Strong Population Bottlenecks Research Articles

Genomic insights into the evolutionary and demographic histories of the extinct Hoopoe Starling (Fregilupus varius)

The Hoopoe Starling Fregilupus varius is an extinct species of the Sturnidae that was endemic to Réunion Island in the Indian Ocean. The species rapidly disappeared in the middle of the 19th century, primarily because of overexploitation by humans. We generated an approximately 11× coverage genome to reconstruct the demographic history of the Hoopoe Starling and compared these results with the demographic histories of other starlings and myna species. Our analyses confirmed the close affinities of the Hoopoe Starling with the genera Sturnia, Leucopsar and Sturnornis, and revealed that it went through a strong population bottleneck during its evolutionary history, but that its effective population size was not particularly low when compared with other extinct or critically endangered species of birds.

Loss of West Nile virus genetic diversity during mosquito infection due to species-dependent population bottlenecks

Vector competence (VC) refers to the efficiency of pathogen transmission by vectors. Each step in the infection of a mosquito vector constitutes a barrier to transmission that may impose bottlenecks on virus populations. West Nile virus (WNV) is maintained by multiple mosquito species with varying VC. However, the extent to which bottlenecks and VC are linked is poorly understood. Similarly, quantitative analyses of mosquito-imposed bottlenecks on virus populations are limited. We used molecularly barcoded WNV to quantify tissue-associated population bottlenecks in three variably competent WNV vectors. Our results confirm strong population bottlenecks during mosquito infection that are capable of dramatically reshaping virus population structure in a non-selective manner. In addition, we found that mosquitoes with differing VC uniquely shape WNV population structure: highly competent vectors are more likely to contribute to the maintenance of rare viral genotypes. These findings have important implications for arbovirus emergence and evolution.

Small-scale genetic structure of populations of the bulb mite Rhizoglyphus robini.

Bulb mites are an economically significant pest of subterranean parts of plants and a versatile laboratory animal. However, the genetic structure of their populations remains unknown. To fill this gap in our knowledge of their biology, we set up a field experiment in which we allowed mites to colonize onion bulbs, and then determined the genetic structure of colonisers based on a panel of microsatellite loci. We found moderate but significant population structure among sites separated by ca. 20m (FST range 0.03-0.21), with 7% of genetic variance distributed among sites. Allelic richness within some bulbs was nearly as high as that in the total population, suggesting that colonisation of bulbs was not associated with strong population bottlenecks. The significant genetic structure we observed over small spatial scales seems to reflect limited dispersal of mites in soil.

Leishmania allelic selection during experimental sand fly infection correlates with mutational signatures of oxidative DNA damage

Trypanosomatid pathogens are transmitted by blood-feeding insects, causing devastating human infections. These parasites show important phenotypic shifts that often impact parasite pathogenicity, tissue tropism, or drug susceptibility. The evolutionary mechanisms that allow for the selection of such adaptive phenotypes remain only poorly investigated. Here, we use Leishmania donovani as a trypanosomatid model pathogen to assess parasite evolutionary adaptation during experimental sand fly infection. Comparing the genome of the parasites before and after sand fly infection revealed a strong population bottleneck effect as judged by allele frequency analysis. Apart from random genetic drift caused by the bottleneck effect, our analyses revealed haplotype and allelic changes during sand fly infection that seem under natural selection given their convergence between independent biological replicates. Our analyses further uncovered signature mutations of oxidative DNA damage in the parasite genomesafter sand fly infection, suggesting that Leishmania suffers from oxidative stress inside the insect digestive tract. Our results propose a model of Leishmania genomic adaptation during sand fly infection, with oxidative DNA damage and DNA repair processes likely driving haplotype and allelic selection. The experimental and computational framework presented here provides a useful blueprint to assess evolutionary adaptation of other eukaryotic pathogens inside their insect vectors, such as Plasmodium spp, Trypanosoma brucei, and Trypanosoma cruzi.

Whole-genome sequencing analysis of wild house mice (Mus musculus) captured in Madagascar.

