Divergent Fate and Roles of Dissolved Organic Matter from Spatially Varied Grassland Soils in China During Long-Term Biogeochemical Processes.

Terrestrial dissolved organic matter (DOM) is critical to global carbon and nutrient cycling, climate change, and human health. However, how the spatial and compositional differences of soil DOM affect its dynamics and fate in water during the carbon cycle is largely unclear. Herein, the biodegradation of DOM from 14 spatially distributed grassland soils in China with diverse organic composition was investigated by 165 days of incubation experiments. The results showed that although the high humified fraction (high-HS) regions were featured by high humic-like fractions of 4-25 kDa molecular weight, especially the abundant condensed aromatics and tannins, they unexpectedly displayed greater DOM degradation during 45-165 days. In contrast, the unique proteinaceous and 25-100 kDa fractions enriched in the low humified fraction (low-HS) regions were drastically depleted and improved the decay of bulk DOM but only during 0-45 days. Together, DOM from the high-HS regions would cause lower CO2 outgassing to the atmosphere but higher organic loads for drinking water production in the short term than that from the low-HS regions. However, this would be reversed for the two regions during the long-term transformation processes. These findings highlight the importance of spatial and temporal variability of DOM biogeochemistry to mitigate the negative impacts of grassland soil DOM on climate, waters, and humans.

Comparative analysis of molecular and morphological diversity in two diploid Paspalum species (Poaceae) with contrasting mating systems.

KEY MESSAGE: Interspecific comparison of two Paspalum species has demonstrated that mating systems (selfing and outcrossing) contribute to variation (genetically and morphologically) within species through similar but mutually exclusive processes. Mating systems play a key role in the genetic dynamics of populations. Studies show that populations of selfing plants have less genetic diversity than outcrossing plants. Yet, many such studies have ignored morphological diversity. Here, we compared the morphological and molecular diversity patterns in populations of two phylogenetically-related sexual diploids that differ in their mating system: self-sterile Paspalum indecorum and self-fertile P. pumilum. We assessed the morphological variation using 16 morpho-phenological characters and the molecular diversity using three combinations of AFLPs. We compared the morphological and molecular diversity within and among populations in each mating system. Contrary to expectations, selfers showed higher morphological variation within populations, mainly in vegetative and phenological traits, compared to outcrossers. The high morphological variation within populations of selfers led to a low differentiation among populations. At molecular level, selfing populations showed lower levels of genotypic and genetic diversity than outcrossing populations. As expected, selfers showed higher population structure than outcrossers (PhiST = 0.301 and PhiST = 0.108, respectively). Increased homozygous combinations for the same trait/locus enhance morphological variation and reduce molecular variation within populations in selfing P. pumilum. Thus, selfing outcomes are opposite when comparing morphological and molecular variation in P. pumilum. Meanwhile, pollen flow in obligate outcrossing populations of P. indecorum increases within-population molecular variation, but tends to homogenize phenotypes within-population. Pollen flow in obligate outcrossers tends to merge geographically closer populations; but isolation by distance can lead to a weak differentiation among distant populations of P. indecorum.

Expert Consensus on Pathological Diagnosis of Intrahepatic Cholangiocarcinoma (2022 version).

Intrahepatic cholangiocarcinoma (iCCA) can originate from the large bile duct group (segment bile ducts and area bile ducts), small bile duct group (septal bile ducts and interlobular bile ducts), and terminal bile duct group (bile ductules and canals of Hering) of the intrahepatic biliary tree, which can be histopathological corresponding to large duct type iCCA, small duct type iCCA and iCCA with ductal plate malformation pattern, and cholangiolocarcinoma, respectively. The challenge in pathological diagnosis of above subtypes of iCCA falls in the distinction of cellular morphologies, tissue structures, growth patterns, invasive behaviors, immunophenotypes, molecular mutations, and surgical prognoses. For these reasons, this expert consensus provides nine recommendations as a reference for standardizing and refining the diagnosis of pathological subtypes of iCCA, mainly based on the 5th edition of the World Health Organization Classification of Tumours of the Digestive System.

Treponema pallidum Subspecies Pallidum Intrapatient Homogeneity at Various Body Locations in Men with Infectious Syphilis.

