Epichloë festucae is a common symbiont of the perennial and widely distributed cool season grass, Festuca rubra. The symbiosis is highly integrated involving systemic growth of the fungus throughout above-ground host parts and vertical transmission from plant to its offspring via host seeds. However, the nature of symbiosis is labile ranging from antagonistic to mutualistic depending on prevailing selection pressures. Both the loss of fungus in the maternal host lineage and horizontal transmission through sexual spores within the host population may partly explain the detected variation in symbiosis in wild grass populations. Epichloë species are commonly considered as pathogens when they produce sexual spores and partly castrate their host plant. This is the pathogenic end of the continuum from antagonistic to mutualistic interactions. Here we examined the population genetic structure of E. festucae to reveal the gene flow, importance of reproduction modes, and alkaloid potential of the symbiotic fungus in Europe. Epichloë-species are highly dependent on the host in survival and reproduction whilst benefits to the host are largely linked to defensive mutualism attributable to fungal-origin bioactive alkaloids that negatively affect vertebrate and/or invertebrate herbivores. We detected decreased genetic diversity in previously glaciated areas compared to non-glaciated regions during the last glacial maximum period and found three major genetic clusters in E. festucae populations: southern, northeastern and northwestern Europe. Sexual reproduction may have a higher role than expected in Spanish E. festucae populations due to the predominance of unique genotypes and presence of both mating types in the region. In contrast, asexual reproduction via host seeds predominates in the Faroe Island and Finland in northern Europe due to the presence of biased mating-type ratios and large dominant genotypes in the E. festucae populations within the region. A substantially larger variation of alkaloid genotypes was observed in the fungal populations than expected, although the variability of the alkaloid genotypes within populations is considerably lower in northern than Spanish populations in southern Europe. E. festucae populations consist of different combinations of alkaloid classes from the gene clusters of ergot alkaloid and indole-terpenes, and from pyrrolopyrazine alkaloid gene. We suggest that the postglacial distribution history of the host grass, prevailing reproduction strategies of E. festucae, and local selection pressures likely explain a large part of the genetic variation observed in fungal populations among geographic regions. The identified alkaloid genotypes can be used by turfgrass breeders to improve resistance against herbivores in red fescue varieties and to develop new sustainable cultivars in Europe. Microbes are ubiquitous and involved in all biogeochemical processes supporting the evolutionary trajectories from the origin of life. Earliest free-living microbes enabled terrestrial life on Earth by producing atmospheric oxygen , and sinEpichloë festucae Leuchtmann, Schardl, and Siegel , commonly associated with the widely distributed cool-season perennial grass, Festuca rubra L.   to an unfertilized fruiting body, a stroma, of opposite mating type vectored by phylogenetically distinct clade of anthomyiid flies (Botanophila ssp.)  . Transming seeds . In addiulations . A similulations , 1997. Ala ssp.) . This alla ssp.) . The strla ssp.) . A maturla ssp.) . The choin Spain , and a sin Spain . Thus, Eperation , 2016. Hronments .Epichloë-species are highly dependent on the host grasses whilst the benefits from the interaction remains conditional to the host. In most cases the specific Epichloë species or isolate is either directly or indirectly linked to defensive mutualism attributable to alkaloids  markers as well as mating type and alkaloid gene markers. Nuclear microsatellite markers enable us to make inferences about population structure, gene flow and genetic drift based on the patterns of genetic diversity within and among populations and regions. Moreover, SSR and mating type gene markers provide estimates of recombination potential and reproduction modes  based on the genotype frequencies, the structure of multilocus genotypes and mating type ratios , from six populations located in two different habitat types, meadows (n = 73) and riverbanks (n = 30), in Finland, and from three populations located in two different habitat types, Mediterranean oak forest (n = 22) and semiarid oak grassland (n = 45), in Spain (n = 66). More detailed information on the plants, occurrence of associated Epichloë-fungus and collection sites, including population geographic locations, coordinates, altitudes, habitat features and estimate of grazing intensity, can be found in Epichloë- individuals in the studied populations ranged from 5% to 81% and the number of individual isolates varied accordingly . Plates were stored at room temperature until mycelium emerged, after which a small sample of mycelium were transferred to a new PDA plate on a piece of sterilized cellophane. Total genomic DNA was extracted from pure cultures of mycelium growth using the E.Z.N.A Plant DNA Kit  according to the procedures described in E. festucae isolates were investigated using 14 polymorphic SSR markers, which were developed based on the searches for ≥10 mono- and dinucleotide repeats, and for ≥8 tri-, tetra-, penta-, and hexanucleotide repeats in the unplaced genomic scaffold sequences of E. festucae , Finland (six isolates), and Spain (six isolates), the samples were not included in the study.Genetic structure and the amount of clonality in the 240 EAS), indole-diterpenes (IDT) and lolines (LOL) are complex gene clusters in Epichloë taxa, whereas the pyrrolopyrazine alkaloids  production is dependent on the alleles of the perA gene  were examined in a total of 198 E. festucae isolates originating from six populations in the Faroe Islands (n = 60), six populations in Finland (n = 91) and three populations in Spain (n = 46)  .A or B) and key genes present at each alkaloid loci (IDT genes (idtK and idtF) used for mating type and alkaloid gene profiling, expected product sizes and six different multiplex sets are described in Supplementary Material in idtF gene were determined in isolates that contained the IDT genes required for terpendole C or lolitrem B productions. The primers idtF-M-F (5′-GGGCCATCCTATCTTACAC-3′) and idtF-M-R (5′-ACGAAGCCTTGAATCCAC-3′) were designed based on the idtF gene sequences with and without the mutation (GenBank accession numbers: EU530694 and MF464362). The PCR product of each alkaloid gene locus was sequenced using the following methods: the PCR products were separated on a 1.2% agarose gel, extracted from the gel and purified with E.Z.N.A. Gel Extraction Kit . The purified PCR products were submitted to Macrogen Inc., for Sanger sequencing with both upstream and downstream primers. The obtained sequences were visualized and manually corrected using E. festucae and the alkaloid gene region in question. The sequences were submitted to the European Nucleotide Archive. Accession numbers are available in idtF gene in the set of samples.A multiplex PCR method was used to determine the mating type  produced PCR bands of the expected size, and chanoclavine was expected if only dmaW and easC were present. Peramine, a pyrrolpyrazine-1-one, was assumed to be produced, if all three markers including perA5′, perAT2 and perAR, produced PCR bands, and if the reductase domain (perAR) was absent, then pyrrolpyrazine-1, 4-dione (PPZ-1 diones) were expected (F. rubra – E. festucae symbionts in the present study (n = 27) determined by idtG and idtQ are both present. Isolates that can produce early pathway terpendoles, such as terpendoles E and I, also contain a functional idtF, and isolates that can produce late pathway terpendoles, such as terpendole C, contain idtF and idtK in addition to idtG and idtQ. Lolitrem B (LTB), the end product of IDT biosynthesis in E. festucae, was predicted to be produced, if in addition to the genes mentioned above also idtJ produced a PCR band. The prerequisite of the production of terpendole C and lolitrem B was also that the sequence of idtF-gene was functional, without the deletion in the first exon of the gene that causes an inframe stop codon  produced PCR bands.The presence or absence of a set of key alkaloid genes within the alkaloid class pathway detected here are described in expected . Ergovalop codon . In addiPT values were used to estimate population pairwise differentiation levels within regions, two populations, FAS4 and RBS3, were excluded from the analysis, because of low numbers (<5) of isolates per population using the GenAlex version 6.5.  