First report of cedar-quince rust Gymnosporangium clavipes on fruit of dwarf hawthorn Crataegus uniflora in Florida, USA.

The dwarf hawthorn Crataegus uniflora Münchh. (Rosaceae, Maloideae) is a small deciduous tree species native to the central and eastern US and south into northern Mexico. Dwarf hawthorn is drought tolerant and commonly found in disturbed areas (e.g., hedges and roadsides). In May 2021, we observed several individuals of dwarf hawthorn growing on the border of an empty field in the Natural Area Teaching Laboratory at The University of Florida main campus in Gainesville, Alachua County, Florida, USA (N29.633382, W82.368350) that were severely infected by fruit galls with visible, whitish aecia (e-Xtra Fig.1). The affected fruit were collected and transported to the Florida Department of Agriculture and Consumer Services - Division of Plant Industry headquarters in Gainesville for identification (FDACS-DPI, 2021-107788). The conspicuous rust fungus, occurring on the fruit (fructicolous), consisted of tubular aecia (roestelioid), 4 - 5 mm in length × 0.5 mm in diameter, with whitish peridia containing bright orange spores in masses. Aeciospores were semigloboid to globoid, some with an angular side, with bright orange contents, 26 - 31 µm in diameter (n= 20). The wall was densely verrucose, hyaline, 3 - 4 µm wide. Side and wall ornamentation are considered diagnostic features (EPPO Bulletin, 2006). Peridial cells of the aecia were hyaline, angular (pentagonal to hexagonal) to irregular, with a thin, convoluted wall, 41- 57 × 30 - 35 µm (n =10). Aeciospores were detected on blackish, mummified berries five months after the initial collection and aecium disappearance. This persistence demonstrates one of the adaptations allowing the pathogen to remain in a given location (e-Xtra Fig.1). The morphological characteristics are consistent with those of Gymnosporangium clavipes Cooke & Peck (Gymnosporangiaceace, Pucciniaceae, Pucciniomycotina) described by Kern (1973). A voucher was deposited in the DPI Herbarium (PIHG, specimen number 15618). The morphological identification was confirmed by molecular identification: following DNA extraction (DNeasy Plant Pro extraction kit, Qiagen Corporation, Hilden, Germany), we amplified a fragment of the internal transcribed spacer (ITS) and the large subunit (LSU) via PCR using the primer pairs Rust2inv/LR6 and Rust28S/LR5 (OK337508) (Aime, 2006); amplicons were then Sanger sequenced. NCBI megaBlast searches (Chen et al. 2015) of the resulting fungal sequences revealed high identity (ITS and LSU) to two G. clavipes vouchers: NYBG461394 (99.86%, Genbank accession no. MN605691) sequenced in the latest publication addressing species delimitation in Gymnosporangium (Zhao et al., 2020), and PPST 2020-104160 (99.72%, GenBank MW148514): the first report of this rust occurring on Crataegus marshallii Eggleston (McVay et al., 2021), also recently found in Gainesville, Florida. Phylogenetic analyses were carried out in the phylogenetic package RAxMLv8.0.0 (Stamatakis, 2014) (e-Xtra Fig. 2) further supports placement of 2021-107788 within G. clavipes. The heteroecious nature of this rust fungus precludes Koch's postulates. Based on exhaustive reviews of collection indices and literature, a specimen of G. clavipes on C. uniflora exists at the U.S. National Fungus collections (BPI 117783A) collected in Newfield, New Jersey in 1888 (Farr & Rossman, 2022); this rust fungus has a host range of at least 18 other species of Crataegus (Farr and Rossman, 2022; McVay et al., 2021; Zhao et al., 2020). This report represents the first published record of G. clavipes on dwarf hawthorn, and the first report in Florida.

Smilax auriculata: A new host for Orchid fleck dichorhavirus identified in Florida, USA.