Syphilis, caused by Treponema pallidum subspecies pallidum (TP), is a complex multistage infectious disease. Systematic dissemination occurs within a few hours of transmission. We determined the molecular variation of TP at various body locations and peripheral blood within patients in different stages of syphilis to assess the distribution of TP strains at these locations. We included 162 men who have sex with men (MSM) with syphilis visiting the Sexual Health Center in Amsterdam between 2018 to 2019, who had TP DNA detected in at least one sample type (anal swab, urine sample, peripheral blood, pharyngeal swab, and/or ulcer swab). TP DNA was detected in 287 samples using a qPCR targeting the polA gene. With multilocus sequence typing (TP-MLST) based on partial sequence analysis of three genetic regions (tp0136, tp0548, tp0705), we characterized all TP DNA positive samples. Samples could be typed (119/287) from at least one anatomical location or peripheral blood from 93/162 (57%) patients in the following stages: 48 (52%) primary, 35 (38%) secondary, and 10 (11%) early latent stage syphilis. The TP-MLST type was identical within each of the 12 patients with typed samples at ≥2 different body locations. The most prevalent TP strains were 1.3.1 (39/93, 42%) and 1.1.1 (17/93, 18%) belonging to the SS14 lineage; 80% (74/93) of the patients carried a SS14 lineage TP strain and 20% (19/93) Nichols lineage. The distribution of TP-MLST types did not differ between patients by syphilis stage. We found intrapatient TP strain homogeneity and no TP strain variation between anatomical location or syphilis stages. More early latent samples should be typed and added in future studies to investigate this in more detail. IMPORTANCE Syphilis, caused by Treponema pallidum subspecies pallidum, is a complex multistage infectious disease. Systematic dissemination is known to occur within a few hours of transmission. Despite the effective antibiotic penicillin, syphilis remains prevalent worldwide. Men who have sex with men are disproportionally affected in high income countries like the Netherlands where 96% of the syphilis cases in 2020 were among this population. The inability to in vitro culture T. pallidum directly from patient samples limits whole-genome sequencing efforts. Fortunately, in 2018 a multilocus sequence typing technique was developed for T. pallidum allowing the monitoring of circulating strains. The significance of our research is in the investigation of T. pallidum molecular variation at various body locations and blood within patients in different stages of syphilis in order to assess the distribution of strains at these locations.

Phylogeographic analyses of poplar revealed potential glacial refugia and allopatric divergence in southwest China.

The distribution pattern and genetic structure of plant species have been profoundly influenced by climate oscillations. Phylogeographic analyses have been numerously conducted in biodiversity hotspot regions and some general conclusions have been documented. However, other regions have received less attentions and these places may serve as potential glacial refugia for plant species to survive the Quaternary glaciation. Here, we used six nuclear and three cpDNA markers to estimate the phylogeographic pattern of Populus davidiana, a widespread species distributed in southwest China. As a widely distributed species in southwest China, the nucleotide diversity of P. davidiana was relatively high (Na = 6.28, HO = 0.534, and HE = 0.658). Genetic differentiation (FST) between the two main distribution regions, Yunnan and Guizhou provinces, was 0.21221. According to the composition of chloroplast haplotypes and the result of structure in these populations, we clearly distinguished two distantly sublineages corresponding to two distribution regions. Results of the Mantel test showed that there was a significant correlation between genetic distance and geographical distance (R2 = 0.8252, p<.05). The topographically heterogeneous regions and the low dispersal ability of seed and pollen may lead to high genetic differentiation between these two regions. A potential glacial refugia for P. davidiana located in adjacent regions to the Hengduan range was revealed and allopatric divergence in separated glacial refugia may directly lead to the present phylogeographic pattern of this species.

Identification of High Molecular Variation Loci in Complete Chloroplast Genomes of Mammillaria (Cactaceae, Caryophyllales).

In plants, partial DNA sequences of chloroplasts have been widely used in evolutionary studies. However, the Cactaceae family (1500-1800 species) lacks molecular markers that allow a phylogenetic resolution between species and genera. In order to identify sequences with high variation levels, we compared previously reported complete chloroplast genomes of seven species of Mammillaria. We identified repeated sequences (RSs) and two types of DNA variation: short sequence repeats (SSRs) and divergent homologous loci. The species with the highest number of RSs was M. solisioides (256), whereas M. pectinifera contained the highest amount of SSRs (84). In contrast, M. zephyranthoides contained the lowest number (35) of both RSs and SSRs. In addition, five of the SSRs were found in the seven species, but only three of them showed variation. A total of 180 homologous loci were identified among the seven species. Out of these, 20 loci showed a molecular variation of 5% to 31%, and 12 had a length within the range of 150 to 1000 bp. We conclude that the high levels of variation at the reported loci represent valuable knowledge that may help to resolve phylogenetic relationships and that may potentially be convenient as molecular markers for population genetics and phylogeographic studies.