based on the presence (1) or absence (0) of each key alkaloid gene was determined using haploid binary data and multilocus options within populations, across populations within regions and across the whole data set. For the alkaloid gene data set, pairwise PHiion 6.5. , 2012.A and rd; clone corrected data, 999 permutations) were computed for each population, across Finnish, Faroese and Spanish populations within regions and across the whole data set. Tests of random association among loci were not performed for the Faroese and Finnish populations due to lack of statistical power because of a low sample size after clone correction , the number of expected genotypes based on rarefaction (eMLG) and random association among loci . The partition of optimal K numbers, which refer to number of groups into which the SSR data can be clustered, was conducted by performing 150 iterations of K from 2 to 30, which resulted in the number of genetically diverged clusters in optimal partition to be 15 . Fixed K model was used, because BAPS identified several small clusters . The number of clusters for the fixed K model was determined based on the uppermost hierarchical levels of genetic structure shown in a UPGMA tree based on the Kullback-Leibler divergence matrix. The individual level mixture clustering analysis with linked option was conducted using the fixed K mode with 150 iterations of K = 4.To study the genetic relationships of isolates, a Bayesian Analysis of Population Structure (BAPS) software, version 6.0, was used by applying a non-spatial mixture clustering analysis at individual level (sampling unit) with linked loci option  using mun = 240) and using all unique MLGs within regions (n = 103) with haploid data option and are based on the numbers of allele frequencies at each locus. The percentage of polymorphic loci (P%), the average effective numbers of alleles (Ne), the average numbers of unique alleles (Np) and unbiased genetic diversity (uh) estimates were calculated for each locus (n = 14), over the entire sample set (n = 240), over the MLGs (n = 103), for each region (n = 3), for each population (n = 15) and for each genetic group obtained by BAPS analysis (K = 4). A principal coordinate analysis (PCoA) was used to plot the major patterns in SSR data sets based on the whole data set (n = 240) and separately for each region  without clone corrections in relation to the genetic similarities of the isolates using pairwise individual-by-individual haploid genetic distance matrixes. Tests for significance were run with 999 random permutations. Analyses were conducted using GenAlex v. 6.5.Genetic diversity indices of the SSR were calculated with two data sets by using all isolates (K = 4) in SSR data set . Moreover, the degree of differentiation among regions were also estimated with the data set with unique MLGs (n = 103). The analysis was conducted using Arlequin software, version 3.5  value = −2194.4]. At K = 4, the isolates are mainly distributed according to the geographical regions (Fin-group (n = 104) represents mainly Finnish isolates (n = 101) and three isolates from the Faroe Islands (FAS3). Far-group (n = 69) contains mainly Faroese isolates (n = 67) and two isolates from Finnish populations . The Spanish isolates were partitioned in one large and one smaller genetic group. Sp1-group includes fourteen isolates from SPGD population. Sp2-group (n = 53) represents all isolates from SPLV (n = 23) and SPPOR (n = 21) but also eight isolates from SPGD and one isolate from the Faroe Islands (FAS6).The Bayesian mixture analysis was conducted using fixed K mode of  regions . Fin-gron = 240) according to the principal coordinate analyses based on pairwise individual-by-individual haploid genetic distance matrixes, in which the first and second axes explained 44.5% and 18.6% of the variation, respectively , the AMOVA analysis estimated that 66.6% of the genetic variation occurs among regions, 36.1% within populations and only 7.3% among populations within regions indicating high differentiation among the regions and low differentiation among the populations (n = 103) for the regions, 44% of the genetic variation occurs among populations and 56% within populations , most of the variation (73.1%) was detected within populations and moderate levels of genetic differentiation (26.9%) among the six populations . The popn = 3) and riverbank (n = 3) habitats , majority of the variation (95.5%) was within populations and only low level (4.5%) genetic differentiation was found among the six populations . No signp < 0.001, see In Spain , most of the variation (73.6%) was detected within populations and moderate level genetic differentiation (26.4%) observed among the three populations . A signiK = 4, including all the isolates, n = 240), AMOVA analysis revealed that 75% of the genetic variation is distributed among the genetic groups and 25% within clusters indicating very high genetic differentiation among the groups (n = 103), 53.3% of the genetic variation occurs among populations and 46.7% within populations (Among the genetic groups (e groups . Using tulations . The pain = 240) in fifteen populations collected from Spain, the Faroe Islands and Finland (n = 56) located in different Faroese populations, three largest clone sizes are 22,11 and 8 isolates/nMLG. In Finland, fifteen (78.9%) nMLGs were unique in the region and four common nMLGs consist of 85.4% of the isolates (n = 88) located in different Finnish populations, two largest clone sizes are 64 and 19 isolates/nMLG.The clonal structure of the isolates according to populations within regions is visualized in  Finland . High fr Finland . In the A and rd) were utilized to estimate the relative importance of reproduction types, i.e., either sexual or asexual reproduction predominated in the regions (The indices of associations (I regions . Signifi regions .K = 4) are shown in A and B was 0.92, therefore the proportions of each mating type are close to equal among populations in Spain. In SPLV and SPPOR populations most of the isolates were of mating type B, whereas most of SPGD isolates were of mating type A. In contrast, majority of the isolates in Finland and the Faroe Islands were of mating type A, although both mating types were present in half of the populations in both Finland and Faroe Islands. Mating type genes were not detected in four of the isolates. Based on the genetic groups (K = 4) Fin-, Far-, and Sp2 -groups shared the mating types A and B, but only mating type A occurred in Sp1-group , indole-diterpene (IDT), pyrrolpyrazine (PPZ), and loline (LOL) production  are predicted to produce at least one end product of EAS, PPZ and/or IDT alkaloid class. Most  of the isolates had all the EAS genes targeted, and thus, they are likely to have a functional EAS pathway and are predicted to produce ergovaline. About one fifth  of the isolates contained all PER markers and they are expected to produce peramine, the pyrrolopyrazine-1-one PPZ-1-ones (n = 150) that lack the reductase domain, are predicted to make pyrrolopyrazine-1, 4-diones (PPZA2). Chemotypically determined ergovaline and peramine production in the same F. rubra-E. festucae-symbiosis revealed that 72.7% (n = 11) and 100% (n = 10) of the isolates corresponded with the predicted alkaloid profiles, respectively , 43.9% (n = 87), and 8.6% (n = 17) of the isolates, respectively.The alkaloid gene profiles of different aMLGs with mating types, isolate numbers and predicted and confirmed chemotypes are presented in n = 198; . Common Z-1-ones . Isolateectively . Three iThe numbers of isolates within the aMLGs differed largely from unique genotypes (one isolate/aMLG) to common genotypes (2-71 isolates/aMLG) . Most ofn = 19) and considerably higher amounts of predicted chemotypes were detected in three Spanish populations compared to northern populations  (t-40 (n = 15), contained about one third (31.9%) of the Spanish isolates and occurred in all populations  , but not between the dehesa populations of SPLV and SPPOR   . In cont (gt-21) . HoweverR n = 3) . Gt-40 wpendoles . Signifi= 0.106) .n = 12). Overall, five unique aMLGs and seven common aMLGs with 91.7% of the fungal isolates were present in the region. Several IDT genes were absent in the Faroese isolates (gt-27 (n = 30) was observed in half of the Faroese isolates in five populations and tested positive for seven out of thirteen markers including all EAS genes and lacked perAR marker and all five IDT markers (gt-33 (n = 9) contained 15% of Faroese isolates in four populations and tested positive of eight out of thirteen markers including all EAS genes and lacked perAR marker and all IDT genes except idtG (p > 0.05) except between FAS1 and FAS5   of the isolates possessed different aMLGs (isolates . Most (8isolates . The mos markers . Second ept idtG . No sign= 0.039) .n = 14). Overall, seven unique and seven common aMLGs with 92.3% of the isolates were detected in the region  and they are expected to produce ergovaline and/or terpendole I, respectively (gt-39 (n = 70) was found in 76.9% of Finnish isolates in all six populations . Thus, gt-39 is likely to produce ergovaline due to presence of all five detected EAS genes, pyrrolopyrazine-1, 4-diones (PPZA-2) due to presence of perA5 and perAT2 markers and the indole-diterpenes pathway intermediate terpendole I due to presence of idtG, idtQ and idtP  were distributed unevenly in the examined two habitats. The genetically more diverse Sp2-group predominated in Mediterranean savannah-like grasslands, dehesa, located higher in altitudes near Salamanca (populations SPLV and SPPOR). Sp2-group was detected also in Mediterranean oak forest (population SPGD) to a lesser extent. In contrast, the genetically less diverse Sp1-group was prevalent in Mediterranean oak forest and absent in dehesa. The alkaloid gene assemblage differed also among the habitat types and larger variation of alkaloid chemotypes occurred in dehesa compared to Mediterranean oak forest. Nearly all examined fungal lineages had the potential to produce ergot alkaloids with known anti-vertebrate and anti-invertebrate properties as well as peramine or PPZA-2 with anti-invertebrate properties (In Spain, we found the highest genetic diversity that can be explained by the occurrence of populations near glacial refugia and biodiversity center of fine fescues  and occaoperties . More thoperties . These roperties .E. festucae in the Faroe Islands do not support the same for the symbiotic fungus. Most Faroese isolates fell into locally adapted Far-group, had lost several IDT genes and were predicted to produce the same alkaloids across islands. Only three non-local SSR isolates were detected, two isolates from the Fin-group in FAS3 population and one isolate from Sp2-group in FAS6 population. This suggests that the long-distance co-dispersal of microbes with their hosts occur but not as efficiently into the region as its host F. rubra which possess a contact zone of various maternal lineages in the Faroe Islands especially since gene flow from the other locations have been found to be more effective in maritime than inland locations as seeds might arrive by floating