Tracking the transition to agriculture in Southern Europe through ancient DNA analysis of dental calculus.

Archaeological dental calculus, or mineralized plaque, is a key tool to track the evolution of oral microbiota across time in response to processes that impacted our culture and biology, such as the rise of farming during the Neolithic. However, the extent to which the human oral flora changed from prehistory until present has remained elusive due to the scarcity of data on the microbiomes of prehistoric humans. Here, we present our reconstruction of oral microbiomes via shotgun metagenomics of dental calculus in 44 ancient foragers and farmers from two regions playing a pivotal role in the spread of farming across Europe-the Balkans and the Italian Peninsula. We show that the introduction of farming in Southern Europe did not alter significantly the oral microbiomes of local forager groups, and it was in particular associated with a higher abundance of the species Olsenella sp. oral taxon 807. The human oral environment in prehistory was dominated by a microbial species, Anaerolineaceae bacterium oral taxon 439, that diversified geographically. A Near Eastern lineage of this bacterial commensal dispersed with Neolithic farmers and replaced the variant present in the local foragers. Our findings also illustrate that major taxonomic shifts in human oral microbiome composition occurred after the Neolithic and that the functional profile of modern humans evolved in recent times to develop peculiar mechanisms of antibiotic resistance that were previously absent.

Dental calculus as a proxy for animal microbiomes.

The field of dental calculus research has exploded in recent years, predominantly due to the multitude of studies related to human genomes and oral pathogens. Despite having a subset of these studies devoted to non-human primates, little progress has been made in the distribution of oral pathogens across domestic and wild animal populations. This overlooked avenue of research is particularly important at present when many animal populations with the potentiality for zoonotic transmission continue to reside in close proximity to human groups due to reasons such as deforestation and climatic impacts on resource availability. Here, we analyze all previously available published oral microbiome data recovered from the skeletal remains of animals, all of which belong to the Mammalia class. Our genus level results emphasize the tremendous diversity of oral ecologies across mammals in spite of the clustering based primarily on host species. We also discuss the caveats and flaws in analyzing ancient animal oral microbiomes at the species level of classification. Lastly, we assess the benefits, challenges, and gaps in the current knowledge of dental calculus research within animals and postulate the future of the field as a whole.

Integrative approach using Yersinia pestis genomes to revisit the historical landscape of plague during the Medieval Period.

Over the last few years, genomic studies on Yersinia pestis, the causative agent of all known plague epidemics, have considerably increased in numbers, spanning a period of about 5,000 y. Nonetheless, questions concerning historical reservoirs and routes of transmission remain open. Here, we present and describe five genomes from the second half of the 14th century and reconstruct the evolutionary history of Y. pestis by reanalyzing previously published genomes and by building a comprehensive phylogeny focused on strains attributed to the Second Plague Pandemic (14th to 18th century). Corroborated by historical and ecological evidence, the presented phylogeny, which includes our Y. pestis genomes, could support the hypothesis of an entry of plague into Western European ports through distinct waves of introduction during the Medieval Period, possibly by means of fur trade routes, as well as the recirculation of plague within the human population via trade routes and human movement.

The repeat structure of two paralogous genes, Yersinia ruckeri invasin (yrInv) and a "Y. ruckeri invasin-like molecule", (yrIlm) sheds light on the evolution of adhesive capacities of a fish pathogen.