In Madagascar, the house mouse (Mus musculus) is widely believed to have colonized with human activities and is now one of the most abundant rodents on the island. However, its genetic background at the genomic level remains unclear, and clarifying this would help us to infer the timing of introduction and route of migration. In this study, we determined the whole-genome sequences of five Madagascar house mice captured from an inland location in Madagascar. We examined the genetic background of samples by analyzing the mitochondrial and autosomal genomes. We confirmed that the mitochondrial genome lineages of collected samples formed a single clade placed at one of the most basal positions in the Mus musculus species. Autosomal genomic sequences revealed that these samples are most closely related to the subspecies M. m. castaneus (CAS), but also contain a genetic component of the subspecies M. m. domesticus (DOM). The signature of a strong population bottleneck 1,000-3,000 years ago was observed in both mitochondrial and autosomal genomic data. In a comparison with global samples of M. musculus, the Madagascar samples showed strong genetic affinity to many CAS samples across a wide range of Indian Ocean coastal and insular regions, with divergence time estimated as around 4,000 years ago. These findings support the proposition that the ancestors of these animals started to colonize the island with human agricultural activity and experienced a complex history during their establishment.

Detecting Selection in the HIV-1 Genome during Sexual Transmission Events.

Little is known about whether and how variation in the HIV-1 genome affects its transmissibility. Assessing which genomic features of HIV-1 are under positive or negative selection during transmission is challenging, because very few virus particles are typically transmitted, and random genetic drift can dilute genetic signals in the recipient virus population. We analyzed 30 transmitter–recipient pairs from the Zurich Primary HIV Infection Study and the Swiss HIV Cohort Study using near full-length HIV-1 genomes. We developed a new statistical test to detect selection during transmission, called Selection Test in Transmission (SeTesT), based on comparing the transmitter and recipient virus population and accounting for the transmission bottleneck. We performed extensive simulations and found that sensitivity of detecting selection during transmission is limited by the strong population bottleneck of few transmitted virions. When pooling individual test results across patients, we found two candidate HIV-1 genomic features for affecting transmission, namely amino acid positions 3 and 18 of Vpu, which were significant before but not after correction for multiple testing. In summary, SeTesT provides a general framework for detecting selection based on genomic sequencing data of transmitted viruses. Our study shows that a higher number of transmitter–recipient pairs is required to improve sensitivity of detecting selection.

Informing conservation strategies with museum genomics: Long-term effects of past anthropogenic persecution on the elusive European wildcat.

Like many carnivore species, European wildcats (Felis silvestris) have suffered severe anthropogenic population declines in the past, resulting in a strong population bottleneck at the beginning of the 20th century. In Germany, the species has managed to survive its near extinction in small isolated areas and is currently recolonizing former habitats owing to legal protection and concerted conservation efforts. Here, we SNP‐genotyped and mtDNA‐sequenced 56 historical and 650 contemporary samples to assess the impact of massive persecution on genetic diversity, population structure, and hybridization dynamics of wildcats. Spatiotemporal analyses suggest that the presumed postglacial differentiation between two genetically distinct metapopulations in Germany is in fact the result of the anthropogenic bottleneck followed by re‐expansion from few secluded refugia. We found that, despite the bottleneck, populations experienced no severe genetic erosion, nor suffered from elevated inbreeding or showed signs of increased hybridization with domestic cats. Our findings have significant implications for current wildcat conservation strategies, as the data analyses show that the two presently recognized wildcat population clusters should be treated as a single conservation unit. Although current populations appear under no imminent threat from genetic factors, fostering connectivity through the implementation of forest corridors will facilitate the preservation of genetic diversity and promote long‐term viability. The present study documents how museum collections can be used as essential resource for assessing long‐term anthropogenic effects on natural populations, for example, regarding population structure and the delineation of appropriate conservation units, potentially informing todays' species conservation.

Identification of management units in threatened populations of Arnica mallotopus Makino (Asteraceae) using novel EST-SSR markers.

Arnica mallotopus is a perennial herb endemic to the snowy regions of Japan. At the southern edge of its distribution, in Kyoto Prefecture, overgrazing by sika deer and decreased snowfall have resulted in the rapid decline of A. mallotopus populations. Therefore, there is an urgent need for a conservation genetic analysis of the remaining local populations. In this study, we first developed 13 EST-SSR markers to evaluate genetic variation in A. mallotopus. The average number of alleles per locus was 5.33. Genetic analysis using these markers showed that the investigated samples were classified into two groups corresponding to landscape structure. One group isolated from a tributary of the Yura River showed a strong population bottleneck signal, likely resulting from founder effects and subsequent drifts. On the other hand, the genetic diversity of the second group in the main distribution along the Yura River was higher and less inbred. Overall, our assessment suggested recognizing the two genetic groups as management units in conservation programs for the threatened populations.