and by birds (F. rubra individuals in Faroese populations may reflect the process of infection losses. In addition, similarly to Spanish populations, the Faroese populations were more differentiated from each other revealed by SSR markers than the Finnish ones, although predominantly two relatively large identical genotypes were present in most of the populations indicating fungal gene flow via host seeds among the islands. The observed genetic differentiation of the populations may have been caused by varying selection pressures driving populations in different directions, gene flow from other locations and predominance of vertical transmission of E. festucae in mainly clonally dispersing F. rubra in Faroe Islands (In concordance with the presumption that diversity should decrease with the distance to the source regions, some genetic diversity appears to be lost during the colonization of exposed land following retreating ice sheet in North Europe. However, our previous study on the host grass supports the hypothesis that some of the grass individuals may have survived on nunataks in Faroe Islands . The preby birds . The genby birds . Thus, r Islands .E. festucae populations present distinct genetic Fin-group with relatively low genetic diversity compared to the Southern Spanish populations. Similarly to its host populations of F. rubra, one large genotype were observed also in the fungal populations indicating longevity and expansion of local host-fungus genotypes (F. rubra clones have been detected to be centuries old and occupying large areas (E. festucae strains (In the species northernmost distribution range in subarctic Finland, the examined enotypes . F. rubrge areas  as selecge areas . Only twge areas , which cge areas . Non-loc strains . Moreove strains . In contEpichloë (E. festucae isolates and other related Epichloë species revealed considerable diversity within the genus based on the presence or absence of the alkaloid genes (perA gene, transposable elements have disrupted the gene causing the loss of the reductase domain, which results in production of pyrrolopyrazine-1, 4-diones rather than the expected peramine (F. rubra-E. festucae symbiotum and only mentioned in E. festucae may provide greater insights into the evolution of these biosynthetic genes. The more limited predicted chemotypes found in Finland and the Faroe Islands versus that of Spain, may be due to selection pressure since the Spanish populations may have an advantage of many different chemotypes.The alkaloid genotyping has proven to be very powerful to identify potential bioactive alkaloids produced by populations of Epichloë . Genome id genes . In the peramine , 2019. IE. festucae and mating type distribution likely explain large part of the differences among geographic regions in genetic diversity among the populations within the regions. Low genetic diversity in Finland and Faroe Islands, and lack of differentiation between distinct habitats in Finland, appears to be attributable to the extremely rare production of sexual structures, detectable as symptoms called “choke disease” on the host inflorescences, and dominance of one mating type. For example, we have intensively monitored F. rubra populations in northern Finland during the last 20 years but never detected choke disease (Reproductive strategy of  disease . FurtherE. festucae populations. In contrast, although past studies have detected choke disease in less than 1% of Epichloë symbiotic F. rubra plants in Spain (E. festucae in northern Europe.Thus, we assume that postglacial distribution history of the host, founder effect, genetic drift and local adaptation of symbiotum largely explain the detected genetic structure of northern in Spain , the higThe datasets presented in this study can be found in MH and KS designed the collection of the data and performed the sampling. MC, HK, CY, and PL designed the genetic study. MC genotyped the isolates and analyzed the data. CY detected and confirmed the alkaloid profiles. MC and KS wrote the manuscript with contributions by all authors.CY was employed by the Noble Research Institute, LLC, United States. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Southeast Asia (SEA) has one of the longest records of modern human habitation out-of-Africa. Located at the crossroad of the mainland and islands of SEA, Peninsular Malaysia is an important piece of puzzle to the map of peopling and migration history in Asia, a question that is of interest to many anthropologists, archeologists, and population geneticists. This review aims to revisit our understanding to the population genetics of the natives from Peninsular Malaysia and Borneo over the past century based on the chronology of the technology advancement: 1) Anthropological and Physical Characterization; 2) Blood Group Markers; 3) Protein Markers; 4) Mitochondrial and Autosomal DNA Markers; and 5) Whole Genome Analysis. Subsequently some missing gaps of the study are identified. In the later part of this review, challenges of studying the population genetics of natives will be elaborated. Finally, we conclude our review by reiterating the importance of unveiling migration history and genetic diversity of the indigenous populations as a steppingstone towards comprehending disease evolution and etiology. Homo erectus (Southeast Asia (SEA) is believed to be one of the earliest regions of hominin habitation recorded outside Africa nearly 2 million years ago, following the arrival of the ancient “Java Man” known as the  erectus . Archeol erectus , Callao  erectus , and Nia erectus , suggestSEA was once a great landmass bridging the Eurasia during the last glacial maximum peaked ∼20 kya . This laMalaysia has been an important piece of puzzle to the global map of peopling and migration history, owing to its strategic location at the crossroad of the mainland and islands of SEA. It is a multi-ethnic, multi-lingual, and multi-cultural nation with diverse socio-economic practices. The Malay ethnic group is the predominant native in Malaysia, making up ∼61% of the total population; whereas the natives from Borneo comprise ∼9% of the total population . The indThe Malaysian section of Borneo comprises the states of Sabah and Sarawak. The Sabah state (formerly known as North Borneo) is home to culturally diverse populations, comprising of more than 40 ethnicities that are broadly categorized into five major groups: Dusunic, Paitanic, Murutic, Ida’anic, and Sama-Bajaw , with KaThe SEA populations are enriched with their linguistics spectrum, primarily represented by five language families: Austroasiatic, Austronesian, Tai-Kadai, Hmong-Mien and Sino-Tibetan . NonetheGiven such rich ethnological diversity and complex prehistorical events in this landmass, addressing migration and peopling history of Peninsular Malaysia and Borneo is therefore a pertinent question.In this article, we attempt to summarize an overview on our understanding of migration and peopling histories of the natives from Peninsular Malaysia and Borneo over the past century, based on five chronological eras of genomic technological advancement: 1) Anthropological and Physical Characterization; 2) Blood Group Markers; 3) Protein Markers; 4) Mitochondrial and Autosomal DNA Markers; and 5) Whole Genome Analysis. We also provide some hints to the “missing puzzles” of the peopling history in SEA yet to be uncovered, and finally reiterate the implications of migration and peopling history of SEA populations on human health and diseases.The study of human populations in Malaysia began in the 19th century when the colonial scholars attempted to classify “race”. Since genetic knowledge was rather limited, hypotheses were built merely based on physical and anthropological observations. The population of the Peninsular Malaysia was first grossly classified into two geographical races: “Negrito-like” and “East Asian-like” . PopulatThe basic “three-way division” framework was raised by the British anthropologists The Negritos from the SEA was once argued to be the remnants of the wrecked slave ship from Africa , yet theThe population classification for the Bornean native populations has been far more complicated because most of these populations have indistinguishable physical appearance and economic activities; yet a rich variety of cultural heritage and linguistics. These populations were classified as the East Asian populations, as exemplified by several native populations in the Philippines and Taiwan . The claThe distinct phenotypic characteristics of these SEA inhabitants along with a handful of archaeological and linguistic evidence thence provided hints to the early “Two-Layer” migration hypothesis, which argued that SEA was occupied (the first layer) by the direct descendants of the early modern humans out-of-Africa, and subsequently admixed with the later immigrants (the second layer) from North and/or East Asia leading to the present day SEA human diversity .It was not until the 1950’s that a new form of anthropological measurement was introduced into the consideration of the “race” classification – the blood group . The stuThe first known blood group test on Orang Asli was carried out in the 117 Semai sub-tribe, which showed a higher frequency of the “O” blood group . This reFurther investigations into blood groups were carried out, attempting to address some contentious questions like classifications of the Orang Asli; relationships between the Orang Asli Negrito with the Negritos from Africa, SEA, and Australia, and the links between the Orang Asli groups with the rest of the populations in the world. Many of these studies were carried out by Ivan Polunin . The catThe period between the 1960s–1980s marked the advancement of population genetics with the popularity of protein electrophoretic and isozyme variations from erythrocytes, serum, cerumen, human placenta, and saliva. The majority of these works were carried out by Lie-Injo Luan Eng and Tan Soon-Guan  