Gut microorganisms are crucial for many biological functions playing a pivotal role in the host’s well-being. We studied gut bacterial community structure of marine iguana populations across the Galápagos archipelago. Marine iguanas depend heavily on their specialized gut microbiome for the digestion of dietary algae, a resource whose growth was strongly reduced by severe “El Niño”-related climatic fluctuations in 2015/2016. As a consequence, marine iguana populations showed signs of starvation as expressed by a poor body condition. Body condition indices (BCI) varied between island populations indicating that food resources  are affected differently across the archipelago during ‘El Niño’ events. Though this event impacted food availability for marine iguanas, we found that reductions in body condition due to “El Niño”-related starvation did not result in differences in bacterial gut community structure. Species richness of gut microorganisms was instead correlated with levels of neutral genetic diversity in the distinct host populations. Our data suggest that marine iguana populations with a higher level of gene diversity and allelic richness may harbor a more diverse gut microbiome than those populations with lower genetic diversity. Since low values of these diversity parameters usually correlate with small census and effective population sizes, we use our results to propose a novel hypothesis according to which small and genetically less diverse host populations might be characterized by less diverse microbiomes. Whether such genetically depauperate populations may experience additional threats from reduced dietary flexibility due to a limited intestinal microbiome is currently unclear and calls for further investigation.The online version contains supplementary material available at 10.1007/s00114-020-01714-w. A wide variety of microbial communities exist in the gastrointestinal tract of vertebrates, constituting the gut microbiome  represents a suitable natural system to study the influence of diet and starvation on the gut microbiome. Marine iguanas forage in the tidal and intertidal zones where they consume macrophytic algae  and red algae (Centroceras sp. and Gelidium sp.)  —displaying markedly different genetic population parameters . A sample representative of the gut microbiome was also taken by gently introducing a sterile swab into the cloaca and the gastrointestinal tract. This is a valid method to sample gut microbial communities in reptiles   × 106) . BCI values were compared for matching pairs of islands between the years and as the median across populations of each sampling period. Furthermore, differences between year and matching pair of sites were tested by performing a GLM with BCI values as dependent variable and site and year and the interaction between both site and year as factors. Tukey post hoc tests were carried out to explore formal differences between matching pairs of sites from both years.Marine iguanas were measured for total length (TL) and weighed after being captured; TL was measured as the distance from the tip of the snout to the tip of the tail using a metric tape. Body mass was determined on a scale (± 5 g). The residuals of a linear regression between the values of both body mass and TL  were used as the body condition index (BCI). This and similar analyses were carried out with the GLM procedure in Statistica software . This approach results in similar estimates of BCI based on the formula (body mass/snout-vent length) Laurie . It offe) Laurie , we also) Laurie  associatA. cristatus, up to 50 individuals were randomly pruned from the large dataset, while sampling sites with less than 20 genotyped individuals were discarded , mean observed heterozygosity (Ho), and rarefied mean allelic richness (AR), in the software packages GenAlEx 6.5  was used to calculate a rarefied mean allelic richness.Genetic diversity of marine iguana host populations was estimated from a previous comprehensive dataset based on 12 polymorphic nuclear microsatellite loci downloaded from the Dryad data repository [10.5061/dryad.pp6bm] , based  Jombart  and PopGRVAideMemoire , we performed partial Mantel tests with the package vegan  was performed, followed by a pairwise comparison with the software PRIMER 7 and R package re Hervé . HeterogP value (P < 0.05). This analysis included 328 individuals—as sex was not available for four of the samples (see Table HS), showing that genetic diversity and starvation are independent parameters .Bacterial diversity was calculated to explore the impact of starvation as well as genetic population parameters on gut bacterial diversity. We used the calculated species richness  as obtained from QIIME. Differences in species richness among populations and sex categories were explored by carrying out a GLM followed by post hoc Tukey tests if a factor showed a significant ape package  of observed bacterial OTUs into account, we calculated Shannon’s effective number of species and performed similar regression analyses as for observed species richness Jost . To explUnless stated otherwise, all statistical analyses were performed in Statistica v. 7 (StatSoft) and R.14,311 = 28.264, P < 0.001, adjusted R squared = 0.54; factor sites, F12,311 = 32.22, P < 0.001; factor sex, F2,311 = 9.516, P < 0.001). Concerning sex categories, post hoc comparisons revealed that females had statistically higher BCI than males (Tukey test: P = 0.009), but all the other comparisons  remained non-significant (P > 0.32). As demonstrated by the distinct values of body condition index  had the highest body condition value , while Santa Fe iguanas (A. c. trillmichi) were the ones most affected by starvation  . On the other hand, Sphingobacterium appeared to be common only in iguanas from Genovesa and Marchena. A detailed overview is provided in Fig. 16S metabarcoding of the V4 region from 332 marine iguana samples revealed a total of 2711 OTUs after filtering and rarefying. Marine iguana gut bacterial communities were dominated by members of the phyla Firmicutes (36%), Proteobacteria (19%), Bacteriodetes (18%), and Actinobacteria (18%). Other phyla were also present in marine iguanas, such as Tenericutes (4%) as well as Lentisphaerae (0.7%) and Verrucomicrobia (0.6%) together with other taxa found in minor amounts , showing that both variance and location effects may be responsible for the observed differences in bacterial community structure. A MRM revealed that interpopulation differences in bacterial beta diversity were more correlated with Nei’s genetic distance  than with geographical distance ; however, the overall variation explained by this model was low . Partial Mantel tests supported the results obtained in the MRM. A relatively strong relationship between beta diversity and genetic distance was observed when controlling for geographic distance . However, beta diversity did not correlate significantly with geographical distance when controlling for the effects of genetic distance .Bacterial communities differed statistically among particular island populations . It was lowest in the Punta Pitt population  and on Santa Fe , and highest on Fernandina (192.93 OTUs) and Isabela (184.42 OTUs)—both populations belong to the subspecies A. c. cristatus. A detailed overview of species richness found for the distinct island populations (subspecies) is shown in Table F1,324 = 0.24; p = 0.624; see Table Species richness differed among populations but not among sex categories  and diversity  as estimated in PICRUSt. Explorative analysis on specific taxonomic phyla involved on fermentation and digestion did not show a clear association with marine iguana body condition. The abundance of Firmicutes showed a weak, but significant relationship with BCI , while marine iguanas from Pinta  and Punta Pitt displayed the lowest values  (Table Hs) than populations with less diverse microbiomes  as well as allelic richness  . Bacterial species richness also tended to be related to observed heterozygosity (Ho) .Among all island populations, we found the highest level of genetic diversity on Fernandina  only tended to be significant  during “El Niño” 2015/2016) and non-“El Niño” (2004) years revealed that iguanas displayed lower BCI values in 2015/2016 and thus suffered from starvation on several islands  and Isla Lobos (negative BCI) on San Cristóbal. These locations are only separated by roughly 10 km coastline and belong to the same subspecies, i.e., Periods of fasting and food limitation represent a special physiological situation and can become extremely stressful for individuals when extended during periods of starvation  showed a lack of host genetic influence on bacterial gut community structure  of the marine iguana microbiome and the diversity of food plants and foraging mode relative to population sizes in marine iguanas. As a relevant observation for conservation management, we found that populations with a more diversified bacterial community did not perform better during “El Niño”-dependent starvation than populations with lower species richness of their gut bacterial community. This could indicate that digestive performance is dependent on a core microbiome accounting for most of its functionality and calls for future analysis of the marine iguana gut microbiome from an experimental and functional perspective.ESM 1(DOCX 22.4 mb)


Larix laricina  is a transcontinental North American conifer with a prominent disjunction in the Yukon isolating the Alaskan distribution from the rest of its range. We investigate whether in situ persistence during the last glacial maximum (LGM) or long‐distance postglacial migration from south of the ice sheets resulted in the modern‐day Alaskan distribution. We analyzed variation in three chloroplast DNA regions of 840 trees from a total of 69 populations (24 new sampling sites situated on both sides of the Yukon range disjunction pooled with 45 populations from a published source) and conducted ensemble species distribution modeling (SDM) throughout Canada and United States to hindcast the potential range of L. laricina during the LGM. We uncovered the genetic signature of a long‐term isolation of larch populations in Alaska, identifying three endemic chlorotypes and low levels of genetic diversity. Range‐wide analysis across North America revealed the presence of a distinct Alaskan lineage. Postglacial gene flow across the Yukon divide was unidirectional, from Alaska toward previously glaciated Canadian regions, and with no evidence of immigration into Alaska. Hindcast SDM indicates one of the broadest areas of past climate suitability for L. laricina existed in central Alaska, suggesting possible in situ persistence of larch in Alaska during the LGM. Our results provide the first unambiguous evidence for the long‐term isolation of L. laricina in Alaska that extends beyond the last glacial period and into the present interglacial period. The lack of gene flow into Alaska along with the overall probability of larch occurrence in Alaska being currently lower than during the LGM suggests that modern‐day Alaskan larch populations are isolated climate relicts of broader glacial distributions, and so are particularly vulnerable to current warming trends. Larix laricina (larch) is a transcontinental North American conifer with a disjunction in the Yukon isolating its Alaskan distribution. Using chloroplast‐microsatellite analysis and species distribution modeling, we provide evidence for the glacial persistence and long‐term isolation of larch in Alaska.Range disjunctions represent natural laboratories to examine the interplay between long‐distance migration and refugial persistence in biome development.  