Parallel adaptation to climate above the 35th parallel.

Independent or parallel evolution of similar traits is key to understanding the genetics and limitations of adaptation. Adaptation from the same genetic changes in different populations defines parallel evolution. Such genetic changes can derive from standing ancestral variation or de novo mutations and excludes instances of adaptive introgression. In this issue of Molecular Ecology, Walden et al.(2020) investigate the scale of parallel climate adaptation from standing genetic variation between two North American Arabidopsis lyrata lineages, each formed by a distinct evolutionary history during the last glacial cycle. By identifying adaptive variants correlated with three ecologically significant climatic gradients, they show that instead of the same genetic variants or even genes, parallel evolution is only observed at the level of biological processes. The evolution of independent adaptive variants to climate in two genetically close lineages is explained by their different post-glacial demographic histories. Separate glacial refugia and strong population bottlenecks were probably sufficient to change the landscape of shared allele frequencies, hindering the possibility of parallel evolution.

On the origins of the Sakhas' paternal lineages: Reconciliation of population genetic / ancient DNA data, archaeological findings and historical narratives

Sakhas are Turkic-speaking people from Northeastern Siberia, constituting the largest ethnic population in Yakutia. According to popular legends, two heroes who arrived from the Asian Steppe during the late medieval ages, namely Elley Bootur and Omogoy Baay, are the progenitors of all Sakhas. While there is ample historical evidence towards the existence of such legendary characters, archaeological findings and ancient DNA studies provide further insights on actual Sakha ethnogenesis. This study aims to establish the genetic basis of the legendary characters Elley and Omogoy, at least through their paternal lineages, and then to reveal the prevalence of these Y-chromosomes among the contemporary Yakut population. To this end, an attempt was made to delineate fact from fiction with respect to the Sakhas’ paternal lineages through a reconciliation of population genetics data on contemporary and ancient Sakhas, along with archaeological evidence and well-recorded historical narratives. To achieve this, 17-loci Y-chromosomal STR and haplogroup analyses were conducted on a contemporary Sakha who was presumably a direct descendant of Elley’s paternal line. Furthermore, 367 Sakha Y-chromosomal STR haplotypes were compiled from the literature and elsewhere, and searched against the Y-chromosome STR Haplotype Reference Database to find potential matches with non-Sakha populations. Sakhas’ paternal lineages were found to comprise 6 major descent clusters, each corresponding to an ancient clan. The most prevalent haplotype indeed corresponded to that of the contemporary Elley descendant. Furthermore, data presented in the current work suggests a Khitan origin for this paternal line. As shown before, Sakhas’ paternal lineages were found to be very homogenous and exhibit signs of a strong population bottleneck. Reconciled genetic and archaeological data agree well with Sakhas’ historical narratives, whereby, at least from a paternal lineage perspective, only a few individuals may have arrived from Central Asia and had reproductive success that led to the Sakha Y-chromosomal diversity today.

Coalescent models characterize sources and demographic history of recent round goby colonization of Great Lakes and inland waters.

The establishment and spread of aquatic invasive species are ecologically and economically harmful and a source of conservation concern internationally. Processes of species invasion have traditionally been inferred from observational data of species presence/absence and relative abundance. However, genetic‐based approaches can provide valuable sources of inference. Restriction site‐associated DNA sequencing was used to identify and genotype single nucleotide polymorphism (SNP) loci for Round Gobies (Neogobius melanostomus) (N = 440) from 18 sampling locations in the Great Lakes and in three Michigan, USA, drainages (Flint, Au Sable, and Cheboygan River basins). Sampled rivers differed in size, accessibility, and physical characteristics including man‐made dispersal barriers. Population levels of genetic diversity and interpopulation variance in SNP allele frequency were used in coalescence‐based approximate Bayesian computation (ABC) to statistically compare models representing competing hypotheses regarding source population, postcolonization dispersal, and demographic history in the Great Lakes and inland waters. Results indicate different patterns of colonization across the three drainages. In the Flint River, models indicate a strong population bottleneck (<3% of contemporary effective population size) and a single founding event from Saginaw Bay led to the colonization of inland river segments. In the Au Sable River, analyses could not distinguish potential source populations, but supported models indicated multiple introductions from one source population. In the Cheboygan River, supported models indicated that colonization likely proceeded from east (Lake Huron source) to west among inland locales sampled in the system. Despite the recent occupancy of Great Lakes and inland habitats, large numbers of loci analyzed in an ABC framework enable statistically supported identification of source populations and reconstruction of the direction of inland spread and demographic history following establishment. Information from analyses can direct management actions to limit the spread of invasive species from identified sources and most probable vectors into additional inland aquatic habitats.