Supplem. Some fiOther intriguing observations included the frequencies Hb E, G6PD deficiency, and ovalocytosis among the native populations from Malaysia that were parallel with past distributions of malaria posted the theory that malaria is selective for these hematological traits .Although the questions pertaining to the history of peopling and migrations in Malaysia and SEA were not noticeably addressed in these early studies, the clarification of population relationships using these conventional technologies has essentially laid an important foundation for future population genetics, or subsequent studies in Malaysia could not have been made successful.The availability of molecular markers in particular maternal lineage mitochondrial DNA (mtDNA) markers, and subsequently molecular clocking approach have completely changed the way we appreciate the human migration and peopling history in Peninsular Malaysia. Several postulations on the migration history of SEA were initially proposed but posited considerable doubts. For instance, Despites the said controversial, several hallmark studies had provided further insights into the prehistorical peopling in SEA. Based on mtDNA variation, The completion of the human genome project has revolutionized and accelerated the progress of population genetic studies at a tremendous pace. Genome-wide genotyping array (SNP array) and next-generation sequencing technologies were subsequently made available for large genome mapping initiatives to be carried out, including Human Genome Diversity Project , the IntSurprisingly however, the indigenous populations from SEA were underrepresented in these large initiatives. Acknowledging this notion, the HUGO Pan-Asian SNP (PASNP) Consortium initiated the mapping of the Asian human genetic diversity with ∼50,000 genome-wide autosomal SNPs, and revealed that the ancestry of Asian populations harbors genetic contributions derived from five language groups, and that the migrations of SEA populations were more complex than the anticipated. Most notably, the study supports an initial single migration wave of the ancestors of East Asian (EA) populations via “Southern route” into SEA, followed by multiple subsequent migrations thence shaped the complex genetic diversity of SEA . The groSubsequently a growing body of evidence thenceforth refined the peopling history in Malaysia. The SEA Austronesian populations generally carried ancestry components derived from four primary admixture events: 1) aboriginal Taiwan; 2) Austroasiatic; 3) Melanesian; and 4) Negrito ; whilst When dissecting the genetic architecture of the modern Malays from Peninsular Malaysia, population substructure was observed, suggesting plausible different origins from neighboring regions of SEA . Finer iThe notion of multiple prehistorical migration waves to SEA was further supported by ancient genomes analyses , revealiWhilst the peopling history of Austronesian is seen clearer, the genetic affinity between the Orang Asli Negrito and ancestors of the Austroasiatic language-speaking populations remains controversial. Growing body of evidence confirm that the Orang Asli Negrito once shared common origins with the East Asian populations . HoweverIntriguingly, the Orang Asli Negrito (Jehai sub-tribe) showed a shared genetic drift with ancient genomes from Hoabinhian ancestry , suggestRecent investigation postulates that pre-Neolithic ancestors of today’s Austroasiatic language-speaking populations were widespread in South Asia and SEA, and the populations from these regions were the result of multiple migrations of East Asian farmers during the Neolithic period . The autWe acknowledge however, some of these findings should be interepreted with caution, because many of these studies were carried out with a rather small sample size, which may have introduced sampling biasness hence compromised conclusion.Whilst some old questions are addressed, peopling history in SEA is complicated by the genomic introgression between modern human and archaic hominin. Denisovan genome introgression was found significantly higher in the Papuan and the Philippine Negrito (Mamanwa); but not in the native populations from Peninsular Malaysia Borneo, and the Andamanese Negrito . TherefoWhat is more intriguing, is that genome analysis of a human remained dated ∼7 kya surprisingly expressed a substantial East Asian ancestry component, with a mixture of Denisovan gene flow, indicating an East Asian ancestry present in Wallacea much earlier than Austronesian expansion. This discovery has overthrown our common understanding of SEA human peopling history, thence shows that the peopling of SEA was much more complex than has previously been appreciated , and theThe cumulative evidence presented above thenceforth implies “multi-layer” migration to SEA, and Peninsular Malaysia and Borneo to be more specific , insteadFor the ease of reference, a summary of the published genotyping and sequencing data for the natives of Peninsular Malaysia and Borneo is tabulated in (i) The ancestors of SEA populations migrated out of Africa via “Southern Route” along the coastal line to East Asia .(ii) The Negrito from Peninsular Malaysia once shared a common ancestry with East Asian populations .(iii) Both mtDNA molecular clocking and inference of divergence time using autosomal DNA support the notion that the ancestors of Peninsular Malaysia Negrito may be the earliest inhabitant of SEA at least 50 kya .(iv) The native populations from Peninsular Malaysia and Borneo are genetically distinct, each with a unique population history .(v) Austronesian expansion occurred at least in the SEA region, southwards to the Philippines, towards the Northern Borneo .(vi) There are at least four prehistorical gene flows events that occurred in Peninsular Malaysia and Borneo thus concurs the “multi-layer” migration model. The first migration was likely to had derived from the earliest ancestors of the Negrito-like populations; subsequently the Austroasiatic migration southwards replacing the descendants of the early inhabitants. The third migration being the Austronesian expansion, and the last likely being the East Asian or islands of SEA .(vii) The native populations from both Peninsular Malaysia and Borneo received minimal gene flow from the archaic Denisovan hominin .With the advent of genomic technology, many long-standing questions were addressed with renewed vigor, some possibly with refined insights. Based on the collective findings to date, the following section summarizes, what we know about the peopling and migration history of the native populations from Malaysia:Based on our current understandings along with earlier proposed models , we redr(i) The genetic link between the Negrito-like populations from SEA, including Negrito from Peninsular Malaysia, the Philippines, the Andaman, Papuan and Australian aborigines, and possibly the African pygmies, remain inconclusive. Whilst it has been shown that these populations shared an ancient basal Asian ancestry component , they ha(ii) The pre-historical migrations of the Negrito into this landmass remain fragmentary. If the Orang Asli Negrito were postulated to be the direct descendants of the modern human out of Africa and that the divergence time between Australia aborigines and Eurasia occurred 65 kya , we woul(iii) What are the relationships between the Orang Asli Negrito and Senoi populations? Different postulations have been made on the origins of Senoi. Some related them to the Veddoids; others argued that they were the descendants of admixture between Negrito and Austronesian . However(iv) Reason(s) of the absence of Negrito-like populations in Borneo. Archaeological evidence suggests that Borneo was first inhabited by the ancestor of SEA modern human who were believed to be the direct descendants from the out of Africa exodus . It was (v) It is intriguing to observe a close genetic affinity between the modern Malays and Cambodian population . Further investigation should be carried out to explain the links between the modern Malay, Cambodian and Yunnan Migration Hypothesis; or to dispute this hypothesis.(vi) What remained to be answered, we think, are:The population genetics study in Malaysia is not without challenge. Owing to their long period of isolation and high inbreeding rate, population genetic studies of the native populations – specifically referring to the Orang Asli and the Bornean natives – are often restricted to limited number of unrelated samples. This hinders the statistical power thence affects the strength of the evidence to support or dispute a hypothesis, resulting in a rather limited impact publication. In addition, although the cost for genome-wide analysis has reduced dramatically, it is still unaffordable for many low- and middle-income countries. Limited research funding available for such less prioritized science has always been a challenge to carry out high-throughput genome sequencing initiatives.Nonetheless, the more pressing challenge is the ethical and integrity concerns that have been raised over the years, particularly issues on accessing the samples and data. A thorough process of ethics approval has been established especially for the genetic study of native populations in SEA countries, such as Malaysia, the Philippines and Indonesia. Efforts are spent in engaging and building trusts with the native populations, and collecting biological materials and data. Often such a process takes years before seeing success. Therefore, the contribution from the local scientists merits scientific acknowledgments. Unfortunately, we see occasionally, scientific publications or reports on the marginalized populations from SEA without appropriately acknowledging the local scientists. Such practice has raised some arguments over the years. We, therefore, call for close collaborations between investigators locally and abroad to warrant the advancement of science in mutual respect and ethical manner.The complex