesting sites, and sociality, played no role in models that included a phylogeny of these bees. Doing best are spring-flying, city-dwelling species. Building on these results, we here investigate changes in bee diversity from the 1990s to 2018 at three protected sites within the city perimeter of Munich, focusing on the correlates of flight season (spring or summer), flight duration (in months), and number of habitats (one or two vs. three to six). Local species pools were assessed against the total species pool from 1795 onwards. Twenty years ago, 150 species were present at one or more of the sites, while in 2017/2018, this was true of 188 species, with the increase at two sites being of similar proportion. In two of the three areas, broad habitat use was positively correlated with persistence. Flight season or duration had no statistical effect. These results underscore the function of urban protected sites in bee conservation and imply that summer food shortages, which negatively affect bees in agricultural areas, play no role in urbanized regions so that late-season flight is not an extinction handicap.

AIM: Trees need to avoid frost damage to their young leaves by leafing out after the occurrence of the last frost, yet they also need to start photosynthesis early in the season to achieve sufficient growth. This trade‐off leads to the hypothesis that ‘safety margins’ against spring frost should become shorter, the longer the winter duration, perhaps reaching an asymptotic limit where frost damage would occur in most years. Physiologically, shorter safety margins in high‐latitude ecotypes might be achieved by lower degree‐day requirements for leaf‐out, compared to low‐latitude ecotypes. LOCATION: Europe. TIME PERIOD: 1902–2009. MAJOR TAXA STUDIED: Temperate trees. METHODS: Using herbarium collections of Acer platanoides, Carpinus betulus, Fagus sylvatica and Prunus spinosa made over 108 years at 40° to 60° N latitude, we related historic leaf‐out dates to winter and spring temperatures (chilling and degree‐days), winter duration, and date of last frost occurrence in the relevant years and locations. RESULTS: In all species, frost safety margins decreased towards high‐latitude regions with long winters, with each day increase in winter duration reducing frost safety margins by 0.48 days in Fagus and 0.32–0.21 days in Prunus, Acer and Carpinus. These latitudinal differences correlate with northern ecotypes’ shorter degree‐day requirements for leaf‐out. MAIN CONCLUSIONS: The decline in spring frost safety margins in regions with long winters supports the new hypothesis that species may reach their geographic range limit where they ‘bump up’ against experiencing regular frost injury to their young leaves. Larger datasets are necessary to further corroborate our hypothesis and future efforts should thus be directed toward increasing the latitudinal range of existing phenological databases.

Background and Aims Unlike other nuclear genes in eukaryotes, rDNA genes (5S and 35S loci) are present in numerous copies per cell and, when stained, can therefore provide basic information about genome organization. In tracheophytes (vascular plants), they are usually located on separate chromosomes, the so-called S-type organization. An analysis of 1791 species of land plants suggested that S-type arrays might be ancestral in land plants, while linked (L-type) organization may be derived. However, no outgroup and only a handful of ferns and bryophytes were included. Methods We analysed genome sizes and the distribution of telomere, 5S and 35S rDNA FISH signals in up to 12 monoicous or dioicous species of liverworts from throughout a phylogeny that includes 287 of the 386 currently recognized genera. We also used the phylogeny to plot chromosome numbers and the occurrence of visibly distinct sex chromosomes. Key Results Chromosome numbers are newly reported for the monoicous Lejeunea cavifolia and for females of the dioicous Scapania aequiloba. We detected sex-related differences in the number of rDNA signals in the dioicous Plagiochila asplenioides and Frullania dilatata. In the latter, the presence of two UU chromosomes in females and additional 5S-35S rDNA loci result in a haploid genome 0.2082 pg larger than the male genome; sex-specific genome differences in the other dioicous species were small. Four species have S-type rDNA, while five species have mixed L-S rDNA organization, and transitions may have occurred multiple times, as suggested by rDNA loci not being conserved among closely related species of Pellia. All species shared an Arabidopsis-like telomere motif, and its detection allowed verification of the chromosome number of Radula complanata and chromosome rearrangements in Aneura pinguis and P. asplenioides, the latter also showing sex-specific interstitial telomere repeats. Conclusions The S and L rDNA arrangements appear to have evolved repeatedly within liverworts, even in the same species. Evidence for differential accumulation of rDNA between the sexes so far is limited.