Elevated Proportions of Deleterious Genetic Variation in Domestic Animals and Plants.

A fraction of genetic variants segregating in any population are deleterious, which negatively impacts individual fitness. The domestication of animals and plants is associated with population bottlenecks and artificial selection, which are predicted to increase the proportion of deleterious variants. However, the extent to which this is a general feature of domestic species is unclear. Here, we examine the effects of domestication on the prevalence of deleterious variation using pooled whole-genome resequencing data from five domestic animal species (dog, pig, rabbit, chicken, and silkworm) and two domestic plant species (rice and soybean) compared with their wild ancestors. We find significantly reduced genetic variation and increased proportion of nonsynonymous amino acid changes in all but one of the domestic species. These differences are observable across a range of allele frequencies, both common and rare. We find proportionally more single nucleotide polymorphisms in highly conserved elements in domestic species and a tendency for domestic species to harbor a higher proportion of changes classified as damaging. Our findings most likely reflect an increased incidence of deleterious variants in domestic species, which is most likely attributable to population bottlenecks that lead to a reduction in the efficacy of selection. An exception to this pattern is displayed by European domestic pigs, which do not show traces of a strong population bottleneck and probably continued to exchange genes with wild boar populations after domestication. The results presented here indicate that an elevated proportion of deleterious variants is a common, but not ubiquitous, feature of domestic species.

Low mitochondrial diversity in native Italian pig breeds is consistent with the occurrence of strong population bottlenecks.

In this study, we have analysed the variation of 81 Italian pigs from the Calabrese, Casertana, Cinta Senese, Sarda and Nero Siciliano breeds as well as 54 Italian wild boars by using a dataset of mitochondrial control region sequences generated by us and others. Diversity parameters were rather low in Italian native pigs, with haplotype and nucleotide diversities ranging between 0.35-0.79 and 0.0013-0.0061, respectively. This result is consistent with the strong population bottlenecks that traditional Italian breeds have suffered due to competition with more productive foreign pig varieties. Moreover, median-joining network analysis showed that the majority of Italian pig sequences are distributed in two main clusters and that all of them belong to the E1 haplogroup. Conversely, Italian wild boars were more diverse than their domestic counterparts and they harboured the E1 and E2 haplogroups. The absence of the E2 haplogroup in Italian pigs and its moderate frequency in wild boars might suggest that this haplogroup was rare at the time that wild boars were domesticated in Italy.

The Near East as a cradle of biodiversity: A phylogeography of banded newts (genus Ommatotriton) reveals extensive inter- and intraspecific genetic differentiation

The banded newt (genus Ommatotriton) is widely distributed in the Near East (Anatolia, Caucasus and the Levant) – an understudied region from the perspective of phylogeography. The genus is polytypic, but the number of species included and the phylogenetic relationships between them are not settled. We sequenced two mitochondrial and two nuclear DNA markers throughout the range of Ommatotriton. For mtDNA we constructed phylogenetic trees, estimated divergence times using fossil calibration, and investigated changes in effective population size with Bayesian skyline plots and mismatch analyses. For nuDNA we constructed phylogenetic trees and haplotype networks. Species trees were constructed for all markers and nuDNA only. Species distribution models were projected on current and Last Glacial Maximum climate layers. We confirm the presence of three Ommatotriton species: O. nesterovi, O. ophryticus and O. vittatus. These species are genetically distinct and their most recent common ancestor was dated at ∼25Ma (Oligocene). No evidence of recent gene flow between species was found. The species show deep intraspecific genetic divergence, represented by geographically structured clades, with crown nodes of species dated ∼8–13Ma (Miocene to Early Quaternary); evidence of long-term in situ evolution and survival in multiple glacial refugia. While a species tree based on nuDNA suggested a sister species relationship between O. vittatus and O. ophryticus, when mtDNA was included, phylogenetic relationships were unresolved, and we refrain from accepting a particular phylogenetic hypothesis at this stage. While species distribution models suggest reduced and fragmented ranges during the Last Glacial Maximum, we found no evidence for strong population bottlenecks. We discuss our results in the light of other phylogeographic studies from the Near East. Our study underlines the important role of the Near East in generating and sustaining biodiversity.