demographic history of modern humans out-of-Africa has created different genetic architectures across populations from different continents, hence disease susceptibility. With genomic data from diverse populations and ancestries, we can compare these information over time and geography to better understand the origins and evolution of both individual genetic variants and human populations.FY) against Plasmodium vivax infection, and the selection signal was derived from the Asian ancestry.From global perspective, studying the genomics of the SEA marginalised populations, in this particular case, the natives from Peninsular Malaysia and Borneo, complements the catalogue of genetic variations that is known to be strikingly bias to European populations. Particularly, it allows characterizations of rare, and population (or ancestry) specific genetic variations . A classFrom regional perspective, many tropical diseases that are uncommon in many developed countries, for instance Dengue and nasopharyngeal carcinoma. In addition, owing to different demographic history, climate, diet and lifestyles, pathophysiology for many complex diseases, for instance, cardiovascular diseases and metabolic syndromes, in the SEA populations may vary from the developed countries, and manifest in different intermediate phenotypes. Therefore, population genomic studies of the native will be utmost pertinent to the disease mapping efforts in SEA.The population genetic study of the native populations in Malaysia has begun since the 19th century with the intention of racial classification. Postulations and arguments on human migration and peopling history especially to the SEA region were merely based on anthropology, archaeological, and linguistics evidence. With the advancement of genomics and life science technology, the picture of the peopling and migration history of the natives from Malaysia, and their attributions with other neighboring populations has become clearer. The natives from Malaysia were originated from multiple waves of migration events. It began with the direct descendants of the exodus of out-of-Africa, followed by the early Austroasiatic primitive farmers from Indochina that occupied the Peninsular Malaysia region and interacted with the existing inhabitants; subsequently with multiple waves of migrations of Austronesian populations from Southern China (and Taiwan) and other islands of SEA. However, we acknowledge that the migration model is far more complex than anticipated, and there are doubts yet to be addressed. We reiterate here, the importance of unveiling migration history and genetic diversity of the indigenous populations, not only to address the fundamental question of the origin of modern humans, but to complement the catalog for human genome variation, and to provide a stepping stone towards comprehending disease evolution and physiology . Increas


C. ryukyuensis sp. nov. The taxonomic complexity of east Asian mandarins then collapses to a satisfying simplicity, accounting for tachibana, shiikuwasha, and other traditional Ryukyuan mandarin types as homoploid hybrid species formed by combining C. ryukyuensis with various mainland mandarins. These hybrid species reproduce clonally by apomictic seed, a trait shared with oranges, grapefruits, lemons and many cultivated mandarins. We trace the origin of apomixis alleles in citrus to mangshanyeju wild mandarins, which played a central role in citrus domestication via adaptive wild introgression. Our results provide a coherent biogeographic framework for understanding the diversity and domestication of mandarin-type citrus through speciation, admixture, and rapid diffusion of apomictic reproduction.The origin and dispersal of cultivated and wild mandarin and related citrus are poorly understood. Here, comparative genome analysis of 69 new east Asian genomes and other mainland Asian citrus reveals a previously unrecognized wild sexual species native to the Ryukyu Islands:  Citrus ryukyuensis native to the Ryukyu islands and a new population of wild mainland Asian mandarin explains the origin and diversity of mandarins and their ability to reproduce apomictically.To explore the nature of wild and cultivated mandarins, the authors carry out genomic analysis of diverse east Asian citrus. The discovery of a wild species  Because of their consumer-friendly attributes, mandarins have seen the greatest percentage increase in global citrus production3.The phenotypic and genomic diversity of mandarin types in the Nanling region of southern China has driven speculations that mandarins first arose and were domesticated in this region4. Yet the nature of the domestication process, and the relationships among these mainland Asian types remain poorly understood.Mandarin-type citrus comprise a heterogeneous group of east Asian citrus with small and typically easily peelable fruit5, including: tachibana [C. tachibana (Makino) Yu Tanaka], a culturally significant ornamental citrus grown throughout mainland Japan; shiikuwasha [C. depressa Hayata], grown in the Ryukyus and renowned for its health promoting qualities6; and other traditional and wild Ryukyuan citrus of unknown ancestry  by generating maternal clones from somatic tissue through the process of nucellar embryony21  seeds.Most strikingly, we identified a new wild citrus species native to the Ryukyu islands that we designate rus Fig. . These arus Fig.  that werC. ryukyuensis as a pure species  is supported by three findings  and C. maxima . Based on sequence comparisons, we estimate that C. ryukyuensis diverged from mainland Asian mandarins around ~2.2–2.8 Mya . One sub-species, which we call ‘common mandarin,’ is the predominant contributor to domesticated mandarin. Many domesticated types, however, also contain admixture from the newly recognized second mandarin sub-species and from pummelo  and common mandarin (MA) populations are sufficiently differentiated from each other (FST~0.49) that they should be considered as at least distinct sub-species  and two distinct F1 hybrids of mangshanyeju with nearly pure common mandarins (M01 and M04) , which is a distantly related citrus species from the same region21.We identify the second mainland mandarin subspecies with types that are colloquially referred to as “mangshanyeju”, i.e., wild mandarin (“ju”) from the Mangshan region of the Nanling mountain range. Although Wang et al.r FST~0.4 that theanyeju MS and MS2 anyeju MS and MS2 C. ryukyuensis and C. reticulata (2.2–2.8 Mya), and between mangshanyeju and common mandarins (1.4–1.7 Mya) are consistent with the paleogeology of the region  and Pleistocene, with intervals of connectivity to the south near present day Taiwan and north to the Japanese islands. This variable connectivity and associated climatic variation led to the emergence of new species in this region in many plant30 and animal34 taxa, coinciding with our estimates for the divergence of the distinct C. ryukyuensis from mainland Asian mandarin. The emergence of C. ryukyuensis by allopatric speciation was accompanied by a population bottleneck, suggested by its reduced heterozygosity relative to mainland mandarins  (coefficient of relatedness 0.41), but contains a single 2.4 Mbp introgressed pummelo segment.rus Fig. . The shints Fig. . Unexpecnts Fig. . RK3 is nts Fig. . Sequenc36, either matching tachibana mandarins (here recognized as C. ryukyuensis type), or matching certain mainland Asian mandarins (here recognized as a C. reticulata type). Evidently, RK3 mandarin served as both seed and pollen parent in the numerous hybridization events that generated shiikuwasha. The hybrid nature of shiikuwasha accounts for its previously noted genotypic and phenotypic diversity12, and is consistent with previous suggestions that shiikuwashas are hybrids based on high levels of nucleotide polymorphism37. More detailed understanding was elusive since C. ryukyuensis had not been recognized or characterized.The observation that shiikuwasha form a large half-sib family explains the previously puzzling finding that shiikuwasha chloroplast DNAs (cpDNAs) are of two distinct typesC. ryukyuensis and mainland Asian mandarins, but distinct from shiikuwasha  can be organized into a third hybrid family that we named yukunibu, meaning “sour citrus” in Okinawan dialect. Yukunibu citrus are F1 hybrids with a kunenbo-mikan seed parent and diverse C. ryukyuensis pollen parents  from Indochina sometime between the 8th and 12th centuries45. Our collection also contains other members of the extended yukunibu family as well as other unrelated hybrid genotypes with C. ryukyuensis ancestry  reproduce apomictically by nucellar embryony. This implies that the apomixis trait was transmitted to these three hybrid species by their migrant mainland mandarin parents. Apomictic reproduction of shiikuwasha46 and tachibana22 enabled the rapid establishment and dispersal of these new hybrid species after their formation by hybridization with the pre-existing locally adapted C. ryukyuensis population. Notably, the mainland mandarin parents of shiikuwasha and yukunibu (RK3 and kunenbo-mikan) both produce polyembryonic seed16.While CitRKD118 gene  that dominantly confers an apomictic phenotype. We find that this MITE insertion arose in the mangshanyeju population in the early Pleistocene and spread into other mandarins, oranges, grapefruits, and lemons as well as shiikuwasha, tachibana, and yukunibu  occur in different combinations  in diverse apomictic citrus, highlighting the complex history of this critical genomic region  DNA transposon insertion in the promoter of the ibu Fig. . The fouCitRKD1 gene 25. Alternatively, their seeds could have arrived via the Kuroshio Current57, a powerful south-to-north warm current that passes by Taiwan, the Ryukyu Arc and mainland Japan. In contrast, kunenbo-mikan (the mainland Asian parent of yukunibu) was introduced to the Ryukyus by trade between the 8th and 12th centuries42. Other imported mainland Asian citrus