Diverse forms of cultivation have evolved across the tree of life. Efficient farming requires that the farmer deciphers and actively promotes conditions that increase crop yield. For plant cultivation, this can include evaluating tradeoffs among light, nutrients, and protection against herbivores. It is not understood if, or how, nonhuman farmers evaluate local conditions to increase payoffs. Here, we address this question using an obligate farming mutualism between the ant Philidris nagasau and epiphytic plants in the genus Squamellaria that are cultivated for their nesting sites and floral rewards. We focused on the ants' active fertilization of their crops and their protection against herbivory. We found that ants benefited from cultivating plants in full sun, receiving 7.5-fold more floral food rewards compared to shade-cultivated plants. The higher reward levels correlated with higher levels of crop protection provided by the ants. However, while high-light planting yielded the greatest immediate food rewards, sun-grown crops contained less nitrogen compared to shade-grown crops. This was due to lower nitrogen input from ants feeding on floral rewards instead of insect protein gained from predation. Despite this tradeoff, farming ants optimize crop yield by selectively planting their crops in full sun. Ancestral state reconstructions across this ant-plant clade show that a full-sun farming strategy has existed for millions of years, suggesting that nonhuman farmers have evolved the means to evaluate and balance conflicting crop needs to their own benefit.

Coriariaceae comprise only Coriaria , a genus of shrubs with nine species in Australasia (but excluding Australia), five in the Himalayas, Taiwan, the Philippines, and Japan, one in the Mediterranean, and one ranging from Patagonia to Mexico. The sister family, Corynocarpaceae, comprises five species of evergreen trees from New Guinea to New Zealand and Australia. This distribution has long fascinated biogeographers as potential support for Wegener's theory of continental drift, with alternative scenarios invoking either Antarctic or Beringian range expansions. Here, we present the discovery of pollen grains from Early Campanian (ca. 82 Mya) deposits in Antarctica, which we describe as Coriaripites goodii sp. nov., and newly generated nuclear and plastid molecular data for most of the family's species and its outgroup. This greatly expands the family's fossil record and is the so far oldest fossil of the order Cucurbitales. We used the phylogeny, new fossil, and an Oligocene flowering branch assigned to a small subclade of Coriaria to generate a chronogram and to study changes in chromosome number, deciduousness, and andromonoecy. Coriaria comprises a Northern (NH) and a Southern Hemisphere (SH) clade that diverged from each other in the Paleocene (ca. 57 Mya), with the SH clade reaching the New World once, through Antarctica, as supported by the fossil pollen. While the SH clade retained perfect flowers and evergreen leaves, the NH clade evolved andromonoecy and deciduousness. Polyploidy occurs in both clades and points to hybridization, matching weak species boundaries throughout the genus.

Predicting the timing of autumn leaf senescence in northern trees remains challenging because the seasonal interplay in the effects of day length, climate, and plant productivity is not well understood. This severely limits our ability to forecast vegetation activity and carbon uptake in temperate and boreal ecosystems. Here we present a new framework for predicting autumn senescence dates based on the idea that day length mediates the effects of climate on autumn phenology, with early-season (pre-solstice) growth and late-season temperatures constituting antagonistic forces. To test these predictions, we used a combination of satellite-derived vegetation productivity across Northern Hemisphere forests, ground-sourced European phenology observations of four widespread tree species, and a climate-manipulation experiment on European beech. Our results reveal important constraints on the late-season carbon uptake potential of northern trees, improving our understanding of vegetation dynamics in response to climate change.

Linnaeus's very first opus, written when he was 22 years old, dealt with the analogy that exists between plants and animals in how they ‘propagate their species’, and a revised version with a plate depicting the union of male and female Mercurialis annua plants became a foundational text on the sexuality of plants. The question how systems with separate males and females have evolved in sedentary organisms that appear ancestrally bisexual has fascinated biologists ever since. The phenomenon, termed dioecy, has important consequences for plant reproductive success and is of commercial interest since it affects seed quality and fruit production. This theme issue presents a series of articles that synthesize and challenge the current understanding of how plants achieve dioecy. The articles deal with a broad set of taxa, including Coccinia , Ginkgo , Mercurialis , Populus , Rumex and Silene , as well as overarching topics, such as the field's terminology, analogies with animal sex determination systems, evolutionary pathways to dioecy, dosage compensation, and the longevity of the two sexes. In this introduction, we focus on four topics, each addressed by several articles from different angles and with different conclusions. Our highlighting of unclear or controversial issues may help future studies to build on the current understanding and to ask new questions that will expand our knowledge of plant sexual systems. This article is part of the theme issue ‘Sex determination and sex chromosome evolution in land plants’.

Microscopically dimorphic sex chromosomes in plants are rare, reducing our ability to study them. One difficulty has been the paucity of cultivatable species pairs for cytogenetic, genomic and experimental work. Here, we study the newly recognized sistersCoccinia grandisandCoccinia schimperi, both with large Y chromosomes as we here show forCo. schimperi. We built genetic maps for male and femaleCo. grandisusing a full-sibling family, inferred gene sex-linkage, and, withCo. schimperitranscriptome data, tested whether X- and Y-alleles group by species or by sex. Most sex-linked genes for which we could include outgroups grouped the X- and Y-alleles by species, but some 10% instead grouped the two species' X-alleles. There was no relationship between XY synonymous-site divergences in these genes and gene position on the non-recombining part of the X, suggesting recombination arrest shortly before or after species divergence, here dated to about 3.6 Ma.Coccinia grandisandCo. schimperiare the species pair with the most heteromorphic sex chromosomes in vascular plants (the condition in their sister remains unknown), and future work could use them to study mechanisms of Y chromosome enlargement and parallel degeneration, or to test Haldane's rule about lower hybrid fitness in the heterogametic sex.This article is part of the theme issue ‘Sex determination and sex chromosome evolution in land plants’.