Mitochondrial DNA D-Loop diversity and evolutionary relationship of wild Punjab Urial sheep (Ovis vignei punjabiensis) with closely related taxa

The taxonomy and systematics of wild sheep is complex with several classifications and revisions proposed on the basis of morphology, geographic distribution and chromosome number. Punjab Urial (O. vignei punjabiensis), an important sub-species of O. vignei is distributed in Pakistan and is classified under vulnerable category. The taxonomic status of this sub-species remains uncertain and the present study is an attempt to assess its mitochondrial DNA diversity and evolutionary relationship with closely related taxa. Analysis of mitochondrial DNA D-Loop of O. vignei punjabiensis revealed wide variations in the number and pattern of tandem repeats as compared to other Moufloniform wild sheep. With the exception of tandem repeat regions, a total of 117 sequences coveringan overlapping region of 748bp from nine wild and domestic sheep populations were analyzed. The nucleotide and haplotype diversity was low to moderate within different sheep populations and Punjab Urial sheep exhibiting a mean of 0.0043 and 0.829 respectively. The average pairwise differences within population varied from 0.67 (O. musimon) to 23.69 (Kachi) while the average differences between Urial sheep and other Moufloniform wild sheep populations was high indicating the distinctness of O. vignei. Bayesian and maximum likelihood phylogeny clearly established the divide between the lineages of O. vignei and O. orientalis populations. Also, the average pairwise differences between O. vignei bochariensis and O. vignei punjabiensis were significantly higher (P<0.01) despite both the populations being classified as sub-species of Urial type sheep. To compare O. vignei punjabiensis with Argaliform and Pachyceriform wild sheep, pairwise FST between populations were utilized to perform principal components analysis (PCA). PCA revealed clustering of Asiatic (O. orientalis), European (O. musimon) and Captive Mouflon type sheep together, while O. nivicola and O. canadensis clustered separately. O. ammon and O. vignei bochariensis were observed to be clustering together while O. vignei punjabiensis was located distinctly. The tests for selective neutrality indicated purifying selection in Punjab Urial sheep while Asiatic Mouflon appears to have experienced a strong population bottleneck in the recent past. Considering the declining population trend and loss in genetic diversity due to possible genetic bottleneck events, there is an urgent need to implement strong conservation measures for O. vignei punjabiensis in the region.

High adaptive variability and virus-driven selection on major histocompatibility complex (MHC) genes in invasive wild rabbits in Australia

The rabbit haemorrhagic disease virus (RHDV) was imported into Australia in 1995 as a biocontrol agent to manage one of the most successful and devastating invasive species, the European rabbit (Oryctolagus cuniculus cuniculus). During the first disease outbreaks, RHDV caused mortality rates of up to 97% and reduced Australian rabbit numbers to very low levels. However, recently increased genetic resistance to RHDV and strong population growth has been reported. Major histocompatibility complex (MHC) class I immune genes are important for immune responses against viruses, and a high MHC variability is thought to be crucial in adaptive processes under pathogen-driven selection. We asked whether strong population bottlenecks and presumed genetic drift would have led to low MHC variability in wild Australian rabbits, and if the retained MHC variability was enough to explain the increased resistance against RHD. Despite the past bottlenecks we found a relatively high number of MHC class I sequences distributed over 2–4 loci. We identified positive selection on putative antigen-binding sites of the MHC. We detected evidence for RHDV-driven selection as one MHC supertype was negatively associated with RHD survival, fitting expectations of frequency-dependent selection. Gene duplication and pathogen-driven selection are possible (and likely) mechanisms that maintained the adaptive potential of MHC genes in Australian rabbits. Our findings not only contribute to a better understanding of the evolution of invasive species, they are also important in the light of planned future rabbit biocontrol in Australia.

A case of complete loss of gill parasites in the invasive cichlid Oreochromis mossambicus.