Amplification was performed by implementing a 3‐step program: (a) a 2‐min denaturation at 94°C; (b) 35 cycles of 30‐s denaturation at 94°C, 30‐s annealing at 55°C, and 2‐min elongation at 72°C; and (c) a 5‐min final elongation at 72°C.We sampled 251 da Table . At eachThe three target cpSSR loci were amplified for all larch samples and migrated in an ABI 3730xl automated sequencer (Applied Biosystems) with 50‐cm capillary arrays using LIZ500 fluorophores  by the W.M. Keck Center at the University of Illinois . Fragments were then scored using the GeneMapper program (Applied Biosystems). Chlorotypes were obtained by concatenating alleles obtained at the three cpSSR loci.L. laricina across 589 individuals from 45 populations sampled across eastern and western Canada , which is the equivalent to expected heterozygosity for diploid data  seeking to maximize the differentiation among groups by considering the number of requested groups and their allele frequencies as varying parameters. The optimal k‐value  is determined by running 10 replicates of k = 2 through 10 and then selecting the scenario where log marginal likelihood first reaches a plateau. Between the identified groups, an analysis of molecular variance (AMOVA) and pairwise FST among populations were implemented in ARLEQUIN version 3.5  was used to simulate modern‐day potential range and hindcast the LGM distribution for 33.1L. laricina uncovered five previously unknown chlorotypes (N1–N5) of which three  are endemic to Alaska  indicated that mean within‐population chlorotype diversity (HS) is significantly lower in Alaska compared with western Canadian populations east of the range discontinuity . According to an analysis of molecular variance , whereas the molecular variance among the populations within these two regions is not statistically significant . Alaskan populations exhibited dominance of a single chlorotype (XV), whereas greater evenness of the chlorotypes was found in Canada, across the disjunction : group 1 in eastern Canada, group 2 in western Canada (from Lake Winnipeg to the Yukon range discontinuity), and group 3 in Alaska  but significantly higher than that of the Alaskan larch populations . When compared to the Alaskan group, BAPS groups from Canada display greater evenness in the relative abundance of chlorotypes . A mere 4% of the variation is explained by populations within the BAPS groups (FSC = 0.055), and the remaining 66% pertains to variance among individuals within the populations (FST = 0.334).Population differentiation among the three BAPS clusters varied greatly, as demonstrated by the distribution of pairwise s Figure . The twoR2 = 0.7, p < .0001). Our data provide no evidence that chlorotype V ever crossed the Yukon discontinuity westward into Alaska, although the frequency of this haplotype remains high (42 ± 20%) across Canada and does not vary significantly along a longitudinal domain from 130°W to 55°W .Chlorotypes XV and V are the two most common haplotypes found across the transcontinental range of tamarack, and their frequency distribution might provide insight on the direction and extent of postglacial gene flow Figure . The relins high 2 ± 20% a3.2L. laricina, 368 meet the criterion to be considered useful (TSS > 0.6). We retained the 50 best models for the final ensemble SDM, with TSS values ranging between 0.70 and 0.81. The ensemble SDM for L. laricina predicts a modern distribution that matches well with the observed modern‐day range. The model suggests higher probability of occurrence in interior Alaska and throughout western and eastern Canada, and lower probability of presence in the United States (except for the Great Lake states and New England states where the species is indeed present), British Columbia, and Yukon , and south of the Laurentide ice sheet , are largely dominated by a single chlorotype (XV; Figure L. laricina populations between eastern Canada, western Canada, and Alaska, as indicated by the cpDNA Bayesian analysis suggests the existence of three distinct ice‐age refugial lineages (Figure Results from the 24 s Figure . The twos Figure . Our ress Figure .L. laricina in Alaska, especially when compared to the modern‐day conjoined populations forming the Canadian distribution, probably reflects long‐term isolation and erosion of genetic diversity due to drift (i.e., Frankham, The low genetic diversity of L. laricina existed in central Alaska, which was unglaciated during the LGM (Kaufman & Manley, L. laricina has been overlooked due to inadequate or missing data. Despite advances in arctoboreal palynology (Oswald, Brubaker, Hu, & Kling, Our species distribution model corroborates the genetic data, supporting three LGM refugia for larch in North America Figure . FurtherOur results join a growing body of research demonstrating ice‐age persistence in areas previously thought to be climatically unsuitable for tree species during the LGM (e.g., Napier et al., The proposed “reverse‐refugial dynamic” and asymmetrical gene flow may help explain the current distribution of Alaskan larch. In contrast with the broad habitat availability and larger effective population sizes during the LGM Figure , the emeThe authors have no conflict of interest to declare.F.S.H. and G.dL. designed the research. J.D.N. and G.dL. planned the fieldwork, while F.S.H. and G.dL. conducted it. J.D.N., M.C.F., and G.dL. performed laboratory work and analyzed data. All authors contributed to manuscript writing. Click here for additional data file.


Chuetas are a group of descendants of Majorcan Crypto-Jews  who were socially stigmatized and segregated by their Majorcan neighbours until recently; generating a community that, although after the seventeenth century no longer contained Judaic religious elements, maintained strong group cohesion, Jewishness consciousness, and endogamy. Collective memory fixed 15 surnames as a most important defining element of Chueta families. Previous studies demonstrated Chuetas were a differentiated population, with a considerable proportion of their original genetic make-up. Genetic data of Y-chromosome polymorphism and mtDNA control region showed, in Chuetas’ paternal lineages, high prevalence of haplogroups J2-M172 (33%) and J1-M267 (18%). In maternal lineages, the Chuetas hallmark is the presence of a new sub-branching of the rare haplogroup R0a2m as their modal haplogroup (21%). Genetic diversity in both Y-chromosome and mtDNA indicates the Chueta community has managed to avoid the expected heterogeneity decrease in their gene pool after centuries of isolation and inbreeding. Moreover, the composition of their uniparentally transmitted lineages demonstrates a remarkable signature of Middle Eastern ancestry—despite some degree of host admixture—confirming Chuetas have retained over the centuries a considerable degree of ancestral genetic signature along with the cultural memory of their Jewish origin. With the Christian conquest of Majorca in 1229, their physical survival was guaranteed, despite social and religious pressures forcing their conversion to Christianity between 1391 and 1435. Consequently, there were officially no more Jews in Majorca nearly 60 years before the Edict of Expulsion by the Catholic Kings in 1492. Many of these converted Jews were integrated in the general population; however, a few families remained in the ghetto and secretly adhered to Judaism, forming a Crypto-Jewish community which was persecuted by the Inquisition (fifteenth–seventeenth centuries)2. The last “Autos de Fe” in 1691 put a stop to their hidden Jewish religious practices, and this population of convicts and their descendants came to be known as Chuetas, a word probably derived from the Catalan for Jew3, with their social stigma and segregation (imposed by their Majorcan neighbours) continuing until the mid-twentieth century. There was a definitive point of inflection when Majorca opened to tourism, as the arrival of newcomers (Spaniards or foreigners) who had no knowledge of the status of Chuetas led to a decrease in anti-Chueta prejudice. Therefore, Chuetas were an isolated population with very scarce intermarriage with the Majorcan host population until recently4. One of the most important defining elements of this group is that they bear one of the 15 surnames of converso lineages  targeted by the inquisitorial sentences for Crypto-Judaism in the last quarter of the seventeenth century5. Some of these surnames are common in other Spanish regions, where they are not related to Judaism. In Majorca, however, they have been fixed in the collective memory by their identification as Chueta families.Jewish communities in the Balearic Islands date back to the fifth century AD6. Since then, populations with Jewish origin have been analysed by means of uniparental and recombining markers12, and more recently also through genome-wide SNP arrays18. The combined analysis of millions of polymorphic markers along the genome have led to greater precision in the clustering of different Jewish groups and to the ability to estimate the Middle Eastern, European, and African components in each group. These analyses reflect that each of today’s Jewish populations is the result of the blending of Middle Eastern and host populations . Regarding haploid markers, on the one hand, male lineages indicate that most Jewish communities share a common Middle Eastern ancestral origin, and remained relatively isolated from neighbouring non-Jewish communities during the Diaspora. On the other hand, mtDNA studies lead to conclude that there are differences in the demographic history of the widespread communities resulting from the Jewish Diaspora in terms of maternal ancestries, indicating different maternal founder effects.The rise of modern Population Genetics in the second half of the twentieth century enabled geneticists to endeavour to define the origins and relatedness of Jewish people21, or owing to their isolation and differentiation from their neighbours, such as the Portuguese Crypto-Jew communities in Belmonte and Bragança24 or the Chuetas in Majorca, the subject of the present study. Chuetas have been previously studied by means of autosomal and X-chromosome markers29. The results conducted to date demonstrate that Chuetas are a differentiated population that has kept a considerable proportion of its original genetic make-up, especially clear