In June 2020, Orchid fleck virus (OFV) was detected in a species of Liriope in Leon and Alachua County, Florida (Fife et al; 2021). In October of the same year, four adjacent dune/ear-leaf greenbrier vines, Smilax auriculata (Smilaceae: Liliales), showed yellowing and mottling symptoms (Figure 1). Infected and healthy S. auriculata leaves samples were collected in Alachua County by the Florida Department of Agriculture and Consumer Services, Gainesville, Florida. OFV primers successfully detected in four Smilax samples by conventional RT-PCR assay. Amplicon sequences (Acc. No. MZ645935 and MZ645938) shared 99% nucleotide identity with OFV infecting orchids (LC222629) and citrus (MK522804). The OFV subgroup I (OFV-Orc1) and subgroup II (OFV-Orc2) specific primers (Kondo et al 2017) were utilized to confirm the presence of OFV type strains infecting Smilax. Sanger sequencing of subgroup I specific amplicons (MZ645934) shared 99% nucleotide identity with OFV-Orc1 (LC222629) whereas subgroup II specific amplicon sequence (MZ645930) shared 98-99 % nucleotide identity with OFV-Orc2 (AB244417). Further confirmation was done by USDA-APHIS-PPQ-Plant Pathogen Confirmatory Diagnostics Laboratory utilizing optimized conventional RT-PCR protocols (Roy et al. 2020) and deep sequencing on a on a NextSeq550 Illumina platform. Assembled reads identified seven non-overlapping viral contigs. Five RNA1 and two RNA2 contigs covered more than 97% of the bipartite OFV genome with average coverage depth of 5297.61 and 5186.04, respectively. Contigs of RNA1 and RNA2 shared 98-99% nt identity to OFV-Orc2-RNA1 (AB244417) and OFV-Orc-RNA2 (AB244418 and LC222630). No other pathogen sequences were identified. This is the first time the genus Smilax has been identified as a natural host of OFV. Very recent findings of OFV-Orc in Florida in Liriope, Aspidistra, and Ophiopogon among the Asparagaceae family members (Fife et al; 2021) and now in the Smilacaceae suggest a broader host range of the virus than previously known; further research should be conducted to better characterize the potential risk of introduction into citrus in Florida.

First Report of Cucurbit Chlorotic Yellows Virus affecting Watermelon in USA.

Watermelon (Citrullus lanatus) is a high nutrient crop, high in vitamins and very popular in the U.S and globally. The crop was harvested from 101,800 acres with a value of $560 million in the U.S (USDA-NASS, 2020). California, Florida, Georgia and Texas are the four-leading watermelon-producing states in the U.S. During the fall season of 2020, plants in two North Florida watermelon fields, one in Levy County (~20 acres) and one in Suwannee County (~80 acres) with varieties Talca and Troubadour, respectively, exhibited viral-like symptoms. The fields had 100% disease incidence that led to fruit quality issues and yield losses of 80% and above. Symptoms observed in the watermelon samples included leaf crumpling, yellowing and curling, and vein yellowing similar to that of single/and or mixed infection of cucurbit leaf crumple virus (CuLCrV; genus: Begomovirus, family: Geminiviridae), cucurbit yellow stunting disorder virus (CYSDV; genus: Crinivirus, family: Closteroviridae) and squash vein yellowing virus (SqVYV; genus: Ipomovirus, family: Potyviridae), although the vine decline symptoms often associated with SqVYV infection of watermelon were not observed. All three viruses are vectored by whiteflies and previously described in Florida (Akad et al., 2008; Polston et al., 2008; Adkins et al., 2009). To confirm the presence of these viruses, RNA was isolated from 20 symptomatic samples using the RNeasy Plant Mini Kit (Qiagen, USA) as per protocol. This was followed by RT-PCR (NEB, USA) using gene-specific primers described for CuLCrV, CYSDV and SqVYV (Adkins et al., 2009). Amplicons of expected sizes were obtained for all the viruses with the infection of CuLCrV in 17/20, CYSDV in 16/20, and SqVYV in 8/20 samples. In addition, the presence of cucurbit chlorotic yellows virus (CCYV; genus: Crinivirus, family: Closteroviridae) in mixed infection was confirmed in 4/20 samples (3 leaves and 1 fruit) by RT-PCR with primers specific to the CCYV coat protein (CP), heat shock protein 70 homolog (HSP70h) and RNA dependent RNA polymerase (RdRp) designed based on the available CCYV sequences (Sup Table. 1). The RT-PCR amplification was performed using a symptomatic watermelon sample and the amplicons of RdRp, HSP70h and CP were directly sequenced by Sanger method, and the sequences of the amplicons were deposited in GenBank under the accession number: MW527462 (RdRp, 952 bp), MW527461 (HSP70h, 583 bp) and MW527460 (CP, 852 bp). BLASTn analysis demonstrated that the sequences exhibited an identity of 99% to 100% (RdRp and HSP70h, 100%; and CP, 99%) with the corresponding regions of the CCYV isolate Shanghai from China (accession number: KY400636 and KY400633). The presence of CCYV was further confirmed in the watermelon samples by ELISA (Loewe, Germany) using crude sap extracted from the RT-PCR-positive, symptomatic watermelon samples. CCYV was first identified in Kumamoto, Japan in 2004 on melon plants (Gyoutoku et al. 2009). The CCYV was previously reported on melon from Imperial Valley, California (Wintermantel et al., 2019), and more recently on squash in Tifton, Georgia (Kavalappara et al., 2021) and cantaloupe in Cameron, Texas (Hernandez et al., 2021). To our knowledge, this is the first report of CCYV on field watermelon production in the U.S. Continued monitoring of the CCYV in spring and fall watermelon crop, and cucurbit volunteers and weeds will be critical toward understanding the spread of this virus and its potential risk to watermelon in Florida and other regions of the U.S.