This study investigates the recent evolution of a rich parasite community associated with one of the world's most invasive species, the cichlid fish Oreochromis mossambicus. Populations from the species' native range (Mozambique) are compared to a population from New Caledonia (Wester Pacific), an island where the species was introduced in 1954. The results support the complete local extinction of the gill parasite community in the course of the invasion process. Up to six gill parasite species per locality were documented in the O. mossambicus native range, and previous surveys consistently reported at least one parasite species introduced along African cichlid species established out of Africa. The absence of parasites in New Caledonia is therefore exceptional. This can be attributed to local factors, such as a strong initial population bottleneck, the likely absence of multiple host introductions, and the frequent occurrence of brackish watersheds that might enhance the probability for natural deparasitation.

Genome-wide view of genetic diversity reveals paths of selection and cultivar differentiation in peach domestication.

Domestication and cultivar differentiation are requisite processes for establishing cultivated crops. These processes inherently involve substantial changes in population structure, including those from artificial selection of key genes. In this study, accessions of peach (Prunus persica) and its wild relatives were analysed genome-wide to identify changes in genetic structures and gene selections associated with their differentiation. Analysis of genome-wide informative single-nucleotide polymorphism loci revealed distinct changes in genetic structures and delineations among domesticated peach and its wild relatives and among peach landraces and modern fruit (F) and modern ornamental (O-A) cultivars. Indications of distinct changes in linkage disequilibrium extension/decay and of strong population bottlenecks or inbreeding were identified. Site frequency spectrum- and extended haplotype homozygosity-based evaluation of genome-wide genetic diversities supported selective sweeps distinguishing the domesticated peach from its wild relatives and each F/O-A cluster from the landrace clusters. The regions with strong selective sweeps harboured promising candidates for genes subjected to selection. Further sequence-based evaluation further defined the candidates and revealed their characteristics. All results suggest opportunities for identifying critical genes associated with each differentiation by analysing genome-wide genetic diversity in currently established populations. This approach obviates the special development of genetic populations, which is particularly difficult for long-lived tree crops.

The Social Biology of Quorum Sensing in a Naturalistic Host Pathogen System

Many microorganisms cooperate by secreting products that are commonly available to neighboring cells. These "public goods" include autoinduced, quorum-sensing (QS) molecules and the virulence factors activated by these signals. Public goods cooperation is exploitable by cheaters, cells that avoid the costs of production but gain an advantage by freeloading on the products of others. QS signals and responses can be cooperative under artificial laboratory conditions, but it remains unclear whether QS is cooperative in nature: little is known about the frequency of cheaters in natural populations, and cheaters may do poorly because of the importance of QS in major transcriptional networks. Here, we investigate the cooperative nature of QS in a natural system: the Gram-positive insect pathogen Bacillus thuringiensis and the larvae of the diamondback moth, Plutella xylostella. Although we find evidence of cooperation, QS null mutants are not effective cheats in vivo and cannot outcompete wild-type strains. We show that spatial structure limits mutant fitness and that well-separated microcolonies occur in vivo because of the strong population bottlenecks occurring during natural infection. We argue that spatial structure and low densities are the norm in early-stage infections, and this can explain why QS cheaters are rare in B. thuringiensis and its relatives. These results contrast with earlier experiments describing the high fitness of Gram-negative QS cheaters and suggest that QS suppression ("quorum quenching") can be clinically effective without having negative impacts on the evolution of virulence.

Cooperation and the evolutionary ecology of bacterial virulence: TheBacillus cereusgroup as a novel study system

How significant is social evolution theory for the maintenance of virulence in natural populations? We assume that secreted, distantly acting virulence factors are highly likely to be cooperative public goods. Using this assumption, we discuss and critically assess the potential importance of social interactions for understanding the evolution, diversity and distribution of virulence in the Bacillus cereus group, a novel study system for microbial social biology. We conclude that dynamic equilibria in Cry toxin production, as well as strong spatial structure and population bottlenecks in hosts are the main ecological factors maintaining the cooperative secretion of virulence factors and argue that collective action has contributed to the evolution of narrow host range. Non-linearities in the benefits associated with public goods, as well as the lack of private secretion systems in the Firmicutes may also explain the prevalence and importance of distantly acting virulence factors in B. cereus and its relatives.