in some markers where Chuetas show polymorphic frequencies of alleles that are very rare in neighbour populations, but not in Middle Eastern populations30. However, a certain degree of admixture from and with the host population has also been detected29.Converted Jews have also been subject of study, either due to their contribution to the host population genetic poolIn this study, we focus on haploid markers in order to investigate the ancestry and demographic history of the maternal and paternal founding lineages of the Chueta population, and to analyse whether cultural isolation has led to the reduction of genetic diversity in mtDNA and Y-chromosome lineages in this population.31. For mtDNA analyses, 183 samples were used: 104 non-maternally related individuals from the Chueta population  and 79 Majorcans. All participants provided appropriate informed consent statements, approved by the "Direcció General de R + D + I" , and the University of the Balearic Islands (procedure AAEE24/2014), following the procedures approved by the Ethics Committee of the University of Porto (N102/CEUP/2012). Anonymity of the recruited samples was preserved during the study. All methods were carried out in accordance with the guidelines and regulations of the Declaration of Helsinki.For the study of the Y-chromosome, samples from 146 unrelated males were obtained: 100 from the Chueta population, and 46 from Majorca , included as the host population of Chuetas. The Majorcan samples constitute a subset of a larger sample previously genotyped for 12 Y-STRsDNA was extracted by standard phenol–chloroform method and quantified on a NanoVue Plus spectrophotometer .35 , following the manufacturer’s recommendations. Thirty-eight SNPs were typed to define the major male lineages. Thirty-two of them were genotyped using SNaPshot kit (Applied Biosystems) in five multiplexes as previously described35 Fig. . M1 and 37, and Y-SNP haplogroups according to the latest ISOGG update (2019).Y-STR amplification products and Y-SNP purified minisequencing products were separated in an ABI PRISM 3130 Genetic Analyser, and electropherograms were analysed using GeneMapper ID software v3.2 and Peak Scanner software (Applied Biosystems). Y-STR alleles were designated according to ISFG recommendations38. The amplified product was purified with the MBS Spin PCRapace kit , and subsequent sequencing reactions were carried out using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), following the manufacturers’ procedures. Finally, products were run in an ABI prism 3130 analyser.The mitochondrial DNA control region, or D-loop (16024-576), was amplified with two overlapping fragments using mtDNA-specific primers http://www.phylotree.org/) using HaploGrep2 tool40, and assigned haplotypes were validated by EMPOP (http://empop.org/) curators.Sequences were assembled and compared to the revised Cambridge Reference Sequence (rCRS) (NC_012920) using Geneious software version 7.1.3 . Haplogroups were classified following the updated mtDNA phylogeny, PhyloTree, mtDNA tree Build 17  plot of the pairwise FST matrix was represented using SPSS v.15.0 .Haplogroup frequencies were estimated by gene counting. Allele and haplotype frequencies, molecular diversity indices, Analysis of Molecular Variance (AMOVA), as well as the corresponding non-differentiation p-values, and Y-chromosome pairwise F22 was to consider haplogroups with a frequency greater than 5% and TMRCA prior to 2 Kya as founder lineages.To attain statistical significance for frequencies of putative Jewish founding lineages, Bayesian 0.90 credible region (90% CR) was calculated using SAMPLING software . Furthermore, another criterion established by Behar et al.www.fluxus-technology.com)44.Median joining networks of Y-STR haplotypes were constructed using Network 4.6.1.1 (http://www.goldensoftware.com/products/surfer).In order to visualise the distribution of mtDNA haplogroup R0a frequencies in the Mediterranean geographic context, an isometric spatial frequency distribution map was constructed with the program Surfer 9 , indicating the differentiation of the Chuetas with respect to their host population. SAMPLING software identified three differential haplogroups between Chuetas and Majorcans: R1b1a1a2-M269 (hereafter R1b-M269), J1-DYS458.2, and J2-M172, pointing towards the lack of R1b-M269 and the presence of J1-DYS458.2 and J2-M172 as Chuetas' putative founding lineages. The frequency in Chuetas of haplogroups rarely found in neighbouring populations—E1b-M78, Q1-P36.2, G-M201, and R1a1a-M17 —could also mean that they might have been present in the original Jewish Majorcan gene pool.AMOVA analyses at both haplogroup and haplotype (Y-STRs) level showed significance when taking Chuetas and Majorcans as a whole  and Cortés was the most diverse with 4 haplogroups and an h value of 0.691; although the sample size would need to be enlarged to confirm these results. Putative founding haplogroups in the Chueta population, J1-M267 and J2-M172, were found to be associated to the surnames Picó and Aguiló (J1-M267) and Segura, Fortesa, and Fuster (J2-M172). The haplotypes carried by the individuals of most surnames show a star-like distribution with only one or two mutational steps between them. Foundation of each Chueta surname by one or a very few individuals in the Christian conversions (fourteenth–fifteenth centuries) could explain these results. In a few cases, the same haplotype is shared by different surnames or, contrarily, individuals within a surname belong to very distant Y-lineages, although the scarcity of historical documents with the Christian names that converted Jews adopted does not allow us to assess the different scenarios that could explain these cases further.Although studying the relationship between haplogroups and surnames was not an initial aim of our work on the Y-chromosome in Chuetas and, therefore, the sampling was not designed for it, with a median joining network we assigned and compared the haplotypes and haplogroups found with the surnames of the individuals Fig. . Fourtee49. Haplogroup H together with haplogroups U, K, and HV (frequencies ranging from 13 to 14%) accounted for 78% of total diversity. Other haplogroups found in this population were I, J, L, N, T, V, and X. In contrast with the Majorcan population, in the Chueta samples, haplogroup H only accounted for 17% of total diversity. The modal haplogroup in Chuetas (~ 20%) was found to be the Middle Eastern haplogroup R0a + 60.1 T, followed by haplogroups T, K, U, and J . Together these 5 haplogroups represented 64% of all variation. The remaining lineages were observed at frequencies ranging from ~ 1 to 5% . By means of the SAMPLING analyses, and also with the Behar et al.22 criterion, two haplogroups showed up as the founder lineages in the Chueta population: R0a + 60.1 T (~ 20%) and T1a (~ 6%).All in all, the mitochondrial haplogroup composition indicated that Chuetas are statistically different from their host population (Majorca) —could also mean that they might have been present in the original Jewish Majorcan gene pool.50. Ancient DNA studies carried out in recent years54 have shown that this lineage is associated with the spread of Steppe ancestry during the Bronze Age. In Iberia former Y-chromosome lineages were nearly completely replaced by haplogroup R1b-M26953, and a west-to-east gene flow from Iberia could have introduced these haplogroups into Western Mediterranean islands54. Majorca showed similar values (63%) to the rest of Iberian populations46, whereas the Chueta population (4%), had similar values to Middle Eastern and North African populations56. In Jewish populations, values range between 5% and 11.5%58, with the highest frequencies in Sephardim. These results support some degree of Iberian admixture in Sephardic Jews19 and important gene flow from the host population in Bragança Crypto-Jews (with an R1b-M269 frequency of 28%), as suggested by Nogueiro et al.23.The most frequent subclade of haplogroup R in Europe is R1b-M269, with frequencies ranging from 41 to 83%59, shows an East to West gradient in Europe. Although the phylogeography of haplogroup J is complex, its radiation seems to be concentrated mainly in the Bronze Age, an essential period for the establishment of the modern European genetic pool60. It contains two major branches, one of them, J2-M172, has its high frequencies in the Levant and is the most frequent sub-haplogroup in Europe, mainly throughout the Mediterranean basin. It was long considered to have spread across Europe with the demic diffusion of Neolithic agriculturalists62, but ancient DNA studies contradict this hypothesis, since in early Middle Eastern farmers, and also in European Neolithic remains, haplogroup J2-M172 is only detected sporadically63. Signs of population movements from the East, mainly maritime, such as the Phoenicians63 have been linked to J260 and also to the other main branch, J1-M267. This lineage has its maximum frequency in Arabia, but also high frequencies in the Middle East and in Jewish groups64. Some of its derived lines have purportedly been associated with Arabisation in North Africa62 while others have been implicated with different expansions of Middle Eastern populations through the Mediterranean Sea56. In most Jewish populations, J2-M172 reaches considerable frequencies57  to 15% (North African Jews)61; and in Middle East non-Jewish populations, from 10.0 to 17.0%57. Haplogroup Q1-P36.2 (xM346) is practically absent in Europe and Africa66. In Chuetas it showed a frequency of 8%, while in Jewish populations, percentages ranging from < 1% to 5% have been found66, with one branch typical of Ashkenazi Jews66. One of the main sub-branches of haplogroup G-M201, G2a, was the predominant male lineage in early European farmers, although the important Y-chromosome turnover in the Bronze Age nearly completed replaced it with the R1b lineage53. Nowadays haplogroup G-M201 is most common in Caucasus where the maximum frequencies are observed (> 70%), but it also occurs in the rest of the Middle East and south-western European countries at frequencies ranging from 5 to 15%67; while in North Africa it is far lower than in any European Mediterranean population46. In Jewish populations, Moroccan and Sephardic Jews have the highest values, 16% and 19%, respectively57  and is also present in other non-Ashkenazi Jewish populations, but at a much lower frequency (4.4%)70. In Chuetas, R1a1a-M17 was found in 4% of the individuals analysed, but not in the Majorcan samples. It could be interesting to assess whether the Chueta R1a1a chromosomes belong to the subclade shared by Ashkenazim and other Middle Eastern populations (M582), but absent in Eastern Europeans70.Other Y-haplogroups with differential frequency between Chuetas and their host population were E1b-M78, Q1-P36.2, G-M201, and R1a1a-M17. E1b-M78 seems to have originated in north-eastern Africa and several lines of evidence suggest that some E1b-M78 derived lines have been involved in