Applied Human Genetic Epidemiology.

This chapter covers the study of human epidemiology, including family studies in genetic epidemiology, linkage analysis, genetic Mapping in human diseases, human genetic influences on diseases, genetic relationships in familial aggregation, and derivation of familial risk.An Illustration is provided of a research project in genetic epidemiology research which included (1) Heritability Analysis (2) Molecular Variation Study Methods (3) Genomics for Human Genetic Epidemiology Complex Traits and Mendelian Inheritance Mendel's Laws Hardy-Weinberg Principle Gene Structure and Genetic Code Genetic Linkage and Linkage Disequilibrium Study Designs for of Rare Genetic Variations Spectrum of Variation Familial Factors in Human Genetic Epidemiology *Human Genetic Association Genetic Epidemiology Owing to Population Stratification Environmental Effects on Genetic Epidemiology Genetic Epidemiology and Public Health.

Species and cell-type properties of classically defined human and rodent neurons and glia.

Determination of the molecular properties of genetically targeted cell types has led to fundamental insights into mouse brain function and dysfunction. Here, we report an efficient strategy for precise exploration of gene expression and epigenetic events in specific cell types in a range of species, including postmortem human brain. We demonstrate that classically defined, homologous neuronal and glial cell types differ between rodent and human by the expression of hundreds of orthologous, cell specific genes. Confirmation that these genes are differentially active was obtained using epigenetic mapping and immunofluorescence localization. Studies of sixteen human postmortem brains revealed gender specific transcriptional differences, cell-specific molecular responses to aging, and the induction of a shared, robust response to an unknown external event evident in three donor samples. Our data establish a comprehensive approach for analysis of molecular events associated with specific circuits and cell types in a wide variety of human conditions.

Phylogeographic analyses of a widely distributed Populus davidiana: Further evidence for the existence of glacial refugia of cool-temperate deciduous trees in northern East Asia.

Despite several phylogeographic studies had provided evidence to support the existence of glacial refugia of cool-temperate deciduous trees in northeast China, the species used in these studies were limited by the species ranges, which could not exclude the possibility that northern populations were the colonists from southern refugial populations during the last glacial maximum (LGM). Here, we estimated the nucleotide variation in Populus davidiana, a widespread species distributed in Eurasia. Three groups in northeast, central, and southwest China were constructed according to the simulation results from SAMOVA, composition of chloroplast haplotypes and structure results. We revealed that the northeast China had endemic haplotypes, the haplotypes and nucleotide diversity in northern regions were not lower than that in southern China, and this species has not experienced population expansion base on the estimation of Bayesian skyline plots. Ecological niche modeling (ENM) indicated that the northeast China had a high suitability score during the last glacial maximum. The combined evidence clearly demonstrated that northeastern and southwestern refugia were maintained across the current distributional range of P. davidiana during the LGM. The genetic differentiation between these two refugia might be mainly caused by differences of climate among these areas. The phylogeographic analyses of a widely distributed P. davidiana provided robust evidence to clarify the issue of refugia in northeast China, and these results are of great importance for understanding the influence of Quaternary glaciations on the distribution and evolution of species in East Asia.

Hubby and Lewontin on Protein Variation in Natural Populations: When Molecular Genetics Came to the Rescue of Population Genetics.

The 1966 GENETICS papers by John Hubby and Richard Lewontin were a landmark in the study of genome-wide levels of variability. They used the technique of gel electrophoresis of enzymes and proteins to study variation in natural populations of Drosophila pseudoobscura, at a set of loci that had been chosen purely for technical convenience, without prior knowledge of their levels of variability. Together with the independent study of human populations by Harry Harris, this seminal study provided the first relatively unbiased picture of the extent of genetic variability in protein sequences within populations, revealing that many genes had surprisingly high levels of diversity. These papers stimulated a large research program that found similarly high electrophoretic variability in many different species and led to statistical tools for interpreting the data in terms of population genetics processes such as genetic drift, balancing and purifying selection, and the effects of selection on linked variants. The current use of whole-genome sequences in studies of variation is the direct descendant of this pioneering work.