such as sour orange (daidai) and pummelo also hybridized with C. ryukyuensis and shiikuwasha to produce rokugatsu and Ryukyu sour orange (deedee), respectively  of tachibana had substantial mangshanyeju admixture  and no pummelo admixture. On the C. ryukyuensis side, genetic differentiation among tachibana and shiikuwasha haplotypes (Fst =0.17–0.20) is consistent with separate mainland Japanese and Ryukyuan C. ryuykyuensis populations that diverged ~220,000–350,000 years ago  on one haplotype, but only limited variation from their apomictic mainland parent(s) on the other. These hybrid genetic configurations are stably propagated by ongoing apomictic reproduction, which also reproductively isolates the hybrids from their parental species. This scenario provides a new model for homoploid hybrid speciation involving apomixis that could also apply to other plant taxa.We have shown that shiikuwasha, tachibana, and yukunibu are distinct homoploid  hybrid species. Homoploid hybrid species are rare and are generally complex mixtures of parental species due to the absence of reproductive isolation between F1 hybrids and their parents50, and results in the appealing white flowers celebrated in ancient poetry compared with the ancestral purple color seen in other citrus. While elegant genetic analyses have shown that this recessive trait is due to defective alleles of the MYB transcription factor Ruby50, its evolutionary origin is unknown. Since the same deletion allele is fixed in both mangshanyeju and C. ryukyuensis, it was likely already fixed in the common Pleistocene ancestor of all mandarins. . It follows that loss of anthocyanin in mandarins preceded domestication  the myriad of crosses between those ancestral hybrids and admixtures, that gave rise to the current basic types of edible citrus58  the rise of apomixis in the mainland mandarin lineage during Pleistocene, (2) pummelo introgression that incorporated desirable traits into the ancestral mandarin genomeC. reticulata) and related citrus are  concentrated on (1) cultivated varieties21 and (2) wild varieties from southeastern China, the center of diversity for mandarin citrus4. The extensive wild and cultivated diversity in the Ryukyu islands and southern mainland Japan has been largely neglected with only two tachibana accessions included in previous collections20.Currently available genomic data for mandarin  local island-grown trees of known or uncertain provenance from Okinawa World Theme Park, and (5) wild and cultivated tachibana from southern mainland Japan. Our collection also includes two distant relatives of citrus in the Rutaceae family  that are not used in the present analysis. In total, 71 samples including 69 citrus were collected with appropriate permissions. More detailed information about these collections is provided in Supplementary Note To sample diverse east Asian citrus, we collected (1) named varieties of shiikuwasha and other traditional cultivated varieties  from local Okinawan growers and the Okinawa Prefectural Agricultural Research Center, 2) unnamed local mandarin-type citrus trees from personal gardens and small farms, including the Katsuyama Shiikuwasha Co., Ltd., (3) wild citrus trees from various sources including naturally forested areas, including samples later identified as belonging to the new species  unnamed ® Plant Mini Kit . After libraries had been prepared with a KAPA HyperPlus Library Preparation Kit  and NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina , 150 and 250 bp pair-end reads were obtained using a HiSeq 2500 Rapid v2, HiSeq 4000 and NovaSeq 6000 SP . Each sample was sequenced at more than 30-fold redundancy.Shoots or young leaves were collected from each sample in the field. Leaves were frozen with liquid nitrogen, and then crushed in a mortar. Total DNA was extracted from the frozen powder of leaves using a DNeasy21) were mapped to the haploid Clementine reference sequence v1.021 using BWA-MEM (version 0.7.8-r455)66. We used the Clementine because it is the best quality available mandarin-type reference genome, and therefore suitable for exploring variation in mandarin. It has previously been established21 that Illumina data from diverse citrus species can be readily aligned to this reference sequence, and the average mapping rate across samples sequenced in this study is 97.4%. Duplicate reads were removed using picard MarkDuplicates (version 2.13.2). Variants were called using GATK HaplotypeCaller (version 3.7-0-gcfedb67)67 with filtering based on read map quality, base quality, read depth and allele balance  taking one accession from each population, along with the heterozygosity of each genome. Genome wide values of D defined above). We first identified clones  function cmdscale based on pairwise genomic distances , mangshanyeju (MS), pummelo (PU), citron, Fortunella (kumquat), and Ichang papeda . The tree was rooted with Chinese box orange . The highly supported topology of this species tree . We required complete coverage across all representative individuals  and recovered a total of 209,124 single nucleotide variable sites. Using these characters we constructed a maximum likelihood tree with RAxML70. This is an appropriate reference for genome wide pan-citrus comparisons. We constructed maximum likelihood phylogenetic trees using RAxML69 under the general time-reversal model of nucleotide substitution with 1000 bootstrap replicates.  and a two-epoch model shows that the two-epoch model with a population bottleneck provides a better fit. Though the strength of the bottleneck cannot be determined based on the AFS alone due to the small sample size  for the Okinawa C. ryukyuensis, mangshanyeju and common mandarins  using pure, or mostly pure, individuals that were identified by sliding window analysis of pairwise genomic distance (D) and multidimensional scaling. AIMs were derived using three pure pummelos, three pure C. ryukyuensis accessions, two pure mangshanyeju, and three common mandarins as exemplars for the four ancestral populations. We note that our dataset only includes two pure mangshanyeju. We also found that common mandarins generally contained sub-chromosomal genomic segments with MS or PU ancestry. Segments of these exemplars with evidence for admixture were excluded.  for four ancestral populations  relative to PU and RK. In this way, we obtained a total of 397,887 ancestry informative markers: 268,383 for PU, 54,325 for RK, 35,067 for MA, 22,482 for MM, and 17,630 for MS.AIMs for each target population were defined as single nucleotide variants that are fixed in the target population exemplars relative to the other population exemplars, i.e., homozygous in the target exemplars but not found in the others. Since MS and MA are more weakly differentiated than other population pairs, we introduced a super-population MM to represent markers that are fixed in 20. To call ancestry within a window, we required at least 5 AIMs for each ancestral population, otherwise the ancestry was assigned as Unknown. We note that our local ancestry method is in general agreement with but more sensitive than ADMIXTURE68, which may fail to detect short blocks of admixture at the a few percent level.We used these genome-wide AIMs to carry out local ancestry analysis in our collection of east Asian citrus Figs. b, 3b, 4cr = ½ IBD1 + IBD2. Following21, we infer identify by descent over non-overlapping 200 kb windows using (1) the genetic distance D defined above and (2) the identity-by-state ratio,73. Importantly, if two individuals share the same haplotype across a window, then IBS0=0 and IBSR=1. We infer the IBD state for each window using the following criteria20: If IBSR < 0.95, the genomic window is assigned IBD0. If IBSR > =0.95 and D < 0.05, the window is assigned IBD2. If IBSR > =0.95 and D > 0.05 the window is assigned IBD1.Genetic relatedness between a pair of diploid individuals can be quantified by the proportion of their genomes that share zero, one or two haplotypes that are ‘identical by descent’ . The familiar coefficient of relatedness is defined from these quantities via 21. For such regions, we inferred the IBD state by comparing phased haplotypes instead of diploid genotypes. We performed inter-specific phasing using representative accessions from the two parental populations20. To allow errors from SNP calling and phasing, we consider two haplotypes identical if the mismatch rate is below 2 × 10−4.Genomic windows for two interspecific hybrids need to be treated differently, since in these cases the IBSR value is inflated by species-specific alleles and does not reflect shared haplotypesC. ryukyuensis haplotypes of various shiikuwashas and yukunibus are found to be unrelated by direct comparison.By this method we find that the mainland Asian mandarin RK3 shares one haplotype with all shiikuwasha across the entire genome. Since shiikuwasha are F1 interspecific hybrids, this implies that RK3 is the common parent of shiikuwasha . We found that the elite Nakamoto seedless shiikuwasha SH4) is a somatic mutant of one of the six basic shiikuwasha genotypes (SH2)   across a collection of 55 citrus accessions including mandarins and interspecific mandarin hybrids and admixtures derived from six citrus species . We carried out genetic admixture analysis for this 200 kb window using ADMIXTURE68 for K=3–11. For each value of K, we performed twenty independent runs and used the run with lowest cross-validation error. Figure K = 8, with additional plots for K = 8–10 included in Supplementary Fig. K = 8 all six citrus species are resolved with further differentiation of mandarins (C. reticulata) into three sub-populations, namely, common mandarin (MA), and two mangshanyeju sub-populations h1 and h2 across this window. Progressively finer resolution is observed for K = 9 and 10. At K = 9, two sub-populations  are differentiated within common mandarins. With ten ancestral populations (K = 10), further differentiation within pummelos  is revealed. Importantly, the two mangshanyeju  sub-population ancestry compositions remain unchanged for K = 8–10.To investigate the genetic ancestry of the ne Ciclev010497m (Further information on research design is available in the Supplementary InformationPeer Review FileSupplementary Data 1Supplementary Data 2Supplementary Data 3Description of additional supplementary filesReporting Summary