trans-Mediterranean migrations directly from Africa to Europe71.All in all, the Y-chromosome haplogroup profile in Chuetas is clearly dissimilar from their neighbouring population, Majorca, and quite similar to the haplogroup frequencies found in Sephardic and other Jewish populations, evidencing a considerable frequency of J2 + J1 haplogroups and low values of R1b Fig. . TherefoA comparison with available populations in the literature, based on both STRs and SNPs, Fig.  position74. As shown in Fig. 75. The highest frequencies of haplogroup R0a are found in the Arabian Peninsula and the Horn of Africa, reaching values as high as ~ 25% in Soqotra Island in Yemen73. Frequencies in Jewish groups76 on the whole show similar frequencies to their host populations  and R0a2′3 (~ 21 Kya). R0a2′3 is defined by the insertion 60.1 T and most of the Jewish populations where information is available show this branch. Only Yemenite Jews show both22.The origin in terms of location and timescale of the Chuetas' modal haplogroup R0a has been under debate in recent years due to the geographic distribution of its frequencies74 to ~ 1.4 Kya, is found in just three samples in the literature, two of them from Jewish origin and the other with unknown ethnicity. In Fig. Taking into account the high prevalence of this haplogroup in the Chueta population, it was considered important to delve into the phylogeny of their possible main maternal founder. Thus, the complete mtDNA genome was obtained for 6 out of the 21 R0a + 60.1 T samples, which classified the Chueta samples as R0a2m. The R0a2m branch, dated by Gandini et al.77. Haplogroup J has higher frequencies in the Middle East and Arabia than in Europe (13–20% vs. 9%), while haplogroup T shows the opposite behaviour (10% in Europe and 8% in the Middle East)78. No significant differences were found between modern and ancient Majorcan populations regarding haplogroup frequencies since at least the Bronze Age79. T1a (5.8%) is considered one of the founders of the Chueta population, and originated in the Near East ~ 17 Kya, although most of its sub-branches seem to be European77.The time of the appearance of haplogroup JT can be estimated at ~ 58 Kya, before the settlement of the Fertile Crescent. It has been suggested that haplogroups J and T diverged during the settlement ~ 40 Kya and ~ 30 Kya, respectively80. The U1a sub-haplogroup is dated at ~ 13–15 Kya and is present in Southwest and South Asia, the Caucasus, and Europe. Five U1a1a1 samples were found in the Chueta population, whereas no U1 sub-haplogroup was observed in the host population. Haplogroup K origin has been dated to ~ 36 Kya and, although the place of origin is still under discussion82, a Levantine origin seems the most likely. Sub-haplogroup K1a1b1a (dated to ~ 4.4 Kya82), found in four Chueta samples, is a founder lineage in Ashkenazim81 and also present in Sephardic communities22, but not in non-European Jews, which can be seen as evidence of its European origin82.Regarding the other mtDNA haplogroups with differential frequencies between Chuetas and their host population, Haplogroup U is the second most frequent in modern Europeans and was predominant in pre-agricultural Europe83 and one of its most frequent lineages in West-Central Africa is L3e2b (7%)84. Five Chueta samples showed haplogroup L3e2b, while in the host population just one sample presented one African haplogroup L3, but belonging to another subclade, L3d.Haplogroup L3e is widespread in Africa but practically absent in Eurasia (except in neighbouring areas due to genetic exchange). Its origin is situated in Central or Eastern Africa about 46 Kya24, this haplogroup is absent or very uncommon in Chuetas and other Sephardic groups; additionally, haplogroup K1a1b1a, which is a founder (8.5%) in the Iberian Exile Jewish community from Bulgaria, has a lower frequency in other populations with Sephardic origin (4% in Chuetas and 0.8% in Turkish Jews)22.Founding lineages in Chuetas, R0a + 60.1 T (~ 20%) and T1a (~ 6%), are different from those of other Sephardic populations, which also show important dissimilarity between each other. For instance, while HV0b is found as a founder in the Portuguese Crypto-Jewish communities from Belmonte and Bragança24. These results reveal that demographic processes more complex than the loss of genetic diversity expected under conditions of extreme inbreeding and drift, have shaped the gene pool of both isolated populations. Ongoing data from recombinant markers, together with classical genealogical studies, will help to explain what mating strategies were undertaken by these communities to avoid the expected reduction of diversity, and also whether other factors, such as high heterogeneity in founder populations, could have contributed to the diversity observed.Genetic diversity in both paternal and maternal lineages in the Chueta population was higher than expected for a small, endogamous population. Comparable high diversity values were observed in the Portuguese Crypto-Jewish communities of BragançaThe composition of the Chuetas’ uniparentally transmitted lineages indicates a remarkable signature of Middle Eastern ancestry. In recent years, archaeogenetic research has shed light on the history of European and Middle Eastern populations, revealing a greater degree of population movements and interactions in the past than previously considered. In this context, from the analysis of current populations, it is difficult to infer what the genetic profile of the parental populations that gave rise to the Chuetas was exactly, and therefore precise inferences of the original source of the haplogroups found in this population, since there are no reasons to assume or assess the degree of genetic continuity. However, the most reasonable explanation for the differences found between Chuetas and their host population seems to be the Jewish origin of the Chuetas, considering the unquestionable historical evidence relating them with the Jewish populations who settled in Majorca long ago in the past. Therefore, our results would confirm that Chuetas have kept not only the cultural memory of their Jewish origin over centuries, but also a substantial degree of ancestral genetic signature.In terms of paternal lineages, the results show that most Jewish communities are more similar to each other and to Middle Eastern populations than to their host populations. The Chueta population has the same behaviour, which can be observed by the high prevalence of haplogroups J2-M172 and J1-M267, and the lack of R1b-M269. Haplogroup distribution in Chuetas is very similar to other Sephardic communities, although in their gene pool there might be signatures of other Jewish communities’ contribution, such as North African and Ashkenazim, which can be inferred from the presence of haplogroups such as E1b1b1a1-M78, Q1-P36.2, and R1a1a-M17.22. Various scenarios could explain this dissimilarity found in maternal founder lineages in distinct current populations with Sephardic origin: differences in gene flow from and admixture with other populations resulting from the contrasting history of each community; a lack of homogeneity in maternal lineages of the original Sephardic groups that settled in different areas of the Iberian Peninsula ; or genetic drift in the current populations, resulting in a lack of lineages that do not allow us to infer the original mtDNA composition of the Sephardic Jews that lived in Spain and Portugal in the middle ages from the surviving lines. Tests based on genome-wide data that enable a greater genetic resolution, such as analysis of Runs of Homozygosity (ROH), together with ancient DNA analysis, could help to solve this question.The hallmark in the maternal gene pool in Chuetas is the presence of a new sub-branching of the rare Middle Eastern haplogroup R0a + 60.1 T as their modal haplogroup, and the low frequency of H. The presence of other haplogroups found in Jewish/Middle Eastern populations (K1a1b1a and U1a1a1) is also noteworthy. Current Jewish populations do not usually share modal maternal lineages, unlike the situation on the paternal side; not even populations with a supposed common ancestry, such as the Sephardic branchSupplementary Information.


Dipteronia sinensis in the Qinling Mountains (QM) and adjacent areas in East Asia based on variations in the complete chloroplast genomes (cpDNA) and reduced‐genomic scale single nucleotide polymorphisms (SNPs). Population structure and phylogenetic analysis based on the cpDNA variations suggested that D. sinensis could be divided into two intraspecific genetic lineages in the eastern and western sides of the QM  in East Asia. Molecular dating suggested that the intraspecific divergence of D. sinensis occurred approximately 39.2 million years ago during the later Paleogene. It was significantly correlated with the orogeny of the QM, where the formation of this significant geographic barrier in the region may have led to the divergence of independent lineages. Bayesian clustering and demographic analysis showed that intraspecific gene flow was restricted between the EQM and WQM lineages. Isolation‐with‐migration analysis indicated that the two genetic lineages experienced significant demographic expansions after the Pleistocene ice ages. However, the genetic admixture was determined in some populations between the two lineages by the large scale of SNP variations due to DNA incompatibility, the large significant population size, and rapid gene flow of nuclear DNA markers. Our results suggest that two different conservation and management units should be constructed for D. sinensis in the EQM and WQM areas. These findings provide novel insights into the unprecedented effects of tectonic changes and climatic oscillations on lineage divergence and plant population evolution in the QM and adjacent areas in East Asia.Geological and climatic events are considered to profoundly affect the evolution and lineage divergence of plant species. However, the evolutionary histories of tree species that have responded to past geological and climate oscillations in central China's mountainous areas remain mostly unknown. In this study, we assessed the evolutionary history of the endangered and relict tree species  Dipteronia sinensis plant species in the Qinling Mountains in central China. The readers will take help to understand the mechanism of lineage divergence due to the mountain's orogenesis and repeated climatic changes.This novel study describes the unprecedented details of geological and climatic changes profoundly shaped the lineage divergence and population evolution of  Detailed information for all of the sampled populations is provided in Table D. sinensis population (preserved in silica gel) before DNA extraction with the CTAB method  . We then used the default parameters in NGSQC Toolkit v 2.3.3 to extract the clean data . We used quantitative real time‐PCR to assess the quality of the DNA fragments before nuclear DNA library construction. Restriction endonuclease