Molecular and morphological variation of Paragonimus westermani in Vietnam with records of new second intermediate crab hosts and a new locality in a northern province.

Paragonimus westermani is one of the most medically important lung flukes and is widely distributed in Asia. It exhibits considerable variation in morphological, genetic and biological features. In central provinces of Vietnam, a high prevalence of metacercariae of this species has been reported from the crab intermediate host, Vietopotamon aluoiense. In this study, we detected P. westermani metacercariae in two additional crab hosts, Donopotamon haii in Quang Tri Province, central Vietnam and Indochinamon tannanti in Yen Bai Province in the north. The latter is a new locality for P. westermani in a northern region of Vietnam where P. heterotremus is the only species currently known to cause human paragonimiasis. Paragonimus westermani metacercariae found in Vietnam showed considerable morphological variation but slight genetic variation based on DNA sequences from the nuclear ribosomal ITS2 region and the mitochondrial 16S gene. Co-infection of the same individual crabs with P. westermani and P. heterotremus and/or some other Paragonimus species was found frequently, suggesting potential for co-infection in humans. The findings of the present study emphasize the need for highly specific molecular and immunodiagnostic methods to differentially diagnose between P. westermani and P. heterotremus infections.

Characterization of a Novel Polerovirus Infecting Maize in China.

A novel virus, tentatively named Maize Yellow Mosaic Virus (MaYMV), was identified from the field-grown maize plants showing yellow mosaic symptoms on the leaves collected from the Yunnan Province of China by the deep sequencing of small RNAs. The complete 5642 nucleotide (nt)-long genome of the MaYMV shared the highest nucleotide sequence identity (73%) to Maize Yellow Dwarf Virus-RMV. Sequence comparisons and phylogenetic analyses suggested that MaYMV represents a new member of the genus Polerovirus in the family Luteoviridae. Furthermore, the P0 protein encoded by MaYMV was demonstrated to inhibit both local and systemic RNA silencing by co-infiltration assays using transgenic Nicotiana benthamiana line 16c carrying the GFP reporter gene, which further supported the identification of a new polerovirus. The biologically-active cDNA clone of MaYMV was generated by inserting the full-length cDNA of MaYMV into the binary vector pCB301. RT-PCR and Northern blot analyses showed that this clone was systemically infectious upon agro-inoculation into N. benthamiana. Subsequently, 13 different isolates of MaYMV from field-grown maize plants in different geographical locations of Yunnan and Guizhou provinces of China were sequenced. Analyses of their molecular variation indicate that the 3' half of P3-P5 read-through protein coding region was the most variable, whereas the coat protein- (CP-) and movement protein- (MP-)coding regions were the most conserved.

Coevolution of venom function and venom resistance in a rattlesnake predator and its squirrel prey.

Measuring local adaptation can provide insights into how coevolution occurs between predators and prey. Specifically, theory predicts that local adaptation in functionally matched traits of predators and prey will not be detected when coevolution is governed by escalating arms races, whereas it will be present when coevolution occurs through an alternate mechanism of phenotype matching. Here, we analyse local adaptation in venom activity and prey resistance across 12 populations of Northern Pacific rattlesnakes and California ground squirrels, an interaction that has often been described as an arms race. Assays of venom function and squirrel resistance show substantial geographical variation (influenced by site elevation) in both venom metalloproteinase activity and resistance factor effectiveness. We demonstrate local adaptation in the effectiveness of rattlesnake venom to overcoming present squirrel resistance, suggesting that phenotype matching plays a role in the coevolution of these molecular traits. Further, the predator was the locally adapted antagonist in this interaction, arguing that rattlesnakes are evolutionarily ahead of their squirrel prey. Phenotype matching needs to be considered as an important mechanism influencing coevolution between venomous animals and resistant prey.