Genomic landscape of the global oak phylogeny.

The tree of life is highly reticulate, with the history of population divergence emerging from populations of gene phylogenies that reflect histories of introgression, lineage sorting and divergence. In this study, we investigate global patterns of oak diversity and test the hypothesis that there are regions of the oak genome that are broadly informative about phylogeny. We utilize fossil data and restriction-site associated DNA sequencing (RAD-seq) for 632 individuals representing nearly 250 Quercus species to infer a time-calibrated phylogeny of the world's oaks. We use a reversible-jump Markov chain Monte Carlo method to reconstruct shifts in lineage diversification rates, accounting for among-clade sampling biases. We then map the > 20 000 RAD-seq loci back to an annotated oak genome and investigate genomic distribution of introgression and phylogenetic support across the phylogeny. Oak lineages have diversified among geographic regions, followed by ecological divergence within regions, in the Americas and Eurasia. Roughly 60% of oak diversity traces back to four clades that experienced increases in net diversification, probably in response to climatic transitions or ecological opportunity. The strong support for the phylogeny contrasts with high genomic heterogeneity in phylogenetic signal and introgression. Oaks are phylogenomic mosaics, and their diversity may in fact depend on the gene flow that shapes the oak genome.

Uncovering the genomic signature of ancient introgression between white oak lineages (Quercus).

Botanists have long recognised interspecific gene flow as a common occurrence within white oaks (Quercus section Quercus). Historical allele exchange, however, has not been fully characterised and the complex genomic signals resulting from the combination of vertical and horizontal gene transmission may confound phylogenetic inference and obscure our ability to accurately infer the deep evolutionary history of oaks. Using anchored enrichment, we obtained a phylogenomic dataset consisting of hundreds of single-copy nuclear loci. Concatenation, species-tree and network analyses were carried out in an attempt to uncover the genomic signal of ancient introgression and infer the divergent phylogenetic topology for the white oak clade. Locus and site-level likelihood comparisons were then conducted to further explore the introgressed signal within our dataset. Historical, intersectional gene flow is suggested to have occurred between an ancestor of the Eurasian Roburoid lineage and Quercus pontica and North American Dumosae and Prinoideae lineages. Despite extensive time past, our approach proved successful in detecting the genomic signature of ancient introgression. Our results, however, highlight the importance of sampling and the use of a plurality of analytical tools and methods to sufficiently explore genomic datasets, uncover this signal, and accurately infer evolutionary history.

Sympatric parallel diversification of major oak clades in the Americas and the origins of Mexican species diversity.

Oaks (Quercus, Fagaceae) are the dominant tree genus of North America in species number and biomass, and Mexico is a global center of oak diversity. Understanding the origins of oak diversity is key to understanding biodiversity of northern temperate forests. A phylogenetic study of biogeography, niche evolution and diversification patterns in Quercus was performed using 300 samples, 146 species. Next-generation sequencing data were generated using the restriction-site associated DNA (RAD-seq) method. A time-calibrated maximum likelihood phylogeny was inferred and analyzed with bioclimatic, soils, and leaf habit data to reconstruct the biogeographic and evolutionary history of the American oaks. Our highly resolved phylogeny demonstrates sympatric parallel diversification in climatic niche, leaf habit, and diversification rates. The two major American oak clades arose in what is now the boreal zone and radiated, in parallel, from eastern North America into Mexico and Central America. Oaks adapted rapidly to niche transitions. The Mexican oaks are particularly numerous, not because Mexico is a center of origin, but because of high rates of lineage diversification associated with high rates of evolution along moisture gradients and between the evergreen and deciduous leaf habits. Sympatric parallel diversification in the oaks has shaped the diversity of North American forests.