enzymes were applied to amplify each genome sample. All of the samples were mixed after PCR before selecting good quality fragments with the desired length based on the results of capture arrays, which were then used to construct the DNA library. The genotyping‐by‐sequencing technique was employed to sequence the selected nDNA library fragments. After sequencing, the raw reads were filtered based on the paired‐end reads.Dipteronia sinensis samples. Burrows–Wheeler aligner software was used to align the clean data , we also obtained the GC contents of the 23 2.4Dipteronia sinensis, we used the complete chloroplast genomes for Acer buergerianum (NC_034744.1), A. morrisonense (NC_029371.1), A. miaotaiense (NC_030343.1), A. griseum (NC_034346.1), A. palmatum (NC_034932.1), A. truncatum (NC_037211.1), Sapindus mukorossi (NC_025554.1), D. dyeriana (NC_031899.1), Litchi chinensis (NC_035238.1), Dodonaea viscosa (NC_036099.1), Koelreuteria paniculata (NC_037176.1), and Dimocarpus longan (NC_037447.1) as outgroup taxa. jModelTest and PAUP* v 4.0 software were used to obtain the general time‐reversible model and gamma (G) distribution for the rate variation among sites based on Akaike's information criterion  were reconstructed by using MEGA v 7.0 and MrBayes v 3.2.3  was used to construct a neighbor‐joining phylogenetic tree based on 1,000 bootstrap values.We used 23 2.5Dipteronia sinensis lineages  algorithm steps, with 5,000,000 steps as the burn‐in, and collected the parameters after every 5,000 steps. We used Tracer v 1.5 software  was used for STRUCTURE analysis based on 23 Dipteronia sinensis populations. A PLINK input file (‐Ped file) was prepared to construct the population genetic structure and lineage information. The DJ population was used as the reference genome.STRUCTURE analysis based on nDNA SNP data was conducted to determine the genetic variation in a non‐random distribution of a species or population. According to the geographical distribution, a population or group of populations can be divided into several sub‐groups. Different individuals with strong genetic relationships lie closer and within the same group. By contrast, the populations with weak genetic relationships are located at greater distances. Population structure analysis is useful for understanding the evolutionary process and for associated studies of genotypes and phenotypes. The online program PLINK  to analyze the demographic history and divergence time (generations). The genetic diversity parameters were also investigated for each lineage by using DnaSP v 5.0, including the nucleotide diversity Pi (π) and theta (θ)  model for are Hey, . All of Principal component analysis (PCA) was conducted based on nDNA SNP data to visualize the significant axes of variation in the population genetics using the adegenet package Jombart,  in R sofn” = XX autosomal data, while ignoring more than two alleles and inconsistent data. In the equation above, “i” is the number of individuals, “k” is the position of an SNP, ikd indicates that if an individual “i” and the reference allele are homozygous, then ikd = 0, whereas if they are heterozygous, then ikd = 1, and if the “i” individual is homozygous for the non‐reference allele, then ikd = 2. In this study, the eigenvectors and eigenvalues were calculated using GCTA (http://cnsgenomics.com/software/gcta/pca.html) software and the PCA plot was drawn using R software.PCA calculates the number of individuals “33.1Dipteronia sinensis in terms of their structure, tRNAs, mRNAs, and gene contents ) and from its sister species D. dyeriana about 51.3 Mya . The first divergence time for D. sinensis into the EQM and WQM was estimated at 39.21 Mya  in the late Eocene era. The EQM lineage populations diverged about 38.12 Mya , and the estimated divergence time for the WQM lineage populations was 30.2 Mya   Figure . The phyDipteronia sinensis populations by genotyping to produce a maximum of >744 million reads for each population. The estimated percentage of GC contents ranged from 41.47% to 39.05%. In total, 28,246 SNP loci were generated for further population genetics analyses. Phylogenetic analysis based on the nDNA SNPs identified three D. sinensis clades, where two clades contained most of the EQM populations and WQM populations. However, one clade contained a mixture of the EQM and WQM populations  based on nDNA SNP data obtained a different genetic structure than that with the cpDNA results  and Theta (θ) by using DnaSP software based on the SNP loci (cpDNA). We estimated that the nucleotide diversity was much higher for the EQM lineage (π = 0.00026 and θ = 0.00031) than the WQM lineage (π = 0.00009 and θ = 0.00011).We calculated the nucleotide diversity Pi (p < .001).IMa software was used to analyze the gene flow migration between the EQM and WQM lineages. The plot obtained contained a sharp peak for the posterior density (results not shown). The mean effective population size estimated for the EQM lineage  was much higher than that for the WQM lineage (q2 = 81.25 95% HPD 286.81–39.88)  identified the same genetic pattern as STRUCTURE analysis based on the nDNA data. Three different genetic groups were determined by PCA, which contained the EQM and WQM populations, and a mixture of the populations from both sides, as shown in Figure 44.1Dipteronia sinensis lineages based on the complete chloroplast genomes (cpDNA) and large‐scale SNP variations in the QM and adjacent areas. Phylogenetic analysis based on cpDNA clearly determined the bi‐phyletic clades within the species (EQM versus. WQM)  , which is consistent with a previous phylogenetic analysis based on transcriptome data sets  of the QM might have been an important factor responsible for the genetic alteration of D. sinensis. Recently, the divergence time of the genus Sinothela was estimated at between 23–2.6 Mya or 10–2.6 Mya in the QM   based on nDNA SNP data comprised two large admixed population groups  also suggested little gene flow between the two lineages. Previously, it was reported that divergence due to geographical tectonics or climatic isolation could produce a strong signature because of highly restricted gene flow    of A) Table , which sas Table . Previouas Table  to promoas Table . Phylogeas Table . Based o4.3Dipteronia sinensis is a naturally endangered and relict tree species included in the Red List, and thus, there is a critical need to design effective conservation strategies for this plant ; Methodology (lead); Project administration ; Software (lead); Writing‐original draft (lead); Writing‐review & editing . Mi‐Li Liu: Methodology ; Writing‐review & editing . Yu‐He Zhao: Methodology ; Software ; Writing‐review & editing . Ting‐Ting Zhang: Methodology ; Writing‐review & editing . Jian‐Ni Liu: Data curation ; Supervision ; Writing‐review & editing . Zhonghu Li: Project administration ; Software ; Supervision ; Writing‐original draft ; Writing‐review & editing .


Pseudocercospora musae, causal agent of Sigatoka leaf spot, or yellow Sigatoka disease, is considered a major pathogen of banana (Musa spp.). Widely disseminated in Brazil, this study explored the genetic diversity in field populations of the pathogen from production areas in the Distrito Federal and the States of Bahia, Minas Gerais, and Rio Grande do Norte. Resistance to demethylation inhibitor (DMI) fungicides was also examined. For 162 isolates from 10 banana growing regions, analysis of mating type idiomorph frequency was conducted, together with estimation of genetic diversity at 15 microsatellite loci. A total of 149 haplotypes were identified across the examined populations, with an average genetic diversity of 4.06. In general, populations displayed 1:1 proportions of idiomorphs MAT1-1 and MAT1-2, providing evidence for sexual recombination. Multilocus linkage disequilibrium also indicated asexual reproduction contributing to the genetic structure of certain populations. AMOVA revealed that 86.3% of the genetic differentiation of the pathogen occurred among isolates within populations. Discriminant Analysis of Principal Components (DAPC) identified six most probable genetic groups, with no population structure associated with geographic origin or collection site. Although genetic similarity was observed among certain populations from different states, data revealed increasing genetic differentiation with increasing geographic distance, as validated by Mantel’s test . On the basis of DMI fungicide sensitivity testing and CYP51 gene sequence polymorphism, isolates from the Distrito Federal separated into two main groups, one with generally higher EC50 values against eight DMI fungicides. A clear phenotype-to-genotype relationship was observed for isolates carrying the CYP51 alteration Y461N. Conventionally adopted fungicides for control of Sigatoka leaf spot are likely to be overcome by combined sexual and asexual reproduction mechanisms in P. musae driving genetic variability. Continued analysis of pathogen genetic diversity and monitoring of DMI sensitivity profiles of Brazilian field populations is essential for the development of integrated control strategies based on host resistance breeding and rational design of fungicide regimes. Pseudocercospora musae (Zimm.) Deighton [sexual morph: Mycosphaerella musicola R. Leach ex J. L. Mulder], causal agent of Sigatoka leaf spot, or yellow Sigatoka disease, is one of the main pathogens affecting banana fruit production worldwide  which likely affect azole binding. Resistance in this species is also associated with a 19 bp repeat elements in the CYP51 promoter region linked to gene over-expression , which encodes a protein containing an alpha domain. The latter isolates possess a MAT1-2-1 gene, encoding a protein with a high-mobility group domain (HMG). Both genes encode transcription factors which are known to regulate the sexual cycle through control of signal transduction involved in mating identity   , amplifiA (RAPD) , ERIC-PCA (RAPD) , inter-sA (RAPD)  and micrA (RAPD) . Whilst A (RAPD) , applica limited .P. musae from important banana production areas in Brazil across the Distrito Federal and the States of Bahia, Minas Gerais, and Rio Grande do Norte, ranging in distance from 22 to 1900 km. Analyses of dispersal and genetic recombination processes were based on mating type idiomorph frequencies and SSR-based genetic diversity within and among populations. Additional investigation of sensitivity to DMI fungicides and analysis of potential mutations in the CYP51 gene was conducted on isolates from sampled populations.This study was conducted to determine the genetic diversity and structure in populations of Pseudocercospora musae isolates were collected from infected banana leaf material with typical Sigatoka leaf spot symptoms . Material originated from 10 different zones across Brazil, distributed in the Distrito Federal and surroundings (DF), as well as the states of Bahia (BA), Minas Gerais (MG) and Rio Grande do Norte (RN)  and distilled water. Leaf lesions were incubated in Petri dish