Road networks and human density are major factors contributing to habitat fragmentation and loss, isolation of wildlife populations, and reduced genetic diversity. Terrestrial mammals are particularly sensitive to road networks and encroachment by human populations. However, there are limited assessments of the impacts of road networks and human density on population‐specific nuclear genetic diversity, and it remains unclear how these impacts are modulated by life‐history traits. Using generalized linear mixed models and microsatellite data from 1444 North American terrestrial mammal populations, we show that taxa with large home range sizes, dense populations, and large body sizes had reduced nuclear genetic diversity with increasing road impacts and human density, but the overall influence of life‐history traits was generally weak. Instead, we observed a high degree of genus‐specific variation in genetic responses to road impacts and human density. Human density negatively affected allelic diversity or heterozygosity more than road networks ; increased road networks and human density also positively affected allelic diversity and heterozygosity in 15 and 6–9 genera, respectively. Large‐bodied, human‐averse species were generally more negatively impacted than small, urban‐adapted species. Genus‐specific responses to habitat fragmentation by ongoing road development and human encroachment likely depend on the specific capability to (i) navigate roads as either barriers or movement corridors, and (ii) exploit resource‐rich urban environments. The nonuniform genetic response to roads and human density highlights the need to implement efforts to mitigate the risk of vehicular collisions, while also facilitating gene flow between populations of particularly vulnerable taxa. Indeed, life‐history traits can increase mammalian susceptibility to the ecological effects of RNHD  across microsatellite loci per study extracted from the MacroPopGen database . Furthermore, microsatellites are largely selectively neutral , specifically data on the mean number of alleles (MNA) and observed heterozygosity  and South America (283 populations) as most mammal populations from MacroPopGen were located in North America and the paucity of openly available road and traffic data for Central/South America limits the reliability of analyses for mammal populations in these regions.Data from 1444 populations used in this study originated from 45 species, 27 genera, and 12 families, and were based on 76,682 individual genotypes from 206 studies  to avoid fine‐scale patch effects   as the measure of genetic diversity. Disturbances to population structure take longer to accumulate changes in HO than MNA, and thereby, HO represents a different time scale that roads and human encroachment are fragmenting populations. GLMMs with MNA as the response variable were fit using a gamma distribution with a log‐link, because measured values of MNA values are always positive, continuously distributed, and often positively skewed. Conversely, GLMMs with HO as the response variable were fit using a beta distribution with a logit link because HO values are continuously distributed between zero and one.We used two separate sets of generalized linear mixed models (GLMMs) for each metric of genetic diversity, for a total of four separate model selection analyses, to test our hypotheses concerning the effects of RNHD , life‐history traits, and taxon specificities on genetic diversity of North American mammalian populations. The first set of models included MNA per locus as the measure of genetic diversity, because reductions in population size by habitat fragmentation can cause strong, rapid, and detectable changes in MNA  in R. Multicollinear variable(s) with the highest VIF scores (>3) were removed stepwise until all other variables had VIF scores <3  and human density with taxonomic grouping. Taxonomic grouping was a fixed effect because we were specifically interested in directly identify the slope of the interaction between RNDH and taxa, instead of using taxa as a random effect to account for variation between groups. Support for models that include an interaction between taxon level and RNHD would indicate the taxonomic rank that RNHD impacts predominately manifest in mammal population genetic diversity, while allowing direct identification of relative taxa‐specific trends. More complex models that included both taxonomic grouping and life‐history traits were excluded from model selection for both MNA and HO), we used the information‐theoretic approach (AIC) to compare relative support of alternative models based on fit and complexity  across 25 genera; however, two genera were unique to each of the HO and MNA subsets.Of the 1444 genetically distinct populations, a total of 1054 populations had data for analysis with a mean MNA value of 6.15 (SD =2.7) across 25 genera. Comparatively, there were 1032 populations with 3.22, with a mean of 0.057 (SD = 0.055). Road density for highways, primary, and secondary roads was, respectively, 1.8 times, 3.9 times, and 8.8 times higher than small local roads. Vehicular traffic based on AADT from the Highway Performance Monitoring System indicated that highways, primary roads, and secondary roads have, respectively, 15.3, 3.75, and 1.37 times as much vehicular traffic as local roads. Based on these ratios, mammalian populations experienced road impact values ranging from 0 to 0.772 traffic*km /km2, with a mean of 0.204 (SD = 0.18). Comparatively, human density experienced by mammalian populations was more variable and ranged from 0 to 168.7 persons/km2, with a mean of 16.15 (SD = 24.40).Within a 250 km radius, mammal populations in this study experienced total road densities ranging from 0 to 0.242 km/km3.3HO that modeled positive spatial autocorrelation in the spatial structure of mammalian populations from a continental scale to a local geographic level. Forward variable selection retained three dbMEMs for both MNA and HO as significant predictors of spatial structure for analysis in subsequent GLMMs. For both metrics of genetic diversity, the first dbMEM explained a large proportion of variation in spatial structure at a broad continental scale, likely due to the unequal distribution of genetic studies of mammalian populations, or large geographical features, such as mountain ranges . The validation set approach further identified that the overall fit of MNA models was improved by excluding the interaction term between home range size and human population density. Similarly, HO models were improved by excluding the interaction term between body mass and human population density . MNA increased with increased human density for six of 25 genera, decreased in 13 genera, and did not vary for six genera , Peromyscus (deer mice), and Gulo (wolverine), genetic diversity did not vary with increasing road impacts or human density; comparatively, HO diversity of Canis (wolves and coyotes) did not vary with increasing road impact or human density .For examining the influence of taxonomic rank on the genetic impact of RNHD on terrestrial mammals, the best‐fit model for each metric of genetic diversity included road impact–genera and human density–genera interaction terms, and not family or species  had consistently negative slopes for both road impact and human density, supporting the prediction that taxa that exhibit skittish, human, and road‐avoidant behavior should be the most severely impacted. Conversely, taxa familiar with urban settings, such as Ursus (Bears) and Procyon (Raccoons), which may use road corridors for travel and scavenging, had consistently positive road impact slopes.Visualizing the relative effects of road impacts and human density on mammal genera into four quadrats reveals that genus‐specific population genetic diversity was more negatively affected by human density than by road impacts Figure . Namely,4Based on an unprecedented amount of genetic data from 1444 populations, our results show that RNHD affects population genetic diversity, similar to past work , Rangifer (Caribou), and Ursus (bears). These taxa often require large habitable areas, which may have been affected by land conversion and human encroachment, resulting in habitat loss and disruption of population connectivity , may be able to exploit linear features of road edges as corridors for hunting or patrolling  or Tamias (chipmunks), can mitigate negative effects and potentially benefit from increased population connectivity and habitable area provided by roads and human structures  suggests that habitat fragmentation caused by roads can both positively and negatively impact wildlife populations. These effects can function separately from habitat loss by land‐use conversion and expansion of human population densities, which largely negatively affect wildlife populations (Fahrig, Our quantitative analysis of nuclear DNA in North American terrestrial mammal populations reveals that taxon‐specific responses to road impacts and human population density are highly variable. The relatively high number of genera positively affected by road impacts ( Fahrig, . Our resWe emphasize that our synthesis has focused on neutral genetic diversity, for which the most data are currently available in the scientific literature: The consequences of roads, human density, habitat fragmentation, and habitat loss on adaptive genetic variation and differentiation remain understudied (Brady & Richardson, The authors have no conflicts of interest to declare.Supplementary MaterialClick here for additional data file.