Inverse autotransporters comprise the recently identified type Ve secretion system and are exemplified by intimin from enterohaemorrhagic Escherichia coli and invasin from enteropathogenic Yersiniae. These proteins share a common domain architecture and promote bacterial adhesion to host cells. Here, we identified and characterized two putative inverse autotransporter genes in the fish pathogen Yersinia ruckeri NVH_3758, namely yrInv (for Y. ruckeri invasin) and yrIlm (for Y. ruckeri invasin-like molecule). When trying to clone the highly repetitive genes for structural and functional studies, we experienced problems in obtaining PCR products. PCR failures and the highly repetitive nature of inverse autotransporters prompted us to sequence the genome of Y. ruckeri NVH_3758 using PacBio sequencing, which produces some of the longest average read lengths available in the industry at this moment. According to our sequencing data, YrIlm is composed of 2603 amino acids (7812bp) and has a molecular mass of 256.4kDa. Based on the new genome information, we performed PCR analysis on four non-sequenced Y. ruckeri strains as well as the sequenced. Y. ruckeri type strain. We found that the genes are variably present in the strains, and that the length of yrIlm, when present, also varies. In addition, the length of the gene product for all strains, including the type strain, was much longer than expected based on deposited sequences. The internal repeats of the yrInv gene product are highly diverged, but represent the same bacterial immunoglobulin-like domains as in yrIlm. Using qRT-PCR, we found that yrIlm and yrInv are differentially expressed under conditions relevant for pathogenesis. In addition, we compared the genomic context of both genes in the newly sequenced Y. ruckeri strain to all available PacBio-sequenced Y. ruckeri genomes, and found indications of recent events of horizontal gene transfer. Taken together, this study demonstrates and highlights the power of Single Molecule Real-Time technology for sequencing highly repetitive proteins, and sheds light on the genetic events that gave rise to these highly repetitive genes in a commercially important fish pathogen.

Mediterranean Y-chromosome 2.0-why the Y in the Mediterranean is still relevant in the postgenomic era.

CONTEXT: Due to its unique paternal inheritance, the Y-chromosome has been a highly popular marker among population geneticists for over two decades. Recently, the advent of cost-effective genome-wide methods has unlocked information-rich autosomal genomic data, paving the way to the postgenomic era. This seems to have announced the decreasing popularity of investigating Y-chromosome variation, which provides only the paternal perspective of human ancestries and is strongly influenced by genetic drift and social behaviour. OBJECTIVE: For this special issue on population genetics of the Mediterranean, the aim was to demonstrate that the Y-chromosome still provides important insights in the postgenomic era and in a time when ancient genomes are becoming exponentially available. METHODS: A systematic literature search on Y-chromosomal studies in the Mediterranean was performed. RESULTS: Several applications of Y-chromosomal analysis with future opportunities are formulated and illustrated with studies on Mediterranean populations. CONCLUSIONS: There will be no reduced interest in Y-chromosomal studies going from reconstruction of male-specific demographic events to ancient DNA applications, surname history and population-wide estimations of extra-pair paternity rates. Moreover, more initiatives are required to collect population genetic data of Y-chromosomal markers for forensic research, and to include Y-chromosomal data in GWAS investigations and studies on male infertility.

Bight of Benin: a Maternal Perspective of Four Beninese Populations and their Genetic Implications on the American Populations of African Ancestry.

The understanding of the first movements of the ancestral populations within the African continent is still unclear, particularly in West Africa, due to several factors that have shaped the African genetic pool across time. To improve the genetic representativeness of the Beninese population and to better understand the patterns of human settlement inside West Africa and the dynamics of peopling of the Democratic Republic of Benin, we analyzed the maternal genetic variation of 193 Beninese individuals belonging to Bariba, Berba, Dendi, and Fon populations. Results support the oral traditions indicating that the western neighbouring populations have been the ancestors of the first Beninese populations, and the extant genetic structure of the Beninese populations is most likely the result of admixture between populations from neighbouring countries and native people. The present findings highlight how the Beninese populations contributed to the gene pool of the extant populations of some American populations of African ancestry. This strengthens the hypothesis that the Bight of Benin was not only an assembly point for the slave trade during the Trans-Atlantic Slave Trade but also an important slave trapping area.

Pig domestication and human-mediated dispersal in western Eurasia revealed through ancient DNA and geometric morphometrics.