Phylogenomics reveals a complex evolutionary history of lobed-leaf white oaks in western North America.

Species within the genus Quercus (oak) hybridize in complex patterns that have yet to be fully explored with phylogenomic data. Analyses to date have recovered reasonable divergent patterns, suggesting that the impact of introgression may not always be obvious in inferred oak phylogenies. We explore this phenomenon using RADseq data for 136 samples representing 54 oak species by conducting phylogenetic analyses designed to distinguish signals of lineage diversification and hybridization, focusing on the lobed-leaf species Quercus gambelii, Q. lobata, and Q. garryana in the context of a broad sampling of allied white oaks (Quercus section Quercus), and particularly the midwestern Q. macrocarpa. We demonstrate that historical introgressive hybridization between once sympatric species affects phylogeny estimation. Historical range expansion during periods of favorable climate likely explains our observations; analyses support genetic exchange between ancestral populations of Q. gambelii and Q. macrocarpa. We conclude that the genomic consequences of introgression caused the attraction of distant lineages in phylogenetic tree space, and that introgressive and divergent signals can be disentangled to produce a robust estimate of the phylogenetic history of the species.

A genetic legacy of introgression confounds phylogeny and biogeography in oaks.

Oaks (Quercus L.) have long been suspected to hybridize in nature, and widespread genetic exchange between morphologically defined species is well documented in two- to six-species systems, but the phylogenetic consequences of hybridization in oaks have never been demonstrated in a phylogenetically diverse sample. Here, we present phylogenomic analyses of a ca 30 Myr clade that strongly support morphologically defined species and the resolution of novel clades of white oaks; however, historical hybridization across clade boundaries is detectable and, undiagnosed, would obscure the imprint of biogeographic history in the phylogeny. Phylogenetic estimation from restriction-site-associated DNA sequencing data for 156 individuals representing 81 species supports two successive intercontinental disjunctions of white oaks: an early vicariance between the Eurasian and American white oaks, and a second, independent radiation represented by two relictual species. A suite of subsampled and partitioned analyses, however, supports a more recent divergence of the Eurasian white oaks from within the American white oaks and suggests that historic introgression between the Eurasian white oaks and a now-relictual lineage biases concatenated phylogenetic estimates. We demonstrate how divergence and reticulation both influence our understanding of the timing and nature of diversification and global colonization in these ecologically and economically important taxa.

The evolution and diversification of the red oaks of the California Floristic Province (Quercus section Lobatae, series Agrifoliae).

PREMISE OF THE STUDY: The California Floristic Province (CA-FP) is a unique and diverse region of floral endemism, yet the timing and nature of divergence and diversification of many lineages remain underexplored. We seek to elucidate the evolutionary history of the red oaks of the CA-FP, the Agrifoliae. METHODS: We collected PstI-associated RAD-seq data as well as morphometrics from individuals of the four species across their ranges, including varieties and hybrids. Phylogeny and divergence times were estimated. We analyzed morphological differentiation in over 70 plants using PCA and assessed species delimitation and admixture using genotype clustering analysis in over 40 plants. KEY RESULTS: We find that the Agrifoliae are monophyletic and sister to all other red oak species. Within the Agrifoliae, all species are supported, with Quercus kelloggii sister to a clade of subevergreen taxa: (Quercus agrifolia - (Q. parvula + Q. wislizeni)). Molecular and morphometric analyses are equivocal for named varieties. Notably, Q. parvula var. tamalpaisensis appears to be part of a hybrid swarm between Q. parvula and Q. wislizeni. Dating estimates were concordant with previous hypotheses and geological evidence, with diversification occurring between 10 and 20 million years ago. CONCLUSIONS: The Agrifoliae represent a geographically discrete, early-diverging red oak lineage that diversified during the period of drying and warming associated with Sierran uplift during the middle Miocene. Molecular differentiation within the clade supports the current taxonomy, including an east-west species level pattern (Q. parvula and Q. wislizeni) and north-south intraspecific patterns to some degree, although the latter require additional study.