moist chambers for 48 h then observed under stereoscope microscopy to check for sporodochia. In order to obtain monosporic isolates, conidia were transferred aseptically from sporodochia to slides containing water-agar blocks (1 cm2) and subsequently to solid V8 agar medium . All pure cultures were preserved in sterile distilled water (rte (RN) . Isolateon areas . At eached water  and on sP. musae, P. fijiensis, and Pseudocercospora eumusae) using the “Muscle” algorithm in the software MEGA, v.7. Phylogenetic analyses were conducted based on Bayesian inference, with the evolution model for each single region chosen using hierarchical likelihood tests considering the Akaike Information Criterion (AIC) within the program MrModeltest, v.2.3. For each target region, consensus trees were generated using the program MrBayes, v.3.2, with 5,000,000 generations (convergence 0.001) through three independent runs; each using four Markov Monte Carlo Chains (MCMC), two hot and two cold chains, a sampling of a tree every 1,000 generations, and burn-in of the initial 1,250,000 generations. Concatenated data was used to conduct a multilocus phylogenetic analysis, based on the same criterion for the Bayesian inference described above. Trees were visualized and edited using the program FigTree, v.1.3.1. . All thermocycling reactions were carried out using the amplification program: 94°C for 5 min, 39 cycles of 95°C for 1 min, primer annealing at 63°C for 40 s and 72°C for 40 s, followed by a final extension of 7 min at 72°C. To confirm specific amplification of mating type idiomorphs, representative amplicons were purified with ExoSAP , sequenced on an ABI 3730 sequencer , and analyzed via BLASTn for comparison with the sequence of alpha and HMG conserved domains of P. musae available at NCBI1.In order to determine the distribution of mating type idiomorphs in each population, the P. musae populations (POPs) was analyzed using 15 SSR loci . Following 20 days growth on V8 medium, mycelial fragment suspensions were prepared by macerating two mycelial areas, of approximately 1 square cm diameter per isolate, in 3 mL of sterile distilled water using a mortar and pestle. As inoculum, 100 μL aliquots of mycelial suspensions (20-fold dilutions in water) were added to each well containing liquid medium. Plates were incubated for 7 days at 25°C and growth measured by absorbance readings at 630 nm using a FLUOstar OPTIMA microplate reader  in well-scanning mode with a 2 × 2 matrix of scanning points of 3-mm diameter. Fungicide sensitivities were determined as 50% effective concentration (EC50 in μg ml–1) using a dose-response relationship (4-parameter fit) determined with the OPTIMA Software.P. musae CYP51 gene target of the DMI fungicides were verified in complete and partial sequences of the gene, amplified using the primer pair combinations: MMFOR2 (CAA TGG GA/TC TCC TCC AGG A)/MMREV1 (TTG ACG CTG CCA/G TCC/G ACA TTG TA), MMFOR1 (GGA GGA GCG CTT TTG CTT CGG CTT)/MMREV5 (TAT GGA CAC GCC GAT AAA GAT GCT) and MMFOR8 (CGA CAC AGG TGC ATC GGT GAG CA)/MMREV8 (ATT TCT TTT CTC TCC TCT CCC ATC). Primers were designed based on CYP51 gene sequences available from Z. tritici and P. fijiensis reference genomes2. PCR reactions were carried out on a Biometra T3000 thermocycler  in a final volume of 50 μL containing 50 ng of fungal template DNA. For primer pair MMFOR2/MMREV1, PCRs contained 0.5 μM for each primer and 200 μM dNTP, 1× Phusion HF buffer, and 1.0 unit of Phusion High Fidelity DNA polymerase . Amplification conditions comprised 98°C for 30 s, followed by 40 cycles at 98°C for 10 s, 50°C for 20 s, and 72°C for 1 min with a final DNA extension at 72°C for 5 min. For primer pairs MMFOR1/MMREV5 and MMFOR8/MMREV8, PCR reactions contained 1.0 μM for each primer and 200 μM dNTP, 1× of Easy-A reaction buffer and 2.5 units of Easy-A High Fidelity PCR cloning enzyme . Amplifications were conducted at 95°C for 2 min, followed by 40 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 2 min, with a final DNA extension at 72°C for 10 min. PCR products were sequenced by MWG Eurofins Genomics GmbH . Sequences were assembled and aligned with Geneious v.6.1.4 software , and amino acid substitutions determined after sequence analysis.Potential genetic alterations in the DEST) as an appropriate estimator for Wright’s FST or related GST. Variance components and genetic differentiation values were tested by 1000 permutations for the haplotypes among the 10 populations, to estimate significance levels (P ≤ 0.05). The number of migrants per generation (Nm) were calculated using the formula Nm = 0.5 (1 − DEST)/DEST. Genotypic diversity, richness and evenness were measured using the Shannon Wiener (H’) and Hill’s (N1) indexes. In order to determine the contribution of sexual reproduction or clonal spread in the populations, multilocus linkage-disequilibrium analyses were conducted across microsatellite loci using the index of association (IA) and a standardized version (r–d) that accounts for number of loci   test to determine significant deviation from the expected 1:1 ratio   were per:1 ratio  using th:1 ratio .P. musae isolates were obtained from different geographic regions in Brazil, from the Distrito Federal and the states of Bahia, Minas Gerais, and Rio Grande do Norte. Sample locations were separated from each other by distances ranging from 22 to 1900 km. Across the locations, P. musae isolates were obtained from Musa cultivars Maravilha, Fhia 17, Fhia 02, Cavendish Grande Naine, Thap Maeu, Prata, Prata comum, Pavocan, Maça, Prata anã, Prata comum, and Terra , the lowest allelic richness was found in Pop 7, with only 5.87 expected haplotypes. Lowest genotypic diversity was also observed in this population, with a Shannon Wiener index value of 1.95. Pops 4, 6, 8, 9, and 10 presented intermediate diversity values, with Pops 1, 2, 3, and 5 showing the highest diversity for the Shannon–Weiner and Hill indexes, with evenness also closest to 1. According to AMOVA-based analysis, greatest genetic differentiation occurred among individuals within populations, accounting for 86% of the total variation , and high genetic differentiation between geographically distant populations . A lack of differentiation, however, was also apparent among certain populations, despite their considerable geographical distance. For example, for the two most geographically separate populations , which are approximately 1900 km apart, a value of zero was observed for the D statistic.Analysis of the on pairs  generallk = 6)  . Clusterk = 6) .P. musae, with a single amplicon produced for each isolate (400 bp for MAT1-1-1 and 700 bp for MAT1-2-1). Sequences for 155 of the isolates  test revealed that, in general, the studied populations did not show significant deviation from the expected 1:1 frequency of MAT1-1-1 and MAT1-2-1 idiomorphs, consistent with regular cycles of sexual reproduction in these populations  and 5 (STO) (Sensitivity of  5 (STO) .P. fijiensis CYP51B gene coding sequence (GenBank XM_007930561), it was evident that this fragment covered CYP51 codon positions 7 to 507, after removal of primer sequences. This region of the protein shows a high level of homology with P. fijiensis CYP51B (96.2%) and spans all positions that have been reported to affect azole binding  associated with CYP51 Y461N were 2.2, 3.0, 3.3, 3.4, 4.1, 4.2, 7.6, and 9.1 for epoxiconazole, fluquinconazole, cyproconazole, prochloraz, triadimenol, propiconazole, pyrifenox and tebuconazole, respectively. Using primer pairs MMFOR1/REV5 and MMFOR8/REV8, which amplified fragments of 671 and 187 bp, respectively, the CYP51 gene was further sequenced in two DMI fungicide-sensitive isolates (8CPAC and 9SJA) and two insensitive isolates (2SJA and 18STO). Assembly and alignment of sequences from 539 bp upstream of the start codon to the last codon revealed no further nucleotide alterations in either the CYP51 promoter region or the coding sequence among the comparative isolates.Using primer pair MMFOR2/REV1, a fragment of 1702 bp was amplified and sequenced in all isolates (see nucleotide positions 550-2251 in GenBank accessions MF521833 and MF521834). Given the high nucleotide sequence similarity (89%) to the  binding . When se binding . Based oSigatoka leaf spot has been present in Brazil for almost 70 years, causing significant impact on banana production across the country . An incrP. musae populations  also ranged from 0.142 to 0.360, from smallest to largest populations, respectively (P. fijiensis from Columbia (0.46) (Z. tritici populations from Saskatchewan (0.44)   , as welln (0.44)  and the n (0.44) . Recent a (0.71) .DEST values and high values of migrants per generation, distributing new alleles and contributing to the homogenization of certain populations. This is in agreement with what is expected for the disease, with dissemination able to occur locally (by rain splash) and over long distances (by air) , rather than among populations (5.8%). The lack of population structure and low differentiation of populations, as also observed by (by air) . In prevustralia . Similar to 0.26 . FST valountries , with thP. musae from Minas Gerais in Brazil  fungicides  has been linked with high levels of DMI insensitivity  and or Y137 (Y136 in P. musae), which are key residues for azole binding, further from the docked azoles (Z. tritici and P. fijiensis, it is likely that multiple mutations will evolve which, in combination with each other and with other resistance mechanisms, such as overexpression of CYP51 and efflux pumps, will result in higher levels of resistance to fungicides (P. musae in different geographic regions in Australia (P. musae field populations, to enable baseline information on resistance emergence and distribution across Musa growing regions.Selected ngicides . Data ree clonal . The presitivity . An idenutations . The codtructure . Moleculd azoles . Single ngicides . With Qoustralia , it is tAn increased understanding of the genetic diversity of pathogen populations, together with monitoring of the evolution and spread of DMI and QoI resistance, is fundamental for the development of optimal integrated management strategies for Sigatoka leaf spot, based on host resistance breeding and rational design of fungicide resistance management strategies involving agrochemical mixtures or alternation regimes.P. musae is available in GenBank under accession numbers: KP996493, KP996494, and KP996495.Representative sequence data employed in molecular identification of RM, FB, and JS planned the experiments. FB and JS conducted the isolate sampling, molecular identification, and characterization of mating types and haplotypes. FB, VA, YP, SO, CF, FH, and EA participated in data analysis. FB and BF performed fungicide sensitivity bioassays. RM conceived the study, participated in characterization of mating types and haplotypes, and drafted the manuscript. All authors have contributed to, read, and approved the final manuscript.The 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.