Zooarcheological evidence suggests that pigs were domesticated in Southwest Asia ~8,500 BC. They then spread across the Middle and Near East and westward into Europe alongside early agriculturalists. European pigs were either domesticated independently or more likely appeared so as a result of admixture between introduced pigs and European wild boar. As a result, European wild boar mtDNA lineages replaced Near Eastern/Anatolian mtDNA signatures in Europe and subsequently replaced indigenous domestic pig lineages in Anatolia. The specific details of these processes, however, remain unknown. To address questions related to early pig domestication, dispersal, and turnover in the Near East, we analyzed ancient mitochondrial DNA and dental geometric morphometric variation in 393 ancient pig specimens representing 48 archeological sites (from the Pre-Pottery Neolithic to the Medieval period) from Armenia, Cyprus, Georgia, Iran, Syria, and Turkey. Our results reveal the first genetic signatures of early domestic pigs in the Near Eastern Neolithic core zone. We also demonstrate that these early pigs differed genetically from those in western Anatolia that were introduced to Europe during the Neolithic expansion. In addition, we present a significantly more refined chronology for the introduction of European domestic pigs into Asia Minor that took place during the Bronze Age, at least 900 years earlier than previously detected. By the 5th century AD, European signatures completely replaced the endemic lineages possibly coinciding with the widespread demographic and societal changes that occurred during the Anatolian Bronze and Iron Ages.

First archaeological evidence for ginger consumption as a potential medicinal ingredient in a late medieval leprosarium at St Leonard, Peterborough, England.

Leprosy was one of the most outwardly visible diseases in the European Middle Ages, a period during which leprosaria were founded to provide space for the sick. The extant documentary evidence for leprosy hospitals, especially in relation to diet, therapeutic, and medical care, is limited. However, human dental calculus stands to be an important source of information as it provides insight into the substances people were exposed to and accumulated in their bodies during their lives. In the present study, microremains and DNA were analysed from the calculus of individuals buried in the late medieval cemetery of St Leonard, a leprosarium located in Peterborough, England. The results show the presence of ginger (Zingiber officinale), a culinary and medicinal ingredient, as well as evidence of consumption of cereals and legumes. This research suggests that affected individuals consumed ingredients mentioned in medieval medical textbooks that were used to treat regions of the body typically impacted by leprosy. To the authors' knowledge, this is the first study which has identified Zingiber officinale in human dental calculus in England or on the wider European continent.

A dietary perspective of cat-human interactions in two medieval harbors in Iran and Oman revealed through stable isotope analysis.

Cats are hypercarnivorous, opportunistic animals that have adjusted to anthropogenic environments since the Neolithic period. Through humans, either by direct feeding and/or scavenging on food scraps, the diet of cats has been enriched with animals that they cannot kill themselves (e.g., large mammals, fish). Here, we conducted carbon and nitrogen stable isotope ratio analysis to reconstruct the diet of medieval cats and investigate cat-human interactions in two medieval harbor sites (Qalhât, Oman and Siraf, Iran). The analysis included 28 cat individuals and 100 associated marine and terrestrial faunal samples pertaining to > 30 taxa. The isotopic results indicate a high marine protein-based diet for the cats from Qalhât and a mixed marine-terrestrial (C4) diet for the cats from Siraf. Cats at these sites most likely scavenged on both human food scraps and refuse related to fishing activities, with differences in the two sites most likely associated with the availability of marine resources and/or the living conditions of the cats. By shedding light on the dietary habits of cats from two medieval harbors in the Arabian Gulf and Gulf of Oman, this study illustrates the potential of stable isotope analysis in reconstructing human-cat interactions in the past.

Adulis and the transshipment of baboons during classical antiquity.

Adulis, located on the Red Sea coast in present-day Eritrea, was a bustling trading centre between the first and seventh centuries CE. Several classical geographers-Agatharchides of Cnidus, Pliny the Elder, Strabo-noted the value of Adulis to Greco-Roman Egypt, particularly as an emporium for living animals, including baboons (Papio spp.). Though fragmentary, these accounts predict the Adulite origins of mummified baboons in Ptolemaic catacombs, while inviting questions on the geoprovenance of older (Late Period) baboons recovered from Gabbanat el-Qurud ('Valley of the Monkeys'), Egypt. Dated to ca. 800-540 BCE, these animals could extend the antiquity of Egyptian-Adulite trade by as much as five centuries. Previously, Dominy et al. (2020) used stable isotope analysis to show that two New Kingdom specimens of Papio hamadryas originate from the Horn of Africa. Here, we report the complete mitochondrial genomes from a mummified baboon from Gabbanat el-Qurud and 14 museum specimens with known provenance together with published georeferenced mitochondrial sequence data. Phylogenetic assignment connects the mummified baboon to modern populations of P. hamadryas in Eritrea, Ethiopia, and eastern Sudan. This result, assuming geographical stability of phylogenetic clades, corroborates Greco-Roman historiographies by pointing toward present-day Eritrea, and by extension Adulis, as a source of baboons for Late Period Egyptians. It also establishes geographic continuity with baboons from the fabled Land of Punt (Dominy et al., 2020), giving weight to speculation that Punt and Adulis were essentially the same trading centres separated by a thousand years of history.