Poor fit to the multispecies coalescent is widely detectable in empirical data.

Model checking is a critical part of Bayesian data analysis, yet it remains largely unused in systematic studies. Phylogeny estimation has recently moved into an era of increasingly complex models that simultaneously account for multiple evolutionary processes, the statistical fit of these models to the data has rarely been tested. Here we develop a posterior predictive simulation-based model check for a commonly used multispecies coalescent model, implemented in *BEAST, and apply it to 25 published data sets. We show that poor model fit is detectable in the majority of data sets; that this poor fit can mislead phylogenetic estimation; and that in some cases it stems from processes of inherent interest to systematists. We suggest that as systematists scale up to phylogenomic data sets, which will be subject to a heterogeneous array of evolutionary processes, critically evaluating the fit of models to data is an analytical step that can no longer be ignored.

Testing monophyly without well-supported gene trees: evidence from multi-locus nuclear data conflicts with existing taxonomy in the snake tribe Thamnophiini.

Ideally, existing taxonomy would be consistent with phylogenetic estimates derived from rigorously analyzed data using appropriate methods. We present a multi-locus molecular analysis of the relationships among nine genera in the North American snake tribe Thamnophiini in order to test the monophyly of the crayfish snakes (genus Regina) and the earth snakes (genus Virginia). Sequence data from seven genes were analyzed to assess relationships among representatives of the nine genera by performing multi-locus phylogeny and species tree estimations, and we performed constraint-based tests of monophyly of classic taxonomic designations on a gene-by-gene basis. Estimates of concatenated phylogenies demonstrate that neither genera are monophyletic, and this inference is supported by a species tree estimate, though the latter is less robust. These taxonomic findings were supported using gene tree constraint tests and Bayes Factors, where we rejected the monophyly of both the crayfish snakes (genus Regina) and the earth snakes (genus Virginia); this method represents a potentially useful tool for taxonomists and phylogeneticists when available data is less than ideal.

Phylogeographic analysis and environmental niche modeling of the plain-bellied watersnake (Nerodia erythrogaster) reveals low levels of genetic and ecological differentiation.

Species that exhibit geographically defined phenotypic variation traditionally have been divided into subspecies. Subspecies based on phenotypic features may not comprise monophyletic groups due to selection, gene flow, and/or convergent evolution. In many taxonomic groups the number of species once designated as widespread is dwindling rapidly, and many workers reject the concept of subspecies altogether. We tested whether currently recognized subspecies in the plain-bellied watersnake Nerodia erythrogaster are concordant with relationships based on mitochondrial markers, and whether it represents a single widespread species. The range of this taxon spans multiple potential biogeographic barriers (especially the Mississippi and Apalachicola Rivers) that correspond with lineage breaks in many species, including other snakes. We sequenced three mitochondrial genes (NADH-II, Cyt-b, Cox-I) from 156 geo-referenced specimens and developed ecological niche models using Maxent and spatially explicit climate data to examine historical and ecological factors affecting variation in N. erythrogaster across its range. Overall, we found little support for the recognized subspecies as either independent evolutionary lineages or geographically circumscribed units and conclude that although some genetic and niche differentiation has occurred, most populations assigned to N. erythrogaster appear to represent a single, widespread species. However, additional sampling and application of nuclear markers are necessary to clarify the status of the easternmost populations.

Evidence of multiple paternity in Morelet's Crocodile (Crocodylus moreletii) in Belize, CA, inferred from microsatellite markers.

Microsatellite data were generated from hatchlings collected from ten nests of Morelet's Crocodile (Crocodylus moreletii) from New River Lagoon and Gold Button Lagoon in Belize to test for evidence of multiple paternity. Nine microsatellite loci were genotyped for 188 individuals from the 10 nests, alongside 42 nonhatchlings from Gold Button Lagoon. Then mitochondrial control region sequences were generated for the nonhatchlings and for one individual from each nest to test for presence of C. acutus-like haplotypes. Analyses of five of the nine microsatellite loci revealed evidence that progeny from five of the ten nests were sired by at least two males. These data suggest the presence of multiple paternity as a mating strategy in the true crocodiles. This information may be useful in the application of conservation and management techniques to the 12 species in this genus, most of which are threatened or endangered.