Molecular variations in Vibrio alginolyticus and V. harveyi in shrimp-farming systems upon stress

A study was performed to investigate the genomic variations in the shrimp farm isolates of Vibrio alginolyticus and V. harveyi when the isolates were subjected to environmental stress. Samples of shrimps, water and sediment were collected from Southern Indian coastal shrimp farms. Vibrio isolates were biochemically identified and confirmed using 16S rDNA and gyrB gene specific PCR. The bacterial strains were genotyped by PCR fingerprinting using GTG(5) and IS (Insertion Sequence) primers. Seven strains each of V. alginolyticus and V. harveyi were subjected to 10 passages through trypticase soya broth (TSB), which contained different NaCl concentrations (3, 6 and 8%) and trypticase soya agar (TSA). V. alginolyticus was also passaged through TSB with a 12% NaCl concentration. PCR fingerprinting, which was performed on the strains that were passaged through different salt concentrations, confirmed that V. alginolyticus and V. harveyi could affect the genomic variations, depending on the environmental conditions of the culture. The study highlights the complex genotypic variations that occur in Vibrio strains of tropical aquatic environment because of varied environmental conditions, which result in genetic divergence and/or probable convergence. Such genetic divergence and/or convergence can lead to the organismal adaptive variation, which results in their ability to cause a productive infection in aquatic organisms or generation of new strains.

Multiple glacial refugia for cool-temperate deciduous trees in northern East Asia: the Mongolian oak as a case study.

In East Asia, temperate forests are predicted to have retracted southward to c. 30° N during the last glacial maximum (LGM) based on fossil pollen data, whereas phylogeographic studies have often suggested glacial in situ survival of cool-temperate deciduous trees in their modern northern ranges. Here we report a study of the genetic diversity and structure of 29 natural Mongolian oak (Quercus mongolica) populations using 19 nuclear simple sequence repeat (nSSR) loci and four chloroplast DNA fragments. Bayesian clustering analysis with nSSRs revealed five groups, which were inferred by approximate Bayesian computation (ABC) to have diverged in multiple refugia through multiple glacial-interglacial cycles. Analysis of chloroplast DNA variation revealed four lineages that were largely but incompletely geographically disjunct. Ecological niche modelling (ENMs) indicated a southward range shift of the oak's distribution at the LGM, although high suitability scores were also evident in the Changbai Mts. (Northeast China), the Korean Peninsula, areas surrounding the Bohai Sea, and along the coast of the Russian Far East. In addition, endemic chloroplast DNA haplotypes and nuclear lineages occurred in high-latitude northern areas where the ENM predicted no suitable habitat. The combined evidence from nuclear and chloroplast DNA, and the results of the ENM clearly demonstrate that multiple northern refugia, including cryptic ones, were maintained across the current distributional range of the Mongolian oak during the LGM or earlier glacial periods. Though spatially limited, postglacial expansions from these refugia have led to a pattern of decreased genetic diversity with increasing latitude.

Development of an overlapping PCR method to clone the full genome of infectious hypodermal and hematopoietic necrosis virus (IHHNV).

Decapod Penstyldensovirus 1, previously named as infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an economically important pathogen that causes shrimp diseases worldwide. However, a rapid method for cloning full-length IHHNV genome sequences is still lacking, which makes it difficult to study the genomics and molecular epidemiology of IHHNV. Here, a novel and rapid PCR technique was developed to determine the complete genomic sequences of IHHNV. The IHHNV genome was amplified in two overlapping fragments which each yielded a 2kb PCR product covering the first half or the second half of IHHNV genome, respectively. Using this method, six complete genomic sequences of IHHNV, which were collected from different regions of Zhejiang province in China, were cloned and sequenced successfully. The new cloning method will greatly facilitate the study on the genomics and molecular epidemiology of IHHNV.

Molecular variation and horizontal gene transfer of the homocysteine methyltransferase gene mmuM and its distribution in clinical pathogens.

The homocysteine methyltransferase encoded by mmuM is widely distributed among microbial organisms. It is the key enzyme that catalyzes the last step in methionine biosynthesis and plays an important role in the metabolism process. It also enables the microbial organisms to tolerate high concentrations of selenium in the environment. In this research, 533 mmuM gene sequences covering 70 genera of the bacteria were selected from GenBank database. The distribution frequency of mmuM is different in the investigated genera of bacteria. The mapping results of 160 mmuM reference sequences showed that the mmuM genes were found in 7 species of pathogen genomes sequenced in this work. The polymerase chain reaction products of one mmuM genotype (NC_013951 as the reference) were sequenced and the sequencing results confirmed the mapping results. Furthermore, 144 representative sequences were chosen for phylogenetic analysis and some mmuM genes from totally different genera (such as the genes between Escherichia and Klebsiella and between Enterobacter and Kosakonia) shared closer phylogenetic relationship than those from the same genus. Comparative genomic analysis of the mmuM encoding regions on plasmids and bacterial chromosomes showed that pKF3-140 and pIP1206 plasmids shared a 21 kb homology region and a 4.9 kb fragment in this region was in fact originated from the Escherichia coli chromosome. These results further suggested that mmuM gene did go through the gene horizontal transfer among different species or genera of bacteria. High-throughput sequencing combined with comparative genomics analysis would explore distribution and dissemination of the mmuM gene among bacteria and its evolution at a molecular level.