Stable isotopes unveil one millennium of domestic cat paleoecology in Europe.

The domestic cat is the world's most popular pet and one of the most detrimental predators in terrestrial ecosystems. Effective protection of wildlife biodiversity demands detailed tracking of cat trophic ecology, and stable isotopes serve as a powerful proxy in dietary studies. However, a variable diet can make an isotopic pattern unreadable in opportunistic predators. To evaluate the usefulness of the isotopic method in cat ecology, we measured C and N isotope ratios in hundreds of archaeological cat bones. We determined trends in cat trophic paleoecology in northern Europe by exploiting population-scale patterns in animals from diverse locations. Our dataset shows a high variability of isotopic signals related to the socio-economic and/or geomorphological context. This points toward regularities in isotopic patterns across past cat populations. We provide a generalized guide to interpret the isotopic ecology of cats, emphasizing that regional isotopic baselines have a major impact on the isotopic signal.

Deep into the roots of the Libyan Tuareg: a genetic survey of their paternal heritage.

Recent genetic studies of the Tuareg have begun to uncover the origin of this semi-nomadic northwest African people and their relationship with African populations. For centuries they were caravan traders plying the trade routes between the Mediterranean coast and south-Saharan Africa. Their origin most likely coincides with the fall of the Garamantes who inhabited the Fezzan (Libya) between the 1st millennium BC and the 5th century AD. In this study we report novel data on the Y-chromosome variation in the Libyan Tuareg from Al Awaynat and Tahala, two villages in Fezzan, whose maternal genetic pool was previously characterized. High-resolution investigation of 37 Y-chromosome STR loci and analysis of 35 bi-allelic markers in 47 individuals revealed a predominant northwest African component (E-M81, haplogroup E1b1b1b) which likely originated in the second half of the Holocene in the same ancestral population that contributed to the maternal pool of the Libyan Tuareg. A significant paternal contribution from south-Saharan Africa (E-U175, haplogroup E1b1a8) was also detected, which may likely be due to recent secondary introduction, possibly through slavery practices or fusion between different tribal groups. The difference in haplogroup composition between the villages of Al Awaynat and Tahala suggests that founder effects and drift played a significant role in shaping the genetic pool of the Libyan Tuareg.

Wild cereal grain consumption among Early Holocene foragers of the Balkans predates the arrival of agriculture.

Forager focus on wild cereal plants has been documented in the core zone of domestication in southwestern Asia, while evidence for forager use of wild grass grains remains sporadic elsewhere. In this paper, we present starch grain and phytolith analyses of dental calculus from 60 Mesolithic and Early Neolithic individuals from five sites in the Danube Gorges of the central Balkans. This zone was inhabited by likely complex Holocene foragers for several millennia before the appearance of the first farmers ~6200 cal BC. We also analyzed forager ground stone tools (GSTs) for evidence of plant processing. Our results based on the study of dental calculus show that certain species of Poaceae (species of the genus Aegilops) were used since the Early Mesolithic, while GSTs exhibit traces of a developed grass grain processing technology. The adoption of domesticated plants in this region after ~6500 cal BC might have been eased by the existing familiarity with wild cereals.