Adoption of diets based on some cereals, especially on rice, signified an iconic change in nutritional habits for many Asian populations and a relevant challenge for their capability to maintain glucose homeostasis. Indeed, rice shows the highest carbohydrates content and glycemic index among the domesticated cereals and its usual ingestion represents a potential risk factor for developing insulin resistance and related metabolic diseases. Nevertheless, type 2 diabetes and obesity epidemiological patterns differ among Asian populations that rely on rice as a staple food, with higher diabetes prevalence and increased levels of central adiposity observed in people of South Asian ancestry rather than in East Asians. This may be at least partly due to the fact that populations from East Asian regions where wild rice or other cereals such as millet have been already consumed before their cultivation and/or were early domesticated have relied on these nutritional resources for a period long enough to have possibly evolved biological adaptations that counteract their detrimental side effects. To test such a hypothesis, we compared adaptive evolution of these populations with that of control groups from regions where the adoption of cereal‐based diets occurred many thousand years later and which were identified from a genome‐wide dataset including 2,379 individuals from 124 East Asian and South Asian populations. This revealed selective sweeps and polygenic adaptive mechanisms affecting functional pathways involved in fatty acids metabolism, cholesterol/triglycerides biosynthesis from carbohydrates, regulation of glucose homeostasis, and production of retinoic acid in Chinese Han and Tujia ethnic groups, as well as in people of Korean and Japanese ancestry. Accordingly, long‐standing rice‐ and/or millet‐based diets have possibly contributed to trigger the evolution of such biological adaptations, which might represent one of the factors that play a role in mitigating the metabolic risk of these East Asian populations. In this regard, the Yangtze River valley in Eastern China is the geographical area where the oldest archaeological evidence of habitual wild rice consumption  was found  and foxtail millet , whose domestication was completed 8,000–6,000 years ago in the Hebei and Manchuria provinces of Northern China  has been identified also in the Indo‐Gangetic Plain crossing northern regions of the Indian subcontinent   or where the adoption of cereal‐based diets occurred only several thousand years later  Table . We then22.1N = 36), Bangladeshi (N = 29), and Vietnamese (N = 20) populations was sampled using the Oragene DNA OG‐500 kit  and according to ethnographic information. Informed consent was also obtained from all individuals, and on 04/08/2013 the University of Bologna ethics committee released approval for the present study (within the framework of the ERC‐2011‐AdG 295733 project), which was designed and conducted in accordance with relevant guidelines and regulations and according to ethical principles for medical research involving human subjects stated by the WMA Declaration of Helsinki. DNA was extracted from saliva samples using the prepit‐L2P protocol  and quantified with the Quant‐iT dsDNA Broad‐Range Assay Kit . DNA samples were genotyped for ~720,000 genome‐wide SNPs by means of the HumanOmniExpress v 1.1 chip  at the Center for Biomedical Research & Technologies of the Italian Auxologic Institute .Saliva from 85 individuals representative of Korean (2.2p > 1.41 × 10−8) were retained. Individuals showing more than 5% of missing data were excluded, together with ambiguous strand single nucleotide polymorphisms (SNPs) with A/T or G/C alleles. The obtained dataset was then submitted to linkage‐disequilibrium (LD) pruning, and the identified SNPs in approximate LD with each other (r2 < .1) were used to estimate the degree of recent shared ancestry (IBD) for each pair of subjects by calculating the genome‐wide proportion of shared alleles . Individuals showing IBD kinship coefficient higher than 0.1 were removed. A “high‐quality” dataset of 82 samples typed for 688,987 SNPs was thus generated. An “extended” dataset of 4,356 samples belonging to 162 worldwide populations typed for 231,947 SNPs was also obtained by merging the “high‐quality” dataset with publicly available data retrieved from the 1,000 Genomes Project phase 3 (r2 < .2) and by removing sites with a minor allele frequency (MAF) below 0.01. Haplotypes phasing was performed with SHAPEIT2 v2.r790  procedures were performed on the generated genome‐wide data using PLINK v.1.9  errors for each K to identify the most plausible number of ancestry components.Principal component analysis (PCA) was applied first to the “extended” dataset to check for the presence of potential genotypes inconsistency due to errors occurred in the merging procedure and then to a “Pan‐Asian” subset of 2,379 individuals belonging to 124 Asian populations. To compute PCA, the 2.4We computed the number of segregating sites by length (nSL) statistics  that might be ascribable to stochastic variation in allele frequency due to genetic drift, we applied a 200,000 bp sliding windows procedure on the obtained genome‐wide nSL distribution. Derived allele frequency (DAF) of each SNP was computed to enable their classification into bins based on DAF  toward extreme signet values were searched for in the distribution of scores observed within annotated pathways to identified gene networks pervasively subjected to positive selection. Networks of candidate adaptive genes were finally plotted using Cytoscape v3.6.0 , we obtained an “extended” dataset of 4,356 unrelated samples belonging to 162 worldwide populations characterized for 231,947 SNPs. When we submitted it to PCA, we observed the expected cline “V‐shaped” distribution of samples that reflects the main differentiation between populations of sub‐Saharan African ancestry  and non‐African groups Figure , as poinTo focus on the overall genomic landscape of long‐term cereal‐eating populations, we repeated PCA by retaining a “Pan‐Asian” subset of 2,379 individuals belonging to 124 populations from South and East Asia. Accordingly, PC1 was found to explain 4.3% of the investigated genomic variation, enabling to distinguish between populations from the Indian subcontinent and groups from East Asian/South East Asian regions Figure . PC2 ins3.2K = 2 to K = 10 potential ancestral groups  were assumed  and that grouped together populations showing also close geographical proximity, similar cultural patterns, and largely shared demographic histories ; the North West Indian cluster ; the Central South Indian cluster ; the Bangladeshi cluster (N = 114); the Tibeto‐Buman cluster ; the Dai‐Vietnamese cluster (N = 239); the Japanese cluster (N = 132); the Han‐Tujia cluster (N = 262); the Korean cluster (N = 36); and the South East Asian cluster .Distribution of ancestry components depicted by ADMITXUTRE analysis at s Figure . In part3.3By assuming that the ancestors of populations belonging to the same cluster have been plausibly subjected to similar selective pressures due to their largely shared environmental and cultural settings, we calculated genome‐wide distributions of the nSL statistics for each group of populations to detect the main selective sweeps having mediated their adaptive histories. To minimize false‐positive results, we searched for the most plausible candidate chromosomal intervals to have undergone positive selection by retaining top 1% genomic regions in the distribution of 200 kb windows ranked according to their proportion of outlier SNPs showing unusual nSL scores. We finally investigated functional relationships among genes laying in these top 1% windows according to information retrieved from the STRING protein–protein interactions database (see Methods).Large genomic intervals enriched for signatures of positive selection thus turned out to be differentiated especially between clusters from the Indian subcontinent or South East Asia and groups from the northernmost East Asian regions covered by the dataset.FBXW2 ring finger proteins class (RNF), LTN1, SPSB4, ZBTB16, and ubiquitin‐conjugating enzyme class (UBE) genes were some of the several targets of selection shared among the majority of South Asian and South East Asian clusters  was observed in people of Han or Tujia ancestry Figure . For insALDH9A1, GPX3, and MGST3 , a TNFAIP3‐interacting protein 3 (TNIP3), whose overexpression inhibits NF‐kappa B‐dependent gene expression in response to lipopolysaccharide , a glyoxylate reductase 1 homolog (GLYR1) and ADH7  were found to participate to the same biological processes. Even if it is a member of the alcohol dehydrogenase family, the enzyme encoded by ADH7 is most active as a retinol dehydrogenase. Its expression is greatly concentrated in the esophagus . The ALDH1A2 locus encodes for an enzyme that catalyzes the synthesis of retinoic acid from retinaldehyde. Similarly to ADH7, it has been associated to Barrett's esophagus, a premalignant precursor of esophageal adenocarcinoma  and LPIN1, which play important roles in controlling fatty acids metabolism at different levels.Some of these genes were pointed out as candidate targets of positive selection also in the Korean cluster Figure , as is t3.4After having identified large genomic regions plausibly subjected to positive selection, along with the associated biological functions, we prioritized the genes contained in them (see Methods) and we focused on the loci showing the strongest selective signatures in each cluster.DENND1A, MCC, SCRN1, and WIPF3) were found to be included in the top 1% windows of all clusters , with a limited overlap also with signatures observed in the Bangladeshi group .Again, we observed appreciably different adaptive profiles among the considered clusters, especially between South Asian/South East Asian groups and East Asian people from more northerly latitudes. In fact, only four genes  and phospholipases (PLCB), or in the response to dietary stimuli, such as the insulin‐like growth factor 1 receptor (IGF1R) and FOXO genes , PPARGC1A, and PRKACG, were found to have played a putative adaptive role only in JPT  and compared it with that of control groups from South East Asian and South Asian regions where cereals agriculture spread only more recently. For this purpose, we took advantage from newly generated and literature genomic data for 2,379 individuals belonging to 124 Asian populations.4.1To frame patterns of genetic diversity observable at both candidate and control populations within the overall South Asian and East Asian genomic landscape and to test for representativeness of the selected samples with regard to their ethnic group, we applied PCA and ADMIXTURE algorithms to the assembled “Pan‐Asian” dataset. These population structure analyses confirmed the well‐known genetic differentiation between groups from the Indian subcontinent and from East Asian or South East Asian regions, along with gradients of variation observable within these macro‐geographical areas Figure .In detail, genetic affinity among populations of South Asian ancestry was found to follow a cline that roughly reflects both their latitudinal dislocation across the Indian subcontinent and the distribution of different language groups, as previously attested by several studies  plays a role in the development of polycystic ovaries, anovulation, and hyperandrogenism that mainly characterize the polycystic ovary endocrine syndrome  as pointed out by gene‐network analyses performed on CHB and JPT genome sequences  located in their promoter regions, while FOXP1 acts as a transcriptional repressor that inhibits them by directly interacting with the FOXO1 gene or by competing with it to bind IREs of target genes  implicated in the oxidation of retinol to retinaldehyde and in the subsequent synthesis of retinoic acid from it (Figure Finally, putative adaptive events shared by all candidate clusters were observed at loci encoding for alcohol and aldehyde dehydrogenases, particularly at genes (e.g., t Figure . This pat Figure . In factt Figure , being et Figure .In conclusion, by using genome‐wide SNP and whole genome sequence data to reconstruct the adaptive history of a large “Pan‐Asian” set of populations, we provided evidence for selective sweeps and polygenic mechanisms having possibly contributed to the biological adaptation to rice‐ and/or millet‐based diets of people of Han/Tujia, Korean and Japanese ancestries. Actually, inferring the specific environmental or cultural stresses having triggered a given biological adaptation is not straightforward and we cannot rule out the possibility that other selective pressures in addition to those represented by cereal‐based diets have played a role in shaping the detected signatures of adaptive evolution. For instance, climate conditions differ between the geographical areas occupied by candidate and control populations, with temperate/continental climate being typical of northern China, northern Japan, and Korea in contrast to the subtropical or tropical climates of most Indian regions. Accordingly, some metabolic adaptations observed in the East Asian candidate populations might have been evolved also in response to a more energy demanding environment than that in which South Asian people live, as previously suggested for other adaptive loci (Sazzini et al., None declared.Supplementary MaterialClick here for additional data file.Table S1‐S2Click here for additional data file.