Motivation Phylogenetic and phylogenomic analyses require multi-gene input files in different formats, but there are few user-friendly programs facilitating the workflow of combining, concatenating or separating, aligning and exploring multi-gene datasets. Results We present Concatenator, a user-friendly GUI-driven program that accepts single-marker and multi-marker DNA sequences in different input formats, including Fasta, Phylip and Nexus, and that outputs concatenated sequences as single-marker or multi-marker Fasta, interleaved nexus or Phylip files, including command files for downstream model selection in IQ-TREE. It includes the option to (re)align markers with MAFFT and produces exploratory trees with FastTree. Although tailored for medium-sized phylogenetic projects, Concatenator is able to process phylogenomic datasets of up to 30 000 markers. Availability and implementation Concatenator is written in Python, with C extensions for MAFFT and FastTree. Compiled stand-alone executables of Concatenator for MS Windows and Mac OS along with a detailed manual can be downloaded from www.itaxotools.org; the source code is openly available on GitHub (https://github.com/iTaxoTools/ConcatenatorGui).

In a commentary on our paper (Renner et al., Oecologia 195:825–831, 2021), Harder and Miksha lay out why they think that our finding of higher honeybee abundances reducing wild bee abundances in an urban botanical garden is not statistically supported. Here, we explain the statistical test provided in our paper, which took advantage of a natural experiment offered by 2019 being a poorer year for bee keeping than 2020.

Although hybridization plays a large role in speciation, some unknown fraction of hybrid individuals never reproduces, instead remaining as genetic dead-ends. We investigated a morphologically distinct and culturally important Chinese walnut, Juglans hopeiensis, suspected to have arisen from hybridization of Persian walnut (J. regia) with Asian butternuts (J. cathayensis, J. mandshurica, and hybrids between J. cathayensis and J. mandshurica). Based on 151 whole-genome sequences of the relevant taxa, we discovered that all J. hopeiensis individuals are first-generation hybrids, with the time for the onset of gene flow estimated as 370,000 years, implying both strong postzygotic barriers and the presence of J. regia in China by that time. Six inversion regions enriched for genes associated with pollen germination and pollen tube growth may be involved in the postzygotic barriers that prevent sexual reproduction in the hybrids. Despite its long-recurrent origination and distinct traits, J. hopeiensis does not appear on the way to speciation.

When flowers provide nectar, they can count on visits, and potential pollen transport, by many kinds of nectar-drinking animals. Yet, some flowers instead offer fatty oils that certain specialized bees gather with their forelegs. A recent study reveals how such a switch occurred and may have contributed to the formation of a new species.

Persian walnut (Juglans regia) is cultivated worldwide for its high-quality wood and nuts, but its origin has remained mysterious because in phylogenies it occupies an unresolved position between American black walnuts and Asian butternuts. Equally unclear is the origin of the only American butternut, J. cinerea. We resequenced the whole genome of 80 individuals from 19 of the 22 species of Juglans and assembled the genome of its relatives Pterocarya stenoptera and Platycarya strobilacea. Using phylogenetic-network analysis of single-copy nuclear genes, genome-wide site pattern probabilities, and Approximate Bayesian Computation, we discovered that J. regia (and its landrace J. sigillata) arose as a hybrid between the American and the Asian lineages and that J. cinerea resulted from massive introgression from an immigrating Asian butternut into the genome of an American black walnut. Approximate Bayesian Computation modeling placed the hybrid origin in the late Pliocene, ∼3.45 My, with both parental lineages since having gone extinct in Europe.

Cytogenetic studies in bryophytes have been limited by the difficulty of obtaining sufficient dividing nuclei and by the absence of modern protocols. The technical difficulties stem from the plants’ small size and lack of roots and pollen mother cells, the main sources of cells in division in vascular plants. In bryophytes instead, tiny sporophytes, antheridia, or phyllid meristems must be used to obtain meiotic or mitotic chromosome spreads. We here describe the preparation of such spreads from phyllids, antheridia, and sporophytes in several species of liverworts and compare available protocols with or without prefixation treatments. We also provide illustrated step-by-step instructions. The three prefixation agents (including colchicine) that we tested failed to improve synchronization of cell divisions. Young sporophytes were the best source of diploid synchronized cells, while antheridia were the best source of haploid cells. For meiotic nuclei, a short fixation of capsule tissue at the right developmental stage with 45% acetic acid sufficed to conserve the DNA for cytological investigations, while for mitotic nuclei, fixation in 3:1 ethanol/glacial acetic acid for a longer period (4–24 h) worked well.