Before humans invented agriculture and the first farmers appeared in southwestern Asia, other ancient foragers (also known as hunter-gatherers) in southeastern Europe had already developed a taste for consuming wild plants. There is evidence to suggest that these foragers were intensely gathering wild cereal grains before the arrival of agriculture. However, until now, the only place outside southwestern Asia this has been shown to have occurred is in Greece, and is dated around 20,000 years ago. In the past, researchers proposed that forager societies in the Balkans also consumed wild cereals before transitioning to agriculture. But this has been difficult to prove because plant foods are less likely to preserve than animal bones and teeth, making them harder to detect in prehistoric contexts. To overcome this, Cristiani et al. studied teeth from 60 individuals found in archaeological sites between Serbia and Romania, which are attributed to the Mesolithic and Early Neolithic periods. Food particles extracted from crusty deposits on the teeth (called the dental calculus) were found to contain structures typically found in plants. In addition, Cristiani et al. discovered similar plant food residues on ground stone tools which also contained traces of wear associated with the processing of wild cereals. These findings suggest that foragers in the central Balkans were already consuming certain species of wild cereal grains 11,500 years ago, before agriculture arrived in Europe. It is possible that sharing knowledge about plant resources may have helped introduce domesticated plant species in to this region as early as 6500 BC. This work challenges the deep-rooted idea that the diet of hunter-gatherers during the Palaeolithic and Mesolithic periods primarily consisted of animal proteins. In addition, it highlights the active role the eating habits of foragers might have played in introducing certain domesticated plant species that have become primary staples of our diet today.

Multipronged dental analyses reveal dietary differences in last foragers and first farmers at Grotta Continenza, central Italy (15,500-7000 BP).

This paper provides results from a suite of analyses made on human dental material from the Late Palaeolithic to Neolithic strata of the cave site of Grotta Continenza situated in the Fucino Basin of the Abruzzo region of central Italy. The available human remains from this site provide a unique possibility to study ways in which forager versus farmer lifeways affected human odonto-skeletal remains. The main aim of our study is to understand palaeodietary patterns and their changes over time as reflected in teeth. These analyses involve a review of metrics and oral pathologies, micro-fossils preserved in the mineralized dental plaque, macrowear, and buccal microwear. Our results suggest that these complementary approaches support the assumption about a critical change in dental conditions and status with the introduction of Neolithic foodstuff and habits. However, we warn that different methodologies applied here provide data at different scales of resolution for detecting such changes and a multipronged approach to the study of dental collections is needed for a more comprehensive and nuanced understanding of diachronic changes.

Population differences in allele frequencies at the OLR1 locus may suggest geographic disparities in cardiovascular risk events.

BACKGROUND: Several studies have demonstrated a link between cardiovascular disease (CVD) susceptibility and the genetic background of populations. Endothelial activation and dysfunction induced by oxidized low-density lipoprotein (ox-LDL) is one of the key steps in the initiation of atherosclerosis. The oxidized low density lipoprotein (lectin-like) receptor 1 (OLR1) gene is the main receptor of ox-LDL. We have previously characterized two polymorphisms (rs3736235 and rs11053646) associated with the risk for coronary artery disease (CAD) and acute myocardial infarction (AMI). AIM: Given their clinical significance, it is of interest to know the distribution of these variants in populations from different continents. SUBJECTS AND METHODS: A total of 1229 individuals from 17 different African, Asian and European populations was genotyped for the two considered markers. RESULTS: The high frequencies of ancestral alleles in South-Saharan populations is concordant with the African origin of our species. The results highlight that African populations are closer to Asians, and clearly separated from the Europeans. CONCLUSION: The results confirm significant genetic structuring among populations and suggest a possible basis for varying susceptibility to CVD among groups correlated with the geographical location of populations linked with the migrations out of Africa, or with different lifestyle.

Was the Cinta Senese Pig Already a Luxury Food in the Late Middle Ages? Ancient DNA and Archaeozoological Evidence from Central Italy.

The Cinta senese is a pig breed, highly esteemed for its meat and derived products, characterized by a black coat with a typical white "belt" and documented by scant iconography, since the 13th-14th century in Italy. A piece of pottery showing a Cinta pig was found in the Graffignano castle (Northern Latium, Italy) dated 15th-16th centuries, spurring us to investigate the diet of the inhabitants. Ancient DNA analysis was carried out on 21 pig specimens on three nuclear SNPs: (1) g.43597545C>T, on the KIT gene, informative for the identification of the Cinta senese breed; (2) rs81460129, on an intergenic region in chr. 16, which discriminates between domestic pigs and wild boars, and; (3) a SNP on the ZFY/ZFX homologous genes, to determine the sex of the individuals. Our results indicate that the Cinta senese was present in Northern Latium in Late Medieval time, although it was not the only breed, and that pigs, including Cinta, interbred with wild boars, suggesting free-range breeding for all types of pigs. Moreover, the unexpected high proportion of young females may be considered as evidence for the wealth of the family inhabiting the castle.