Analysis of genetic characteristics and molecular variation of human rhinovirus strains isolated in Shenzhen during 2012 / 中华微生物学和免疫学杂志

Objective To analyze the genetic characteristics and molecular variation of human rhi-novirus strains isolated in Shenzhen.Methods RNA samples were extracted from nasopharyngeal swab samples collected from influenza-like subjects in Shenzhen and analyzed by fluorescent RT-PCR.The VP4-VP2 and VP1 gene regions of human rhinovirus strains were amplified by nested RT-PCR.Clustal W and MEGA programs were used to evaluate molecular variation of the human rhinovirus strains.Results Both human rhinovirus A and B were prevalent in Shenzhen during 2012.Human rhinoviruses A was the predomi-nant pathogen, including subtypes A47, A31, A90, A18 and so on.Two recombinant strains of human rhi-noviruses A47 and A31 were detected.The mutations scattered on the VP1 protein and varied in different subtypes.The receptor binding sites ( loop BC, DE and HI) in different subtypes showed polymorphism. Five out of twenty-five drug sensitivity sites ( I121V, L123M, V167I, Y189H and H259G) showed muta-tion.Conclusion Multiple subtypes of human rhinovirus were prevalent in Shenzhen and were in a state of constant recombination and variation.

Epidemic pattern and molecular variation of respiratory syncytial virus strains isolated in Shenzhen from year 2012 to 2013 / 中华微生物学和免疫学杂志

Objective To study the epidemic pattern and molecular variation of respiratory syncy-tial virus ( RSV) strains isolated in Shenzhen from year 2012 to 2013.Methods The clinical samples iso-lated from patients with influenza-like illness were analyzed by fluorescent RT-PCR to screen RSV positive strains.The C-terminal variable regions of genes encoding G proteins were amplified by nested RT-PCR. Molecular variation was analyzed by using Clustal W and MEGA softwares.Results RSV strains were wide-ly prevalent in Shenzhen from 2012 to 2013.Two epidemic peaks usually occurred in spring and summer/au-tumn of each year.The RSV isolates were subtyped into group A belonging to genotype NA1 and group B be-longing to genotype BAⅨ.Most of the mutations scattered at the C-terminal region of G protein.A few mu-tations caused the disappearance of certain glycosylation sites.A novel recombinant virus strain containing 24 inserted amino acids was identified in 2013, which was likely to be introduced into our country from abroad. Conclusion RSV strains were widely and continuously prevalent in Shenzhen, characterized by constant evolution and variation.

Pathways of introduction of the invasive aquatic plant Cabomba caroliniana.

The pathway and frequency of species' introductions can affect the extent, impact, and management of biological invasions. Here, we examine the pathway of introduction of the aquatic plant Cabomba caroliniana (fanwort) into Canada and the northern United States using plastid DNA sequence (intergenic spacers atpF-atpH, trnH-psbA, and trnL-trnF) and DNA content analyses. We test the hypothesis that the spread of fanwort is a result of commercial trade by comparing a Canadian population (Kasshabog Lake, ON) to native populations from southern U.S., introduced populations in northern U.S., and plants from commercial retailers. Thirteen plastid haplotypes were identified throughout North America, including one dominant haplotype, which was present in all C. caroliniana populations. Several rare haplotypes were used to infer shared colonization history. In particular, the Canadian population shared two rare alleles with a population from Massachusetts, suggesting range expansion of C. caroliniana from the northern U.S. However, the possibility of a commercial introduction cannot be excluded, as common alleles were shared between the Canadian population and both commercial and southern U.S. sources. Variation in C. caroliniana genome size was bimodal and populations were classified into "high" and "low" categories. The Canadian population had DNA contents similar to several northern U.S. populations (low DNA content). This may provide additional support for range expansion from these introduced populations rather than from commercial sources or populations in the southern U.S., which had high DNA content.