Aulopyge huegelii, a sole representative of its genus. Being highly distributionally restricted, it faces various threats that promote a critical decline in population abundance and even population extinction. Phenotypic and molecular diversity of the introduced  and natural  populations of Dalmatian barbelgudgeon from Bosnia and Herzegovina was studied by using two mitochondrial genes and morphometric traits (linear and geometric morphometrics). Nonparametric ANOVA showed that two analyzed populations significantly differed in six linear measurements, except snout length and postorbital head length. Contrary to centroid size, two populations were found to be significantly different in body shape. Deformation grids indicated that individuals from Studena River are characterized by wider and slightly shorter body comparing to individuals from Šator Lake. Incongruence in cytochrome c oxidase subunit I (COI) and cytochrome b (cyt b) mitochondrial DNA (mtDNA) variation was observed since a common COI haplotype was observed, while four and three cyt b haplotypes were registered in Šator Lake and Studena River, respectively. Since it was demonstrated that cyt b mtDNA was a faster evolving gene, we encourage its use in intraspecies studies, especially for evaluating the connectivity of fragmented populations and for studying the evolutionary footprint of the processes incorporated into the distinctive evolution of Aulopyge. Finally, findings herewith provide a firm basis for designing a long‐term sustainable conservation strategy for endemic species in Dinaric karst.The complex biogeographical history of the Balkan Peninsula caused remarkable freshwater fish diversity and endemism, among which Cyprinidae fish dominate. The Dinaric karst was a Pleistocene refugium and it harbors ancient and endemic cyprinids, including  Aulopyge huegelii, our results provide deeper insights into the freshwater fish biodiversity of one of the most famous karstic regions of the world.The manuscript deals with the issue of phenotypic and molecular diversity of the introduced  and natural  populations of Dalmatian barbelgudgeon from Bosnia and Herzegovina based on the use of two mitochondrial genes and morphometric traits (linear and geometric morphometrics). Given that the Dinaric karst was a Pleistocene refugium and it harbors ancient and endemic cyprinids, including  Thus, the species is assessed as Endangered B1ab according to IUCN (http://www.iucnredlist.org/details/61350/0). As a consequence, urgent habitat protection and population monitoring of the species were proposed  and Croatia   of the two populations, one of them was recently introduced in Šator Lake (Šator Mt), while the second was autochtonous population from Studena River (Duvanjsko Polje). The data on variation of the two mitochondrial genes was contrasting to evaluate their usefulness in conservation and forensics of this endangered fish species. Indeed, choice of appropriate molecular marker is a prerequisite for population discrimination and identification of divergent units. Given the power of DNA taxonomy in the biodiversity assessment, our additional goal was to fill the gap of DNA barcode database of the vulnerable ichthyofauna in the Mediterranean Hotspot region. Furthermore, regarding that phenotypic variation of A. huegelli has been studied based on linear morphometrics  and two mitochondrial genes  and four COI mtDNA sequences (one from Livno drainage in Bosnia and Herzegovina and three from Cetina River in Croatia) were downloaded from GenBank  and Studena River in Duvanjsko Polje  using sport fishing technique with a fishing net (“čerenac”). Sampling was conducted in 2015 in Šator Lake  by R. Dekić and M. Lukač, and in 2017 in Studena River  by J. Pavličević. Species determination was carried out using morphological keys  and Studena River  were analyzed. Digital images of the right lateral side were photographed with a Nikon D7100 digital camera with 50‐mm f/1.4D objective appointed on a tripod stand and positioned vertical to the surface of the object. Additionally, since the linear measurements and precise position for landmarks could not have been precisely determined on right‐side images, for six specimens left lateral side was captured and use in morphometric analyses. The scale factor is determined for each photo according to the millimeter scale before the digitization process.2.2.1M is the original morphometric measurement, Madj the size‐independent measurement, Ls the overall mean of SL for all fish in both population, Lo the SL of the fish, b the slope  of regression of logM on logLo calculated for both population. Correlation coefficients between adjusted size‐independent variables and SL were calculated in order to confirm that the size effect was eliminated and significant correlation was not found.Linear morphometric characteristics were measured (in mm) on digital images using measurement mode in tpsDig 2.30 Rohlf, . AccordiPrior to statistical analysis, a normal distribution test was performed (for each measure separately) on percentage ratios and size‐adjusted measurements data using both Shapiro‐Wilk test . Also, for matrix of size‐adjusted measurements, Doornik & Hansen omnibus test for multivariate normality was done.Nonparametric ANOVA  was used to determine differences in variances among two populations. Furthermore, principal component analysis (PCA) based on size‐adjusted measurements was performed. All statistical analysis on linear measures was executed in PAST 4.03 , the matrix of shape variables (Procrustes coordinates) was subjected to discriminant function analysis (DFA). The dependability of the discrimination among groups is assessed using leave‐one‐out (jackknifing) cross‐validation procedure. Finally, to estimate the extent of morphological variation of body shape, Procrustes distances between pairs of individuals and morphological disparity were calculated for each population. The morphological disparity was calculated as variance  of fish, a ‐ scaling coefficient, b ‐ length exponent (shape parameter for the body form). Model of LWR was transformed into linear type of data using logarithmic transformation :a) is the intercept and b is the slope of linear regression  of fish body we measured the weight of 11 and 13 individuals from Šator Lake and Studena River, respectively, using digital weighing scale KERN 440‐33 to the nearest 0.01 g. The relationship between standard length (in cm) and weight was estimated according to the formula Froese, :W=aLbwhLe Cren, .r2) that indicates reliability of regression model fit was calculated for each population separately. The value of parameter b shows the type of somatic growth pattern. Isometric growth is represented if b = 3 and allometric (positive or negative) if b ≠ 3. The b value was estimated for each population separately. To assess whether the obtained b values statistically differed from the isometric value (b = 3), t test was used  and following the manufacturer's protocol. The tissue and the extracted DNA were stored at −20°C. A fragment of cyt b mtDNA was amplified using Glu‐F (5′‐GAAGAACCACCGTTGTTATTCAA‐3′)/Thr‐R (5′‐ACCTCCRATCTYCGGATTACA‐3′) primer pair /HCO‐2198 (5′‐TTAAACTTCAGGGTGACCAAAAAATCA‐3′) primer pair  were obtained using MEGA X 10.0.5  for erroneously called bases, while sequence alignment was performed in BioEdit 7.2.5 Hall, . Haplotyθ , were based on the infinite‐allele model implemented in Arlequin version 3.11 , Hθ obtained from the observed homozygosity (H), Sθ obtained from the observed number of segregating sites (S = number of polymorphic sites) and πθ obtained from the mean number of pairwise differences (π). Kθ was estimated from the infinite‐allele mutation model equilibrium relationship between the expected number of haplotypes (K), the sample size (n) and θ using Ewens' sampling formula. Sθ Watterson's estimator is based on the infinite‐site mutation model relationship between the number of segregating sites (S), the sample size (n) and θ. Tajima's estimator of θ (πθ) is also based on the infinite‐site mutation model, but on the relationship between the mean number of pairwise differences (p) and θ.Estimates of p‐distances obtained from cytb mtDNA sequences) and phenotypic distances  Mantel's test with 10,000 permutations was applied.To assess the correlation of genetic distance (33.1p < .05). Also, multivariate normality test indicated significant deviation from normal distribution for size‐adjusted measurements .Univariate analysis of normality suggested that linear measures TL, SL, BD, and CPD, morphometric ratio OL(%HL) and all six size‐adjusted measures deviated from the normal distribution , PHL(%HL), BD(%SL), CPD(%SL)] were higher in Studena River population than values recorded in Šator Lake population. Furthermore, for all size‐adjusted measures, there is a gap in range values between two populations contrary to raw linear and morphometric ratio measures where ranges largely overlapped Table .adj (0.78).Nonparametric ANOVA performed on all types of linear measurements showed that two analyzed populations significantly differed in all but SNL(%HL) and PHL(%HL) Table . PCA on 3.2p < .05). Therefore, nonparametric ANOVA on centroid size was applied and showed no statistically significant differences between two populations . Multivariate regression of shape variables on centroid size showed no significant allometry (p = .34) and accounted for only 2.98% of the overall shape variation.Normality test showed that the size variable (centroid size) deviated from the normal distribution  that were separated with no overlap along discriminant axis .Discriminant function analysis conducted on Procrustes coordinates showed significant body shape difference between populations  and for Studena River is 2.922 (95% confidence limits: 2.443–3.401). The t test revealed statistically significant deviation from 3 for b value for population from Šator Lake (p < .05).The values of 3.4cyt b mtDNA haplotypes  were found among the 39 analyzed individuals (sequences obtained in this study will be submitted to GenBank after the manuscript acceptance)  ranged from 0.09% to 0.38%  Figure . There wes Table , divergies Table . Four haes Table  and 5. I8% Table .COI mtDNA fragment (652‐bp long) of 37 fish encompassed three haplotypes (HA‐HC) with two variable positions in total, resulting in p‐distance 0.15%–0.31%  and three (Studena River) distinct haplotypes, COI mtDNA showed a lack of variation in both populations. Hence, the highest θ values were observed for cyt b mtDNA in the Šator Lake sample (Table The genetic heterogeneity and discordance in variation of the two mtDNA genes in Šator Lake and Studena River is in agreement with the le Table .p‐distance) was found .Using the Mantel's test no significant correlation between morphology (body shape) and genetics (4COI and cyt b mtDNA), and linear and geometric morphometrics in quantification of variation in populations with different evolutionary histories  and selection regimes (under‐ and aboveground ecosystems of sinking Studena River and strictly aboveground environment of Šator Lake) of A. huegelii, the relict cyprinid evolutionary lineage.In this study, we tested the usefulness of molecular markers  and Croatia (Krka and Cetina Rivers)  was in line with the differences obtained for other population comparisons . On the contrary, COI mtDNA as a slowly evolving gene expressed lower variation within A. huegelii. For instance, individuals from Šator Lake and Studena River shared the same haplotype (HA), which was also registered in Cetina River  and HB (p‐distance: 0.31%).Interpopulation differentiation of the two genes was observed in our study in spite of the limited DNA data that was available. Indeed, just a few θ values as well. The genetic heterogeneity at cyt b mtDNA expressed by Kθ, Hθ and Sθ was slightly higher in Šator Lake compared to Studena River. However, introduced population possessed more cyt b haplotypes  than natural populations , which is in contrast to the number of private haplotypes . Since the Šator lake population shares the same haplotypes with Studena River (I), Buško Blato (III), and Krka River (V) we assumed that more than once Dalmatian barbelgudgeon has been introduced to the novel environment. Indeed, data on origin and number of introduced fish is still unknown  linear measurements. So far, linear measurements were used in study of phenotypic variation of the Dalmatian barbelgudgeon from Buško Blato , unlike the introduced trout species that did not manage to survive ; Formal analysis ; Investigation ; Methodology ; Visualization ; Writing‐original draft ; Writing‐review & editing . Ljubinka Francuski: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Visualization ; Writing‐original draft ; Writing‐review & editing . Milica Lukač: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Visualization ; Writing‐original draft ; Writing‐review & editing . Radoslav Dekić: Data curation ; Writing‐review & editing . Vesna Milankov: Conceptualization ; Funding acquisition ; Investigation ; Methodology (supporting); Project administration (lead); Resources ; Supervision (lead); Writing‐original draft ; Writing‐review & editing .Supplementary MaterialClick here for additional data file.