First genetic insight into Libyan Tuaregs: a maternal perspective.

The Tuaregs are a semi-nomadic pastoralist people of northwest Africa. Their origins are still a matter of debate due to the scarcity of genetic and historical data. Here we report the first data on the mitochondrial DNA (mtDNA) genetic characterization of a Tuareg sample from Fezzan (Libyan Sahara). A total of 129 individuals from two villages in the Acacus region were genetically analysed. Both the hypervariable regions and the coding region of mtDNA were investigated. Phylogeographic investigation was carried out in order to reconstruct human migratory shifts in central Sahara, and to shed light on the origin of the Libyan Tuaregs. Our results clearly show low genetic diversity in the sample, possibly due to genetic drift and founder effect associated with the separation of Libyan Tuaregs from an ancestral population. Furthermore, the maternal genetic pool of the Libyan Tuaregs is characterized by a major "European" component shared with the Berbers that could be traced to the Iberian Peninsula, as well as a minor 'south Saharan' contribution possibly linked to both Eastern African and Near Eastern populations.

Human mitochondrial DNA variation in Southern Italy.

BACKGROUND: Since prehistoric times Southern Italy has been a cultural crossroads of the Mediterranean basin. Genetic data on the peoples of Basilicata and Calabria are scarce and, particularly, no records on mtDNA variability have been published. AIM: In this study mtDNA haplotypes of populations from Basilicata, Calabria and Sicily are compared with those of other Italian and Mediterranean populations, so as to investigate their genetic relationships. SUBJECTS AND METHODS: A total of 341 individuals was analysed for mtDNA in order to provide their classification into haplogroups. Multivariate analysis was used to compare the studied populations with other Mediterranean samples; median-joining network analysis was applied to observe the relationship between the major lineages of the Southern Italians. RESULTS: The haplogroup distribution in the Southern Italian samples falls within the typical pattern of mtDNA variability of Western Eurasia. The comparison with other Mediterranean countries showed a substantial homogeneity of the area, which is probably related to the historic contact through the Mediterranean Sea. CONCLUSION: The mtDNA analysis demonstrated that Southern Italy displays a typical pattern of Mediterranean basin variability, even though it appears plausible that Southern Italy was less affected by the effects of the Late Glacial Maximum, which reduced genetic diversity in Europe.

Metagenomic analysis of dental calculus in ancient Egyptian baboons.

Dental calculus, or mineralized plaque, represents a record of ancient biomolecules and food residues. Recently, ancient metagenomics made it possible to unlock the wealth of microbial and dietary information of dental calculus to reconstruct oral microbiomes and lifestyle of humans from the past. Although most studies have so far focused on ancient humans, dental calculus is known to form in a wide range of animals, potentially informing on how human-animal interactions changed the animals' oral ecology. Here, we characterise the oral microbiome of six ancient Egyptian baboons held in captivity during the late Pharaonic era (9th-6th centuries BC) and of two historical baboons from a zoo via shotgun metagenomics. We demonstrate that these captive baboons possessed a distinctive oral microbiome when compared to ancient and modern humans, Neanderthals and a wild chimpanzee. These results may reflect the omnivorous dietary behaviour of baboons, even though health, food provisioning and other factors associated with human management, may have changed the baboons' oral microbiome. We anticipate our study to be a starting point for more extensive studies on ancient animal oral microbiomes to examine the extent to which domestication and human management in the past affected the diet, health and lifestyle of target animals.

pYR4 From a Norwegian Isolate of Yersinia ruckeri Is a Putative Virulence Plasmid Encoding Both a Type IV Pilus and a Type IV Secretion System.

Enteric redmouth disease caused by the pathogen Yersinia ruckeri is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of Y. ruckeri have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian Y. ruckeri isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic Yersiniae, pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other Y. ruckeri plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the Y. ruckeri NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system (tra). In contrast to other Yersinia plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other Y. ruckeri plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the Y. ruckeri 150 and Y. ruckeri ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for de novo genome sequencing. As the tra operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to Y. ruckeri virulence.