Gentiana crassicaulis Duthie ex Burk, an endemic Chinese alpine species in this area, were investigated based on four chloroplast fragments and internal transcribed spacer region of the nuclear ribosomal DNA (nrITS) sequences of 11 populations. The populations with highly diverse chloroplast haplotypes were mainly found at the edge of the QTP. There were two main haplotypes of nrITS clones, one shared by the Yunnan and Guizhou populations, and the other by the remaining populations. The population with the highest diversity was the Gansu population, located at the edge of the plateau. Based on molecular dating, the diversification of G. crassicaulis at the edge of the plateau occurred before the Last Glacial Maximum (LGM), and the species may have completed its expansion from the edge to the platform. Ecological niche models were conducted to predict the distributional ranges of G. crassicaulis at present, during the LGM, and during the last interglacial (LIG) period. The results demonstrated that G. crassicaulis survived on the QTP platform and at the edge during the LGM but afterward retreated from the platform to the southern edge, followed by expansion to the platform.The Himalaya–Hengduan Mountain region is one of the hotspots of biodiversity research. The uplift of the Qinghai–Tibetan Plateau (QTP) and the Quaternary glaciation caused great environmental changes in this region, and the responses of many species in the QTP to the Quaternary climate are still largely unknown. The genetic structure and phylogeographical history of  Gentiana crassicaulis, an alpine gentian endemic to the Himalaya–Hengduan Mountains, could survive on the QTP platform and at the edge during the LGM but afterward retreated from the platform to the southern edge, followed by expansion to the platform. ENMs have been widely used in spread of invasive species  cloning sequences, four chloroplast fragments, and MaxEnt models in three periods  period, during the LGM, and at present) to reveal the species differentiation and migration patterns, with the following purposes: (1) to evaluate the value of chloroplast and ITS data in the systematic analysis of G. crassicaulis, (2) to evaluate the genetic differentiation level of G. crassicaulis, and (3) to investigate how this species expanded and contracted its range during the Quaternary glacial and interglacial periods.Previous studies suggest that the herbaceous plants flourished in China from the Miocene of tertiary Tao, . In the 22.1Gentiana crassicaulis Duthie ex Burk were collected throughout the distribution range, with sample size per population ranging from 3 to 15 depending on the population size. The individuals collected were ≥5 m apart to avoid sampling bias toward closely related individuals , haplotype diversity (Hd), nucleotide diversity (π), average number of nucleotide differences (k), gene flow (Nm), and genetic differentiation (Fst). The mismatch distribution and neutrality tests (Tajima's D) were performed using DNASP5. Total gene diversity (Ht) and average gene diversity within populations (Hs) were calculated. To test whether there was a phylogeographic structure, STG and STN were calculated using PERMUT   analysis was conducted using PAUP 4.0  , and during the last interglacial  period . The data obtained from online databases were double checked. A total of 79 spatially unique localities were used for the analysis (Table http://worldclim.org) with a resolution of 2.5 arcmin. Multivariate collinearity among climate variables was tested by Pearson correlation coefficient using SPSS 20.0. The climate variables with Pearson correlation coefficient less than 0.8 were retained, and among the variables with Pearson correlation coefficient greater than 0.8, the one which was closely related to geographical distribution was selected. Eight climate variables were screened out and used , number of iterations (500), and occurrence localities divided into testing and training datasets .We used ecological niche models (ENMs) based on the maximum entropy method implemented in MAXENT Version 3.4.0 , 11 simple sequence repeat (SSR) differences, and 8 insertion–deletions (InDels) were discovered  in Tibet and was the most widely distributed haplotype. H2 was found in QH (Qinghai), GS (Gansu), and SC1  populations. H5 was detected in SC1 and SC2  populations. H6 was present in YN (Yunnan) and SC2 populations. H3 and H4 were private haplotypes, which were found to exist only in GS and GZ (Guizhou) populations, respectively. GS, SC1, and SC2 populations each had two haplotypes, and there was only one haplotype per population in the other populations. The geographical distribution of each haplotype is shown in Figure G. crassicaulis was constructed based on the chloroplast haplotypes, with Gentiana straminea as an outgroup  value of each population was slightly lower than the average value of 0.040, except for SC1 population (0.046) (Table Hs = 0.086) was much lower than the overall genetic diversity (Ht = 0.774). The genetic differentiation coefficient among the populations was high (Fst = 0.85605), and the gene flow among the populations was limited (Nm = 0.11). There was not a phylogeographic structure for G. crassicaulis because Nst was not significantly greater than Gst . AMOVA based on chloroplast haplotypes revealed that 85.61% of the total variation was partitioned among the populations, whereas 14.39% was within the populations  of ITS per population ranged from 0.515 to 0.968, with an average value of 0.647, and the π value ranged from 0.117 to 1.186, with an average value of 0.543. GS population had the most abundant haplotypes and the highest Hd and π values, showing the highest diversity, followed by XZ1 population. YN population had the lowest diversity , and the Nm was extensive (2.33). Nst (0.145) was smaller than Gst (0.183), indicating no phylogenetic structure. AMOVA based on ITS suggested high‐level within‐population differentiation  and low‐level among‐population differentiation (9.75%) , respectively, indicating that the populations may have not experienced rapid expansion events , the two models of the LGM period both showed a significant southward shift compared with the models of the LIG period, which were mainly concentrated in Yunnan and Guizhou, and the distribution area with a high probability at the present spread throughout.The traditional AUC and TSS methods of estimation of model accuracy of all the models in different periods indicated good predictability for the models. The AUC value of the model in each period ranged between 0.974 and 0.980, and TSS value ranged between 0.765 and 0.793 Table , indicat4G. crassicaulis were studied by using two genomic fragments, namely maternal chloroplast sequences and parental nrITS sequences. Chloroplasts have their own complete set of genomes . However, the analysis of the ITS haplotypes showed that the gene flow among the populations was extensive, and most of the genetic variation existed within the populations (90.25%). This variation was found to be much higher than the genetic variation among the populations. Both ITS and chloroplast sequences are commonly used in phylogeographic studies, and the results in most species are basically consistent ; Data curation (lead); Formal analysis (lead); Funding acquisition ; Investigation ; Methodology ; Resources ; Software (lead); Validation (lead); Writing – original draft (lead). Weitao Li: Data curation ; Formal analysis ; Investigation ; Methodology ; Software (lead); Validation ; Writing – original draft . Zhili Zhao: Conceptualization (lead); Data curation ; Funding acquisition (lead); Investigation (lead); Methodology (lead); Project administration (lead); Resources (lead); Software ; Supervision (lead); Validation (lead); Writing – original draft ; Writing – review & editing (lead). Dorje Gaawe: Conceptualization ; Investigation ; Resources ; Supervision . Tonghua Liu: Funding acquisition ; Project administration ; Resources .Fig S1Click here for additional data file.Fig S2Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.